Conference Abstracts
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Basic Cardiovascular Sciences Conference 2008
Heart Failure: Molecular Mechanisms and Therapeutic Targets
Keystone Conference Center
I
Keystone, Colorado
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July 28 – 31, 2008
my.americanheart.org
Sponsored by the American Heart Association’s Council on Basic Cardiovascular Sciences and the Council on Clinical Cardiology and the American Heart
Association Interdisciplinary Working Group on Functional Genomics and Translational Biology. Co-sponsored by the National Heart, Lung, and Blood Institute.
e36
Circulation Research
Vol 103, No 5
August 29, 2008
BCVS Conference 2008 Abstracts
P1
Incremental Value of Tenascin-C and BNP Levels for Predicting Prognosis
in Patients with Dilated Cardiomyopathy and Decompensated Heart Failure
Michiaki Hiroe, International Med Cntr of Japan, Tokyo, Japan; Katsuya Onishi, Naoki
Fujimoto, Mie Univ, Tsu, Japan; Akira Sato, Tsukuba Univ, Tsukuba, Japan; Toshimichi
Yoshida, Kyoko Imanaka-Yoshida; Mie Univ, Tsu, Japan
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Backgrounds : TN-C is an extracellualr matrix glycoprotein sparsely detected in the healthy
adult heart, but becomes expressed in the pathological myocardium such as myocardial
infarction and some cases of DCM, closely associated with inflammation and tissue remodeling.
Recently, we reported that high serum TN-C levels in patients with acute myocardial infarction
or DCM indicate a greater incidence of ventricular remodeling. Objectives and Methods To
evaluate the prognostic value of TN-C, levels of TNC and plasma BNP, , a well known a strong
predictor of adverse outcome, of one hundred ten patients hospitalized with HF due to DCM
were meaured. After optimal therapy, the patients were discharged and forllowed-up for 22⫾4
months. Results The average LV ejection fraction on admission was 0.37⫾0.14. The average
TN-C and BNP levels were 73⫾38ng/ml and 279⫾414 pg/ml, respectively. After the follow-up
periods, there were 17 re-hospitalizations due to exacerbated HF. The receiver operating
characteristic (ROC) analyses showed that the optimal cut-off values of serum TN-C that
predicted the re-hospitalization due to HF was ⱖ78.4 ng/ml, and that of BNP was ⱖ 219pg/ml.
In multivariate Cox regression analysis, elevated TN-C and BNP levels had incremental
predictive power for re-hospitalization due to decompensated HF. When patients were divided
into four groups according to their serum TN-C and BNP levels, patients with TN-Cⱖ78.4 ng/ml
and BNPⱖ219 pg/ml had significantly higher re-hospitalization rates. Conclusions Combination
of predischarge TN-C and BNP levels is very useful to predict re-hospitalizations due to
decompensated HF.
P2
Effects of Post-Myocardial Infarction Exercise Training and L-Arginine
Treatment on Cardiac Oxidant/Antioxidant System in Rats
Xiaohua Xu, Weiyan Zhao, Shunhua Lao, Bryan S Wilson, Univ of Texas at San Antonio, San
Antonio, TX; Lisa L Ji, Univ of Texas Health Science Cntr at San Antonio, San Antonio, TX;
John Q Zhang; Univ of Texas at San Antonio, San Antonio, TX
Accumulating evidence suggests that increased oxidative stress and reduction in antioxidant
enzymes are involved in the pathophysiology of congestive heart failure subsequent to
myocardial infarction (MI). The objective of this study was to investigate the interaction of
exercise training and L-arginine or nitric oxide synthase inhibitor (NG-nitro-L-arginine methyl
ester, L-NAME) on cardiac oxidant/antioxidant system in rats following MI. L-arginine
(1g/kg/day) and L-NAME (10 mg/kg/day) were administered in drinking water for 8 weeks. The
animals were randomized to the following groups: sham-operated control (Sham); MI-sedentary
(Sed); MI-exercise (Ex); MI-sedentary⫹L-arginine (Sed-LA); MI-exercise⫹L-arginine (Ex-LA);
MI-sedentary⫹L-NAME (Sed-LN); and MI-exercise⫹L-NAME (Ex-LN). The rats were sacrificed
after 8 weeks of exercise training and treatments. Glutathione peroxidase (GPx), catalase and
gp91phox were comparable among all the groups at mRNA level. The mRNA level for
superoxide dismutase (SOD) was significantly increased in the Ex group (5.43⫾0.29) compared
to the Sed group (1.74⫾0.87, P⬍0.05), whereas this effect was pronouncedly down-regulated
by L-NAME intervention (P⬍0.05). The protein level for SOD in the Sed-LN group was
significantly decreased compared to the Sed group, and the level in the Ex-LN group was
robustly lower than in the Ex group as well (P⬍0.05). There were no significant differences for
GPx, catalase, gp91phox and SOD in sedentary groups or exercise training groups compared
with their L-arginine treatment counterparts at either mRNA or protein level. The collagen
volume fraction in the Ex group was significantly lower than in the Sed group (P⬍0.05).
Fractional shortening was significantly preserved in the exercise training groups compared to
their corresponding sedentary groups with or without drug treatments (P⬍0.05), whereas the
beneficial effect was not further improved by L-arginine treatment. Our results suggest that
exercise training exerts antioxidative effects, attenuates myocardial fibrosis, and preserves
cardiac function in the MI rats. In addition, treatment with L-arginine appears to be no effect
on the cardiac oxidant/antioxidant system, while L-NAME treatment may deteriorate it.
P3
Proteomic Analysis of SERCA2a in Setting of Heart Failure
Changwon Kho, Ahyoung Lee, Avelino Teixeira, Roger J Hajjar; Mount Sinai Sch of
Medicine, New York, NY
Heart failure is the leading cause of disability and death in the United States and the western
world. One of the hallmarks in human and experimental heart failure is depressed sarcoplasmic
reticulum (SR) Ca-cycling. Sarcoplasmic reticulum Ca2⫹-ATPase (SERCA2a) plays a major role
in the regulation of Ca2⫹-homeostasis in the cardiac cells. Indeed, gene transfer of SERCA2a
in experimental models of heart failure was associated with greatly enhanced cardiac
Ca-cycling and performance. However, the underlying molecular cause of cardiac dysfunction
and the cellular mechanism affected by SERCA2a in heart failure is still largely unknown. To
understand the cellular alterations in the expression or modification of myocardial proteins
during heart failure, we performed a comparative proteomic and interactome analyses on a
porcine animal model of heart failure in comparison to isogenic wild type, and heart failure pigs
following overexpression of SERCA2a with adeno-associated virus vectors (AAV). In particular,
to increase the detection coverage of the proteome, we applied with pre-fractionation methods
by sub-organelles or diverse pI ranges from heart tissue proteins. Each fractionated proteins
are grouped, separated with the two-dimensional gel electrophoresis (2-DE) analysis. We
detected 270 proteins with altered expression levels by the effect of heart failure and 379
proteins, which were changed by SERCA2a overexpression. Based on our proteomic results, we
suggest that numerous alterations in protein expression dependent upon heart failure or
SERCA2a may exist which maintain a complex functional relationship among proteins involved
in calcium handling, myofibrils, and cardiac energy production.
P4
Blockade of T-Type Ca2ⴙ Channel Corrects Imbalance in Cardiac
Autonomic Nerve Activity and Prevents Sudden Arrhythmic Death in
Chronic Heart Failure in Mice
Hideyuki Kinoshita, Koichiro Kuwahara, Shinji Yasuno, Masao Murakami, Yasuaki Nakagawa,
Satoru Usami, Masataka Fujiwara, Yoshihiro Kuwabara, Kenji Ueshima, Masaki Harada,
Kazuwa Nakao; Kyoto Univ, Kyoto, Japan
Background; Pharmacological interventions for the prevention of arrhythmia with chronic heart
failure (CHF) still remain limited. Accumulating evidence implicates increased cardiac
expression of T-type Ca2⫹ channels (TCC) in the progression of CHF. The efficacy of TCC
blockade in preventing ventricular arrhythmias associated with CHF has never been tested.
Methods and Results; We compared effects of efonidipine or mibefradil, dual T- and L-type
Ca2⫹ channel blockers, with those of nitrendipine, a selective L-type Ca2⫹ channel blocker,
on survival, arrhythmogenicity, and cardiac autonomic nerve activity in transgenic mice
expressing dominant-negative mutant of neuron-restrictive silencer factor in a cardiac
restricted manner (dnNRSF-Tg), an useful mouse model for CHF with lethal arrhythmias. We
revealed that efonidipine but not nitrendipine, corrected imbalance in cardiac autonomic nerve
activity. Efonidipine significantly improved survival among dnNRSF-Tg mice by preventing
arrhythmias. Arrhythmogenicity, evaluated in an electrophysiological study, was dramatically
reduced in dnNRSF-Tg mice treated with efonidipine or mibefradil. Conclusions; TCC blockade
reduced lethal arrhythmias in the mouse model of CHF with correcting the imbalance in cardiac
autonomic function. Our findings suggest therapeutic potential of TCC blockade to prevent
sudden death in patients with heart failure.
P5
Pathological Angiogenesis Concomitant with the Downregulation of
Tenomodulin and Upregulation of VEGF-A Causes Degeneration and
Rupture in Chordae Tendineae Cordis
Naritaka Kimura, Daihiko Hakuno, Keio Univ Sch of Medicine, Tokyo, Japan; Chisa
Syukunami, Yuji Hiraki, Kyoto Univ, Institute for Frontier Med Sciences, Kyoto, Japan;
Ryohei Yozu, Keiichi Fukuda; Keio Univ Sch of Medicine, Tokyo, Japan
[Introduction] Cardiac valves and chordae tendineae cordis (CTC) are avascular tissues,
although their avascularity is abrogated in several valvular heart diseases. We found that
tenomodulin (TEM), a chondromodulin-I-related anti-angiogenic factor, is expressed in normal
CTC. This study investigated whether the disturbance of the balance of TEM and other
angiogenic factors such as VEGF-A is related to the degeneration and rupture of CTC. [Methods
and Results] (1) Samples comprising 16 CTC were collected from 15 patients due with rupture,
and from 1 autopsy patient (8 males and 8 females, mean age; 62.4 ⫾ 12.6 y). For controls,
20 micro- and macroscopically normal mitral CTC were collected from 12 autopsied patients
(10 males and 2 females, mean age; 62.7 ⫾ 16.5 y). (2) TEM was restricted to the elastin-rich
mid-layer, but not in the other layers. Neither VEGF-A nor abnormal vessel formation was found
in the normal CTC. (3) The ruptured area contained abnormal vessels in the mid- and
core-layer. TEM was markedly down-regulated at the ruptured area, while in the remote area
(non-ruptured area) it was normally expressed. The TEM-defected area expressed VEGF-A. (4)
Cell numbers, vWF⫹ cell numbers, and the percentage of VEGF-positive area were markedly
increased and the percentage of TEM positive area was markedly decreased in the ruptured
area of CTC. (5) Expression of matrix metalloproteases (MMP-1,2,3,9,13) was not detected in
the normal CTC or in the non-ruptured area, but MMP-1,2,13 showed strong expression in the
ruptured area. Inflammatory cells positive for CD11b, CD14 and vimentin were infiltrated in the
ruptured area, but not in the normal CTC or the non-ruptured area. (6) CTC of tricuspid valve
was exhibited in anesthetized canine model, and the Tem rich layer was removed by filing.
Immunohistochemical analysis was performed at 1 and 3 months. VEGF, MMP1, MMP2 and
MMP13 were observed, and the expression areas reached approximately 15% and 37% of the
depth of the core layer, respectively. The abnormal neo-vessel formation was observed only at
3 months. [Conclusion] The development of angiogenesis in CTC correlated with the
down-regulation of TEM and with the up-regulation of VEGF-A and MMP expression, which
might be critically involved in the rupture of CTC.
BCVS Conference 2008 Abstracts
P6
miR-133b Is Downregulated in Human Heart Failure and Is Involved in
␤-Adrenergic Receptor-Mediated Activation of Hypertrophic Gene Program
Through Upregulation of Calmodulin
Carmen Sucharov; Univ of Colorado HSC, Denver, CO
miRNAs are small ⬃22 nucleotide regulatory non-coding RNAs that are key regulators of gene
expression programs. We have strong preliminary data that supports an important role for
miRNAs in the failing human heart in that our data show that miR133b is down-regulated in
human heart failure and is critical for ␤-Adrenergic Receptor (␤-AR)-mediated changes in gene
expression. Over-expression of miR-133b prevents changes in gene expression patterns
mediated by ␤-adrenergic receptor stimulation. ␤-AR stimulation of Ca2⫹-calmodulin Kinase II
(CaMKII), a kinase activated by the Ca2⫹-CaM pathway, is a key player in the onset of cardiac
hypertrophy, including changes in gene expression. Our preliminary data show that calmodulin
(CaM) is a target of miR133b, and down-regulation of miR133b results in the up-regulation of
CaM and phosphorylation of CaMKII.
miRNA Expression Profile in Patients Treated with ␤-Blocker Therapy
e37
(ESV)), and myocardial WT. Histopathology was performed at T6 to assess WT and infarct size.
MI induced a significant decrease on the E-UpV on the infarcted wall at T1 (13.2⫾0.7 vs.
6.4⫾0.5; p⬍0.01) and at T6 ( 6.2⫾0.5; P⬍0.01). Infarct size was 15.6%⫾ 3.6 and
overlapped those areas. Interestingly, even though the remote wall E-UpV was higher than the
infarcted wall at T1 (9.5⫾0.5; p⬍0.01) and at T6 (9.1⫾0.7; p⬍0.01), it was decreased
compared to baseline values (13.5⫾0.7, P⬍0.01). The baseline EDV (49⫾1 ml) and ESV
(23⫾1 ml) increased, respectively, at T1 (64⫾1 and 38⫾1; both p⬍0.01), and T6 (71⫾2 and
46⫾2; both p⬍0.01). The infarcted WT increased from T0 to T1 (p⬍0.01) and decreased from
T0 to T6 (p⬍0.01), while the remote WT increased over time (p⬍0.01).Gross pathology at T6
confirmed those findings. At T1, there was an inverse correlation between EDV and ESV with
remote wall E-UpV (r⫽-0.88; r⫽-87), and even stronger correlation was found at T6 (r⫽-0.89;
r⫽-89). Moreover, multiple linear analysis revealed that only LV volume predicted remote wall
E-UPV at T6 (p⬍0.05). Conclusion: These results demonstrate, for the first time, that LV volume
is an independent predictor of remote wall E-UpV in chronic settings of MI. If the results of this
study are extrapolated to patients, then the inverse correlation of LV volume and E-UpV may
reduce the diagnostic sensitivity of LV mapping criteria of myocardial damage if volumetric
changes are not taken into account.
P7
Carmen Sucharov, Brian Lowes, Michael Bristow, David Port; Univ of Colorado HSC, Denver,
CO
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miRNAs are ⬃22 nucleotide non-coding regulatory RNAs that are critical modulators of gene
expression programs. Recently miRNAs have been demonstrated to be essential regulators of
cardiac growth and development with distinct changes in their expression profile being
associated with a heart failure phenotype. Although the molecular basis of the progression of
heart failure has been studied extensively, its fundamental basis remains relatively elusive.
Equally elusive is the molecular basis of “response” to heart failure therapeutics. The BORG
Trial, which examined the response of patients with congestive heart failure to beta-blocker
therapies, affords a unique opportunity to examine serial changes in miRNA and gene
expression profiles in patients that responded (or not) to administered therapy. In this trial,
endomyocardial biopsies and cardiac performance (% Ejection Fraction) were obtained from
patients at three time points: baseline, and at 3 and 12 months post initiation of therapy. miRNA
expression profile was examined by miRNA array (Dharmacon, Sanger 10.1). Here we
demonstrate that miRNA expression profiles are markedly different in patients that respond to
beta-blocker therapy compared to the ones that do not. These results suggest that miRNAs are
not only important biomarkers for progression to and reversion from end stage heart failure,
they also likely target the expression of a number of genes that contribute to the progression
of the disease.
P8
Targeting p53 Against Heart Failure After Myocardial Infarction
Atsuhiko T Naito, Tohru Minamino, Sho Okada, Issei Komuro; Chiba Univ Graduate Sch of
Medicine, Chiba, Japan
Despite the significant therapeutic advances, heart failure after myocardial infarction remains
one of the leading causes of death and it is necessary to develop more efficient treatment. We
have recently reported that p53 plays a critical role in the development of heart failure and
proposed that inhibition of p53 in the heart may be a novel therapeutic strategy for heart failure.
To search the molecule(s) that suppress p53 activity in the heart, we performed expression
screening of human heart cDNA library and identified 5 genes. One cDNA was a chaperoneinteracting protein called STUB1 (Stip1 homology and U-box containing protein) that functions
as a ubiquitin ligase and promotes the degradation of misfolded proteins. We here show a
critical role of STUB1 in cardiac remodeling after myocardial infarction (MI). The in vitro
experiments revealed that STUB1 negatively regulates p53 expression by ubiquitinating and
inducing proteasome-mediated degradation. Abrogation of STUB1 increased p53 expression
both in vitro and in vivo, whereas activation of STUB1 prevented hypoxia-induced p53
up-regulation and p53-induced apoptosis. Upon hypoxic stress and myocardial ischemia,
expression of STUB1 was decreased, and p53 was up-regulated. Activation of STUB1 both
genetically and pharmacologically inhibited p53 accumulation in the MI heart and thereby
reduced p53-mediated apoptosis and cardiac remodeling. These results indicate that STUB1
plays an important role in the pathophysiology of heart failure after MI. Activation of STUB1 may
become a novel therapeutic strategy for heart failure.
P9
Left Ventricular Volume Predicts Remote Wall Endocardial Voltage in
Chronic Settings of Myocardial Infarction
Franca S Angeli, Kanu Chatterjee, Nicolas Amabile, Gina Orcino, Sukesh Burjonroppa,
William Grossman, Yerem Yeghiazarians; UCSF, San Francisco, CA
The extent of myocardial damage after infarction (MI) has been assessed in previously reported
studies by measurements of endocardial signals. It is also known that a direct relationship
exists between left ventricular (LV) volume and QRS amplitude and R wave voltage in acute
settings. The aim of this study is to examine the relationship between, myocardial ischemia,
LV volume, wall thickness (WT), and endocardial unipolar voltages (E-UpV) in chronic settings
of MI. Methods and Results: An antero-apical MI was induced by a 90 minute occlusion of the
left anterior descending artery in 12 pigs. LV E-UpV mapping was performed at baseline (T0),
1 week post-MI (T1), and at 6 weeks post-MI (T6) and 324 ⫹/-72 points were collected per
case. Echocardiography was used to assess volumes (end-diastolic (EDV) and end-systolic
P10
Cytokine Combination Therapy Leads to More Viable Myocardium and
Increased Vascular Density and Prevents Left Ventricular Remodeling in a
Porcine Model of Acute Myocardial Infarction
Franca S Angeli, Charles S Smith, Nicolas Amabile, Gina Orcino, Sarah Jahn, Sukesh
Burjonroppa, Mia Shapiro, Andrew Boyle, Stanton Glantz, Kanu Chatterjee, William
Grossman, Yerem Yeghiazarians; Univ of California San Francisco, San Francisco, CA
Cytokine therapy has been reported to improve left ventricular (LV) function after acute
myocardial infarction (MI) in animal models, but the mechanisms responsible for this benefit
are unclear. We investigated the impact of prolonged combination and monotherapy with
darbepoetin alfa (DARB) and granulocyte colony-stimulating factor (G-CSF) on LV function,
infarct size, and vascular density after MI in a porcine model. Methods and Results: MI was
induced in swine by a 90 minute balloon occlusion of the left anterior descending coronary
artery. Animals were randomized to: 1) combination therapy (IV bolus of DARB 0.9 ı̀g/kg and
G-CSF 10 ␮g/kg at time of reperfusion, followed by G-CSF 5 ␮g/kg/d SC from day 5 to 9, and
DARB 0.45 ␮g/kg SC weekly starting on day 1, n⫽8), 2) G-CSF monotherapy (n⫽8), 3) DARB
monotherapy (n⫽8), or 4) saline (control group, n⫽8). LV function was assessed by
echocardiography and pressure volume (PV) measurements at baseline, 1 and 6 weeks
post-MI. Histopathology was performed 6 weeks post-MI. Enzymatic infarct size was similar in
all groups as was LV ejection fraction (EF) at one week. At week 6, only combination therapy
stabilized LVEF and this was significantly higher compared to the control group (41⫾1% vs.
33⫾1%, p⬍0.01). PV measurements confirmed stabilization of LV function with combination
therapy compared to progressive decline in the control group and non-significant improvement
with DARB and G-CSF monotherapies. Positive dP/dt (1038⫾35 vs. 842⫾35 mmHg/s,
p⬍0.01) and stroke work (2088⫾114 vs. 1450⫾114 ml/mmHg, p⬍0.01) were higher, while
negative dP/dt (-890⫾34 vs. -1035⫾34 mmHg/s, p⬍0.01) and Tau (62⫾1 vs. 56⫾1 ms,
p⬍0.05) were lower in combination therapy animals than in controls. Furthermore, histopathology revealed increased areas of viable myocardium (82⫾5 vs. 67⫾4%), arteriole density
in the infarct zone (19⫾1 vs. 14⫾2 vessels/mm2, p⬍0.05), and capillary density at the infarct
border zone (1809⫾196 vs. 1248⫾83 vessels/mm2, p⬍0.05) in the combination therapy
compared to controls. Conclusion: DARB plus G-CSF combination therapy after acute MI leads
to more viable myocardium and increased vascular density, promoting stabilization of LV
systolic and diastolic function and improved indices of LV remodeling.
P11
Donor Aging and Myocardial Infarction Influence the Therapeutic Effect of
Bone Marrow Cells on the Infarcted Heart
Xiaoyin Wang*, Junya Takagawa*, Shereen A Saini, Robert Su, Richard E Sievers, William
Grossman, Yerem Yeghiazarians, Matthew L Springer; Univ of California San Francisco, San
Francisco, CA
Human and animal experiments involving delivery of bone marrow cells (BMCs) to infarcted
hearts have suggested that BMCs can have therapeutic effects. However, animal experiments
typically involve BMCs from young, healthy donors and may poorly represent clinical autologous
cell therapy, in which delivered cells are from older myocardial infarction (MI) patients. We
hypothesized that donor age would impair BMC therapeutic efficacy regardless of the age of
the recipient heart, and that MI in the donor would improve or impair efficacy. To test this, we
implanted BMCs obtained from healthy or infarcted mice of 3 different ages into infarcted
hearts under ultrasound guidance at 3 days post-MI, a time comparable to that used in clinical
trials. Recipient 10-week-young mice were subjected to MI by permanent left coronary artery
ligation. Donor BMCs were harvested from healthy mice or 3 day post-MI mice, at ages of 10
weeks (young), 1 year (middle-aged), and 2 years (old); with a vehicle control (HBSS). In all
groups, left ventricular ejection fraction (EF) declined from a baseline of 57⫾5% (SD) to a day
2 post-MI EF of 34⫾5%. Injection of HBSS permitted a continued decline of EF to 23⫾5% at
day 28 post-MI. Young and middle-aged healthy donor BMCs preserved EF (36⫾7% and
33⫾7%) at day 28, as did young infarcted donor BMCs (31⫾6%). EF in the middle-aged
infarcted donor group was marginally preserved (27⫾9%). Old healthy and old infarcted donor
BMC implantation allowed a decline in EF (24⫾4% and 21⫾4%) comparable to the negative
control, despite good viability in all BMCs. Our findings suggest that therapeutic efficacy of
donor BMCs from old and/or infarcted donors is impaired. * contributed equally
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Circulation Research
Vol 103, No 5
August 29, 2008
P12
Intracellular Paracrine Factors Mediate the Neovascularization and
Functional Improvement of Stem Cell Therapy Post Myocardial Infarction
Megha Prasad, Univ of California San Francisco, Morgan Hill, CA; Henry Shih, Yan Zhang,
Rich Sievers, Petros Minasi, Shereen Saini, Maelene Wong, Franca Angeli, William
Grossman, Matthew L Springer, Andrew Boyle; Univ of California San Francisco, San
Francisco, CA
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Background: Stem cells may improve cardiac function after myocardial infarction (MI). Cells
may release soluble factors responsible for tissue repair and functional improvement. Methods:
Mice with myocardial infarction (MI) induced by ligation of the left anterior descending artery
were divided into 3 groups: to receive ultrasound guided intramyocardial injection of either
BMCs (n⫽5), BMC lysate (lysate, n⫽5), or HBSS⫹0.5% FBS (HBSS, n⫽6) on the 3rd day post
MI. Six animals in each group were followed to day 28 for functional analysis by
echocardiography. The rest of the animals were sacrificed for histological analysis at Day 6 post
MI. An additional control group included an MI group with no injections (n⫽6). Sections from
the mid-papillary level were stained with antibodies against CD31 and alpha smooth muscle
actin. Images of the infarct border zone (BZ), the infarct zone (IZ) and the remote myocardium
(RM) were analyzed using Image Pro software to measure percentage vessel area. Arterioles
were counted in five high power fields in IZ, BZ and RM. Results: Echocardiography at Day 28
showed significantly improved ejection fraction (EF) for mBMC and lysate vs. HBSS (45.8% ⫹
7.9% and 40.5% ⫹ 4.44% vs. 33.9% ⫹ 9.93%. p⬍0.05) and smaller percentage scar size
(24.4%⫾7.3% and 24.3%⫾12.8% vs. 37.9% ⫾11.1% p⬍0.05). There was no significant
difference between mBMC and the lysate groups. At day 6, lysate injection increased vessel
area at IZ (12.7% ⫾0.04% vs. 4.4⫾0.03%, p⫽0.006) and BZ (12.2⫾3.8% vs. 4.3⫾1.1%,
p⫽0.001) vs. HBSS. Moreover, mBMC and lysate therapies had a significantly higher number
of arterioles in the BZ vs. HBSS (0.60⫾0.28 vs. 0.15⫾0.10, respectively, p⫽0.007; 0.55⫾0.10
vs. 0.15⫾0.10, p⫽0.006). Vessel counts did not differ between any of the treated vs. control
groups at RM. At day 28, neither vessel area, nor artery number was significantly different
between HBSS, Lysate and mBMC. Conclusions: Pre-formed intracellular components within
mBMC can enhance vascularity, reduce infarct size and improve cardiac function following MI.
Intact cells are not necessary for these effects. Further studies are necessary to identify the
factors responsible for these effects.
P14
Cardiomyocytes Can Be Generated from Parthenogenetic Embryonic Stem
Cells
Toshimi Kageyama, Shinji Makino, Takeshi Onizuka, Youhei Ohno, Ruri Kaneda, Shinsuke
Yuasa, Fumiyuki Hattori, Mitsushige Murata, Motoaki Sano, Keio Univ, Tokyo, Japan;
Tomohiro Kono, Tokyo Univ of Agriculture, Tokyo, Japan; Tomoyuki Tokunaga, National
Institute of Agrobiological Sciences, Tsukuba, Japan; Satoshi Ogawa, Keiichi Fukuda; Keio
Univ, Tokyo, Japan
Objective: The purpose of this study is to determine whether parthenogenetic ES (p-ES) cells
can differentiate into cardiomyocytes and to examine the effect of maternal imprinting.
Background: Parthenogenesis is the production manner of a new animal from a female
without the sexual involvement of the male, and is considered to be one of the methods to
produce an immuno-compatible ES cells. Methods and Results: The bi-maternal mouse
embryos were produced after the nuclear transfer of oocytes from fully grown (fg) and non
growing oocytes (ng). The ng oocyte genome can be considered ‘imprint free’. ES(wt),
p-ES(fg/fg) and p-ES(fg/ng) cells were established from reconstricted embryos. To induce
cardiomyogeneis, cells were enzymatically dissociated and cultured without LIF. About five
hundreds of hanging drops were created for evaluation of cardiac induction. At day8 culture
after differenciation, these attched embryo bodies started beating. ES(wt) has ability to
differentiate into cardiomyocyte (67%) at 14 day after induction. P-ES(fg/fg) and p-ES(fg/ng)
cells have lower ability to differentiate into cardiomyocyte (31% and 37% respectively). Heart
related mRNA was analyzed by RT-PCR. Nkx2.5, GATA4, MEF2C, ␣MHC, MLC-2a, MLC-2v were
also expressed in all cell lines. Conclusions: pES cell could differentiate to cardiomyocytes
regardless of genomic imprinting. However, ES(wt) cell could differentiate into cardiomyocytes
easier than pES(fg/fg) and pES(fg/ng).
P15
Nkx2–5 Transcriptionally Activates c-kit-ligand and Regulates Cardiac
Progenitor Cell Populations
Rebecca L Scotland, Xiaozhong Shi, Anwarul Ferdous, Michael Kyba, Daniel J Garry; Univ of
Minnesota, Minneapolis, MN
P13
Effects of Single versus Multiple Injections of Bone Marrow Cell Therapy
on Left Ventricular Function After Acute Myocardial Infarction
Yerem Yeghiazarians, Yan Zhang, Richard E Sievers, Maelene L Wong, Junya Takagawa,
Petros Minasi, Jianqin Ye, Andrew Boyle, Matthew L Springer, William Grossman; Univ of
California, San Francisco, CA
Bone marrow cell (BMC) treatment has been proposed as an effective therapy for ischemic
heart disease. To date, no animal or clinical trial has evaluated the efficacy of multiple BMC
injections compared to monotherapy post myocardial infarction (MI). This study examined both
the importance of timing of BMC therapy and whether multiple injections of BMCs would be
more beneficial than mono-therapy in the mouse MI model. MI was induced in male mice
(10 –12 wk) by permanent mid-LAD ligation. Ultrasound-guided injection of mouse BMCs
(106/10␮l) to the peri-infarct zone was performed (n⫽8/group) at different time points post-MI
(day 3, 7, and 14) as monotherapy, and at days 3⫹7 as “double” and days 3⫹7⫹14 as
“triple” therapy. Control group was injected with vehicle at days 3⫹7⫹14. Left ventricular
ejection fraction (LVEF) was evaluated at day 2 before injection and at day 28 post-MI. LVEF
was uniformly reduced from 56.1⫾0.9% to 37.5⫾1.7% (p⬍0.001) 2 days post-MI in all
groups (Fig.). Injection of BMCs at day 3, days 3⫹7, and days 3⫹7⫹14 led to similar and
significant improvements in LVEF at day 28 compared to day 2 and to the control group
(p⬍0.05). There were no significant differences between these three groups at day 28.
Delaying the injection time to day 7 or day 14 post-MI did not improve LV function compared
to the controls. In the mouse MI model, multiple injections of BMC did not have an additive
effect on improving LVEF post-MI compared to a single therapy at day 3. However, further
delaying the time of cell therapy post-MI resulted in worse outcomes. There appears to be a
critical time window post-MI during which cell-based therapies might be effective. This will
need to be addressed by future trials.
C-kit-ligand, also known as stem cell factor, is expressed broadly and has a functional role
during hematopoesis, gametogenesis, melanogenesis, mast cell growth and differentiation.
Although the receptor for c-kit-ligand, c-kit, has been utilized as a marker to identify cardiac
stem cell and progenitor cell populations, the transcriptional regulation and biological function
of c-kit-ligand during cardiogenesis has not been defined. Here we demonstrate that
c-kit-ligand is a novel downstream target of Nkx2–5. The homeodomain transcription factor,
Nkx2–5, is one of the earliest markers of the cardiac lineage and mice lacking this transcription
factor are nonviable. To identify potential Nkx2–5 downstream target genes, we utilized ES/EBs
that were engineered to overexpress Nkx2–5 and undertook transcriptome analysis of
embyroid bodies with and without Nkx2–5 induction. We observed a significant increase in
c-kit-ligand expression following Nkx2–5 induction suggesting a role for Nkx2–5 in the
activation of c-kit-ligand. Furthermore, analysis of the c-kit-ligand promoter revealed three
evolutionarily conserved Nkx2–5 response elements, supporting the notion that Nkx2–5 is a
transcriptional regulator of gene expression. We undertook transcriptional assays and
transfected the c-kit-ligand promoter-luciferase reporter in the absence and presence of
increasing amounts of Nkx2–5. We observed that Nkx2–5, in a dose dependent fashion, was
a potent transcriptional activator of c-kit-ligand. These studies enhance our understanding of
Nkx2–5 mediated transcriptional networks and further emphasize that Nkx2–5 is an important
transcriptional regulator of cardiac progenitor cell populations.
P16
Paradox Effect of MMP-2 in Experimental Viral Myocarditis
Dirk Westermann, Kostas Savvatis, Heinz-Peter Schultheiss, Cartsen Tschöpe; Charite,
Berlin, Germany
Increased activity of matrix metalloproteinases (MMPs) disturb the integrity of the extracellular
matrix in different cardiomyopathies and it was shown that increased MMP levels in cardiac
BCVS Conference 2008 Abstracts
failure are delirious after myocardial infarction or pressure overload cardiomyopathy. Therefore,
inhibition of MMPs is generally supposed to be beneficial. Nevertheless, the role of MMP-2 in
vivo is unknown. To further investigate the potential role of MMP-2 in inflammatory
cardiomyopathies, we used a viral induced myocarditis model, which is characterized by
invading inflammatory cells and cytokines, and investigated the effect of gene deletion of
MMP-2 after viral myocarditis on cardiac inflammation and cardiac function utilizing knock out
mice. Methods: 80 MMP-2 knockout mice and their wildtypes littermates were used in the
current study. 40 were infected with coxsackievirus B3 (CVB3) to induced acute murine
myocarditis. We investigated the LV function of all mice after 7 days using PV measurements
by conductance catheter in vivo. Myocardial inflammation was analyzed by immunohistochemical detection of inflammatory cells and by measuring the protein and mRNA abundance of
inflammatory cytokines. Cardiac apoptosis was analyzed by TUNEL staining and by measuring
the protein content of bax and bcl-2. Results: Interestingly, MMP-2 gene deletion aggravated
cardiac function after CVB3 myocarditis compared to their CVB3 wildtype littermates, which
was further accompanied with highly increased virus load in the KO mice. This was
accompanied by increased cardiac inflammation with highly increased numbers of inflammatory cells (⫹750% in MMP-2 vs. WT) as well as highly increased abundance of cardiac
cytokines (TNF-a: ⫹350%, IL1-b: ⫹550%) in the cardiac tissue. We measured increased
apoptosis in the cardiac tissue in both knock out mice strains. Moreover, MMP-2 KO mice
showed highly increased mortality after induction of myocarditis compared to WTs. Conclusion:
During viral myocarditis, gene deletion of MMP-2 aggravates cardiac injury. These data suggest
that MMP-2 has a beneficial effect in viral induced cardiomyopathy and might be necessary for
virus control in myocarditis, which is in contrast to their delirious role in non viral induced
cardiomyopathies.
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
␣-Dystroglycan Dissociation and ␣7 Integrin Expression Are Associated
with Myocardial Signal Activation in a Muscular Dystrophy Model of
Dilated Cardiomyopathy
P17
P19
Apoptosis in Acute Myocarditis: Friend or Foe?
Antonio Abbate, Virginia Commonwealth Univ, Richmond, VA; Gianfranco Sinagra, Rossana
Bussani, Univ of Trieste, Trieste, Italy; Nicholas N Hoke, Elena Barresi, Virginia
Commonwealth Univ, Richmond, VA; Furio Silvestri, Univ of Trieste, Trieste, Italy; George W
Vetrovec, Rakesh C Kukreja, Alfonso Baldi; Virginia Commonwealth Univ, Richmond, VA
Background. Recent data have shown the presence of apoptosis in acute myocarditis. To
better characterize the presence and extent of apoptosis, we studied patients with acute
myocarditis undergoing autopsy or endomyocardial biopsy (EMB) to correlate pathology with
clinical findings. Methods. Thirty-three cases of acute myocarditis were studied: 15 fatal at
autopsy (Group 1), and 18 cases by EMB samples (Group 2). Four subjects dying of non-cardiac
causes were selected as controls (Group 3). At autopsy, sections were taken from areas
showing signs of acute inflammation and necrosis (affected areas) as well as unaffected areas.
Echocardiography was performed in 8 subjects from Group 2 on the same day of EMB and at
12-month follow up. Co-staining for DNA fragmentation (ApopTag) and caspase-cleaved
cytokeratin-18 (CytoDeath) was used to detect apoptotic cells. Results. The apoptotic rate
(expressed as % of double positive cardiomyocytes on total number per field) from the affected
areas of patients in Groups 1⫹2 was significantly higher than that of Group 3 (1.4% [0.6 –7.8]
vs 0.01% [0.01– 0.01], P⬍0.001), without any significant difference between the autoptic
(group 1) and bioptic (group 2) samples (P⫽NS). In Group 1 patients, we found no differences
in apoptotic rates comparing the affected and unaffected areas in the left ventricle (P⬎0.05).
In Group 2 patients, apoptotic rates were inversely correlated with NYHA class (R⫽-0.76,
P⫽0.028) and signs of adverse remodeling at baseline and at 12-month follow up (Table).
Conclusions. This study confirms the presence of apoptosis in patients with active myocarditis,
and shows that apoptosis is a diffuse process, not limited to the areas of concomitant necrosis.
The inverse correlation between apoptosis and acute and chronic indices of left ventricular
remodeling and heart failure suggests that apoptosis may represent a protective mechanism in
the heart during acute myocarditis.
Baseline
Stephen Armstrong, Melody A Scott, Kelly Graber; Cardiovascular Rsch Cntr, Sioux Falls, SD
Background: Dystrophin (DYS) associates with a protein complex (DAPC) which includes: 1)
the extracellular, laminin (LMN) binding protein, ␣-dystroglycan (␣DG); 2) the transmembrane
protein, ␤-dystroglycan (␤DG) and the sarcoglycan (SG) complex. ␦SG deficiency manifests as
Limb Girdle Muscular Dystrophy 2F, with dilated cardiomyopathy (DCM) occurring in patients.
A ␦SG deficient hamster (TO2) was identified as a DCM model, in which SG complex
disassembly is observed. Hypothesis: This study addressed the hypothesis that ␣DG/LMN
dissociation reverses an inhibition of integrin mediated, myocardial signal activation. Methods:
We utilized echocardiography and histology to assess the development of cardiomyopathy.
Confocal microscopy of myocardium was employed to examine the co-localization of DAPC
components. Myocardial protein expression and kinase phosphorylation was determined by
immuno-blotting studies. Results: Dilated cardiomyopathy and fibro-necrosis were observed in
the TO-2 group. A high level of ␣DG was observed in myocardial lysates of the F1B control
group, as determined by immuno-blotting. ␣DG expression was abrogated in the TO2 group.
In contrast to the absence of ␣DG, ␣SG and ␦SG; a maintenance of ␤DG, dystrophin and
caveolin-3 was observed in the TO2 group. ␣DG/␤DG; ␣DG/LMN; ␣DG/Dys and ␣DG/Cav3
co-localization was observed as a punctate distribution at the sarcolemma, in the F1B group.
These co-localization events were eliminated in the TO2 group. We observed increased
expression of the LMN receptor, ␣7/␤1 integrin. We demonstrated enhanced phosphorylation
of the MAP kinase cascade: Raf, MEK and ERK; in the TO2 model, as compared to the F1B
group. Increased phosphorylation of: Akt Tyr326, GSK3, JNK, FAK Tyr397 and Src were
observed in the TO2 group. Conclusion: We propose that ␣DG dissociation reverses an
inhibition of signal transduction pathways. ␣DG dissociation and kinase activation was
accompanied by increased expression of the ␣7/␤1 integrin complex, presumably as a
compensatory response. This study provides the first description of increased myocardial
␣7/␤1 integrin expression in a DCM or muscular dystrophy model. We propose that increased
expression of the ␣7/␤1 integrin complex initiates signal activation.
P18
BDNF Protects Cardiac Remodeling After Myocardial Infarction
End-diastolic diameter
End-diastolic volume
End-systolic diameter
End-systolic volume
Ejection fraction (%)
12-month follow up
R
P
R
P
-0.77
-0.77
-0.84
-0.74
⫹0.18
⬍0.05
⬍0.05
⬍0.01
⬍0.05
⬎0.10
-0.80
-0.92
-0.69
-0.78
⫹0.67
⬍0.05
⬍0.001
0.070
⬍0.05
0.068
P20
Interleukin-1 Receptor Antagonist Inhibits Caspase-1-Mediated
Cardiomyocyte Apoptosis
Antonio Abbate, Virginia Commonwealth Univ, Richmond, VA; Aldo Dobrina, Elena Vecile,
Univ of Trieste, Trieste, Italy; Fadi N Salloum, Anindita Das, Nicholas N Hoke, George W
Vetrovec, Rakesh C Kukreja; Virginia Commonwealth Univ, Richmond, VA
Background. Interleukin-1 receptor antagonist (IL-1Ra) is a naturally occurring IL-1 antagonist
synthesized by cardiomyocytes in response to ischemia known to bind the IL-1 membrane
receptor. Caspase-1 (or IL-1 converting enzyme) is a key mediator in IL-1beta synthesis as well
as in the apoptotic cascade. We hypothesize that IL-1Ra may inhibit caspase-1 activity and
down-regulate inflammation and apoptosis. Methods and Results. HL-1 cardiomyocytes were
used in vitro to test for cellular uptake of recombinant human (rh-)IL-1Ra in normoxic and
hypoxic conditions. Thrity-five % of cardiomyocytes showed uptake of fluorescein marked
IL-1Ra in normoxic conditions vs 95% in hypoxic conditions (P⬍0.001). An immune
co-precipitation assay and in vitro caspase-1 enzymatic activity assay were used to detect
physical interaction between rh-IL-1Ra and caspase-1. The co-precipitation assay documented
the presence of a physical interaction and binding between IL-1Ra and caspase-1. Caspase-1
activity was inihibited by approximately 50% when incubated with anakinra (100 nM), this
effect was reversed adding also an anti-IL-1Ra blocking antibody. The anti-apoptotic effect of
rh-IL-1Ra in vitro was tested by incubating cells from a primary rat cardiomyocyte culture in
hypoxia ⫹ ‘ischemia’ buffer, and evaluating the effects of adding rh-IL-1Ra (2.5x10-12 g/ml).
Incubation with rh-IL-1Ra was associated with a significant 36% reduction in apoptosis
(11.2%⫾0.5 vs 17.5%⫾0.1, P⬍0.001). Conclusions. These data show for the first time direct
inhibition of caspase-1 activity by rh-IL-1Ra associated with a significant reduction in
cardiomyocyte apoptosis in response to simulated ischemia, opening the way to the use of
rh-IL-1Ra to prevent ischemic myocardial damage.
Sho Okada, Tohru Minamino, Haruhiro Toko, Jun-ichiro Nishi, Masataka Yokoyama, Yosuke
Kayama, Issei Komuro; Chiba Univ Graduate Sch of Medicine, Chiba, Japan
Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, was originally
found to have neurotrophic functions during the embryonic development. Prior studies showed
that systemic disruption of BDNF led to an early postnatal death due to microvascular leakage
in the heart, suggesting that BDNF may be involved in cardiovascular physiology, but the role
of BDNF in the adult heart is largely unknown. Here we show a protective role of BDNF in
cardiac remodeling after myocardial infarction (MI). We generated conditional BDNF knockout
(CKO) mice, in which expression of BDNF was systemically reduced, by using the inducible
Cre-loxP system and produced the MI model. Two weeks after operation, systolic function was
significantly impaired and cardiac size was markedly increased in the CKO mice compared with
control mice. Likewise, deletion of the BDNF receptor TrkB in the heart exacerbated cardiac
dysfunction after MI. Serum levels of BDNF were markedly increased after MI and this increase
was associated with up-regulation of BDNF in the brain but not in the heart. A high-fat diet
significantly reduced neuronal expression of BDNF and therefore more markedly induced
cardiac remodeling after MI compared with normal diet. Treatment of the mice on the high-fat
diet with BDNF significantly improved cardiac dysfunction after MI, suggesting an intriguing
mechanism underlying cardio-protection conferred by BDNF.
e39
P21
GATA4 Inhibits Doxorubicin-Induced Autophagy and Protects from
Cardiomyocyte Death
Satoru Kobayashi, Paul A Volden, Kai Mao, Tricia Krueger, Troy Lackey, Qiangrong Liang;
Cardiovascular Rsch Cntr, Sioux Falls, SD
Doxorubicin (DOX) is a potent anti-tumor drug known to cause heart failure after chronic
chemotherapy. The transcription factor GATA4 regulates the expression of many cardiacspecific genes and is essential for various adaptive responses of cardiomyocytes. GATA4 has
been shown to protect cardiomyocytes from DOX toxicity. However, the down stream
mechanisms that mediate GATA4 pro-survival effect remain partially understood. Autophagy is
the primary cellular pathway for lysosomal degradation of long-lived proteins and organelles.
Accumulating evidence suggests increased autophagic activity as a double-edged sword that
could be either protective or detrimental depending on the nature of the stimuli and the levels
of autophagy induced. Here we investigated the ability of GATA4 to regulate autophagy as a
potential mechanism underlying its protection against DOX toxicity in cultured neonatal rat
ventricular cardiomyocytes (NRVC). Our results show that DOX dramatically increases
e40
Circulation Research
Vol 103, No 5
August 29, 2008
autophagic flux in NRVC as indicated by the GFP-LC3 (microtubule-associated protein light
chain 3) reporter and the protein levels of p62 or LC3 form II. The enhanced autophagy is
associated with DOX-induced NRVC death as determined by propidium iodide staining, DNA
laddering and poly (ADP-ribose) polymerase cleavage. DOX-induced myocyte death is
aggravated by rapamycin, an mTOR inhibitor that activates autophagy, and beclin1, a protein
required for autophagy initiation. Conversely, DOX toxicity is markedly attenuated by either
autophagy inhibitor 3-methyladenine or beclin 1 gene silencing, suggesting enhanced
autophagy as a contributor to DOX-elicited cardiomyocyte death. DOX treatment depletes
GATA4 protein levels, which may predispose NRVC to DOX toxicity. Indeed, GATA4 gene
silencing increases DOX-induced NRVC death, whereas adenovirus-mediated gene delivery of
GATA4 effectively inhibits DOX-induced autophagy, which is accompanied by reduced
cardiomyocyte death. Together, these findings suggest that activation of autophagy is a novel
mechanism responsible for DOX cardiotoxicity, and the protective effect of GATA4 on
DOX-induced myocyte death is mediated, at least in part, by its ability to inhibit autophagic
activity.
P22
Molecular Identification and Characterization of a Novel Gene that
Regulates Endothelial Cell Apoptosis and Angiogenesis
Koji Ikeda, Hiroaki Matsubara; Kyoto Prefectural Univ of Med, Kyoto, Japan
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Therapeutic angiogenesis is a plausible approach to treat not only ischemic heart diseases but
also heart failure due to maladaptive cardiac hypertrophy. Although administration of growth
factors as well as transplantation of endothelial progenitor cells have been clinically employed
to treat ischemic cardiovascular diseases, their effects were considerably limited so far. To
identify novel genes regulating angiogenesis, we have performed signal sequence trap
screening using cDNA from human microvascular endothelial cells. We successfully identified
a novel gene (clone57) that regulates endothelial apoptosis as well as angiogenesis. Clone57
was expressed in a variety of human cultured endothelial cells as well as in blood vessels of
e9.5d mouse embryos, suggesting its role in angiogenesis. Amino acid sequence analysis
revealed that clone57 was a novel membrane protein with no homology to conserved domain
structures reported before. Knockdown of clone57 in HUVEC using short interference RNA
resulted in significant reduction of endothelial apoptosis. In contrast, clone57 knockdown did
not affect either endothelial cell migration or proliferation. Among factors associated with
apoptosis, inhibitor of apoptosis (cIAP)-1, and cIAP-2 expression was significantly increased in
HUVEC by clone57-knockdown. Simultaneous knockdown of cIAP-1 and cIAP-2 abolished the
anti-apoptotic effect of clone57-knockdown. In vitro tube-formation of HUVEC on Matrigel was
reduced by clone57-knockdown at early time (12h). Nevertheless, significantly more tubes
were preserved at late time (96h) by cloen57-knockdown. Moreover, in vivo angiogenesis
assessed by Matrigel-plugs implanted into mouse flanks was significantly enhanced by
clone57-knockdown. Taken together, clone57 is a novel factor regulating endothelial apoptosis
as well as angiogenesis. Detailed analysis of clone57 will provide invaluable information for the
development of effective therapeutic angiogenesis to treat ischemic and/or nonischemic heart
failure.
P23
Inhibition of Endotoxin-Triggered Myocardial Apoptosis and Dysfunction by
Hsp20 Is Associated with Reduced NF-␬B Activation
Xiaohong Wang, Univ of Cincinnati College of Medicine, Cincinnati, OH; Basilia Zingarelli,
Michael O’Connor, Cincinnati Children’s Hosp Med Cntr, Cincinnati, OH; Guoli Chen,
Evangelia G Kranias, Guo-Chang Fan; Univ of Cincinnati College of Medicine, Cincinnati, OH
Cardiac apoptosis plays a critical role in sepsis-induced myocardial dysfunction, which is
considered to be among the major complications in septic patients. A small heat-shock protein,
Hsp20, has been shown to protect cardiomyocytes against apoptosis, induced by ischemia/
reperfusion injury. To investigate the possible protective role of Hsp20 in sepsis-mediated
cardiac injury, we overexpressed Hsp20 in cultured adult rat cardiomyocytes, followed by
addition of endotoxin (LPS, 1 ␮g/ml, 24 h). Adenovirus-mediated overexpression of Hsp20
significantly increased contractility (FS%: AdHsp20, 10.2 ⫾ 0.8 vs. AdGFP, 7.3 ⫾ 0.9, n⫽3
hearts, 15–20 cells/heart for each group, p⬍0.05), and greatly reduced the degree of apoptosis
upon LPS treatment. Furthermore, transgenic mice over-expressing Hsp20 in the heart (TG) and
wild-type littermate controls (WT) were challenged with LPS (25 ␮g/g, ip). Six hours after LPS
exposure, cardiac function, assessed by Langendorff perfusion, revealed depressed left
ventricular developed pressure (LVDP), as well as rates of contraction (⫹dP/dt) and relaxation
(-dP/dt) in both LPS-treated WT and TG mice, however, the decreases were more prominent
in WTs than in TGs (n⫽8, p⬍0.01). The hearts of LPS-treated WTs, but not those of
LPS-treated TGs, displayed increases in the number of TUNEL-positive nuclei and the cleaved
form of caspase-3. In addition, LPS administration resulted in activation of NF-␬B in WT-hearts,
which was significantly attenuated in TG-hearts, determined by electrophoretic mobility shift
assay (EMSA) and Western blots. Accordingly, the proinflammatory cytokines IL-1␤ and TNF-␣
were greatly reduced in myocardial and serum from LPS-treated TG mice, compared with those
of controls (n⫽5, p⬍0.01). Importantly, overexpression of Hsp20 did not alter the expression
levels of other major Hsps (Hsp90, Hsp70, Hsp60, Hsp27, and ␣B-crystallin) either in
LPS-treated or saline-treated hearts. Thus, the sole increase in Hsp20 levels can lead to
protection against LPS-induced cardiac apoptosis and dysfunction, and these beneficial effects
appear to be associated with inhibition of NF-␬B activity.
P24
ASK1 Regulates Mitochondrial Permeability Transition in Necrotic Cell
Death
Osamu Yamaguchi, Toshihiro Takeda, Kinya Otsu; Osaka Univ Graduate Sch of Medicine,
Suita, Japan
Cardiomyocyte death is a critical process in the pathogenesis of heart failure. Although
apoptotic cardiomyocyte death has been thought to be a major cause of cardiomyocyte injury
in the disease, a recent report implies the possible involvement of necrotic cell death in the
pathogenesis. Cyclophilin D-dependent mitochondrial permeability transition (MPT) mediates
necrotic cell death. However, intracellular signaling pathways connecting necrotic stimuli, such
as oxidative stress and Ca2⫹, with MPT have yet to be clarified. Here, we show that reactive
oxygen species-sensitive apoptosis signal-regulating kinase 1 (ASK1) interacts with cyclophilin
D in mitochondria to induce MPT. Cyclophilin D-deficient mice showed significantly better
cardiac function 4 weeks after treatment with pressure overload when compared with wild-type
control mice. H2O2 treatment induced MPT in wild-type cardiomyocytes, but did not in
cyclophilin D- or ASK1-deficient cardiomyocytes. Overexpression of a constitutively active
mutant of ASK1 (ASK⌬N) resulted in a loss of mitochondrial membrane potential in wild-type
cardiomyocytes, but not in cyclophilin D-deficient cells. H2O2 treatment led to the recruitment
of GFP-tagged ASK1 to mitochondria. Immunoprecipitation analysis revealed the interaction of
ASK1 with cyclophilin D and activation of ASK1 by H2O2 enhanced the protein interactions.
Furthermore, cyclophilin D ablation attenuated left ventricular contractile dysfunction in
transgenic mice expressing ASK⌬N in the heart. Thus, ASK1 is a key signaling molecule
connecting necrotic stimuli with cyclophilin D-dependent MPT, which leads to necrotic cell
death and cardiac injury.
P25
Autophagy Is an Adaptive Response in Desmin-Related Cardiomyopathy
Lan Nguyen, Paul Tannous, Hongxin Zhu, Janet L Johnstone, John M Shelton, Univ of Texas
Southwestern Med Ctr, Dallas, TX; Raj Soorappan, Ivor J Benjamin, Univ of Utah Sch of
Medicine, Salt Lake City, UT; Robert D Gerard, Beth Levine, Beverly A Rothermel, Joseph A
Hill; Univ of Texas Southwestern Med Ctr, Dallas, TX
Background: A missense mutation in the aB-crystallin (CryAB) gene triggers a severe form of
desmin-related cardiomyopathy (DRCM) characterized by accumulation of misfolded proteins.
We hypothesized that autophagy increases in response to protein aggregates and that this
autophagic activity is adaptive. Methods and Results: Mutant CryAB (CryABR120G) triggered a
⬎2-fold increase in cardiomyocyte autophagic activity, and blunting autophagy increased the
rate of aggregate accumulation and the abundance of insoluble CryABR120G-associated
aggregates. Cardiomyocyte-restricted over-expression of CryABR120G in mice induced intracellular aggregate accumulation and systolic heart failure by 12 months. As early as 2 months
(well before the earliest declines in cardiac function), we detected robust autophagic activity.
To test the functional significance of autophagic activation, we crossed CryABR120G mice with
animals harboring heterozygous inactivation of beclin 1, a gene required for autophagy.
Blunting autophagy in vivo dramatically hastened heart failure progression with a 3-fold
increase in interstitial fibrosis, greater accumulation of polyubiquitinated proteins, larger and
more extensive intracellular aggregates, accelerated ventricular dysfunction, and early
mortality. Conclusions: This study is the first to report autophagic activity in DRCM. Further, our
findings point to autophagy as an adaptive response in this proteotoxic form of heart disease.
P26
Phospholamban Knockout Alters Hypertrophic Gene Expression and
Improves Cardiac Function in a HCM ␣-Tropomyosin (Glu180Gly) Mouse
James R Pena, Paul H Goldspink, Lynley S Heinrich, Univ of Illinois at Chicago, Chicago, IL;
Evangelia G Kranias, David F Wieczorek, Univ of Cincinnati, Cincinnati, OH; Beata M Wolska;
Univ of Illinois at Chicago, Chicago, IL
Hypertropic cardiomyopathy (HCM) has been linked to numerous mutations in the contractile
proteins and is known as a disease of the sarcomere. We have previously shown that
transgenic mice expressing mutated ␣-tropomyosin (␣-TM(Glu180Gly)) develop severe cardiac
hypertrophy and diastolic dysfunction that is associated with increased myofilament sensitivity
to Ca2⫹ (Prabhakar et al JMCC, 33(10), 1815–1828, 2001). We hypothesize that long term
improvement of Ca2⫹ regulation by phospholamban (PLB) knockout (KO) may attenuate or
eliminate the development of hypertrophy and cardiac dysfunction in TG-␣TM(Glu180Gly) mice.
Four groups of mice were generated that 1) express normal levels of PLB and either express
mutated TM (PLB/␣TM(Glu180Gly)) or wild type TM (PLB/␣TM) or 2) do not express PLB
(PLBKO) and either express mutated Tm (PLBKO/␣TM(Glu180Gly)) or wild type TM (PLBKO/
␣TM). At 3– 6 months of age baseline contractility was significantly lower in PLB/
␣TM(Glu180Gly) (Emax⫽9.4⫾0.8 mmHg/␮l) compared to PLBKO/␣TM(Glu180Gly) (Emax ⫽
19.2⫾ 2.1 mmHg/␮l), PLBKO/␣TM (Emax ⫽ 25.2 ⫾4.9 mmHg/␮l), and PLB/␣TM (Emax ⫽
21.9⫾2.8 mmHg/ı̀l) mice. There was no difference in baseline contractility between the
PLB/␣TM and both PLBKO/␣TM and PLBKO/TG-␣TM(Glu180Gly) mice. The baseline minimum
dP/dt was significantly higher in the PLBKO/␣TM mice compared to the other three groups.
Both at 3– 6 months and 1 year of age heart to body weight ratio (HW/BW) and expression
levels of ANF and ␤-MHC were significantly higher in PLB/␣TM(Glu180Gly) compared to other
groups as determined by quantitative RT-PCR. However, HW/BW and the expression of ANF and
␤-MHC were significantly decreased in PLBKO hearts expressing mutated TM. Moreover, PLB
levels were significantly higher at 1 year of age than at 3 months in the PLB/␣TM and
PLB/␣TM(Glu180Gly) groups. Our data strongly suggest that PLB may be a useful therapeutic
target in HCM models in which the primary defect results in diastolic dysfunction.
BCVS Conference 2008 Abstracts
P27
Proteomic Profiling and Differential Protein Expression Analysis of
Myocardial Tissue in a Murine Model of Familial Hypertrophic
Cardiomyopathy
Lien Lam, Centenary Institute, Newtown NSW, Australia; Jonathan Arthur, Univ of Sydney,
Sydney, Australia; Christopher Semsarian; Centenary Institute, Newtown NSW, Australia
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
INTRODUCTION: Hypertrophic cardiomyopathy (HCM) is a genetic disorder caused by
mutations in genes encoding sarcomere proteins. The mechanisms involved in the development of cardiac hypertrophy and transition to heart failure remain poorly understood. Although
cardiomyopathic mice have been characterised with respect to transcript expression, the global
expression of cardiac proteins is unknown. This study sought to map the normal murine
myocardial proteome, and to use this map to identify proteins differentially expressed in an
established mouse model of HCM. METHODS: Hearts from three groups of mice (n⫽3/group)
were studied: non-transgenic (NTG), and cardiac-specific transgenic models over-expressing
either the normal (TnIWT) or a mutant troponin I gene (Gly203Ser; designated TnIG203S). TnIG203S
mice develop all the characteristic features of HCM. Two-dimensional gel electrophoresis
(2DGE) coupled with tandem mass spectrometry were used to identify differentially expressed
proteins. Image analysis was completed using Progenesis SameSpots, and statistical analysis
by online tool CARMAweb. Protein spots were identified by searching the mass spectra against
the NCBI database using MASCOT. RESULTS: A total of 840 proteins spots were detected and
matched across triplicate gels of NTG samples. Of these, 460 spots underwent mass
spectrometry with 154 spots identified. These identifications represent 77 distinct proteins and
their respective isoforms or subunits. Our comparative analysis of normal versus cardiomyopathic mice revealed 34 differentially expressed protein spots (ⱖ2-fold change) of which, 31
proteins were identified. The biological function of these differentially expressed proteins can
be broadly categorised to be involved in cell structure and motility (18%), energy production
(13%), cell cycle (11%), protein transport (9%), muscle contraction (7%), carbohydrate
metabolism (7%), and signal transduction (2%). CONCLUSIONS: This study describes the first
2-D gel proteome map of the normal murine myocardium. We have demonstrated the use of
this 2-D gel map in a proteomic comparative analysis to identify proteins that may play an
important role in the pathogenesis of HCM and its complications, including heart failure.
P28
Bin1, an Adapter Protein, Plays a Role in Cardiac Hypertrophy
Lisa D Laury-Kleintop, Lydia Koenig, Prasad Gupta, Alexander J Muller, George C
Prendergast; Lankenau Institute for Med Rsch, Wynnewood, PA
Bin1 (Amphiphysin II) is an adapter protein that has been associated with a variety of
subcellular processes. Reports describe Bin1 as a protein that participates in endocytosis, actin
cytoskeletal organization, signaling due to stress responses, and transcription. These reports
support the concept that Bin1 has a dynamic role facilitating intra-cellular transfer between
subcellular compartments from the plasma membrane to the nucleus. The functional diversity
of Bin1 is supported by alternative splicing from the Bin1 gene that results in multiple isoforms
with both ubiquitous and cell-type specific expression patterns. Previous work investigating the
constitutive deletion of the Bin1 gene led to a new observation that Bin1 is necessary for
cardiac function because Bin1 null neonates died within 24 hours after birth of severe
ventricular cardiac hypertrophy. The objective of this study was to extend the previous findings
by characterizing Bin1 loss after birth specifically in the cardiac myocyte, using the
cardiac-specific ␣MHC-Cre-loxP system. Our observations of 1.5–2 month old Bin1 deficient
animals include increased cardiac-myocyte cell size, increased ventricle size, increased
fibrosis, increased ANP expression in the ventricle and altered cardiac function by echocardiography. These results suggest that, indeed, perturbation of Bin1 expression in cardiac
myocytes negatively affects cardiac function, and importantly, confirms that Bin1 is a
non-sarcomeric gene product necessary for normal heart development and function.
e41
least one study population at P ⬍ 0.001, and 7 SNPs showed associations at P ⬍ 0.0001. No
SNPs reached significance at this level in both populations; however, a number of SNPs showed
strong trends and identification of the same risk allele in both populations (P ⫽ 0.01 to 0.001).
These initial findings suggest that inherited variation in candidate cardiovascular genes
contributes to the pathogenesis of common forms of human heart failure and identifies specific
targets for further study. We will describe ongoing work testing for genomic predictors of
longitudinal outcomes and replication of key findings in additional populations.
P30
Adenosine A3 Receptor Deficiency Exerts Unanticipated Protective Effects
on the Pressure Overloaded Left Ventricle
Xin Xu, John Fassett, Zhongbing Lu, Xinli Hu, Guangshuo Zhu, Joel French, Ping Zhang,
Univ of Minnesota, Minneapolis, MN; Marlene A Jacobson, Merck Rsch Laboratories, West
Point, PA; Jurgen Schnermann, National Institute of Diabetes and Digestive and Kidney
Diseases, Bethesda, MD; Robert J Bache, Yingjie Chen; Univ of Minnesota, Minneapolis, MN
Adenosine A3 receptor deficiency exerts unanticipated protective effects on the pressure
overloaded left ventricle Background: Endogenous adenosine can protect the overloaded
heart against the development of hypertrophy and heart failure, but the contributions of
individual adenosine receptor subtypes (A1, A2A, A2B, and A3) is not known. Methods and
Results: To test the hypothesis that A1R and A3R can protect the heart against systolic overload,
we exposed A3R gene deficient (A3R KO) mice and A1R KO mice to transverse aortic constriction
(TAC). Contrary to our hypothesis, loss of adenosine A3 receptors (A3R KO) attenuated 5 weeks
TAC-induced left ventricular (LV) hypertrophy (ratio of ventricular mass/body weight increased
to 7.6 ⫾0.3 mg/g in WT mice as compared with 6.3⫾0.4 mg/g in KO mice), fibrosis and
dysfunction (LV ejection fraction decreased from 86⫾1.7% to 43⫾2.5% in WT mice, and from
84⫾1.6% to 55⫾4.2% in KO mice). A3R KO also significantly attenuated the TAC-induced
increases of myocardial atrial natriuretic peptide (ANP) and the oxidative stress markers
3-nitrotyrosine (3-NT) and 4-hydroxynonenal (4-HNE). In contrast, A1R KO increased TACinduced mortality, although it did not alter ventricular hypertrophy or dysfunction compared to
WT mice. In mice in which CD73 KO decreased endogenous adenosine production, TAC caused
greater ventricular hypertrophy and dysfunction, and increased myocardial 3-NT, while the
stable adenosine analogue 2-chloroadenosine (CADO) partially rescued the CD73 mice from
excessive TAC-induced hypertrophy and dysfunction. In neonatal rat cardiomyocytes induced
to hypertrophy with phenylephrine, CADO reduced cell area, protein synthesis, ANP and 3-NT.
Antagonism of A3R significantly potentiated the anti-hypertrophic effects of CADO. Conclusions: Adenosine exerts protective effects on the overloaded heart, likely through A1 and A2
receptors, while A3 receptors act counter to the protective effect of adenosine. The data
suggest that selective attenuation of A3R activity might be a novel therapeutic approach to treat
pressure overload induced ventricular hypertrophy.
P31
Withdrawn
P29
Large-Scale Candidate Gene Association with Human Heart Failure in 2
Referral Populations
Thomas P Cappola, Mingyao Li, Jing He, Bonnie Ky, Joan Gilmore, Brendan Keating,
Muredach P Reilly, Univ of Pennsylvania, Philadelphia, PA; Faisel Syed, Univ of Cincinnati,
Cincinnati, OH; Gerald W Dorn, II; Washington Univ Sch of Medicine, St. Louis, MO
Numerous genes contribute to heart failure in animal models, but most have not been tested
in human disease. We sought to determine which among ⬃2100 carefully selected candidate
genes associate with risk of human heart failure in two independent referral populations. As
part of an international consortium initiated at Penn, genes were selected and prioritized based
on evidence for involvement in cardiovascular disorders, and an Illumina SNP-array was
developed that assays ⬃45,000 SNPs across these loci of interest, with high priority loci having
substantially higher genotype density than current whole-genome platforms (http://bmic.
upenn.edu/cvdsnp/index.php). We used this array to genotype Caucasians with heart failure in
the Penn Heart Failure Study (n ⫽ 708) and controls with normal ventricular function (n ⫽ 350),
and to genotype similar cases (n ⫽ 900) and controls (n ⫽ 300) in the Cincinnati Heart Study
(total n ⫽ 1608 cases, 650 controls). Cases had ACC/AHA stage C or D heart failure and
mean⫾sd ejection fraction of 31⫾16%, while controls showed no clinical heart failure and
ejection fraction of 65⫾6%. After extensive quality control including removal of poorly
performing SNP assays, removal of SNPs with minor allele frequencies ⬍ 1%, removal of
samples with poor hybridizations, assessment for cryptic relatedness, and genomic assessment for Caucasian ancestry, ⬃37,000 SNPs remained for analysis. Genotype frequencies
were compared between cases and controls with logistic regression adjusting for age, gender,
hypertension, and type 2 diabetes using PLINK software. 47 SNPs showed associations in at
P32
Wnt2 Has a Key Role in Cardiac Development Through Noncanonical
Pathway
Takeshi Onizuka, Shinsuke Yuasa, Keiichi Fukuda; Keio Univ, Tokyo, Japan
Embryonic stem (ES) cells are a promising source of cardiomyocytes, but their clinical
application has been hindered by the lack of selective differentiation methods. We investigated
the expression of wnt genes and whether those are through b-catenin or not in the early heart
developmental stage, and applied this finding to establish an efficient protocol to induce
cardiomyocytes in vitro. [Methods and Results] (1) We analyzed TOP EGFP mice in order to
e42
Circulation Research
Vol 103, No 5
August 29, 2008
clarify which types of wnt signals function in the developing heart. TOP EGFP mice are
transgenic mice in which EGFP gene is located under b-catenin binding site so that EGFP
protein expresses when canonical pathway is activated. Analyzing these mice we could not find
any GFP proteins in early heart. (2) Whole mount in situ hybridization of wnt2 and nkx2.5
(positive control) was performed at mouse embryo stages E7.5, E8.0, E8.5 and E9.0. Wnt2 was
strongly expressed in the heart-forming area. It was clearly expressed at the cardiac crescent
at E7.5 and at the late crescent stage at E8.0 and in the linear heart tube at E8.5 and especially
strong expressed in both atrium at E9.0 after looping. (3) We applied this embryonic wnt2
expression pattern to ES cell differentiation. Using siRNA we knock-downed wnt2 protein in
various phases, this could inhibit forming beating EB, and decrease cardiac muscle gene and
proteins only around the primitive stage(day 2– 4). Also adding wnt2 protein to embryonic cells
in appropriate phase led to a marked induction of cardiac genes [Conclusions] Wnt2 is strongly
expressed in the heart-forming region. We applied this finding to develop an effective protocol
for obtaining cardiomyocytes from mouse ES cells by adding wnt2 protein and also to inhibit
generating cardiomyocytes by inhibition of wnt2 signaling. We concluded wnt2 has a key role
in cardiac development through non-canonical pathway.
P33
Talin2 in Cardiac Development and Adult Heart
Ana M Manso, Shannon Ong, Steven Padilla, Yuan Yang, Lindy Him, Khanh Nguyen, Robert
S Ross; VA San Diego Healthcare System and UCSD, San Diego, CA
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Talin (tln) is a cytoskeletal linker protein that has been found essential for integrin activation.
In heart, integrin expression, function and signaling is critical for cardiac development and also
can be modulated with cardiac hypertrophy. There are two talin genes: tln1 and tln2. Initial
studies have suggested that both transcripts have high expression in the heart. The aim of this
study was to evaluate the expression pattern of tln1 and tln2 in embryonic and adult mouse
heart. For this we used tln2 gene-trapped mice (tln2Gt/Gt) which leads to expression of a
chimeric protein with the N-terminal portion (AA 1- 1295) of tln2 fused to ␤-galactosidase. Tln2
expression was detected by monitoring ␤-galactosidase activity. Tln2 was detected in heart by
embryonic day (E)8. By E10, and through to the adult heart, tln2 expression was more intense
in heart compared to other organs. Due to the high similarity of tln2 and tln1, there are no
specific antibodies to differentiate these two proteins. Our tln2 gene-trapped mice allowed us
to use antibodies against the C-terminal domain of talin and ␤-galactoside to distinguish tln1
from tln2 expression. Inmunofluorescence analyses at E14 showed that both tln1 and tln2 were
expressed in cardiac myocytes. In contrast, analysis of adult heart tissue showed that tln2 was
mainly expressed in cardiomyocytes (CM) and specifically localize in costameres, whereas tln1
was expressed in non-myocyte cells. Western blot analysis showed that tln1 was only
minimally expressed in isolated adult CM. Protein levels and location of beta1D integrin and
vinculin were not changed in CM from tln2Gt/Gt compared with control mice (tln2 ⫹/⫹), but
paxillin was up-regulated (141⫾6 %* tln2Gt/Gt vs 100⫾ 6% tln2 ⫹/⫹; *t-test⫽ 0.01, n⫽3 each
group). Morphometric and echocardiographic analyses of 8 week old mice showed no
significant differences between tln2 ⫹/⫹ and tln2Gt/Gt mice. These results suggest that: (a) tln1
and tln2 expression levels in the heart vary during development, (b) the N-terminal domain of
tln2 is sufficient to localize tln2 in the costameres and to maintain normal cardiac function
under basal conditions and (c) tln2 likely has functions distinct from tln1 in heart due to its
preferential expression in distinct cell types and locations.
P34
14–3–3⑀ Knockout Leads to Cardiac Defects and Inhibition of Cell Cycle
Progression in the Developing Heart
Katarzyna A Cieslik, The Univ of Texas, Houston, TX; Antonio Baldini, Texas A&M Univ,
Houston, TX; Anthony Wynshaw-Boris, UCSF, San Francisco, CA; Matteo Vatta, Baylor
College of Medicine, Houston, TX; Joseph H Yost, Luca Brunelli; The Univ of Utah, Salt Lake
City, UT
14 –3–3⑀ Knockout Leads to Cardiac Defects and Inhibition of Cell Cycle Progression in
the Developing Heart 14 –3–3⑀ is an isoform of ubiquitous phosphoserine/threonine-binding
proteins which regulate numerous critical intracellular functions, although their role in vivo
remains to be defined. The 14 –3–3⑀ gene is always deleted in Miller-Dieker syndrome (MDS),
a severe congenital brain defect characterized by lissencephaly and associated with a short life
span and heart malformations including ventricular septal defects (VSDs). 14 –3–3⑀ has also
been shown to be essential for normal brain development in mice, but the biologic function of
14 –3–3 proteins and 14 –3–3⑀ during development of other organs like the heart is unknown.
We found that germline deletion of 14 –3–3⑀ in mice causes biventricular dilatation, incomplete
interventricular septum, ventricular hypertrabeculation and thin compact zone. The latter
defects are characteristic of left ventricular noncompaction (LVNC), a human cardiomyopathy.
Heterozygous hearts displayed an intermediate phenotype, with a milder degree of noncompaction and a lower frequency of VSDs. Among alternative isoforms, 14 –3–3␥ transcripts were
greatly upregulated in 14 –3–3⑀-/- hearts, suggesting compensatory mechanisms. Expression of
14 –3–3⑀ during normal development was higher at early embryonic stages and, in 14 –3–3⑀-/hearts, defective ventricles were evident by E12.5, and VSDs by E14.5. Apoptosis in 14 –3–3⑀-/hearts was comparable to wildtypes, but proliferation was severely suppressed. 14 –3–3⑀-/cardiac ventricular cells exhibited a marked delay of G1/S transition during the cell cycle, which
correlated with a selective increase of p27Kip1. This was mediated via transcriptional
mechanisms since active FOXO3a protein and p27Kip1 mRNA were upregulated. Our work
defines 14 –3–3 proteins as novel determinants of mammalian cardiac development and
characterizes the dosage effects of 14 –3–3⑀ in the heart. We clarify the molecular
mechanisms by which 14 –3–3⑀ modulates the cell cycle in cardiac ventricular cells during
heart development. Our findings also suggest that loss of 14 –3–3⑀ may lead to heart defect
in individuals with MDS and they may require screening for VSDs and cardiomyopathy.
P35
P21-Activated Kinase 1 Mediates Compensatory Cardiac Hypertrophy that
Antagonizes Pathological Cardiac Remodeling
Kai Mao, Chastity Healy, Dajun Wang, Paul Volden, Martin Gerdes, Timothy O’Connell,
Sanford Rsch/Univ of South Dakota, Sioux Falls, SD; Jonathan Chernoff, Fox Chase Cancer
Cntr, Philadelphia, PA; Qiangrong Liang; Sanford Rsch/Univ of South Dakota, Sioux Falls, SD
Physiological cardiac hypertrophy does not progress to heart failure. Despite intense research
for several decades, the signaling mechanisms that mediate physiological cardiac hypertrophy
remain poorly defined. P21 activated kinase 1 (PAK1) is a serine/threonine protein kinase that
plays an important role in diverse cellular functions including cell growth and survival. We have
shown that PAK1 activity is increased in cardiomyocytes in response to multiple hypertrophic
agonists. However, the specific function of PAK1 in the heart is unclear. This study investigated
the role of PAK1 in cardiac hypertrophy and survival using both gain- and loss-of-function
models. Overexpression of an active form of PAK1 in transgenic (TG) mice was sufficient to
induce cardiac hypertrophy. Heart to body weight ratio was increased by 13% at 6 weeks, 22%
at 3 months, and 47% at 14 months (n⫽6, p⬍0.05), which was not accompanied by either
fibrosis or activation of fetal gene program. Cardiac function was preserved with unchanged left
ventricular (LV) chamber diameter even in 14-month old TG mice as determined by
echocardiography and hemodynamics. The results suggest that PAK1 induces physiological
hypertrophy. After 8 weeks of transverse aortic constriction (TAC), PAK1 TG mice displayed
better cardiac function than wild type (WT) mice as shown by a higher fractional shortening (FS:
TG-TAC 41% vs WT-TAC 31%, n⫽7–9, P⬍0.01). Despite having baseline cardiac hypertrophy,
PAK1 TG mice showed reduced growth response to TAC as indicated by a smaller LV posterior
wall thickness. These data suggest that PAK1 induces a protective mechanism that antagonizes
TAC-induced pathological cardiac remodeling. Moreover, PAK1 knock out (KO) mice showed LV
chamber dilation and impaired cardiac function compared with WT (FS: KO 35% vs WT 49%,
n⫽3– 4, P⬍0.05). Isolated adult cardiomyocytes from PAK1 KO mice had reduced viability at
baseline. These results suggest an important role for PAK1 in maintaining normal cardiac
structure and function. In conclusion, our study demonstrates that PAK1 mediates physiological
cardiac hypertrophy and is required for cardiac homoeostasis. Manipulation of PAK1 signaling
pathway might be exploited as a therapy in pathological cardiac remodeling.
P36
Expression and Regulation of Osteopontin in the Diabetic Heart
Abigail Goodman, Florentina Pluteanu, Kyle K Henderson, Allen M Samarel, Leanne L
Cribbs; Loyola Univ Chicago, Maywood, IL
Diabetic cardiomyopathy is characterized by left ventricular (LV) remodeling and cardiac
dysfunction that is dependent upon the diabetic state, and can be seen in diabetic patients
without other cardiovascular risk factors. Diabetic cardiomyopathy is associated with hypertrophy and fibrosis of the LV; however, the pathway and mechanisms by which diabetic factors
adversely affect LV function need to be further elucidated. To investigate effects of the diabetic
milieu on LV remodeling, Extracellular Matrix and Adhesion Molecule Microarray (SuperArray)
comparisons were carried out on RNA from the LV of a rat model of Type 2 diabetes (Obese
ZSF vs. Lean control). Microarray screening suggested several possible genes altered in the
diabetic heart, including osteopontin (OPN), which has been implicated previously in tissue
inflammation, remodeling and fibrosis, as well as in cardiac ischemia and hypertrophy. OPN is
a secreted protein and its functions are modified by posttranslational phosphorylation,
glycosylation, and cleavage. To further investigate the role of OPN in diabetes, OPN expression
was studied in neonatal rat ventricular myocytes (NRVM) and fibroblasts (NRVF) in culture in the
presence of elevated glucose (5 mM control, 25 mM and 50 mM). Exposure of NRVM to high
glucose led to increased OPN mRNA expression after 24 and 72 hours. Immunostaining
experiments suggested that OPN was expressed both in cardiomyocytes and fibroblasts, where its
localization shifted from nuclear to cytoplasmic, followed by its secretion upon exposure to diabetic
stimuli of elevated glucose or Ang II. Western blot analysis revealed the expected OPN doublet at
60 – 65 kDa in both NRVM and NRVF, along with the appearance of a putative OPN cleavage product
at ⬃ 20 kDa in the NRVF cell lysate and supernatant after 72 hours of treatment with high glucose.
These results suggest that OPN may be regulated in the diabetic heart as a direct consequence of
hyperglycemia and that its expression may also be affected by other aspects of the diabetic milieu.
We propose that the regulation of OPN and changes in its intracellular localization may contribute
to fibrosis and adverse LV remodeling in the diabetic heart.
P37
Candesartan Regulates Polarization of Th1 and Th2 Cytokines in Lymphoid
Organs of Obese Mice with Acute Myocarditis
Tsugiyasu Kanda, Kazuya Kusaka, Takashi Takahashi, Junji Moriya, Jun-ichi Yamakawa, Rui
Chen, Kanazawa Med Univ, Kahoku Ishikawa, Japan; Hiroyuki Sumino, Takasaki Univ of
Health and Welfare, Takasaki Gunma, Japan; Shigeto Morimoto, Takeshi Nakahashi,
Kuninmitsu Iwai; Kanazawa Med Univ, Kahoku Ishikawa, Japan
Introduction: Angiotensin II has proinflammatory effects. The oral administration of candesartan, an angiotensin II receptor blocker, might regulate The Th1-Th2 balance of lymphoid
organs in obese mice with viral myodarditis. We investigated the effects of Th1-Th2 balance
in lymphoid organs to myocardial injury. Material and methods: KKAy mice (n⫽50) were
divided into candesartan group receiving orally candesartan at daily dose of 10mg/kg 7days
starting simultaneously with EMC viral inoculation and untreated control group. Results:
Candesartan treated group showed reduced myocardial necrosis and inflammation as
compared with control group. In candesartan group on day 7, the thymus weight/body weight
ratio was significantly higher than the control (1.80 ⫾ 0.12 mg/g vs. 1.54 ⫾ 0.2 mg/g,
P⬍0.05) and the spleen weight/ body weight ratio was significantly higher than control (3.89 ⫾
BCVS Conference 2008 Abstracts
0.98 vs. 2.87 ⫾ 0.23, P⬍0.05). In candesartan group, thymic atrophy and splenomegaly were
significantly reduced compared with control. The thymic and splenic expressions of
interferon-␥ (IFN-␥) [[Unsupported Character - ]]and interleukin-10 (IL-10) mRNA
were investigated. On days 4 and 7 after viral inoculation, the heart weight/body weight ratio
was significantly reduced by candesartan group (On day 7, 4.31 ⫾ 0.19 mg/g vs. 4.65 ⫾ 0.40
mg/g, P⬍0.05). Myocardial necrosis and inflammation on days 4 and 7 were significantly
reduced in candesartan group compared with the control. The Th1 (IFN-␥)/Th2 (IL-10) ratio in
the thymus and spleen on days 4 and 7 was significantly (P⬍0.05) higher in the candesartan
group compared with the control (Thymic ratio on day 4; 0.24 ⫾ 0.04 vs. 0.14 ⫾ 0.13, on day
7; 0.94 ⫾ 0.19 vs. 0.72 ⫾ 0.22. Splenic ratio on day 4; 0.74 ⫾ 0.05 vs. 0.39 ⫾ 0.04, on day
7; 0.82 ⫾ 0.14 vs. 0.39 ⫾ 0.17). The histological score of myocardiac necrosis was negatively
(P⬍0.05) correlated with and the Th1/Th2 ratio in the spleen. Conclusion: Candesartan
treatment could regulate the polarization of the Th1/Th2 ratio in lymphoid organs in association
with a reduction of myocardial injury in viral myocarditis.
P38
Akt-Dependent Downregulated Glycogen Synthesis Kinase-3␤ Plays a
Critical Role in Lipid Accumulation and Inflammation in Diabetic
Cardiomyopathy: Prevention of Metallothionein
Yuehui Wang, Lu Cai; Univ of Louisville, Louisville, KY
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Cardiac-specific metallothionein (MT)-overexpressing transgenic (MT-TG) mice were highly
resistant to the development of diabetic cardiomyopathy. Increasing evidence indicates that
lipotoxicity is associated with inflammatory effects and oxidative damage, leading to the
development of diabetic cardiomyopathy. which is associated with heart failure. The present
study was aimed at investigating the signaling mechanism by which MT protects the heart from
diabetes-induced lipid accumulation and inflammation. Both MT-TG and wild-type (WT) mice
were given a single dose of streptozotocin to induce hyperglycemia, then lipid accumulation
and inflammation were examined at different times after the onset of diabetes. To explore the
mechanisms, Akt, glycogen synthesis kinase-3␤ (GSK-3␤), glycogen synthase (GS), hexokinase II (HK II) and peroxisome proliferator activated receptor ␣ (PPAR␣) were also examined.
Results showed that at different times of diabetes, lipid accumulation and inflammatory
cytokines (TNF-␣, PAI-1 and ICAM-1) were significantly increased in the hearts of the WT
diabetic mice, but not in the MT-TG diabetic mice. Abundance of total Akt, GSK-3␤ or GS was
not significantly changed, but phosphorylation of Akt was decreased in the hearts of WT
diabetic mice compared to the control mice. Decreased phosphorylation of Akt in the hearts of
WT diabetic mice was accompanied by a decrease in phosphorylation of GSK-3␤, leading to
the increased phosphorylation of GS and the decreased HK II, which inhibited glycogen
synthesis. Meanwhile, decreased GSK-3␤ was accompanied by an increased PPAR␣ in the
hearts of WT diabetic mice, leading to lipid accumulation. However, neither phosphorylation of
Akt nor GSK-3␤ kinase was decreased in the MT-TG diabetic mice. Administration of GSK-3␤
inhibitor to WT diabetic mice for two months significantly prevented diabetes-induced lipid
accumulation, inflammation and fibrosis. Therefore, these results suggest that Akt-dependent
down-regulated GSK-3␤ plays a critical role in diabetes-induced lipid accumulation and
inflammation. MT prevents diabetes-induced increases in cardiac lipid accumulation and
inflammation, through preservation of the Akt-dependent glucose and lipid metabolism.
P39
The Cardioprotective Role of ERK5 in Diabetes-Mediated Exacerbation of
Cardiac Dysfunction After Myocardial Infarction through Ubiquitin Ligase
CHIP-Mediated ICER Degradation
Changhoon Woo, Tetsuro Shishido, Jun-ichi Abe; Univ of Rochester Med Cntr, West
Henrietta, NY
Epidemiological studies strongly indicate that diabetes mellitus (DM) is an independent risk
factor for both mortality and morbidity following myocardial infarction (MI), especially since
post-MI left ventricular (LV) function is significantly worse in diabetic patients. Although
apoptosis plays a critical role in post-MI cardiac dysfunction since the loss of cardiomyocytes
contributes to the transition from cardiac ischemia to heart failure, there is no well-defined
relationship between diabetes and any of the known regulators of myocytes apoptosis. Recently
we found that the phosphodiesterase 3A (PDE3A)-ICER (inducible cAMP early repressor)
feedback loop that positively regulates apoptosis was inhibited by ERK5 activation through the
acceleration of ICER reduction. Here we showed that ERK5 modification by small ubiquitin-like
modification (SUMOylation) was increased in diabetes, which leads to the inhibition of ERK5
transcriptional activity. Interestingly, ERK5-SUMOylation was significantly increased in post-MI
diabetic mice (DM ⫹ MI). In contrast, activation of MEK5-ERK5 inhibited ERK5-SUMOylation,
ICER induction, and myocyte apoptosis in vivo. Run off reaction assay revealed that ICER was
degraded by the E3 ubiquitin ligase CHIP (C-terminus of Hsp70-interacting protein). In addition,
ERK5 activation enhanced CHIP-binding to ERK5 and CHIP-mediated ubiquitination activity,
which was inhibited by ERK5-SUMOylation. Taken together our data suggest that ERK5mediated CHIP activation is a key modulator of ICER degradation and inhibits myocyte apoptosis
in DM ⫹ MI. We believe that this novel mechanism of ERK5-mediated CHIP activation has great
clinical potential in diabetes-mediated exacerbation of cardiac dysfunction after MI.
e43
P40
Paradoxical Modulation of Stromal-Derived Factor Expression in Diabetes
Following Experimental Myocardial Infarction
Kim A Connelly, Jean F Desjardin, Kerri Thai, Andrew Advani, Richard E Gilbert; St
Michael’s Hosp and Univ of Toronto, Toronto, Canada
Background: Impaired collateral vessel formation is thought to contribute to the excessive
morbidity in diabetic subjects with ischaemic heart disease where following myocardial
infarction patients with diabetes are twice as likely to develop heart failure than non-diabetic
subjects. Since the trafficking of bone marrow derived endothelial progenitor cells are thought
to contribute to new vessel formation in ischemia, we postulated that altered chemokine
expression may contribute to the impaired angiogenic response in diabetes. Methods: Six week
old F344 rats were randomized to vehicle or streptozotocin (35mg/kg I.P.) in order to induce
diabetes. Once diabetes was confirmed (plasma glucose ⬎15 mmol/l), a total of 40 animals
then underwent sham procedure or LAD ligation at age 10 weeks, and sacrificed at day 0
(sham) and days 1, 2 and 7 post LAD ligation (4 per group). Infarct size and presence was
determined by echocardiography 24 hours after ligation. RT q PCR was used to assess mRNA
for the chemokines SDF at each time point, from tissue obtained from the peri-infarction zone.
All data was normalized to the housekeeping gene 18S. Results: In control rats an increase in
SDF-1 gene expression was apparent at Day 2 post LAD ligation, persisting through Day 7. In
contrast, diabetic rats showed high basal expression (6-fold greater than non-diabetic rats) that
fell abruptly after ligation and remained persistently low throughout the 7-day time course.
Conclusions: Diabetes was associated with paradoxical alterations in gene expression of both
SDF with high baseline expression and diminution following an ischemic insult. These findings
suggest that disordered chemokine expression may reduce the diabetic heart’s ability to recruit
endothelial progenitors cells to sites of ischemic injury.
P41
mtDNA Abundance Is Decreased in Laser-Captured Heart Samples from
HIV/AIDS Patients Who Received Zidovudine
Ioan Cucoranu, Aleksey Shapoval, Stanley He, David Johnson, James J Kohler, Seyed H
Hosseini, William Lewis; Emory Univ Sch of Medicine, Atlanta, GA
BACKGROUND Nucleoside Reverse Transcriptase Inhibitors (NRTIs) are central to effective
highly active antiretroviral therapy (HAART) in HIV/AIDS. NRTIs such as Zidovudine (3’-azido-2’,
3’-deoxythymidine; AZT) inhibit replication of mtDNA by inhibiting DNA pol-␥. The pathogenesis
of many of the toxicities associated with HAART is thought to be NRTI-induced mitochondrial
dysfunction. It is known that NRTIs cause CM in mice. Individuals with HIV/AIDS may be
particularly susceptible to clinically significant mitochondrial toxicity due to possible effects of
HIV itself on mitochondria. CM occurs in HIV/AIDS and symptomatic heart failure is reported in
up to 5% of the cases. This study focuses on the relationship between cardiac mtDNA depletion
and associated AZT treatment in HIV/AIDS patients. METHODS Twenty-eight autopsy samples
of cardiac tissue were obtained from the NCI AIDS Cancer Specimen Repository. The majority
were males. Mean age was 36. AZT was administered as part of HAART treatment or alone to
18 patients, while 10 patients did not receive AZT. Total DNA was extracted from heart tissues
isolated by laser capture microdissection (1x106 ␮m2) and mtDNA abundance was quantified
by real-time PCR. RESULTS mtDNA was quantified in the 28 samples. Patients treated with AZT
exhibited 55% decrease in mtDNA abundance (p⬍0.02) compared to those without AZT
treatment. CONCLUSION Data support the hypothesis that AZT contributes to decreased mtDNA
abundance in a susceptible target tissue like heart. This can be related to the development of
CM and heart failure in HIV/AIDS and warrants clinical investigation. Clinical awareness of this
problem is important to ensure early detection of NRTI side effects to allow timely consideration
of changing therapy. Demographic data for HIV patients treated or untreated with AZT *
For 7 patients out of 28 the demographic data is not available
Age (range/mean
years)
all patients*
no AZT treatment
AZT treatment
27–52 / 36
34–39 / 37
27–52 / 36
Gender (% males, %
females)
85.7% M 14.3% F
75% M 25% F
88.2% M 11.8% F
Race (% Caucasian , %
African-American, % Hispanic)
81% C, 14.3% A-A, 4.7% HIS
100% C
76.5% C, 17.6% A-A, 5.9% HIS
P42
TAT Conjugated Frataxin as a Nonviral Therapeutic Delivery System for
Friedreich’s Ataxia
Piyush M Vyas; Indiana Univ, Indianapolis, IN
Friedreich’s Ataxia (FRDA) is the most common cause of inherited human ataxia. It is caused
by a deficiency of the protein, frataxin, most often arising from a triplet GAA expansion in
intron-1 of the FRDA gene. It results in iron accumulation and possible oxidative stress in
mitochondria leading to progressive cardiomyopathy and ataxia. Therapies have not been
effective and patients do not survive due to hypertrophic cardiomyopathy. We have used protein
transduction domains (PTD), notably TAT, to deliver frataxin to mitochondria for cells in culture.
A TAT-frataxin (TAT-frataxin) fusion protein was able to transduce into the mitochondria of
human frataxin deficient fibroblasts and remain there for more than 21 days in culture.
TAT-frataxin treated fibroblasts from FRDA patients also showed reduced caspase-3 induction
and cell death compared to controls when treated with iron as an oxidant stress. An in vitro
assay demonstrated that TAT-frataxin was able to bind free iron and reduce generation of
superoxide radicals. Determining the positive response from cell culture studies, we treated
conditional frataxin knock-out mice with TAT-frataxin. These mice have deficiency of frataxin
in brain and heart. We found that these mice can be rescued and can survive long with the help
of TAT-frataxin treatment. Immunohistochemistry suggested the presence of absorbed
TAT-frataxin in the heart sections of these animals and echocardiography suggested functional
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August 29, 2008
improvement in the ejection and systolic function of these animals as compared to untreated
knock-out mice. In addition, frataxin dependent succinate dehydrogenase (SDH) activity was
found to be improved in the TAT-FRATAXIN treated mice as compared to non treated mice.
These results suggest that a functional protein can be delivered to mitochondria via a PTD and
supports the hypothesis that TAT-frataxin can be developed as a novel therapy for the
treatment of Friedreich’s Ataxia using protein transduction domains
P43
Adenylate Kinase-Mediated Metabolic Monitoring and Interaction with
Metabolic Sensor AMPK in Cardiomyocytes
(RT-PCR). Cell viability was assessed using Trypan Blue stain. For in vivo experiments,
SPIO-labeled ASCs were injected into ten rat hearts. The hearts were monitored using MR
imaging. Survival rate of the ASCs was very high (97%–99%) after exposure to SPIO (see
Table). The SPIO-treated ASCs were induced to express specific markers for the three types of
transdifferentiation (see Figures 1). Expression of the cell factors by the ASCs was not affected
by SPIO (Figure 1). Signal voids on MR images were associated with the living SPIO-labeled
ASCs in the rat hearts (Figure 2). We conclude that SPIO does not affect viability,
transdifferentiation potential, or cell-factor secretion of ASCs. MR imaging highlights living
SPIO-labeled stem cells.
EFFECT OF SPIO ON VIABILITY OF ASCS(%)
ASC Culture Conditions
Petras Dzeja, Andre Terzic; Mayo Clinic, Rochester, MN
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Metabolic monitors sense cellular energy state and respond to imbalances by generating and
delivering signaling molecules to metabolic sensors to produce a regulatory response. Central
in cellular metabolic monitoring is enzyme adenylate kinase (AK) which generates adenosine
monophosphate (AMP) signals and facilitates their delivery to a number of AMP-sensitive
components, including a major metabolic sensor - AMP-activated protein kinase (AMPK). In
metabolic signal transduction cascades co-localization and interaction of components in is
critical for the directionality and specificity of signaling response. Here we demonstrate the
presence of the AK1 activity and protein in the immuno-pull-down complexes of cardiac
AMPK␣2. Furthermore, FRET analysis suggests a co-localization of AK1 and AMPK␣2 in intact
cardiomyocytes. Possible formation of macromolecular complexes between AK1 and AMPK␣2
were further corroborated in an intact cellular environment using double immunostaining and
high-resolution confocal microscopy. Our data suggest formation of a multi-enzyme complex,
where AMPK complexes are surrounded by the AK1 molecular network. In such environment,
AK senses alterations in the cellular ATP/ADP ratio and translates those changes into an
amplified AMP signal in the vicinity of AMPK. Quantitative immunocytochemistry and FRET
analyses also indicate that membrane associated AK1␤ isoform closely colocalizes with AMPK
in sarcolemmal area, where both enzymes can regulate ion channels and metabolic signaling
critical in cardioprotection. In cell analysis of AMPK activation by measuring p-AMPK/AMPK
ratio using double immunostaining and confocal image scanning indicate that in response to
stress AMPK activation coincides with AK localization in nuclear, myofibrillar and sarcolemmal
areas. Thus, the presence of AK in close proximity of AMPK could facilitate metabolic signal
transduction and create a favorable energetic environment for phosphorylation of targeted
proteins and elicitation of regulatory response.
1
2
3
4
1st Passage
97.9 ⫾2.1
98.2⫾1.4
98.7 ⫾0.5
98.1 ⫾1.5
2nd Passage
98.8
99.3
99.4
99.3
⫾0.8
⫾0.3
⫾0.4
⫾0.3
3rd Passage
99.1 ⫾0.3
99.5 ⫾ 0.5
99.7 ⫾0.0
99.4 ⫾0.8
4thPassage
99.5
98.8
99.2
99.0
Note: 1, NCM ⫹ PS ⫹ 100 mg/ml Fe⫹⫹ 2, NCM ⫹ PS ⫹ 50 g/ml Fe⫹⫹; 3, CNM ⫹ 6mg/ml protamine sulfate(PS
P44
Inhibitory Effect of P300 on Cardiac Mitochondrial Gene Expression and
Cell Survival in Postnatal Heart
Yasuaki Nakagawa, Koichiro Kuwahra, Masaki Harada, Masao Murakami, Satoru Usami,
Shinji Yasuno, Hideyuki Kinoshita, Masataka Fujiwara, Kenji Uechima, Kazuwa Nakao; Kyoto
Graduate Sch of Medicine , Kyoto, Japan
The transcriptional coactivator p300 is known to be crucially involved in the differentiation and
growth of cardiac myocytes during development. We have now investigated the physiological
function of p300 in adult hearts by analyzing transgenic mice exhibiting cardiac-specific
overexpression of a dominant-negative p300 mutant lacking the C/H3 domain (p300⌬C/H3-TG
mice). p300⌬C/H3 significantly inhibited p300-induced activation of GATA- and MEF2dependent promoters in cultured ventricular myocytes, and p300⌬C/H3-TG mice showed
cardiac dysfunction that was lethal by 20 weeks of age. The numbers of mitochondria in
p300⌬C/H3-TG myocytes were markedly increased, but the mitochondria were diminished in
size. Moreover, cardiac mitochondrial gene expression and mitochondrial membrane potential
were both significantly disrupted in p300⌬C/H3-TG hearts. Transcription of PGC-1␣, a master
regulator of mitochondrial gene expression, and its target genes was significantly downregulated in p300⌬C/H3-TG mice, and p300⌬C/H3 directly repressed MEF2C-dependent PGC-1␣
promoter activity and disrupted the transcriptional activity of PGC-1␣ in cultured ventricular
myocytes. In addition, myocytes showing features of autophagy were observed in p300⌬C/
H3-TG hearts, suggesting autophagic myocyte death triggered by mitochondrial dysfunction
contributes to the progression of the observed cardiomyopathy. Collectively, our findings
suggest that p300 is essential for the maintenance of mitochondrial integrity and for myocyte
survival in the postnatal ventricle.
P45
SPIO Does Not Affect Viability and Function of Adipose-Derived Stem Cells,
and SPIO-Enhanced MRI Identifies Viable Cells
Jixian Deng, Lei Wang, Jian Wang, Bo Xiang, Tonghua Yang, Marco Gruwel, Institute for
Biodiagnostics, Winnipeg, Canada; Tarek Kashour, St Boniface General Hosp, Winnipeg,
Canada; Boguslaw Tomanek, Institute for Biodiagnostics, National Rsch Council Canada,
Winnipeg, Canada; Randy Summer, Institute for Biodiagnostics, Winnipeg, Canada; Darren
Freed, Davinder Jassal, St Boniface General Hosp, Winnipeg, Canada; Guangping Dai, MGH,
Charlestown, MA; Roxanne Deslauriers, Institute for Biodiagnostics, Winnipeg, Canada;
Rakesh Arora, St Boniface General Hosp, Winnipeg, Canada; Ganghong Tian; Institute for
Biodiagnostics, Winnipeg, Canada
This study was to: 1) determine whether superparamagnetic iron oxide (SPIO) affects viability,
transdifferentiation potential, and cell-factor secretion of adipose-derived stem cells (ASCs); 2)
determine whether SPIO-enhanced magnetic resonance (MR) imaging highlights living stem
cells. Rat ASCs were first exposed to SPIO for two days. The SPIO-treated ASCs were then
subjected to adipogenic, osteogenic, and myogenic transdifferentiation. Expression of vascular
endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor -1 by the
SPIO-treated ASCs was measured using reverse transcription-polymerase chain reaction
⫾0.5
⫾1.7
⫾1.1
⫾1.2
PPAR-␦ Agonist Enhances Vasculogenesis by Regulating Endothelial
Progenitor Cells Through Genomic and Nongenomic Activation of the
PI3K/Akt Pathway
P46
Wonjae Lee, Hyun-Jai Cho, Jun-Bean Park, Jung-Kyu Han, Hyun-Sook Lee, Kyung-Woo
Park, Hae-Young Lee, Hyun-Jae Kang, Byung-Hee Oh, Young-Bae Park, Hyo-Soo Kim; Seoul
National Univ Hosp, Seoul, Republic of Korea
Background: Despite the therapeutic potential of endothelial progenitor cells (EPCs) in
ischemic vascular diseases, their insufficient numbers limit clinical applications. Peroxisome
proliferator-activated receptor delta (PPAR-␦) belongs to the nuclear hormone receptor
superfamily, and its functions in various tissues and cells are almost unexplored, especially
with respect to vascular biology. Methods and Results: PPAR-␦ activation in EPCs
phosphorylated Akt and this phosphorylation was mediated not only by genomic but also by
non-genomic pathways through interaction with the regulatory subunit of PI3K. PPAR-␦
activation with agonist (GW501516 or L-165041) increased the proliferation of human EPCs
and protected them from hypoxia-induced apoptosis. In addition, PPAR-␦ activation enhanced
EPCs functions, such as transendothelial migration, and tube formation. These actions by
PPAR-␦ activation in EPCs were dependent on the PI3K/Akt pathway. In ischemic hindlimb of
mice models, transplantation of PPAR-␦ agonist-treated human or mouse EPCs enhanced blood
flow recovery to ischemic limbs compared with vehicle-treated EPCs. In EPCs from PPAR-␦
knockout mice, however, treatment with PPAR-␦ agonist did not enhance in vivo vasculogenic
potential. Systemic administration of PPAR-␦ agonist increased hematopoietic stem cells in
bone marrow and EPCs in peripheral blood, leading to improved vasculogenesis with
incorporation of bone marrow-derived cells to new vessels in a corneal neovascularization
model, and limb salvage with better blood flow in ischemic hindlimb model. Conclusions: The
results of our study suggest that PPAR-␦ agonist has therapeutic vasculogenic potential for the
treatment of ischemic cardiovascular diseases.
P47
Cardiac Induction by Dorsomorphin, a Selective Small Molecule Inhibitor of
BMP Signaling
Charles C Hong, Jijun Hao, Marie A Daleo, Clare K Murphy, Joshua N Ho, Antonis K
Hatzopoulos; Vanderbilt Univ Sch of Medicine, Nashville, TN
Background Pluripotent embryonic stem (ES) cells show great promise as a versatile source
of cells for regenerative therapy. However, basic mechanisms of lineage specification of
pluripotent stem cells are largely unknown, and directing differentiation of ES cells toward
desired cell types remain a major challenge. Small molecules, particularly those that modulate
key developmental pathways like the bone morphogenetic protein (BMP) signaling cascade
hold promise as tools to study in vitro lineage specification and to direct differentiation of stem
cells toward particular cell types. Methodology/ Principal Findings We utilized dorsomorphin,
a selective small molecule inhibitor of BMP signaling, to robustly induce myocardial
development in mouse ES cells. Quantitative-PCR analyses of differentiating ES cells indicate
BCVS Conference 2008 Abstracts
e45
that dorsomorphin treatment during the initial stages of differentiation dramatically promotes
cardiomyogenesis without inducing the peak expression of the mesodermal marker
Brachyury-T or the precardiac mesoderm marker Mesp1. Moreover, our quantitative-PCR
analyses indicate that dorsomorphin treatment reduces the development of endothelial, smooth
muscle, and hematopoietic lineages. Conclusions/ Significance Administration of dorsomorphin during the initial stages of ES cell differentiation substantially promotes the development
of pluripotent stem cells toward the cardiomyocytic lineage by an unanticipated mechanism
that does not involve significant induction of the mesodermal marker Brachyury-T. Our results
suggest that pharmacological inhibition of BMP signaling during the early critical stage may
promote development of a novel subset of cardiac progenitor cells at the expense of other
mesodermal lineages. Selective small molecule modulators of key developmental pathways like
dorsomorphin show promise as versatile pharmacological tools for stem cell research and
regenerative medicine.
activation (also demonstrated by inhibition with cyclosporine A (CsA), transcripts and
expression levels of Ca2⫹ handling proteins such as SERCA2 and Na⫹/Ca2⫹ exchanger (NCX1)
were found to be higher in myocytes exposed to TG. On the contrary, transcript and expression
levels of SERCA2 and NCX1 were reduced in myocytes undergoing adrenergic hypertrophy. On
the other hand, transcription of Transient Receptor Potential Channel (TRPC3, 4 and 6) proteins
was found to be increased following exposure to PE. TRPC mediated Ca2⫹ entry through the
plasma membrane was also found to be increased, contributing to the observed rise of resting
cytosolic Ca2⫹. Our experiments indicate that the complex transcriptional program triggered by
the adrenergic agonist bypasses and neutralizes calcineurin activation of SERCA2 and NCX1
transcription. Reduced expression of SERCA2 and NCX1 proteins, in conjunction with increased
TRPC protein expression, is a major factor in alteration of Ca2⫹ signaling in neonatal rat cardiac
myocytes undergoing adrenergic hypertrophy.
P48
HIF-2␣ Transactivates Abcg2 and Promotes Cytoprotection in Cardiac SP
Cells
Sex Differences in Connexin 43 in a Model of Pathologic Hypertrophy
Cindy M Martin, Anwarul Ferdous, Yi Ren, Univ of Minnesota, Minneapolis, MN; Hesham
Sadek, Univ of Texas Southwestern Med Cntr, Dallas, TX; Daniel J Garry; Univ of Minnesota,
Minneapolis, MN
Stem and progenitor cell populations occupy a specialized niche and are consequently exposed
to hypoxic and oxidative stresses. We have previously established that the multidrug resistance
protein Abcg2 is the molecular determinant of the SP progenitor cell population. Using dual
wavelength FACS analysis and immunohistological techniques we observed that the cardiac SP
cells increase in number within three days of injury following injury. Transcriptome analysis of
the SP cells isolated from the injured adult murine heart reveals increased expression of
cytoprotective transcripts. Overexpression of Abcg2 results in an increased ability to consume
hydrogen peroxide and is associated with increased levels of ␣-glutathione reductase protein
expression. Importantly, overexpression of Abcg2 also conferred a cell survival benefit following
exposure to hydrogen peroxide. To further examine the molecular regulation of the Abcg2 gene,
we have demonstrated that HIF-2␣ binds an evolutionary conserved HIF-2␣ response element
in the murine Abcg2 promoter. Transcriptional assays reveal a dose-dependent activation of
Abcg2 expression by HIF-2␣. These results support the hypothesis that Abcg2 is a direct
downstream target of HIF-2␣ which functions with other factors to initiate a cytoprotective
program for this progenitor SP cell population that resides in the adult heart. We have recently
constructed Abcg2 mutant proteins that lack the ability to function as an active transporter. We
are further analyzing these mutant proteins to determine their ability to initiate cytoprotective
programs.
Background: Connexin 43 (Cx43) is the predominant gap junction protein in the cardiac
ventricle. Pathologic post-translational modification, dephosphorylation, occurs in this gap
junction protein in disease states such as heart failure and post-myocardial infarction.
Dephosphorylation of Cx43 is associated with arrhythmias and sudden cardiac death in humans
and rodents. There is substantial support for a sex-difference in myocellular adaptation to heart
failure and in ischemia reperfusion. In general, the female myocardium appears more resistant
to pathological adaptation and arrhythmias. A sex difference in Cx43 phosphorylation has been
demonstrated in vivo. It is unknown whether sex-differences in Cx43 phosphorylation are
present in the setting of pathologic cardiac hypertrophy in vitro. Hypothesis: Female
cardiomyocytes will demonstrate less dephosphorylation in the setting of a pathologic growth
stimulus. Methods: Ventricular cardiomyocytes were isolated from male or female adult rats
(ARVM, age: 16 –20 weeks) and cultured in serum-free, phenol red-free DMEM. The
sex-specific ARVMs were treated with phenylephrine (PE, 10␮M) for 24 hours. Protein and
mRNA was isolated from the harvested cells. Cx43 expression was evaluated by real-time
quantitative PCR (RTqPCR) and Western blot. Results: Cx43 expression declined over time in
untreated ARVMs in culture. PE treatment caused the typical mRNA pathologic growth response
with upregulation of fetal genes and downregulation of adult genes in ARVMs of both sexes. PE
treatment prevented and reversed the decline in Cx43 in female ARVMs but had no influence
on Cx43 expression in male ARVMS. Conclusion: Alpha-adrenergic stimulation with PE results
in a maintenance of Cx43 phosphorylation in female but not male AVRMs in culture. Resistance
to Cx43 dephosphorylation may be a mechanism underlying the resistance of the female
myocardium to pathologic change and arrhythmias.
P49
Akt Is Dysregulated in Anthrax Lethal Toxin-Induced Systolic Heart Failure
Interaction of Protein Kinase C with Zinc Transporters
P51
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Honey B Golden, TAMHSC-CVRI, Temple, TX; Linley E Watson, Scott & White Memorial
Hosp, Temple, TX; Hind Lal, Suresh K Verma, TAMHSC-CVRI, Temple, TX; Guangrong Lu,
TAMHSC, Temple, TX; Donald M Foster, Central Texas Veterans Health Care System,
Temple, TX; Art Frankel, Scott & White Cancer Rsch Institute, Temple, TX; David E Dostal;
TAMHSC-CVRI, Temple, TX
Anthrax lethal toxin (LT) has recently been shown to cause decreased heart rate with increased
left ventricular end-diastolic volume, reduced ejection fraction and decreased contractility
within 24 hr after intraperitoneal injection in rats. In vitro studies have demonstrated that
anthrax LT decreases phosphorylation levels of glycogen synthase kinase 3-beta (GSK3␤) in
macrophages and initiates apoptotic pathways in noncardiac tissues. The purpose of this study
was to determine whether LT affects cardiac signaling, including the Akt and insulin pathways.
Results demonstrated that a bolus of LT (50 ␮g/kg LF and 100 ␮g/kg PA in 500 ␮l of
phosphate buffered saline) by intraperitoneal injection caused a ⬎30% decrease in left
ventricular ejection within 18 - 48 hr in adult male Sprague-Dawley rats (300 - 400 g).
Molecular studies from cardiac tissue lysates indicated that the cardiac insulin signaling
pathway was significantly affected in LT-treated rats. There were significant decreases in
phosphorylation levels of Akt (Thr308, Ser473) and GSK3␤ (Ser9), and phosphorylation levels of
p70S6K (Ser411) were significantly increased in heart tissue lysates from LT-treated rats
compared to control samples. Analysis of plasma at 48 hours after toxin injection revealed
significantly lower insulin (0.59 ⫾ 0.04 vs. 2.4 ⫾ 0.16, P⬍0.001) and higher glucose levels
(314.6 ⫾ 8.6 vs. 185.5 ⫾ 5.4, P⬍0.001) among LT-treated rats. To determine whether LT had
direct effects on the myocardium, LT was shown to cleave p38-MAP kinase kinase -3 (MEK-3)
in left ventricles of LT-treated rats. In summary, this is the first study to demonstrate that
anthrax LT has significant effects on serum insulin and glucose levels, as well as the cardiac
Akt/insulin signaling pathway, which may contribute to cardiac contractile dysfunction.
P50
Diverse Responses to Calcineurin Activation in Cardiac Myocytes Following
Exposure to Thapsigargin or Adrenergic Hypertrophy
Brian L Stauffer, Rebecca Sobus; Univ of Colorado, Denver, CO
P52
Helen Khanova, Camie Doyle, Irina Korichneva; UMDNJ, New Brunswick, NJ
Proten kinase C (PKC) is a long-known important signaling kinase that plays role in regulation
of vital cardiac functions. The two novel PKC isoforms ␦, ⑀, are particularly important in
myocardial responses to oxidative stress triggered by ischemia/reperfusion. In addition to the
activation by the classical lipid-mediated pathway, PKC can be triggered by reactive oxygen
species. We were the first to demonstrate that both PMA and hydrogen peroxide trigger the
release of chelated zinc ions from the zinc finger of the regulatory domain of PKC. However,
despite correlation of Zn2⫹ removal from PKC with the enzyme activation, in situ and cellular
models provided evidence that zinc is needed for normal functioning of the kinase. Thus, after
being released the zinc ion should stay in proximity with PKC to assist in re-folding after
activation so that the kinase would respond to the next message. In that respect, we searched
for potential partners of PKC among zinc-binding proteins. Two zinc transporters, ZnT1 and
ZnT5 were found by us to co-precipitate with PKC isoforms ␦ and ⑀. In addition, both PKCs were
detected in the immune complexes with anti-flag antibodies in HEK293 cells expressing
flag-ZnT5, thus confirming specificity of protein interaction and excluding the possibility of
cross-reaction of anti-ZnT5 antibodies with PKC. Complex formation was zinc-dependent:
zinc-depletion with the specific chelator TPEN promoted interaction. We have showed that in
adult rat cardiomyocytes in addition to predicted 60 kDa for ZnT1 and 80 kDa for ZnT5
monomers the zinc transporters exist as dimmers and tetrameres, but also may be processed
to 40 kDa and 60 kDa polypeptides, respectively. The processing has been stimulated by PKC
activators, PMA and H2O2. Interestingly, PKC activators increased ZnT5 association with PKC ⑀
and dissociated PKC ␦ from the complex, while at the same time caused ZnT1 association with
PKC ␦. We conclude that the mechanisms of zinc-dependent PKC interaction with zinc
transporters may serve a biomaker and potential therapeutic target in stressed myocardium.
The “b” Splice Variant of PLC␤1 Exclusively Mediates Gq Responses in
Cardiomyocytes and Causes Hypertrophy and Apoptosis
P53
Anand M Prasad, Giuseppe Inesi; California Pacific Med Cntr, San Francisco, CA
David R Grubb, Theresa M Filtz, Elizabeth A Woodcock; Baker Heart Rsch Institute,
Melbourne, Australia
ABSTRACT Cardiac myocytes were cultured from neonatal rats, and exposed to 20 ı̀M
phenylephrine (PE) to produce adrenergic hypertrophy, or to 10 nM thapsigargin (TG) to inhibit
the sarcoplasmic reticulum Ca2⫹ ATPase (SERCA2). Either treatment resulted in reduction of
Ca2⫹ transport by SERCA2, increase of resting cytosolic Ca2⫹, as well as reduction of cytosolic
Ca2⫹ rise following electrical stimuli. Furthermore in both cases, likely due to the high level of
resting cytosolic Ca2⫹, strong calcineurin activation was demonstrated by NFAT dependent
expression of transfected luciferase cDNA. Consistent with a calcineurin role in transcriptional
Activation of the heterotrimeric G protein Gq in cardiomyocytes leads to pathological
hypertrophy and apoptotic cell death in vivo and in vitro; however, Gq itself is not a good
therapeutic target as it is universally expressed and active. Subtypes of PLC␤, an immediate
downstream effector of Gq, may mediate these responses. Neonatal rat cardiomyocytes (NRVM)
express the two splice variants of PLC␤1, PLC␤1a (150 kDa) and PLC␤1b (140 kDa), which
differ only in their extreme C-termini. PLC␤1a is localized in the cytoplasm of NRVM whereas
PLC␤1b targets to the sarcolemma. The C-terminal amino acid residues of PLC␤1a comprise
e46
Circulation Research
Vol 103, No 5
August 29, 2008
a PDZ-interacting domain, whereas the PLC␤1b sequence contains a unique C-terminal
proline-rich domain. An inhibitory peptide from the C-terminus of PLC␤1b selectively
dissociated PLC␤1b from the sarcolemma and inhibited PLC response to Gq activation, with an
EC50 of 12 ⫾ 1.6 ␮M (mean ⫾ SEM, n ⫽ 3). Adenoviral over expression of PLC␤1b in NRVM
caused substantial hypertrophy followed by apoptosis and caspase-3 activation, however over
expression of PLC␤1a did not. Deletion of the unique C-terminal tail of PLC␤1b prevented its
sarcolemmal localization and activation, and these mutants did not cause hypertrophy or
apoptosis. This study demonstrates a requirement for the unique C-terminal domain of PLC␤1b
for sarcolemmal localization, PLC activity, cardiomyocyte hypertrophy and the induction of
apoptotic cell death. Further PLC␤1b but not PLC␤1a is solely responsible for Gq responses in
NRVM. The Gq/PLC␤1b interface provides a selective target of inhibiting Gq in cardiomyocytes.
P54
Impedes Mitogenic Signal Propagation (IMP) Is Essential for Embryonic
Development but Mediates Detrimental Cardiac MAPK Signaling In Vivo
Tim Seidler, Cornelia Grebe, Peter Schott, Bernhard Unsoeld, Albrecht Schmidt, Katrin Kurz,
Ralph Knoell, Kaomei Guan, Gerd Hasenfuss; Univ of Goettingen, Goettingen, Germany
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
IMP was recently identified as an important modulator of Ras dependent activation of ERK
MAPK signaling in non-cardiac cells. We previously reported IMP to be differentially regulated
upon load induced heart failure in a proteomics study. Thus, the aim of this study was to
analyse the role of IMP in myocardial hypertrophy and heart failure. Adenoviruses for
overexpression of IMP and a constitutively activated Raf mutant (Raf-BXB), were created to
study the effects of IMP in isolated rat cardiac myocytes. Transgenic mice, overexpressing IMP
cardiac-specifically and gene-trap mice derived from ES cells were used to analyse IMP in vivo.
In both adult and neonatal rat cardiac myocytes, IMP overexpression profoundly suppressed
MEK and ERK MAPK activation under baseline conditions and following Raf-BXB-stimulation.
This suggests an inhibitory effect of IMP on both adaptive hypertrophy and anti-apoptotic
signaling. Three independently created IMP-transgenic (TG) mouse lines in two different genetic
backgrounds exhibited grossly enlarged hearts, compared to wildtype littermates (WT).
Echocardiography confirmed dilation (left ventricular end-diastolic diameter 4.57⫾0.17 vs.
3,57⫾0.04 mm; p⬍0.05 IMP vs. WT; p⬍0.05; TG vs WT) and significant reduction of fractional
shortening (14⫾3% vs. 40⫾0.4%; IMP TG vs. WT; p⬍0.001). Invasive hemodynamic
measurements confirmed a phenotype consistent with a severe dilative cardiomyopathy.
Kaplan-Maier analysis showed a significantly decreased survival with 50% of mice dying before
the age of 24 weeks. Higher Bax/Bcl ratio and cytoplasmic cytochrome c release indicated
proapoptotic signalling contributed to the phenotype. Activation of JNK and p38 before the
development of phenotypic heart failure suggest activation of these MAPK triggers functional
deterioration. Gene-targeted IMP mice were not born in mendelian ratios, as homozygous IMP
gene-targeted mice were observed to die at embryonic day 9.5–10.5, while heterozygous mice
survived without phenotypic abnormalities until at least 6 months. Conclusions: These data
indicate that IMP can control cardiac size and function via profoundly altered MAPK signalling
and that IMP is essential to embryonic development.
P55
Stage-Specific Changes in Myofilament Protein Phosphorylation Following
Myocardial Infarction
Lori A Walker, John S Walker, Kelly Ambler, Peter M Buttrick; Univ of Colorado, Denver, CO
The responses of the heart to an insult include changes in the expression of signaling proteins,
growth regulatory molecules and modulators of gene expression, as well as in posttranslational modifications of existing proteins. Some of these are cardioprotective, while others
may contribute to failure of the myocardium. We have previously demonstrated that in models
of heart failure (HF) there is an increase in phosphorylation of key myofilament proteins such
as troponin I and MLC2. Functionally, we have demonstrated that HF is associated with a
decrease in maximum force generation and a decrease in calcium sensitivity. However, other
groups have found an increase in calcium sensitivity in failing hearts and a concomitant
decrease in phosphorylation of myofilament proteins. One difference in experimental paradigms
has been the stage of (mal)adapatation following the insult at which the samples are studied.
Therefore, in order to elucidate the discrepancies in these findings, we examined a mouse
myocardial infarction model at two early time points: two days and two weeks post infarct
(average infarct size ⫽40%). Phosphorylation of myofilament proteins was analyzed using
phosphospecific staining of one-dimensional polyacrylamide gels. Phosphorylation of myosin
binding protein c, MLC2 and troponin I were all significantly decreased relative to sham
operated animals at both time points. However, at two weeks, the decrease in phosphorylation
was blunted. No changes were seen in the phosphorylation of troponin T or desmin.
One-dimensional isoelectric focusing of troponin I coupled with phospho-specific antibody
staining revealed a site-specific redistribution of phosphorylation along the troponin molecule.
These data suggest that temporal changes in phosphorylation of myofilament proteins may
contribute both to the compensatory responses to an insult as well as to myocardial failure and
that a balance between kinase and phosphatase activities in the myocardium may define stage
specific adaptation to imposed stress.
P56
Cardiac Myosin Binding Protein-C Phosphorylation Modulates Relaxation in
Myocardium
Carl W Tong, Univ of Wisconsin, Madison, WI; Xin Wu, Mariappan Muthuchamy, Texas A&M
Univ, College Station, TX; Richard L Moss; Univ of Wisconsin, Madison, WI
Diastolic dysfunction is a difficult to treat component of heart failure due in part to incomplete
understanding of mechanisms underlying myocardial relaxation. Protein kinase A (PKA)
phosphorylation of cardiac myosin binding protein C (cMyBP-C) may regulate crossbridge
kinetics to modulate cardiac contractility and thereby impact diastolic function. We tested this
idea with (1) in vivo echocardiography and (2) simultaneous intracellular calcium [Ca2⫹]in and
force measurements on intact papillary muscle from mice expressing only non-PKAphosphorylatable mutant cMyBP-C. Transgenic expression of cMyBP-C with Ser to Ala
mutations of three known PKA sites onto a cMyBP-C knock out (KO) background produced a
mouse (tS3A) with mutant cMyBP-C expression of 74% when compared to normal wild type
(WT). Expression of WT cMyBP-C (72%) on the same background served as a control, i.e., tWT.
Besides hypertrophy, t3SA hearts exhibited increased isovolumetric relaxation time (IVRT) and
increased (E/e’) ratio, indicating diastolic dysfunction. Papillary muscle were paced at 1 Hz and
2Hz, and at 3 Hz -/⫹ dobutamine. t3SA muscles had [Ca2⫹]in transients similar to control but
a depressed force/frequency response. Following normalization of twitch forces to peak twitch
force at 1 Hz, t3SA myocardium produced a smaller increase in force in response to
dobutamine than observed in tWT muscle, i.e., the increase in twitch force per increase in
[Ca2⫹]in was depressed in t3SA, indicating that crossbridge kinetics were accelerated to a
lesser degree in t3SA. t3SA myocardium exhibited depressed relaxation, i.e., (-dF/dt)Max/(⫹dF/
dt)Max was less, indicating impaired relaxation beyond that expected on the basis of depressed
force generation. These differences remained with dobutamine. Thus, slowed kinetics in the
absence of cMyBP-C phosphorylation and the inability to accelerate cross-bridge kinetics with
dobutamine contributed to diastolic dysfunction in tS3A myocardium.
SUMMARY OF ECHOCARDIOGRAPHY AND PAPILLARY MUSCLE
MEASUREMENTS
Measurement Means⫾SEM
Echocardiography
Left ventricular inner diameter at diastole (mm)
Left ventricular posterior wall thickness at diastole (mm)
IVRT (msec)
E/(e’), peak blood inflow Doppler at mitral valve versus peak
tissue Doppler at mitral valve annulus
Force and Ca2ⴙ in intact papillary muscles
Twitch forces, 3 Hz vs 1 Hz
Twitch force / ⌬[Ca2⫹]in, 3 Hz vs 1 Hz
|(-dF/dt)Max/(⫹dF/dt)Max| at 3 Hz
tWT
t3SA
5
4.0 ⫾ 0.1
1.0 ⫾ 0.1
16.0 ⫾ 1.0
18.1 ⫾ 2.1
5
4.0 ⫾ 0.1
1.5 ⫾ 0.1
22.5 ⫾ 1.6
30.1 ⫾ 3.9
n.s.
p⬍0.05
p⬍0.05
p⬍0.05
6
3.11⫾0.74
2.34⫾0.50
0.64⫾0.05
7
1.41⫾0.17
1.26⫾0.14
0.49⫾0.02
p⬍0.05
p⬍0.05
p⬍0.05
P57
Snf1-Related Kinase Is Upregulated in the Ischemic Heart and Regulates
Cellular Metabolism and Death
Michael A Burke, George Ochenjele, Northwestern Memorial Hosp, Chicago, IL; Richard P
Fernandez, Children’s Memorial Hosp, Chicago, IL; Sathyamangla V Prasad, Cleveland Clinic
Foundation, Cleveland, OH; Hossein Ardehali; Northwestern Memorial Hosp, Chicago, IL
Sucrose non-fermenting (Snf1)-related kinase (SNRK) is a novel kinase homologous to the Snf1
kinase of yeast and the AMP-activated protein kinases (AMPK) of mammals. While AMPK and
Snf1 regulate metabolism, the primary function of SNRK is not known. SNRK is upregulated
during apoptosis in neuronal cells; however, it is unclear whether this promotes or inhibits cell
death. We hypothesized that SNRK plays an active role in cell death and, like AMPK and Snf1,
regulates cellular metabolism. Western blots of protein extracts from several cell types showed
that SNRK is widely expressed and exists in different forms in the nucleus and cytoplasm. We
next assessed the effects of ischemia on SNRK levels in the heart. Dog hearts subjected to
ischemia displayed increased SNRK levels as did explanted hearts of patients with ischemic
cardiomyopathy. We then studied the role of SNRK on cell death by expressing GFP-tagged
SNRK in HEK293 cells. Overexpression of SNRKGFP increased cell death compared to controls
in the absence and presence of H2O2. We subsequently studied the role of SNRK on cellular
metabolism. First, we transfected HEK293 cells with GFP, SNRKGFP, or SNRKGFP containing a
mutation in the nuclear localization sequence (SNRK-nls). The levels of the transcription factors
NRF2␣ and NRF2␤, which regulate mitochondrial biogenesis, were increased by 30% and 90%
respectively, with SNRK overexpression. SNRK-nls did not induce a significant change.
Furthermore, SNRK overexpression resulted in a significant reduction in cellular ATP content
compared to controls. Second, we assessed the response of endogenous SNRK to changes in
metabolic stimuli. When glucose was removed from the media, SNRK mRNA increased and
SNRK protein shifted from the nucleus to the cytoplasm. Similarly, SNRK mRNA levels increased
in response to AMP. These data show that SNRK is upregulated in ischemic heart disease and
regulates cell death. SNRK also appears to be an integral component of the metabolic
machinery of the cell. Via alterations in cellular metabolism, SNRK may play an important role
in the response of the heart to ischemia.
P58
NOS3-Dependent Nitric Oxide Production and Hemodynamic Outcomes in a
Mouse Model of Cardiac Arrest
David Beiser, Gerasim Orbelyan, Kimm Hamann, Terry L Vanden Hoek; Univ of Chicago,
Chicago, IL
OBJECTIVE: To characterize the enzyme-mediated production of nitric oxide following cardiac
arrest and resuscitation. METHODS: Adult female wildtype (WT, n⫽5) and NOS3-/- mice (n⫽8)
were anesthetized, intubated, ventilated, and instrumented with intravascular catheters.
Asystole was induced with a bolus of KCL. After 8 minutes of arrest, animals were resuscitated
with mechanical ventilation and chest compressions. Left-ventricular pressure-volume dynamics were monitored until 120 minutes following return of spontaneous circulation (R120). At
R120, whole blood nitrosylhemoglobin (HbNO), as detected by electron paramagnetic resonance spectroscopy, was used as an indirect measure of circulating nitric oxide equivalents.
HbNO was also measured in groups of animals (WT and NOS3-/- n⫽4 at each time point)
sacrificed at: baseline (BL), following 8 minutes of untreated arrest (CA8) or 20 minutes of
arrest (CA20), and at 60 minutes following resuscitation (R60). RESULTS: In the R120 group,
BCVS Conference 2008 Abstracts
return of spontaneous circulation was achieved in 4 of 5 (80%) WT and 4 of 8 (50%) NOS3-/animals. At BL, NOS3-/- animals displayed higher maximum left-ventricular pressure (LVPmax)
than WT animals (113.3 ⫾ 13.19 mmHg vs. 86.8 ⫾ 4.48 mmHg, p⬍0.05). Following
resuscitation, NOS3-/- animals displayed lower LVPmax (51.97 ⫾ 7.429 vs. 66.95 ⫾ 4.394
mmHg, p⬍0.05) and left ventricular stroke work (132.5 ⫾ 26.28 mmHg䡠␮L vs. 456.5 ⫾ 128.9
mmHg䡠␮L, p⬍0.05) than WT. NOS3-/- animals displayed significantly higher left-ventricular
relaxation time constants (tau ⫽ 25.82 ⫾ 6.471 ms vs. 15.45 ⫾ 2.223 ms, p⬍0.05) at R120.
At BL, whole blood HbNO concentrations were equivalent in WT and NOS3-/- animals (0.75 ⫾
0.10 ␮M vs. 0.86 ⫾ 0.11 ␮M, p⫽0.46). In WT animals, HbNO concentrations increased during
untreated arrest at CA20 (1.42 ⫾ 0.19 ␮M, p⬍0.05) and following resuscitation at R120
(1.66 ⫾ 0.25 ␮M, p⬍0.05) relative to BL. NOS3-/- animals HbNO concentrations were lower
than WT at all time points following cardiac arrest (p⬍ 0.05). CONCLUSIONS: Nitric oxide
equivalents increase following cardiac arrest. Such increases appear to be enzyme-mediated
and may play an important role in preserving cardiovascular function following the return of
spontaneous circulation.
P59
Expression of Akt/Pim-1 Survival Proteins in Myocardial Remodeling
Yang Gao, Kinjal Savai, Tielou Li, Changfu Wu, Meredith Bond, Zhongjun J Wu, Bartley P
Griffith; Univ of Maryland Sch of Medicine, Baltimore, MD
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Background: A cascade of molecular events is involved in the cardiac remodeling process post
myocardial infarction (MI). Most recently, Pim-1 was indentified as a regulator of cardiomyocyte
survival downstream of Akt. The protective effects of Akt in the myocardium have been
intensively studied; but the cardioprotective role of Pim-1 as a signaling protein has not been
fully clarified. The objective of this study was to investigate the expression of Akt/Pim-1 survival
proteins and their association with the degree of myocardial remodeling. Methods: A 25% MI
was created in an ovine model (N⫽9). The in vivo regional deformation of the LV (adjacent and
remote myocardium with respect to the infarct) was monitored by 16 sonomicrometric crystals
sutured to the myocardium free wall. Data were collected pre-infarction, and weekly until
termination (10 –12 weeks post-infarction). At termination, regional myocardial tissues were
collected for analyses of Akt/Pim-1 relevant survival proteins and apoptosis. Results: The
terminal remodeling strains in the remote and adjacent zones of the myocardium were 14⫾3%
and 49⫾5%, respectively. As compared to the remote zone, significant up-regulation of the
major regulatory survival proteins in the Akt/Pim-1 pathway were observed in the adjacent
zone: Akt (1.44-fold), Pim-1 33KD (1.75-fold), Pim-1 44KD (1.34-fold), Bcl-2 (2.94-fold) and
Bcl-XL (1.69-fold). The elevated protein expressions were also found to correlate with the
regional remodeling strains: Akt (R2⫽0.45), Pim-1 33KD (R2⫽0.46), Pim-1 44KD (R2⫽0.37),
Bcl-2 (R2⫽0.51) and Bcl-XL (R2⫽0.46). The adjacent zone had more TUNEL positive cardiac
cells (7.10⫾ 1.24) than the remote zone (1.77⫾ 0.30). The p values for all the comparisons
and correlation analyses are ⬍0.05. Conclusions: A positive correlation between the
Akt/Pim-1 survival protein expressions and the regional remodeling strain is observed. Our data
suggested that the upregulation of these proteins was not enough to protect heart cells from
apoptosis, as evidenced by the TUNEL results. Further studies will focus on elucidating if the
up-regulation of Akt/Pim-1 survival proteins directly result from elevated remodeling strains
and if these survival proteins are antagonized in downstream.
P60
Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 1A Is a NFAT
Kinase Mediating Negative Feedback on Calcineurin/NFAT Signaling in
Cardiac Myocytes
Cornelia Grebe, Theda M Klingebiel, Bernhard Unsoeld, Gerd Hasenfuss, Tim Seidler; Univ of
Goettingen, Goettingen, Germany
Calcineurin/NFAT transcriptional signals play pivotal roles in cardiac hypertrophy and heart
failure. Recently, Dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) were
identified as a group of NFAT priming kinases in non-cardiac cells. We examined DYRK for their
potential role in regulating Calcineurin/NFAT in cardiomyocytes. Expression of DYRK isoforms
was examined in human cardiac tissue samples using primers for DYRK1A, 1B, -2, -3, -4 and
-6 by quantitative real-time PCR. Adenoviruses for overexpression of DYRK1A and a
kinase-dead mutant (DYRKK188R), were created to study the effects of DYRK in vitro in isolated
rat cardiac myocytes. Nuclear translocation of NFAT using GFP-tagged NFATc3 and DYRK
expression in response to Calcineurin overexpression were measured 48h after viral
transduction with the examiner blinded towards the group. Planimetric cell surface calculations
were conducted 48 h after adenovirus transfection. Expression levels of DYRK isoforms relative
to GAPDH in human cardiac tissue was highest for DYRK1A with 3,61⫾0,7x10 –2. Expression
of DYRK1B,2,3,4 and-6 were 4,08⫾0,9 x10 –3; 4,37⫾0,2 x10 –3; 1,18⫾0,026x10 – 4;
7,92⫾0,2 x10 – 4; 2,82⫾0,4x10 –2, respectively; n⫽3. While under basal culture conditions
GFP-tagged NFAT was almost exclusively cytoplasmic (1,3⫾1,0 % nuclear NFAT), neither
co-expression of Ad-DYRK1A (4,0⫾5,1%) nor Ad-DYRK1AK188R (2,6⫾2,4%) changed GFPNFAT localization. Upon adenoviral co-expression of Calcineurin, NFAT-GFP was located
predominantly nuclear (82,03⫾1,94%). In these stimulated cells, co-expression of Ad-DYRK1A
(0,83⫾1,16%), but not kinase-dead DYRK1A (71,96⫾6,13%); p⬍0.01 led to a profound
reduction in nuclear NFAT localization. Finally, expression of Calcineurin in Cardiomyocytes
increased DYRK1A expression significantly by 29,7%, compared to control-transfected cells,
suggesting DYRK1A is part of a negative feedback controlling Calcineurin/NFAT signaling.
DYRK1A represents the DYRK isoform with highest expression in Cardiomyocytes. DYRK1A, but
not kinase-dead DYRK robustly reduces nuclear NFAT localisation in Calcineurin stimulated
cells and is part of a negative feedback, limiting Calcineurin/NFAT signaling in cardiomyocytes.
e47
P61
Upregulation of Thioredoxin-1 Through AMP-Activated Protein Kinase Is
Protective in the Ischemic Heart
Dan Shao, Tetsuro Ago, Chiao P Hsu, Junichi Sadoshima; UMDNJ, Newark, NJ
Thioredoxin-1 (Trx1), a 12K Da protein, plays an essential role in regulating the cellular redox
state, growth, and apoptosis in cardiac myocytes. Although Trx1 is upregulated by stress and
plays a protective role in the heart, underlying mechanisms mediating upregulation of Trx 1 and
the cardioprotective effects of Trx1 remain to be elucidated. Glucose deprivation (GD)
upregulated protein expression of Trx1 1.8 fold at 6 h, and further 2.0 fold at 24 h in neonatal
rat cardiac myocytes in vitro, which was accompanied by activation of AMP activated protein
kinase (AMPK) by 2.7 fold. AICAR, a pharmacological activator of AMPK, upregulated Trx1 2.3
fold at 6 h, and further 2.76 fold at 24 h, mimicking the effect of GD. Inhibition of AMPK activity
by adenovirus harboring dominant negative AMPK (Ad-DN-MPK) attenuated upregulation of
Trx1 in response to GD or AICAR in cardiac myocytes. In vivo, expression of Trx1 was
significantly increased in the ischemic heart, which was attenuated in Dn-AMPK transgenic
mice. These results suggest that AMPK plays an essential role in mediating upregulation of Trx1
by GD or ischemia. Knocking down of Trx1 by adenovirus harboring sh-Trx1 or inhibition of
AMPK by Ad-DN-AMPK increased cell death by GD, which was determined by the cell viability
assays, suggesting that upregulation of Trx1 through activation of AMPK is protective against
GD. A possible underlining mechanism might be the regulation of mitochondria biogenesis by
Trx1. In fact, upregulation of genes involved in mitochondria biogenesis, including cytochrome
C, NRF-1, and t-FAM, under GD was significantly attenuated by downregulation of Trx1 using
adenovirus harboring sh-Trx1 in cardiac myocytes. Furthermore, intracellular ATP content
under GD was further decreased by downregulation of Trx1 compared with the control group.
These results suggest that Trx1 is upregulated by ischemia through AMPK-dependent
mechanisms which in turn upregulates genes involved in mitochondrial biogenesis and
preservation of the ATP content, thereby playing a protective role in the heart under stress.
Cam Kinase II ␦ and ␥ Are Required for Phosphorylation and Nuclear
Export of Histone Deacetylase 4 in Response to ␤-Adrenergic Signaling
P62
Johannes Backs, Thea Backs, Univ of Heidelberg, Heidelberg, Germany; David Patrick,
Joseph A Hill, Eric N Olson; UTSW Med Cntr at Dallas, Dallas, TX
␤-adrenergic receptor (␤-AR) signaling plays a pivotal role in the pathogenesis of congestive
heart failure. Although numerous studies have investigated the mechanisms of ␤-AR signal
transduction, the final steps towards cardiac transcription are not well understood. Class IIa
histone deacetylases (HDAC4, 5, 7 and 9) are signal-responsive repressors of the MEF2
transcription factor, which drives pathological cardiac remodeling. CaM Kinase II (CaMKII) is a
major downstream kinase of ␤-ARs, which we have shown to signal selectively to HDAC4.
However, whether ␤-AR signaling modulates phosphorylation of HDAC4 has not been
previously investigated. Here, we show that ␤-AR signaling, mimicked by isoproterenol (Iso)
treatment of neonatal rat cardiomyocytes, induces phosphorylation of HDAC4 but not to other
HDACs, as measured by binding to the 14 –3–3 chaperone protein. We generated mutant mice
that lack the ␦- and ␥-isoform of CaMKII, the two most abundant CaMKII isoforms expressed
in cardiomyocytes. Using in vitro kinase assays, we show that heart lysates from CaMKII␦
knockouts (KO) lack cardiac HDAC4 kinase activity. Similarly, heart lysates from CaMKII␥ KO
mice display reduced levels of cardiac HDAC4 kinase activity. Moreover, Iso treatment resulted
only in an increase of cardiac HDAC4 kinase activity in wild type (WT) littermates but not in
CaMKII␦ KO mice. CaMKII␥ KO also displayed an attenuated response to Iso. We then
investigated the cardiac hypertrophic response of CaMKII␦ and ␥ KO animals to stress induced
by Iso infusion and transverse aortic constriction (TAC). Iso increased the heart weight/body
weight ratio in WT by 40% but in CaMKII␦ KO only by 18% and in CaMKII␥ KO by 13%. TAC
increased the heart weight/body weight ratio in WT by 52% but in CaMKII␦ KO only by 12%
and in CaMKII␥ KO by 22%. Accordingly, in response to TAC, fetal gene expression (BNP and
Myh7) was up-regulated in WT but not in CaMKII␦ and ␥ KO mice. In conclusion, we
demonstrate by the use of specific gene targeting an essential role of the two major cardiac
CaMKII isoforms for cardiac hypertrophy in response to ␤-AR signaling. CaMKII␦ and ␥ are
required for HDAC4 phosphorylation and nuclear export, suggesting that ␤-ARs mediate cardiac
hypertrophy via the CaMKII-HDAC4 pathway.
PDK-1 Is Required for ␤-Adrenergic Response and Cell Survival in the
Heart
P63
Kaoru Ito, Hiroshi Akazawa, Issei Komuro; Chiba Univ Graduate Sch of Medicine, Chiba,
Japan
Background: The 3-phosphoinositide-dependent kinase-1 (PDK-1) is a ubiquitously expressed
kinase that plays important roles in mediating a variety of effects of growth factors on cells.
In particular, PDK-1 is crucial in the phosphoinositide 3-kinase (PI3-K) signaling pathway
through phosphorylating and activating several kinases including Akt and p70 S6 kinase.
Although PDK-1 was reported to be important for the regulation of cardiomyocyte size, the
significance of PDK-1 in the heart is not well understood. Methods and Results: To elucidate
the roles of PDK-1 in the postnatal heart, we generated tamoxifen-inducible heart specific
PDK-1 knockout mice (PDK-1 MerCre) and disrupted PDK-1 gene at the age of 10 weeks.
PDK-1 MerCre died of heart failure from 5 to 15 weeks after dispruption of PDK-1. At 1 week
after PDK-1 disruption cardiomyocyte size was not reduced, but left ventricular systolic
dysfunction was already observed. Langendorff perfusion indicated impairment of isoproterenol
responsiveness in PDK-1 MerCre hearts. Mechanistically, PI3-K ␥ activity was enhanced, which
might lead to ␤-adrenergic receptor desensitization by activating G-protein coupled receptor
e48
Circulation Research
Vol 103, No 5
August 29, 2008
kinase 2 (GRK2), because cardiac-specific overexpression of PIK-domain of PI3-K ␥ prevented
␤-adrenergic receptor internalization by inhibiting the association between PI3-K ␥ and GRK2.
Furthermore, TUNEL staining and immunohistochemical staining for cleaved caspase-3
revealed an extreme increase in the number of apoptotic cardiomyocytes 1 week after
inactivation of PDK-1. PDK-1 MerCre hearts showed reduction of SGK activity, upregulation of
bax protein and endoplasmic reticulum stress signals, which were potentially involved in
cardiomyocyte apoptosis. The expression levels of PDK-1 protein were decreased in murine
models of heart failure induced by myocardial infarction or systemic administration of
adriamycin. Conclusions: These results suggest that PDK-1 is essential for normal cardiac
function by preserving responsiveness to ␤-adrenergic stimulation and by preventing
cardiomyocyte apoptosis. PDK-1 might be a promising molecular target for treatment of heart
failure.
P64
Ubiquitin Proteasome Dysfunction in Human Cardiomyopathies
Jaime Predmore, Frank Davis, Sarah Bartolone, Margaret V Westfall, Francis Pagani, Univ of
Michigan, Ann Arbor, MI; Ping Wang, Feinstein Med Rsch Institute, Manhasset, NY; Hanna
Osinska, Jeffrey Robbins, Cincinnati Children’s Hosp Med Cntr, Cincinnati, OH; Saul Powell,
Feinstein Med Rsch Institute, Manhasset, NY; Sharlene M Day; Univ of Michigan, Ann Arbor,
MI
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
As the major pathway for intracellular protein degradation in the heart, the ubiquitin
proteasome system (UPS) maintains a dynamic equilibrium of proteins and prevents accumulation of damaged and misfolded proteins. Prior work on UPS function in heart failure and
hypertrophy has produced disparate results and underlying mechanisms have not been
elucidated. The objectives of this study are to characterize and compare measures of UPS
function in human heart failure and hypertrophic cardiomyopathy (HCM), and to explore
potential mechanisms responsible for the aberrations observed. Methods and Results:
Proteasome function was studied in human nonfailing donor (NF) hearts, in explanted failing
hearts (F), and in myectomy samples from HCM patients. Chymotrypsin-like and caspase-like
activities were significantly reduced (⬃50%) in F and HCM hearts vs NF hearts (P⬍0.01). There
was a significant increase in the accumulation of polyubiquinated proteins in F hearts vs NF
(⬃2-fold, P⬍0.05), but no significant difference between HCM and NF. Immunoreactivity to an
antibody that recognizes soluble pre-amyloid oligomers was increased in F and HCM hearts
compared to NF, suggesting accumulation of damaged or misfolded protein aggregates. There
were no changes in the protein content of UPS subunits (i.e. 11S, 20S, and 19S) among groups
to explain decreased UPS activity in HCM and F hearts. Furthermore, active site labeling of the
20S proteolytic subunit ␤-5 with a fluorescent activity-based proteasome probe revealed no
differences among groups, indicating an intact proteolytic core in both disease states. Protein
carbonyls and 4-hydroxynonenated proteins were significantly increased in F hearts vs NF
hearts (P⬍0.05), raising the possibility that proteasome inhibition could be due in part to
oxidative damage to regulatory proteasome subunits. Conclusion: Impaired proteasome
activity and protein aggregate formation in HCM and F human hearts is observed in the absence
of changes in proteasome protein content or availability of proteolytic active sites. We
hypothesize that a defect in regulation of protein substrate processing, and/or oxidative damage
to accessible proteasome subunits, may account for defective protein degradation in these
human disease states.
P65
MicroRNA320 Regulates Ischemia/Reperfusion-Induced Cardiomyocyte
Death and Apoptosis by Targeting Hsp20
Jinghai Wu, Xiaoping Ren, Xiaohong Wang, Persoulla Nicolaou, Meifeng Xu, W Keith Jones,
Evangelia Kranias, Guo-Chang Fan; Univ of Cincinnati College of Medicine, Cincinnati, OH
Recent studies have identified critical roles for microRNAs (miRNAs) in a variety of cellular
processes, including regulation of cardiomyocyte death. However, the signature of miRNA
expression and possible roles of miRNA in the ischemic heart have not yet been reported. Here
we performed microRNA arrays to detect the expression pattern of miRNAs in murine hearts
subjected to ischemia/reperfusion (I/R) in vivo. Surprisingly, we found that only miR320
expression was significantly decreased in the hearts 24 h after a 30-min coronary artery
occlusion. This was further confirmed by stem-loop RT-PCR (67% decrease, n⫽6, p⬍0.01).
Overexpression of miR320 by adenovirus in cultured adult rat cardiomyocytes enhanced cell
death and apoptosis upon simulated ischemia/reperfusion, while knock-down of miR320 by
antisense miR320 (Ad.asmiR320) showed the cytoprotective effects, assessed by MTS
incorporation and DNA/nuclear fragmentation. Furthermore, transgenic mice with cardiacspecific overexpression of miR320 revealed an increased extent of apoptosis and infarction size
in the hearts upon I/R in vivo, relative to the WT controls. Using Target-Scan software, we
identified Hsp20, a known cardio-protective protein, as an important candidate target for
miR320. To test this prediction, we examined the expression levels of Hsp20 in Ad.miR320and Ad.asmiR320-infected cardiomyocytes. Indeed, the protein levels of Hsp20 were decreased
by 40% in Ad.miR320-cardiomyocytes, and were increased by 70% in Ad.asmiR320-cells.
Quantitative RT-PCR also showed reduced mRNA expression of Hsp20 in Ad.miR320cardiomyocytes. To elucidate the mechanisms regulating the expression of Hsp20, we analyzed
the effect of miR320 on Hsp20’s 3’UTR activity via transient transfection using both HEK 293
cells and H9C2 cardiomyocytes. In agreement with the mRNA data, the Hsp20 3’UTR activity
was strongly inhibited (up to 4 fold) by miR320. The in vivo experiments further confirmed that
Hsp20 protein levels were increased by 2.1 fold, consistent with the reduction of miR320 levels
upon I/R. Taken together, our findings suggest that miR320 is involved in the regulation of
I/R-induced cell death via antithetical regulation of Hsp20.
P66
Juvenile Anthracycline Treatment Contributes to Heart Failure in Adulthood
by Impairing Vascularization and Cardiac Stem Cell Function
Chengqun Huang, Lucy Kim, BioScience Cntr SDSU, San Diego, CA; Natalie Gude, Mark A
Sussman, SDSU, San Diego, CA; Roberta A Gottlieb, Asa B Gustafsson; BioScience Cntr
SDSU, San Diego, CA
The anthracycline doxorubicin (DOX) is an effective chemotherapeutic agent used to treat
pediatric cancers. However, it is associated with cardiotoxicity which can manifest many years
after the initial exposure. Very little is known about the mechanisms of this late-onset
cardiotoxicity. To understand this problem, we have developed a model of pediatric DOX
cardiotoxicity where mouse pups were injected with 4 doses of 0.5 mg/kg DOX or saline at 5,
10, 15, and 20 days of age. All animals were healthy and had normal cardiac function as adults.
However, when mice were subjected to myocardial infarction (MI), DOX treated mice developed
severe cardiac fibrosis and had increased mortality compared to saline treated mice (67% vs.
33%), suggesting that these hearts do not tolerate stress well. Assessment of infarct size
revealed that the absolute infarct area was greater in DOX-treated hearts; however, when
calculated as % of area at risk, it was comparable to saline controls. Immunohistochemical
analysis of the vasculature showed a reduced number of vessels in DOX treated hearts,
suggesting that DOX impairs cardiac angiogenesis. Moreover, cardiac progenitor cells (CPC)
have been identified in the heart and these cells are thought to facilitate growth during
adolescence and provide a mechanism for minor repair and ongoing cell turnover. We found
that DOX treatment reduced the number of cells positive for the stem cell marker c-kit in hearts
of juvenile mice. DOX treatment also caused upregulation of the cellular senescence marker
p16INK4a in c-kit⫹ cells. Treatment of isolated CPC with DOX reduced proliferation as assessed
by MTT assay and BrdU incorporation, but did not increase cell death, suggesting that DOX
reduces stem cell number through effects on proliferation rather than cell death. MI is known
to stimulate proliferation of c-kit⫹ cells in the border zone. However, DOX treated mice had
significantly fewer c-kit⫹ cells in the border zone after MI compared to saline, suggesting that
juvenile doxorubicin treatment permanently reduced the number of resident CPCs in these
hearts. Our data suggest that anthracyclines impair stem cell function as well as vascular
development in the young heart, resulting in an adult heart that is more susceptible to stress.
P67
Inherent Capacity of the Heart to Augment Antioxidant Defense via
Activation of Amino Acid Metabolism as a Hormetic Response to
Aldehydes
Motoaki Sano, Jin Endo, Takaharu Katayama, Keiichi Fukuda; Keio Univ Sch of Medicine,
Tokyo, Japan
A loss of specialized antioxidant function results in the increasing prevalence of heart failure
with age. Lipid peroxidation-derived aldehydes have been implicated in most pathogenic effect
associated with oxidative stress and oxidative damage by aldehydes accumulates with age. To
characterize a causative role of aldehydes in the pathogenesis of age-associated diseases in
a tissue-specific manner, we developed a transgenic loss-of-function model of aldehyde
dehydrogenase (Aldh) using over-expression of Aldh2*2. Despite higher levels of aldehydes,
Aldh2*2 transgenic hearts displayed normal contractility and an unexpected tolerance to
oxidative stress imposed by ischemia-reperfusion injury. Integrated analysis of metabolome
and transcriptome revealed that this “hormesis” was accomplished by the metabolic
remodeling. This involved maintenance of high levels of glutathione by the activation of amino
acids (serine and cysteine) biosynthetic pathways that provides precursors for glutathione.
Aldehydes-mediated activation of GCN2-eIF2␣-ATF4 pathway contributed to this metabolism
change. Disruption of 3-phosphoglycerate dehydrogenase, a critical enzyme in serine
biosynthesis and the most prominently induced gene in Aldh2*2 transgenic hearts, decreased
intracellular glutathione and sensitized cardiomyocytes to death by oxidative stress both in vitro
and in vivo. Thus, the activation of amino acid metabolism as a hormetic response to aldehydes
confers upon hearts favorable tolerance to oxidative stress. Mimetic triggers of hormesis
appear to offer a promising and effective approach for preventing and delaying the onset of
age-associated diseases including heart failure.
P68
Combination Renin Angiotensin System Improves Ventricular Lusitropy
Without Affecting Cardiac Fibrosis or Cardiac RAS in the Hypertensive
mRen2.Lewis Rat
Jewell A Jessup, Brian M Westwood, Mark C Chappell, Leanne Groban; Wake Forest Univ
Sch of Medicine, Winston-Salem, NC
Hypertension and left ventricular (LV) hypertrophy often precede diastolic dysfunction and are
risk factors for diastolic heart failure development. Pharmacologic inhibition of the renin
angiotensin system (RAS) improves diastolic function and functional capacity in hypertensive
patients with LV hypertrophy. In hypertensive rats, RAS blockade attenuates LV hypertrophic
development by lowering blood pressure (BP). Whether the lusitropic and remodeling benefits
of RAS blockade are due to direct modulation of opposing cardiac RAS components,
specifically, reduced Ang II, and increased Ang-(1–7), remain unclear. Accordingly, we
assessed the effects of dual RAS blockade on diastolic function and LV structure in the
congenic mRen(2).Lewis male rat, a model of tissue renin overexpression and Ang II-dependent
hypertension. Compared with vehicle (VEH) (n⫽8), combination therapy of angiotensin
converting enzyme (ACE) inhibition (lisinopril) and Ang II type-1 receptor (AT1R) blockade with
losartan (LIS/LOS, 10 mg/kg/day, p.o) in 15-wk-old, age-matched rats (n⫽7), lowered systolic
BP 52% (P⬍0.001), without affecting heart rate or systolic fractional shortening. While RAS
blockade decreased plasma Ang II and increased Ang-(1–7), there were no changes in cardiac
BCVS Conference 2008 Abstracts
tissue expression of ACE, ACE2, AT1, and mas receptor mRNAs or ACE2 activity. Despite 37%
and 44% (P⬍0.05) reductions in cardiac hypertrophy and posterior wall thickness, respectively,
LIS/LOS did not alter cardiac interstitial or perivascular collagen. RAS inhibition improved
Doppler indices of myocardial relaxation; specifically, isovolumic relaxation time decreased
(VEH 0.037⫾0.003 vs. LIS/LOS 0.021⫾0.000 sec, P⬍0.0001) and tissue Doppler mitral
annular descent increased (e⬘) (VEH 2.00⫾0.15 vs. LIS/LOS 4.33⫾0.36 cm/s, P⬍ 0.001). The
improved lusitropy in LIS/LOS rats was associated with a 37% reduction in filling pressure
determined by the ratio of early transmitral filling velocity to early mitral annular velocity (E/e⬘)
(P ⬍ 0.001). We conclude that the lusitropic benefit of dual RAS blockade may be due to
improved vascular hemodynamics and/or cardiac calcium homeostasis rather than the
prevailing levels of the cardiac tissue RAS and its effects on myocardial fibrosis.
e49
(UDCA) (500 ␮g/mL) for 5 hours, following which the cells were lysed and subjected to Western
blot analysis. Macrophages treated with tunicamycin had elevated ER-stress (as evidenced by
an increase in the levels of phosphorylation of eukaryotic translation initiation factor-2 alpha
subunit and glucose reactive protein GRP-78), decreased insulin signaling (as evidenced by
attenuated levels of phospho-Akt and increased insulin resistance (increase in the phosphorylation of c-Jun N-terminal kinase JNK). All these parameters were partly reversed following
cellular treatment with UDCA. Treatment with tunicamycin also caused an increase in the
protein levels of CD-36 which resulted in an enhanced uptake of oxidized LDL by macrophages.
Treatment with UDCA inhibited upregulation of these scavenger receptors and attenuated the
uptake of oxidized-LDL by macrophages. These findings have consolidated that notion that
insulin resistance may predispose macrophages to upregulated capacity in LDL-uptake.
Consequently, inhibition of insulin resistance may have potential utility in inhibiting macrophage
LDL-uptake and thus atherosclerotic plaque formation.
P69
Nonmuscle Myosin Type II Distribution in the Mouse Intestinal Villi Is
Altered by B-Type Natriuretic Peptide
Anteneh Addisu, William R Gower, Jr, John R Dietz; Univ of South Florida, Tampa, FL
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Our prior studies have shown that BNP decreases absorption from the GI tract. Since
non-muscle myosin type II is known to have a role in epithelial tight junction regulation and
paracellular absorption of water and small molecules, we aimed to test whether BNP has an
effect on the distribution pattern of non muscle myosin in intestinal villi. We treated wild type
mice with a 10ng/g intravenous bolus of BNP followed by a 1ng/g/minute infusion for 30
minutes vs. vehicle. The mice were then euthanized and intestinal tissue isolated and sectioned
into jejunum, ileum and colon. The tissue was immunostained for non muscle myosin type II
and examined using fluorescence and electron microscopy. The vehicle was used as
experimental control and a tissue section not treated with antibody to non-muscle myosins was
used as a methodological control to evaluate the non specific background binding. Fluorescence microscopy revealed markedly enhanced distribution of non muscle myosin at the crypts
and villus core of the jejunum and ileum of BNP treated mice as compared to vehicle. Electron
microscopy revealed that the microvilli of BNP treated mice lost the distinctive appearance of
contracting microvilli as compared to vehicle. Such an appearance was previously shown to
lead to decreased paracellular permeability of epithelia. Our observation supports the
hypothesis that elevated plasma BNP to pathophysiological levels (around 500pg/ml) makes the
tight junctions of intestinal microvilli less permeable to water and small molecule solutes. We
postulate that decreased paracellular water and electrolyte movement while allowing continued
trans-cellular transport at the apical membrane of intestinal microvilli may be one way the
failing heart attempts to modulate preload by regulating water and electrolyte absorption from
the intestine. Further characterization of this process and understanding of the specific
mediators involved is likely to lead to the identification of novel therapeutic targets for the
treatment of heart failure.
P70
Comparative Effect of Adenoviral Infection and Exogenous Administration
of GDF-15 on Prevention of Cardiac Hypertrophy
David L Crandall, Orlando F Bueno, Christopher A Davis, Thomas M Antrilli, Viera
Kasparcova, Yijie Gao, Chris Brown, Robert V Martinez; Wyeth Rsch, Philadelphia, PA
GDF-15 is a member of the TGF-␤ superfamily, and its plasma elevation has recently been
shown to have prognostic value as a new biomarker in patients with heart failure,
non-ST-elevation ACS, and pulmonary embolism. In preclinical models, GDF-15 exhibits both
antihypertrophic and cardioprotective effects following either adenoviral administration or
transgenic overexpression of the protein. The antihypertrophic activity of exogenously
administered recombinant GDF-15 has not been extensively investigated; however, these data
are critical prior to establishing the in vivo profile of the protein as a potential biotherapeutic.
Rat neonatal cardiomyocytes infected with human GDF-15 adenovirus exhibited an antihypertrophic response to exogenous phenylephrine (PE), and secreted ⬃500 pg/ml/24 h of GDF-15
into the conditioned medium (CM). Gene expression profiling indicated that in the presence of
adeno-GDF-15 ⫹ PE, hepcidin antimicrobial peptide, IGF-1 (insulin-like growth factor-1), and
cadherin expression were increased, while corin and stanniocalcin were repressed. However,
recombinant human GDF-15 from 3 different sources (R&D Systems, E. coli & CHO derived)
added exogenously to cardiomyocytes (20 –200 ng/ml) was ineffective in inhibiting PE-induced
hypertrophy. Adenoviral GDF-15 effectively blunted the PE-induced expression of ␣-actin in
cardiomyocytes, but again, all exogenous recombinant sources of GDF-15 were unable to
impact fetal gene expression. This is the first report of activity differences between adenoviral
and exogenous recombinant GDF-15. Further characterization of GDF-15 protein processing
and secretion may provide insight into the basis of activity, and be critical to further
understanding the biological role of GDF-15.
P71
Ursodeoxycholic Acid Attenuates LDL Uptake in Macrophages
Yinan Hua, Machender R Kandadi, Jun Ren, Nair Sreejayan; Univ of Wyoming, Laramie, WY
Atherosclerosis is one of the most commonly seen causes responsible for the high
cardiovascular morbidity and mortality in Western world. A key event in the formation of early
vascular lesions is monocyte recruitment and uptake of modified LDL by macrophages resulting
in foam cell formation in atheroma. Recent evidence suggests an essential role of insulin
resistance in promoting LDL-uptake in macrophages. Since endoplasmic reticulum (ER) stress
has been implicated in insulin resistance, the aim of this study was designed to investigate the
effect of the chemical chaperone ursodeoxycholic acid on LDL-uptake in insulin-resistant
macrophages. Cultured human macrophages were subjected to insulin resistance using the
ER-stress inducer tunicamycin (0.5 ␮g/mL) in the presence or absence of ursodeoxycholic acid
P72
Autocrine and Paracrine Roles of Connective Tissue Growth Factor in
Cardiac Fibrosis
Misuk Kang, Yunyu Zhang, Martina Schinke, Mie Abe, Jeyaseelan Raju, Rick Vega; Novartis
Institutes for BioMed Rsch, Cambridge, MA
Hypertrophy of cardiac myocytes involves synthesis of new proteins that contribute to the
cardiac remodeling process. In this study, we identified connective tissue growth factor (CTGF)
as a protein secreted from cardiac myocytes upon prostaglandin F2␣ (PGF2␣) stimulation. We
hypothesized that myocyte produced CTGF induces cardiac fibrosis via autocrine and paracrine
actions. In order to verify myocyte CTGF expression, we tested various hypertrophic stimuli in
neonatal rat ventricular myocytes (NRVMs). Quantitative real-time PCR and western blotting
showed increased CTGF gene expression and protein secretion upon PGF2␣, angiotensin II,
phenylephrine, isoproterenol or endothelin-1 stimulation. In addition, up-regulation of CTGF
expression in cardiac myocytes in a rodent model of cardiac hypertrophy (the Dahl salt sensitive
rat) was observed by immunohistochemistry. The autocrine effect of CTGF in NRVMs was
examined either by overexpressing full length CTGF using adenovirus or treatment with
recombinant CTGF protein. No myocyte hypertrophy was observed in either condition.
Interestingly, CTGF increased osteopontin protein secretion detected by ELISA measurements
implying a possible regulatory role of CTGF in osteopontin mediated fibrosis signaling.
Conditioned media collected from PGF2␣ treated NRVMs increased cardiac fibroblast
proliferation. Treatment with the recombinant C-terminal fragment of CTGF also showed a
similar response in fibroblast proliferation showing paracrine effects of CTGF. Fibronectin
expression was also up-regulated in fibroblasts upon CTGF treatment. To understand the
mechanism of CTGF up-regulation in myocytes, various kinase inhibitors were tested to
interrupt PGF2␣ signaling. None of the tested inhibitors blocked PGF2␣ mediated CTGF
expression completely, however slight inhibitory effects were seen with herbimycin and a JNK
or p38 MAPK inhibitor. In conclusion, for the first time we showed that PGF2␣ increases CTGF
secretion in cardiac myocyte in a time- and dose- dependent manner. We also show here
autocrine and paracrine effects of myocyte-produced CTGF through activation of osteopontin
secretion from myocytes as well as proliferation and fibronectin up-regulation in cardiac
fibroblasts.
P73
P21-Activated Kinase Modulates Connexin43 Phosphorylation Through PP2A
Activation in a HEK293 Cell Line Expressing Wild-Type Connexin43
Aiyang (Allen) Jiang, Weiwei Zhao, Steven M Pogwizd, Xun Ai; Univ of Alabama at
Birmingham, Birmingham, AL
Slow ventricular conduction in the failing heart contributes to the development of ventricular
tachycardia and ventricular fibrillation that are major causes of sudden cardiac death. We have
previously shown impaired cell coupling in heart failure (HF) and increased dephosphorylation
of the gap junctional protein connexin43 (Cx43) that was associated with increased amounts
of protein phosphatase type 2A (PP2A) which colocalized with Cx43. P21-activated kinases
(PAK’s) modulate PP2A activity, and we have found that PAKs are upregulated in HF. To explore
whether PAKs could contribute to Cx43 dephosphorylation and impaired cell coupling in HF, we
defined the effects of adenoviral overexpression of constitutively-active PAK (AdPAK) or inactive
PAK (dominant negative mutation, AdPAKdn, vs AdLacZ controls) on Cx43 phosphorylation
status in an HEK293 cell line expressing wild-type Cx43 cells (HEK293-Cx43). We used Western
blotting, PP2A activity assays, and immunoprecipitation techniques in the studies. Intercellular
communication of cell pairs was assessed by microinjection of Lucifer Yellow dye. We found
that PAK was associated with Cx43 and PP2A proteins, indicating interactions between these
proteins. In HEK293-Cx43 cells, AdPAK increased PP2A activity by 34% (n⫽4,4, p⬍0.05) and
Cx43 dephosphorylation by 49% (n⫽6,6, p⬍0.05). In contrast, AdPAKdn did not change the
level of dephosphorylated Cx43. Intercellular coupling between AdPAK-infected HEK293-Cx43
cells was markedly reduced (dye transfer time constant (ô) of 136.2⫾17.8 s vs. 85.6⫾10.1 s
for AdLacZ controls (p⬍0.05; n⫽4,4)). Moreover, okadaic acid (10 nmol/L, which inhibits
PP2A) improved cell coupling (␶⫽101.0⫾11.6). Thus, PAK proteins contribute to Cx43
dephosphorylation and, ultimately, cell coupling through modulation of PP2A activity. PP2A and
PAKs could represent novel therapeutic targets to improve conduction and decrease the high
incidence of sudden death in HF patients.
e50
Circulation Research
Vol 103, No 5
August 29, 2008
P74
Defective Interdomain Interaction Within the Ryanodine Receptor as a
Common Cause of Heart Failure and Lethal Arrhythmia
Hitoshi Uchinoumi, Masafumi Yano, Takeshi Suetomi, Makoto Ohno, Xiaojuan Xu, Hiroki
Tateishi, Shinichi Okuda, Shigeki Kobayashi, Takeshi Yamamoto, Yasuhiro Ikeda, Masunori
Matsuzaki; Yamaguchi Univ, Ube, Japan
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Two domains within the ryanodine receptor (RyR) of sarcoplasmic reticulum (SR) {N-terminal
(0 – 600) been recently shown to interact with each other as a regulatory switch for channel
gating. Here, we investigated whether the defective inter-domain interaction could be a
common cause of heart failure (HF) and lethal arrhythmia. SR vesicles were isolated from dog
LV muscles (n⫽6), then RyR was fluorescently labeled with methylcoumarin acetate (MCA)
using DPc10 a synthetic peptide corresponding to Gly2460-Pro2495 of RyR (one of mutable
domains in CPVT: R2474S), as a site-directing carrier. Addition of DPc10 (10 –100 ı̀M) induced
an unzipped state of the interacting N-terminal and central domains (competing with native
domain in RyR2), as assessed by the quenching of the MCA fluorescence by a large-size
fluorescence quencher. In DPc10-incorporated (normal) cardiomyocytes, the frequency of Ca2⫹
sparks (SpF:s-1䡠100␮m-1) was markedly increased (2.6⫾0.3, p⬍0.01 vs no DPc10), similarly
as in (4 weeks RV pacing-induced) failing cardiomyocytes (2.8⫾0.2, p⬍0.01 vs normal). To
further assess whether the defective inter-domain interaction also causes CPVT, we developed
knock-in mice model harboring the Arg-to-Ser (R2474S) mutation; same mutable site as
DPc10. The RyR2R2474S/⫹ knock-in (KI) mice (n⫽7) revealed no structural or histological
abnormality in hearts, and also had no cardiac dysfunction at rest, as assessed by
echocardiography. In all KI mice (n⫽6), bidirectional ventricular tachycardia (VT) was observed
by exercise with treadmill, but never observed in wild-type (WT) mice (n⫽6). In intact
cardiomyocytes, the frequency of Ca2⫹ sparks (SpF; s-1䡠100␮m-1) was significantly increased
in KI mice, but not in WT mice (at 2 mM [Ca2⫹]; KI:6.5⫾0.6, WT:0.9⫾0.07, p⬍0.01). When
[Ca2⫹]C was buffered at 100 nM (by 0.5 mM EGTA), the mini Ca2⫹ waves were frequently
observed in KI cardiomyocytes, but much less in WT cardiomyocytes. In conclusion, the
defective inter-domain interaction within the RyR2 may induce the de-stabilized channel gating,
as a common key mechanism of HF and lethal arrhythmia.
domain2114 –2149 and domain2234 –2750 causing domain unzipping, as evidenced by an increased
accessibility of the bound MCA to a large-size fluorescence quencher. In failing cardiomyocytes
(F; n⫽15), the frequency of spontaneous Ca2⫹ spark (CaSF:s-1䡠100␮m-1) was markedly
increased compared with normal (N; n⫽15) cardiomyocytes (F:2.7⫾0.7, N:0.9⫾0.4; p⬍0.01
vs N), whereas incorporation of DP2114 –2149 markedly decreased CaSF (1.3⫾0.5, p⬍0.01 vs F),
the same effect as that produced by K201. Cell survival rate of failing cardiomyocytes (F;
n⫽462 cells) was markedly decreased compared with normal (N; n⫽521 cells) cardiomyocytes (F:31⫾8% at 72 hrs, N:82⫾7%; p⬍0.01 vs N), whereas DP2114 –2149 incorporation
markedly improve survival rate (78⫾7%, p⬍0.01 vs F) of failing cardiomyocytes. In conclusion,
we identified the K201-binding site as domain2114 –2149 of RyR2. Interruption of the inter-domain
interaction between the domain2114 –2149 and central domain2234 –2750 seems to mediate
stabilization of RyR2 in failing hearts, which may lead to a novel therapeutic strategy against
heart failure and perhaps lethal arrhythmia
P77
Withdrawn
P75
p53 Is a New Target to Improve Cardiac Metabolism
Masaki Kimata, Satoaki Matoba, Hideo Nakamura, Atsushi Hoshino, Mikihiko Nakaoka,
Yoshimi Hiraumi, Eri Kanai-Iwai, Tetsuya Tatsumi, Hiroaki Matsubara; Kyoto Prefectural Univ
of Medicine, Kyoto, Japan
Altered energetics play an important role in the mechanism of heart failure. Clinical studies
have shown that partial free fatty acid (FFA) inhibition may exert cardioprotective effects in the
setting of myocardial ischemia. We have demonstrated mitochondrial respiration is dosedependently regulated by p53 in both human cancer cells and mice. Functionally, this leads to
a profound reduction in exercise capacity of p53-deficient mice. However, the role of p53 in
cardiac metabolism is unknown. We examined the effect of p53 on cardiac energetics. In
neonatal rat cardiac myocytes, after the treatment with nutlin-3 (MDM2 inhibitor), p53 was
induced and oxygen consumption was augmented. To examine the pathological effect of p53
in the heart, myocytes were exposed to hypoxia. Hypoxia induced the accumulation of p53 and
increased the percentage of apoptotic myocytes in a time dependent manner. We found
glycolysis was inhibited with the accumulation of p53 prior to the change of Bax/bcl-2 protein.
Inhibition of p53 recovered glycolysis, and reduced apoptosis. Although p53 did not affect the
level of ATP, phosphocreatine was significantly preserved by the inhibition of p53. Expression
of TIGAR (TP53-induced glycolysis and apoptosis regulator) protein was increased with the
activation of p53 in hypoxic myocytes. Enzyme assay revealed that the level of fructose-2,6bisphosphate (Fru-2,6-P2) was decreased after the induction of p53 and TIGAR. Since TIGAR
is known to decrease Fru-2,6-P2 and inhibit the key glycolytic enzyme, 6-phosphofructo-1kinase (PFK-1), our results suggest that p53 inhibits glycolysis via TIGAR. Finally, we confirmed
that mitochondrial membrane potential was preserved by the inhibition of p53 and TIGAR.
These results suggested that p53 accumulation inhibits glycolysis and deteriorates the
energetics of the myocytes, which in turn increases apoptosis. Altering the balance of energy
source by inhibiting p53 preserves the high energy phosphates and protects myocytes. p53 is
a new therapeutic target to improve cardiac metabolism.
P76
Identification of Target Domains of the Cardiac Ryanodine Receptor to
Correct Channel Disorder in Failing Hearts
Takeshi Yamamoto, Masafumi Yano, Takeshi Suedomi, Makoto Ohno, Xiaojuan Xu, Hiroki
Tateishi, Shinichi Okuda, Shigeki Kobayashi, Yasuhiro Ikeda, Masunori Matsuzaki;
Yamaguchi Univ Graduate Sch of Medicine, Ube, Japan
We previously demonstrated that K201(JTV519) inhibits the Ca2⫹ leak by correcting the
defective inter-domain interaction between N-terminal (0 – 600) and central regions (2000 –
2500) of the ryanodine receptor (RyR2) in failing hearts. Here, we identified the K201-binding
domain and characterized the role of this domain on RyR2 channel gating. An assay using a
quartz-crystal microbalance technique revealed that K201 specifically bound to recombinant
RyR2 fragment:1741–2270, but not to other RyR2 fragments (⬃500 amino acid residues) from
the 1–2750 region. By further analysis of the fragment1741–2270, K201 was found to specifically
bind to its sub-fragment2114 –2149. Using the peptide matching this sub-fragment (DP2114 –2149) as
a carrier, the RyR2 was specifically labeled with methylcoumarin acetate (MCA). Moreover, of
several recombinant RyR2 fragments (1–2750), only fragment2234 –2750 was specifically
MCA-labeled; this suggests that the K201 binding domain2114 –2149 binds with domain2234 –2750.
Addition of DP2114 –2149 to the MCA-labeled SR interfered with the interaction between
P78
Withdrawn
BCVS Conference 2008 Abstracts
P79
Neuregulin Treatment of Heart Failure in Rat
Anindita Ganguly, Jennifer F Iaci, Maya Srinivas, Andrea M Vecchione, Craig S Hackett,
Sarah Kasperbauer, Anthony O Caggiano; Acorda Therapeutics, Hawthorne, NY
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
The neuregulins are a family of growth factors structurally related to Epidermal Growth Factor
(EGF) and are essential for the normal development of the heart. Evidence suggests that
neuregulins are a potential therapeutic for the treatment of heart disease including heart failure,
myocardial infarction, chemotherapeutic toxicity and viral myocarditis. The following studies
were performed to select a lead neuregulin and optimize dosing in the left anterior descending
(LAD) artery ligation model of congestive heart failure in the rat. Multiple neuregulin splice
variants were cloned and produced. A neuregulin fragment of consisting of the EGF-like domain
(EGF-ld) from previous reports (Liu et al., 2006) was compared to a full-length neuregulin
known as glial growth factor 2 (GGF2) and the EGF-like domain with the Ig domain (EGF-Ig).
Male and female Sprague-Dawley rats underwent LAD artery ligation. At 7 days post ligation
rats were treated with neuregulin daily (IV). Cardiac function was monitored by echocardiography. The first study compared 10 days of dosing with equimolar amounts of EGF-ld or GGF2
(0.0625 and 0.325 mg/kg). GGF2 treatment resulted in significantly (p⬍0.05) greater
improvement in Ejection Fraction (EF) and Fractional Shortening (FS) than did EGF-ld at the end
of the dosing period. The second study compared 20 days of GGF2 with EGF-ld and EGF-Ig
(equimolar). GGF2 treatment resulted in significantly improved EF, FS and LVESD (p⬍0.01).
Improvements in cardiac physiology were not maintained for this period with either EGF-ld or
EGF-Ig. The third study compared daily (q 24 hour), every other day (q 48 hour) and every fourth
day (q 96 hour) dosing for 20 days with GGF2 (3.25 mg/kg). All three GGF2 treatment regimens
resulted in significant improvements in cardiac physiology. The q 96 hour dosing was not as
effective as the q 24 and 48 hour regimens. The studies presented here confirm GGF2 as the
lead neuregulin compound and begin to evaluate optimal dosing regimens. On-going studies
are determining maximal efficacious doses and the ability to sustain the treatment effect. cGMP
manufacturing methods are currently being developed for GGF2. GGF2 will be advanced to
human trials provided preclinical toxicology demonstrates a reasonable risk profile.
P80
IL-10 Modulates Proinflammatory Cytokine-Mediated Left Ventricular
Remodeling with Effects on Cardiac Function, Fibrosis, and Healing After
Myocardial Infarction
Prasanna Krishnamurthy, Johnson Rajasingh, Erin Lambers, Gangjian Qin, Douglas W
Losordo, Raj Kishore; Northwestern Univ, Chicago, IL
Pro- and anti- inflammatory cytokines play an important role in the pathogenesis of heart
failure. Persistent inflammatory response could have adverse effects on left ventricular (LV)
function and remodeling following myocardial infarction (MI). We hypothesized that IL-10
treatment attenuates pro-inflammatory cytokine mediated cardiac dysfunction and remodeling,
post-MI. Mice underwent MI were injected subcutaneously with mouse recombinant IL-10
(50␮g/kg b.w, MI⫹IL-10 group) on 0,1,3,5 and 7 days post-MI. Inflammatory response was
assessed on 3 days; LV functional and structural remodeling changes on 28 days post-MI.
Infarct size (49%) was associated with increased infarct wall thinning in the MI group. IL-10
treatment led to reduced infarct size (P⬍0.05 vs MI) with increased wall thickness (P⬍0.01 vs
MI). Infiltration of inflammatory (CD68-positive) cells in the myocardium increased 3 days
post-MI, which was significantly decreased after IL-10 treatment (P⬍0.01 vs MI group). MI
increased mRNA expression of various pro-inflammatory mediators (IL-1␤, TNF-␣, IL-6, IP-10,
MCP) and TNF receptor-1 in the myocardium at 3 days post-MI. However, IL-10 treatment
decreased their levels (P⬍0.05 vs MI). Echocardiography showed increase in LV end-diastolic
(LVEDD) and end-systolic diameters (LVESD) and decrease in %FS and EF% in MI versus Sham
(P⬍0.01). IL-10 attenuated these effects post-MI (P⬍0.05). Also, MI increased mRNA
expression and gelatinolytic activity of MMP-9 in the LV (P⬍0.01 vs Sham) associated with
increased fibrosis (P⬍0.01 vs Sham). Interestingly, IL-10 treatment reduced the MMP-9 activity
with decreased fibrosis (P⬍0.05 vs MI). Capillary density (CD31 positive cells) was increased
at 28 days post-MI (P⬍0.05 vs Sham), which was further higher after IL-10 treatment (P⬍0.05
vs MI group). Western blotting demonstrated increased phosphorylation of p38 MAP kinase and
reduced p-STAT3 in the myocardium post-MI. However, IL-10 treatment reversed these effects
(P⬍0.05 vs MI). Taken together, our studies demonstrate that IL-10 reduces severity of
pro-inflammatory responses and contributes to improved heart function and remodeling by
affecting MMP-9 activity, fibrosis and angiogenesis after myocardial infarction.
P81
Nkx2–5 Is a Transcriptional Activator of Etsrp71 During Cardiac
Development and Regeneration
Anwarul Ferdous, Daniel J Garry, Univ of Minnesota, Minneapolis, MN; Michael Kyba, Univ
of Texas-Southwestern Med Cntr, Dallas, TX; Arianna Caprioli, Univ of Texas Southwestern
Med Cntr, Dallas, TX; Richard P Harvey; Univ of New South Wales, Sydney, Australia
The overall goal of our study was to decipher transcriptional networks that promoted cardiac
morphogenesis and regeneration following cardiac injury. We generated an Nkx2–5 enhancerEYFP transgenic mouse and utilized FACS analysis to isolate wildtype and Nkx2–5 null cardiac
progenitor cells (CPCs) from single, age-matched embryos at distinct developmental stages
(E7.75, E8.25 and E9.5). Transcriptome and qRT-PCR analyses of RNA isolated from wildtype
and Nkx2–5 null CPCs were used to identify Ets-related protein71 (Etsrp71), a transcript that
was significantly downregulated in the Nkx2–5 null CPCs. The biological function of Etsrp71
during cardiac morphogenesis and/or regeneration is unknown. Using molecular and biochemical techniques, we established that Nkx2–5 binds to an evolutionarily conserved Nkx2–5
responsive element (NKE) in the Etsrp71 promoter and activates the Etsrp71 gene expression
e51
in a dose-dependent fashion. Furthermore, mutation of the NKE significantly attenuated the
transcriptional activation. Our data suggest that Etsrp71 is a novel downstream target gene of
Nkx2–5. Using RT-PCR, in situ hybridization and transgenic techniques, we further established
that Etsrp71 is transiently expressed in the endocardium of the developing heart (E7.75-E9.5)
and is extinguished during the later stages of development and in the adult heart. This spatial
and temporal expression was significantly abrogated in the Nkx2–5 null heart. Although
Etsrp71 was not expressed in the adult heart, Etsrp71 expression was significantly induced in
the adult heart following cardiac injury. These data imply that Etsrp71 may have an important
role in regeneration/repair of damage tissue/organ. Our results, therefore, extend the
Nkx2.5-Etsrp71 transcriptional network and support the hypothesis that Nkx2–5 transactivates
Etsrp71, which is essential for cardiac morphogenesis and regeneration/repair of the injured
heart.
P82
Brown Adipose Tissue-Derived Cells Differentiate into Spontaneous Beating
Cells with Both Cardiac Conduction and Pacemaker Characteristics
Toshinao Takahashi, Masatoshi Narikawa, Tomomi Oyama, Atsuhiko T Naito, Takehiko
Ogura, Haruaki Nakaya, Toshio Nagai, Issei Komuro; Chiba Univ, Chiba, Japan
An adipose tissue is one of the sources of mesenchymal stem cells, which have the potential
to differentiate into various types of cells including myocytes. However, the character of
differentiated myocytes has not been fully elucidated. Here we report that adipose tissuederived mesenchymal stem cells differentiate into the beating cells, which have several
characters of cardiac conduction and pace-maker cells. Brown adipose tissue (BAT) were
isolated from interscapular area of mice and enzymatically digested before culture. Round or
tube-like cells in some of the colonies showed spontaneous beating at 4 –7 days after culturing
of BAT-derived cells. RT-PCR analyses revealed that BAT-derived cells expressed Nkx2.5,
GATA6, Tbx5, ANF, MEF2C, cardiac alpha actin and MyoD, but not GATA4. Immunocytochemical analysis revealed that the most of round and tube-like cells in beating colonies expressed
sarcomeric alpha actinin (SA) and cardiac troponin T. Some of the tube-like cells showed fine
sarcomeric structures. Small mononuclear round and tube-like cells expreesed ANF but not
MyoD, whereas large multinucleated cells express MyoD but not ANF, suggesting that
BAT-derived cells differentiate into both cardiac and skeletal lineages. Patch-clamp techniques
revealed that spontaneous electrical activity appeared between -40 and -60 mV. The shape of
action potential from beating round and tube like cells showed properties of both cardiac
pacemaker and neuronal cells. BAT-derived cells expressed several cardiac conduction system
and pace-maker cell marker genes, such as Tbx5, HF-1b, connexin 40, connexin 45, MinK, and
HCN1 to 4, which are necessary for pace-maker activity. The expression of these genes were
observed 4 days after culture and peaked at 7 days in agreement with the time course of the
appearance of beating colonies. When BAT-derived cells were cultured with 500 nM of
endothelin and 100 ␮M of Cytosine ␤-D-arabino furanoside, SA and HCN positive colony size
was increased. These findings suggest that BAT-derived cells differentiate into cardiac
conduction system and pacemaker-like cells and may become a useful cell source for
arrhythmia therapy.
P83
Adult Murine Heart-Derived Clonal Expanded Sca-1 Positive Cell Sheet
Ameliorates Cardiac Dysfunction After Myocardial Infarction
Katsuhisa Matsuura, Atsushi Honda, Tokyo Women’s Med Univ , Tokyo, Japan; Toshio
Nagai, Chiba Univ, Chiba, Japan; Noritoshi Fukushima, Tatsuya Shimizu, Teruo Okano,
Hiroshi Kasanuki, Tokyo Women’s Med Univ , Tokyo, Japan; Issei Komuro, Chiba Univ,
Chiba, Japan; Nobuhisa Hagiwara; Tokyo Women’s Med Univ , Tokyo, Japan
Cardiac progenitor cells have been thought to be the promising source of cell therapy. Recently
we have established the novel cardiac progenitor cell line, that is clonal expanded Sca-1
positive cells (cSca-1 cells) which are capable of differentiation into cardiomyocytes from adult
murine hearts, but the contribution to cardiac function when in transplantation has not been
fully elucidated. Here we show, using cell sheet techniques, that cSca-1 cells ameliorates
cardiac dysfunction after myocardial infarction. We cultured cSca-1 cells labeled with RFP or
adipose tissue derived mesenchymal cells (ATMC) isolated from GFP mice by using temperature
responsive culture dishes and transplanted each of monolayered cell sheets onto the infarcted
region just after coronary ligation. We randomly placed mice into three groups: mice with
transplantation of monolayered cSca-1 cells (cSca-1 group; n⫽10), mice with transplantation
of monolayered ATMC (ATMC group; n⫽10) and mice without transplantation (untreated group;
n⫽10). Echocardiographic analysis revealed that at 4 weeks after transplantation, LVDd and
LVDs were decreased and FS was significantly improved compared with untreated group and
ATMC group, suggesting that cSca-1 cell sheet transplantation inhibited cardiac remodeling
and improved cardiac contractility after myocardial infarction. Capillary density in the border
zone was significantly increased in cSca-1 group compared with untreated and ATMC group
(cSca-1, 28.0⫾1.4, untreated, 12.2⫾0.9, ATMC, 12.6⫾1.2, p⬍0.01). Cytokine antibody array
showed several secreting angiogenic factors were abundant in cSca-1 compared to ATMC.
Confocal laser microscopic analysis revealed many of RFP(⫹) cSca-1 cells existed in infarcted
areas at 4 weeks after transplantation, while none of GFP(⫹) ATMC existed. Furthermore
⬃30% of RFP(⫹) cells expressed sarcomeric ␣-actinin with a fine striated pattern or formed
vessels in infarcted areas. These findings suggest ex vivo expanded cardiac progenitor cell
sheet transplantation ameliorates cardiac dysfunction after myocardial infarction via cardiomyogenesis, vasculogenesis and secreted factors mediated angiogenesis and then is thought to
be a feasible source for the cardiac regenerative therapies.
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Circulation Research
Vol 103, No 5
August 29, 2008
P84
Mechano-Electrical Feedback During Early Progression of Hypertrophy
Defines Arrhythmic Potential
Hongwei Jin, Elie R Chemaly, Changwon Kho, Ahyoung Lee, Roger J Hajjar, Fadi G Akar;
Mount Sinai Sch of Medicine, New York, NY
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Despite a clear association between LV dysfunction in end stage heart failure (HF) and the
incidence of arrhythmias, the majority of sudden deaths occur at earlier stages. Previously, we
investigated the relationship between electrophysiological (EP) remodeling and arrhythmias in
HF. However, the dynamic nature of EP remodeling during early disease progression has not
been systematically investigated. We hypothesized that cross talk between mechanical,
structural and molecular properties modulate the EP substrate during early disease progression
caused by pressure overload hypertrophy (PoH). Methods: Aortic banding for 0 (sham), 2, 4 or
8 weeks in rats (n⫽26) produced variable degrees of PoH. LV mechanical remodeling was
assessed in vivo using M mode 2D echocardiography and correlated with conduction and
repolarization using optical action potential (AP) mapping in ex vivo perfused hearts. Results:
In contrast to EP remodeling in non ischemic HF, AP prolongation in PoH was predicted by non
invasive metrics of LV function. Namely, AP duration exhibited a strong linear correlation with
changes in LV anteroseptal and posterior wall dimensions in diastole but not systole. Unlike AP
prolongation, conduction velocity (CV) slowing did not consistently correlate with changes in LV
wall thickness but rather with complex remodeling of Cx43. Cx43 underwent increased
expression at intermediate stages of PoH caused by a rise in a PKC phosphorylated (P2)
isoform. Surprisingly, this coincided with moderate but significant (p⫽0.04) CV slowing.
Following 8 wk of PoH, CV underwent major (⬃3X, p⫽0.018) reduction, coinciding with a 10
fold rise in the dephosphorylated (P0)/P2 ratio and decreased physical interaction between
Cx43 and the cytoskeletal protein ZO-1 by Co-IP. Conclusions: AP prolongation in PoH is
correlated with changes in LV wall thickness. CV slowing is not caused by reduced Cx43
density but rather by disrupted balance between the P2 and P0 isoforms. Intermediate stages
of PoH are associated with increased P2 Cx43 which is completely abolished at late stages,
resulting in reduced interaction with ZO-1 and severe CV slowing. Elucidating the mechanistic
links between EP, mechanical and molecular changes in PoH will help identify novel targets for
therapy.
transients. Application of exogenous apelin to myocytes (10nM) increased velocity of shortening
and relaxation but produced inconsistent effects on shortening in unloaded myocytes. To
investigate this further, we explored force-length relations using a novel carbon fiber stretch
apparatus. There was a 14% greater relative slope in force development across a range of
preloads in the apelin treated cells. Endogenous apelin-APJ signaling contributes to tonic
myocardial function in vitro and in vivo. Changes in force-length relationships in the absence
of consistent effects on LV shortening in unloaded myocytes suggest a complex control of
cardiovascular function that may be stretch dependent.
P87
Preferential Increase in CUG-Initiated FGF-2 Isoform Expression in Human
Heart-Derived Fibroblasts Undergoing Apoptosis
Xin Ma, Jon-Jon Santiago, Rakesh Arora, Elissavet Kardami; Institute of Cardiovascular
Sciences, Winnipeg, Canada
FGF-2 is a multifunctional and ubiquitous protein translated as high molecular weight (hi) FGF-2
or low molecular weight (lo, 18 kDa) FGF-2 versions. Overexpression experiments in neonatal
rat cardiac myocytes or HEK293 cells have shown that increases in nuclear hi-FGF (but not
lo-FGF-2) caused chromatin compaction and apoptotic cell death. It is not known if endogenous
human hi-FGF-2 plays a similar role. As a first step in addressing this question, we examined
the relationship between endogenous hi-FGF-2 expression and cell death, in human heartderived primary fibroblasts cultures. We also developed, and affinity-purified, a polyclonal
antibody against hi-FGF-2-specific epitopes, to enable detection in vivo. Western blotting
analysis demonstrated robust expression of both hi- (22–25 kDa) and lo-FGF-2(18 kDa)
isoforms in human atrial tissue and atria-derived human fibroblasts. Relative expression of
hi-FGF-2 was higher in adult-derived compared to immature cardiac fibroblasts. Immunohistochemistry of human tissue sections. or immunofluorescence of human fibroblasts, showed
predominantly nuclear localization of hi-FGF-2. In fibroblast cultures, a small fraction of cells
displayed intense anti-hi-FGF-2 staining. The same population of cells showed condensed
chromatin and stained positive for TUNEL, indicating an apoptotic phenotype. Our data are
consistent with the notion that endogenous hi-FGF-2 has a pro-apoptotic role in human cells,
as suggested by overexpression studies. Understanding translational regulation of FGF-2
isoforms may provides a new strategy for regulating cell survival and cell death.
P85
Withdrawn
P88
Sulfaphenazole Mediates Cardioprotection Through Preconditioning and
Autophagy
Chengqun Huang, Yayoi Tetsuo Tsukada, Cynthia N Perry-Garza, San Diego State Univ, San
Diego, CA; Robert M Mentzer, Jr, Wayne State Univ Sch of Medicine, Detroit, MI; Roberta A
Gottlieb; San Diego State Univ, San Diego, CA
Previously, we showed that sulfaphenazole (SUL) and other cytochrome P450 monooxygenase
inhibitors attenuate ischemia/reperfusion (I/R) injury. In this study we evaluated its effects on
post-ischemic function, signal transduction pathways, mitochondrial function, and autophagy.
Isolated rat hearts were exposed to 30’ global I/120’ R. Hearts pretreated with SUL recovered
80% of pre-ischemic hemodynamic values compared to only 52% for untreated controls;
infarct size was reduced from 53% to 6% of the area at risk (AAR). When SUL was administered
5 minutes after the onset of reperfusion, infarct size was reduced to 9% of the AAR. In a
separate series of experiments using cardiomyocytes subjected to simulated I/R, SUL
decreased cytochrome c release, decreased reactive oxygen species (ROS) production,
suppressed Bax activation and translocation to mitochondria, and suppressed phosphorylation
of p38 MAPK. The cardioprotective effect of SUL was blocked by chelerythrine,
5-hydroxydecanoate, and N-acetylcysteine. Finally, SUL treatment was associated with a
marked increase in autophagy similar to that induced by diazoxide and adenosine. These
observations suggest that the cardioprotective effects of SUL are mediated, in part, by: (1)
activation of preconditioning pathways involving protein kinase C and the mitochondrial K(ATP)
channel; and (2) the stimulation of autophagy. The results suggest that SUL may represent a
new class of agents that could significantly reduce myocardial injury associated with I/R.
P89
P86
The Apelin-APJ Pathway Is an Endogenous Regulator of Cardiac Function
Michael Y Ho, Giovanni Fajardo, Stanford Univ, Stanford, CA; Christian Bollensdorff, Oxford
Univ, Oxford, United Kingdom; Joshua Anderson, David Charo, Hyung Chun, Ramendra
Kundu, Stanford Univ, Stanford, CA; Peter Kohl, Oxford Univ, Oxford, United Kingdom; Daniel
Bernstein, Thomas Quertermous, Euan Ashley; Stanford Univ, Stanford, CA
The peptide ligand apelin and its cognate G-protein coupled receptor APJ constitute a
promising signaling pathway for therapeutic intervention in heart failure. While effects of
exogenous apelin are well characterized, the role of endogenous signaling is less clear. To
investigate the tonic effects of apelin-APJ in contractile function, we generated apelin and APJ
null mice by homologous recombination. Knockout mice were viable and fertile. Inheritance
was mendelian for apelin null mice but not for APJ mice. Compared to littermate controls, adult
apelin null mice manifest modest decreases in exercise capacity, left ventricular ejection
fraction, and end-systolic elastance. Isolated cardiomyocytes lacking apelin exhibit reduced
sarcomeric shortening despite similar calcium transients. These findings are recapitulated in
APJ null mice. Compared to wild-type controls, APJ deficient mice have significantly reduced
exercise capacity (treadmill runtime: 20.30 ⫾ 0.974 vs. 23.64 ⫾ 0.737, P⫽0.009) and LV
ejection fraction (29.88 ⫾ 0.927% vs. 44.93 ⫾ 2.66%, P⬍0.0001). Isolated myocytes from
APJ knockouts exhibit reduced sarcomeric shortening (5.070 ⫾ 0.413% vs. 6.978 ⫾ 0.405%,
P⫽0.002) as well as velocity of contraction and relaxation, with no difference in calcium
The CCN1 Action on Cardiovascular Cell Survival
Fan Mo, National Cheng Kung Univ, Tainan, Taiwan; Chih-Chiun Chen, Lester F Lau; College
of Medicine, Univ of Illinois at Chicago, Chicago, IL
CCN1/CYR61 is an extracellular matrix (ECM) signaling molecule. Since it plays regulatory
rather than structural role in ECM, CCN1 is thought to be a matricellular protein. Identified as
an immediate-early gene after serum growth factor stimulation, CCN1 is highly expressed in
the cardiovascular, skeletal and neuronal systems during development. Its expression is usually
silenced in the adult, but is redeployed in response to injury, inflammation, and diseases, such
as atherosclerosis, restenosis, hypertension, and after myocardial infarction. As an ECM
molecule, CCN1 binds to several integrins to mediate its function in cell adhesion, proliferation,
gene expression, and regulation of cell survival/death. We demonstrated its role in cardiovascular development through gene targeting mutation in mice. Ccn1-null mice suffer embryonic
lethality due to undervascularization of the placental labyrinth (resulting from defective vessel
bifurcation) and loss of vascular integrity in the embryo (excessive cell death in mutant aortae).
Moreover, haploinsufficiency in Ccn1 causes atrioventricular septal defects in Ccn1⫹/- hearts
due to precocious apoptosis in the endocardial cushion tissue. These findings establish CCN1
as a pro-angiogenic and pro-survival factor during development. Surprisingly, CCN1 also
induces apoptosis in fibroblasts, even while serving as an adhesion substrate. CCN1 binds to
integrin ␣6␤1 to mediate its apoptotic activity. The cardiovascular phenotype resulting from
null-mutation in mice is rescued by replacing genomic Ccn1 locus with a ␣6␤1-binding-
BCVS Conference 2008 Abstracts
defective mutant, Ccn1-dm, suggesting that ␣6␤1 is not required for CCN1 function during
development. Together, these observations suggest that CCN1 binds to different integrin
receptors to regulate cell survival/death. Cells of different types, in different contexts, can
respond to CCN1 and determine to live or die through binding to specific integrins.
P90
Intracellular Protein Aggregation Is a Proximal Trigger of Cardiomyocyte
Autophagy
Paul Tannous, Hongxin Zhu, Andriy Nemchenko, Jeff M Berry, Janet L Johnstone, John M
Shelton, Univ of Texas Southwestern Med Ctr, Dallas, TX; Francis J Miller, Jr, Univ of Iowa,
Iowa City, IA; Beverly A Rothermel, Univ of TX Southwestern Med Ctr, Dallas, TX; Joseph A
Hill; Univ of Texas Southwestern Med Ctr, Dallas, TX
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Background: Recent reports demonstrate that multiple forms of cardiovascular stress, including
pressure overload, chronic ischemia, and infarction-reperfusion injury provoke an increase in
autophagic activity in cardiomyocytes. However, nothing is known regarding molecular events
that stimulate autophagic activity in stressed myocardium. As autophagy is a highly conserved
process through which damaged proteins and organelles can be degraded, we hypothesized
that stress-induced protein aggregation is a proximal trigger of cardiomyocyte autophagy.
Methods and Results: Here, we report that pressure overload promotes accumulation of
ubiquitinated protein aggregates in left ventricle (LV), development of aggresome-like
structures, and a corresponding induction of autophagy. To test for causal links, we induced
protein accumulation in cultured myocytes by inhibition of proteasome activity, finding that
aggregation of polyubiquitinated proteins was sufficient to induce cardiomyocyte autophagy.
Further, attenuation of autophagic activity dramatically enhanced both aggresome size and
abundance, consistent with a role for autophagic activity in protein aggregate clearance.
Conclusions: We conclude that protein aggregation is a proximal trigger of cardiomyocyte
autophagy and that autophagic activity functions to attenuate aggregate/aggresome formation
in heart. Also, findings reported here are the first to demonstrate that protein aggregation
occurs in response to hemodynamic stress, situating pressure-overload heart disease in the
category of proteinopathies.
P91
Mst1 Is a Negative Regulator of Autophagy in Cardiac Myocytes
Shiori Kyoi, Junichi Sadoshima; UMDNJ, Newark, NJ
Damaged proteins and organelles accumulate under condition of stress and impaired clearance
of them causes formation of protein aggregates and cellular malfunction. Autophagy is a
mechanism to degrade protein and damaged organelles, and plays an important role in
mediating protein quality control in cells. However, signaling mechanisms regulating autophagy
are poorly understood in the heart. Although autophagy is activated in the failing heart, it was
markedly inhibited in a mouse model of dilated cardiomyophathy induced by cardiac specific
overexpression of mammalian STE 20 like-kinase1 (Tg-Mst1). Autophagosome formation
induced by starvation, as evaluated by GFP-LC3 dots, was suppressed in Tg-Mst1 compare to
in non-transgenic (NTg) mice (42.6⫾3.2, 268.6⫾7.3, p⬍ 0.01). Accumulation of p62, a protein
subject to degradation through autophagy, was greater in Tg-Mst1(3.40⫾0.09 fold vs NTg,
p⬍0.01) than in NTg. Phosphorylation of AMPK at Thr 172 (0.56⫾0.01 fold vs NTg, p⬍0.01)
and eukaryotic elongation factor-2 (eEF2) at Thr 56 (0.34⫾0.1 fold vs NTg, p⬍0.01), positive
mediators of autophagy, were suppressed, whereas phosphorylation of mTOR at Ser 2448
(1.28⫾0.02 fold vs NTg, p⬍0.01), a negative regulator of autophagy was activated in Tg-Mst1
mice. The kinase activity of Mst1 was suppressed by glucose deprivation (GD) in culture cardiac
myocytes (0.28⫾0.01 fold vs GD, p⬍0.01) and restoration of Mst1 activity inhibited induction
of autophagy during GD as determined by LC3-II/LC3-I (1.00⫾0.03 vs 1.48⫾0.02 vs
1.12⫾0.04, Control, GD and GD with Mst1, p⬍0.01). Suppression of autophagy in Tg-Mst1
was accompanied by accumulation of protein aggregate, as evidenced by an increase in
vimentin aggregates (4.80⫾0.37, 1.60⫾0.25, p⬍0.01). These results suggest that Mst1 is a
negative regulator of autophagy in cardiac myocytes. Activation of Mst1 during cardiac stress
inhibits, whereas inhibition of Mst1 during starvation stimulates, autophagy in cardiac
myocytes. Furthermore, Mst1 negatively regulates positive mediators of autophagy, such as
AMPK and eEF2, and positively regulates a negative regulator of autophagy, namely mTOR.
Thus, activation of Mst1 under stress negatively regulates protein turnover through suppression
of autophagy.
P92
Perinatal Cardiomyocyte-Restricted Knockout of the COP9 Signalosome
Subunit 8 Gene Compromises Proteasome Proteolytic Function, Triggers
Necrosis, and Causes Heart Failure in Mice
Huabo Su, Jie Li, Univ of South Dakota, Vermillion, SD; Suchithra Menon, Yale Univ, New
Haven, CT; Kathleen Horak, Faqian Li, Univ of South Dakota, Vermillion, SD; Ning Wei, Yale
Univ, New Haven, CT; Xuejun Wang; Univ of South Dakota, Vermillion, SD
Ubiquitin-proteasome system (UPS) dysfunction was observed in animal models of many forms
of cardiomyopathy and implicated in failing human hearts. Understanding molecular mechanisms that regulate the UPS is extremely important to the search for new measures to prevent
or better treat heart failure. The COP9 signalosome (CSN) comprising of 8 subunits (CSN1
through CSN8) is potentially a critical regulator of the UPS, likely through coordinating the
activity of cullin-based ubiquitin ligases and the proteasome for efficient degradation of specific
substrates. The physiological role of the CSN has not been investigated in the heart. To address
this question, we generated mice in which the csn8 gene was conditionally ablated in the
cardiomyocytes (CR-Csn8KO) at the peri-natal stage using the Cre-loxP system. CR-Csn8KO
destabilized other CSN subunits and impaired CSN deneddylation activity. CR-Csn8KO mice
e53
developed cardiac hypertrophy at 2 weeks, as characterized by increases in the heart weight
to body weight ratio and the ventricle weight to body weight ratio, increased cardiomyocyte
cross-sectional area, and re-activation of the fetal gene program. At 3 weeks, CR-Csn8KO mice
showed multifocal interstitial fibrosis, increased expression of collagenase, and impaired
cardiac function with chamber dilatation and reduced fractional shortening, which led to
premature death of the mice by 4 weeks of age. Electron microscopy revealed marked
ultrastructural changes, including wavy z-line, loss of normal sarcomere alignments, mitochondria degeneration, and increases in autophagosome-like structures. Using a in vivo UPS
proteolytic function reporter, we found severe impairment of UPS proteolytic function
associated with the observed cardiac malfunction. Loss of cardiomyocytes in CR-Csn8KO mice
was caused by necrosis not apoptosis, as supported by a marked increase in Evans blue dye
permeable cardiomyocytes in live animals and the absence of an increase in the TUNELpositive cardiomyocytes at 3 weeks. This study demonstrates for the first time that CSN8/CSN
is essential to maintaining postnatal cardiac structure and function. Impaired CSN and UPS
function can induce cardiomyocyte necrosis, thereby causing the heart to fail.
P93
Polymorphisms of MMP-2 Gene Are Associated with Systolic Heart Failure
Prognosis
Dongfeng Gu, Yihong Hua, Li Song, Naqiong Wu, Gaoqiang Xie, Xiangfeng Lu, Xianmin
Meng, Yuejin Yang; Cardiovascular Institute and Fuwai Hosp, Beijing, China
Background Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes responsible
for myocardial extracellular protein degradation. MMP-2 is an important member of MMPs.
MMP-2 has been demonstrated to play a pivotal role in myocardial remodeling process that
occurs in congestive heart failure. We hypothesized MMP-2 genetic variations could influence
the prognosis of systolic heart failure (SHF). Methods To test our hypothesis, we performed a
follow-up study of 605 patients with SHF. Three single nucleotide polymorphisms (SNPs) of
MMP-2 (rs243864, rs243866, rs17859821) were analyzed by restriction fragment length
polymorphism (RFLP) methods. Results 526 patients (86.9%) were followed up. At follow up
(0 – 47 month, median 24 months), 116 patients (22.1%) died, 102 patients (19.4%) were
readmitted because of heart failure, 3 patients (0.6%) had recurrent myocardial infarction, 24
patients (4.6%) had repeat revascularization. One, two, three and four year survival rate was
86.9%, 81%, 77.9% and 77.9%. MMP-2 rs17859821 A allele carriers had lower all cause
death rate, cardiac death rate and MACE rate than did GG genotype carriers (OR⫽0.655, 0.580,
0.705; P⫽0.030, 0.008, 0.011). After adjustment for age, bundle branch block, LVEF and NYHA
grade by using cox regression analysis, MMP-2 A allele carriers had lower cardiac death rate
and MACE rate than did GG genotype carriers (OR⫽0.643 and 0.746; P⬍0.05). Haplotype
analysis had confirmed the above results. MMP-2 rs243866, rs243864 had no association with
systolic heart failure prognosis. Conclusion The findings of the present study suggest that
MMP-2 rs17859821 A allele confers better prognosis of systolic heart failure in the northern
Han Chinese population.
P94
Profiling Gene Expression During Heart Stress
Huey Tse Goh, Sheila Gardiner, David Brook; Univ of Nottingham, Nottingham, United
Kingdom
Heart disease is the major cause of death in many developed countries. The most effective
treatment for heart failure is heart transplantation but this is limited by the number of available
donors. This study set out to investigate key genetic factors for response and recovery of
cardiac cells in response to an in vivo endotoxic challenge, as a model system for cardiac
stress, using microarray profiling. The endotoxic challenge involved a low dose infusion of
lipopolysaccharide (LPS) which mimics the hyperdynamic phase of sepsis with high cardiac
output and peripheral vasodilatation. The heart is therefore under hemodynamic stress.
Conscious, male Sprague Dawley rats (400 – 450g) were intravenously infused with either
saline (0.4ml/h) or LPS (15␮g/kg/h) for 2h, 6h or 24h (n⫽4/group). At the end of each time
point, the animals were terminally anesthetised and hearts were removed for RNA extraction.
The RNA samples were then amplified, labelled and hybridised onto a mouse 30K spotted
arrays to allow direct comparison of the gene expression profile of the control heart against the
stressed heart. The raw data were processed and analysed using GeneSpring GX software.
1316 genes show a 2 fold, or more, change in at least one time point. By performing one way
ANOVA with a significance cut off of p⬍0.05, 615 genes out of 1316 genes were found to have
significantly different gene expression between 3 time points. Of the 615 genes, 266 were
involved in known biological pathways such as interleukin signaling pathways, myometrial
relaxation and contraction pathways, TNF-␣ NF␬B signaling pathways and Jak-STAT signaling
pathways, which might be differentially regulated in this study. Genes involved in these
pathways include IL 6, Jak2, Stat3, Stat1, Akt1, Nfkbia, Gja1, Ramp2 and Ccl2. Many of these
genes have been implicated to have a role in other heart disease such as ischemic heart
disease and cardiac hypertrophy. Determining what factors regulate the expressions of these
genes might provide an alternative treatment option for heart disease.
P95
The Novel BTB/POZ Protein, Rhobtb3, Regulates Mitochondrial Function via
the Ubiquitin Proteasome System
Shinsuke Yuasa, Takeshi Onizuka, Kenichiro Shimoji, Yohei Ohno, Mie Hara, Satoshi Ogawa,
Keiichi Fukuda; Keio Univ, Tokyo, Japan
Although BTB/POZ family proteins play a critical role in the development of various organs, and
are recently known as a substrate adaptor of CUL3 ubiquitin ligases, the involvement of these
proteins in heart development remains unknown. We recently isolated the novel protein,
e54
Circulation Research
Vol 103, No 5
August 29, 2008
RhoBTB3, an 83-kD protein which comprises an N-terminal Rho GTPase resembling domain
and two tandem BTB domains. The purpose of this study was to investigate the molecular and
functional characteristics of this protein in mice heart. [Methods and Results] (1) RhoBTB3 was
faintly expressed in the embryonic heart, but was strongly expressed in the adult heart. It was
weakly expressed in adult brain and testis. (2) RhoBTB3-GFP fusion protein cDNA was
transfected into primary cultured rat cardiomyocytes using adenovirus. Fluorescence microscopy revealed that RhoBTB3 was located at the cytoplasm of living cardiomyocytes. (3)
HA-tagged RhoBTB3 and MYC-tagged CUL family proteins (1 - 5) were co-transfected into
COS7 cells. RhoBTB3 was co-immunoprecipitated with CUL3 in vitro, but not with other CUL
proteins. (4) The various mutant RhoBTB3 and wild type CUL3 protein were examined by
immunoprecipitation assay. Both the first BTB domain and second BTB domain can bind to
CUL3 protein independently. (5) It was difficult to detect RhoBTB3 in protein analysis. To
determine whether or not RhoBTB3 itself could be the Cul3 ubiquitin target, RhoBTB3 protein
was measured under the presence of proteasome inhibitor MG132 and protein synthesis
inhibitor cycloheximide. The RhoBTB3 protein was immediately degraded and dropped below
the detection level within 2 hours. (6) The transgenic mice which overexpressed the
heart-restricted RhoBTB3 were healthy at a young age. But after 3 months, they gradually
showed heart failure and finally fell into sudden death. Interestingly before heart failure,
electron microscopy demonstrated thar mitochondria dramatically increased in number and
showed deformity in RhoBTB3 transgenic hearts. [Conclusions] RhoBTB3 is mainly expressed
in the adult heart, and its protein was rapidly degraded with the CUL3-mediated ubiquitin
proteasome system in the heart. The overexpression of RhoBTB3 caused a marked increase in
abnormal mitochondria, followed by severe heart failure.
P96
The bHLH Transcription Factor CHF1/Hey2 Regulates Progression to Heart
Failure During Pressure Overload
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Yonggang Liu, Michael T Chin; Univ of Washington Sch of Medicine, Seattle, WA
The role of the cardiovascular transcription factor CHF1/Hey2 in regulation of adult heart
function is poorly understood. To investigate whether CHF1/Hey2 loss of function influences the
progression to heart failure under conditions of pressure overload, we performed transaortic
constriction on 12 week old male wild type and heterozygous mice lacking one functional
CHF1/Hey2 allele. We performed echocardiography preoperatively and 1 week after the aortic
constriction to assess wall thickness and ventricular function and then assessed ventricular
weight to body weight ratio and cardiac fibrosis by Masson-Trichrome staining at sacrifice.
After aortic banding, both WT and heterozygous mice showed increased left ventricular wall
thickness compared with baseline (11.01mm vs. 7.45mm in WT, p⬍0.001; 11.50mm vs.
7.56mm in heterozygous mice, p⬍0.001). Heterozygous knockout mice, however, developed
increased left ventricular end-diastolic dimension compared with WT (40.53mm vs. 36.15mm,
p⫽0.012), which suggested eccentric hypertrophy in heterozygous knockout mice compared
to the concentric hypertrophy in WT mice. Gravimetric analysis demonstrated a greater
increase in ventricular weight to body weight ratio in heterozygous knockout mice compared
with WT (7.132mg/g vs. 6.045mg/g, p⫽0.025). Heterozygous knockout mice also demonstrated increased susceptibility to heart failure. One week after aortic banding, WT mice
showed no decrement in ejection fraction (62% vs. 65%, p⫽0.41). Heterozygous knockout
mice, however, showed decompensated heart function, as evidenced by significantly decreased EF (46% vs. 63% baseline, p⫽0.003 and vs. 62% WT, p⫽0.008). Histological analysis
demonstrated greater cardiac myocyte hypertrophy in heterozygotes (cardiac myocyte cross
sectional area: 3.59um2 in heterozygous vs. 2.95um2 in WT mice, p⫽6.07E-27) along with
more extensive fibrosis. Our findings demonstrate that CHF1/Hey2 loss of function facilitates an
exaggerated hypertrophic response to pressure overload and accelerates development of
fibrosis and progression to heart failure.
P97
Vinculin Scaffolds Connexin 43 and Zonula Occludens-1 in Cardiac
Myocytes: Combined Reduction of Vinculin, Zonula Occludens-1, and
Connexin 43 at the Intercalated Disk Predispose to Sudden Cardiac Death
Alice E Zemljic-Harpf, VA San Diego Healthcare System, UCSD Sch of Medicine, La Jolla,
CA; Elizabeth K Asfaw, UCSD Sch of Medicine, La Jolla, CA; Ana M Manso, Robert S Ross;
VA San Diego Healthcare System, UCSD Sch of Medicine, La Jolla, CA
Vinculin (Vcl) is an actin binding, membrane-associated protein found in cell-to-cell as well as
cell-to-matrix junctions. Cardiac specific inactivation of the Vcl gene by MLC2v-Cre (cVclKO)
caused mal-distribution of Connexin (Cx) 43 in the myocardium with a predisposition of the
cVclKO towards sudden death by ventricular tachycardia. The etiology of this phenotype
remains unclear. We hypothesized that with loss of myocyte Vcl, mal-distribution of other Cx43
associated proteins might occur. Results: Zonula-occludens 1 (ZO-1), a membrane-associated
guanyl kinase has been suggested to function as an adapter to correctly localize Cx43 to the
intercalated disk (ICD) of cardiac myocytes (CMC). Immunoflucorescent microscopy of murine
cardiac tissue showed that Vcl and ZO-1 co-localize at the ICDs of CMC but no co-localization
of ZO-1 and Vcl was seen along the lateral CMC membrane. Cross sectional analysis revealed
strong ZO-1 expression in capillaries and missing ZO-1 expression on the lateral CMC cell
border, indicating that a major component of ZO-1 protein expression in whole heart is
attributable to vascular expression. Analysis of cVclKO hearts found ZO-1 expression absent at
ICDs of CMCs null for Vcl, with abnormal redistribution of ZO-1 to the lateral CMC membrane.
Co-immunoprecipitation (IP) of protein lysates from wild-type whole hearts or isolated CMCs
showed Vcl to IP with both Cx43 and ZO-1. IP was confirmed by also using anti- ZO-1 or
anti-Cx43 antibodies respectively, followed by immunoblotting for Vcl. Western Blotting of
lysates from cVclKO hearts showed no difference in absolute ZO-1 or Cx43 protein expression.
Conclusion: This data is the first to show direct interaction of the cytoskeletal protein Vcl with
ZO-1 and Cx43 in CMCs. In cVclKO hearts, ZO-1 and Cx43 expression is lost from the ICD along
with Vcl. This suggests that Vcl may play a major role in regulating Z0 –1 and Cx43 localization
in CMCs. Since ZO-1 is proposed to be critical for generation of normal functional gap junctions,
we suggest that reduced Vcl protein expression destabilizes the Vcl/Zo-1/Cx43 scaffolding
complex and with its mutation or loss, may predispose to cardiac arrhythmias and sudden
death as was seen in the cVclKO mice.
P98
Systems Biology Approach to Elucidate Fundamental Mechanisms of Heart
Growth and Maturation
Fulvia Ferrazzi, Univ of Pavia, Pavia, Italy; Florian Diehl, Chinmoy Patra, Machteld van
Amerongen, Tatyana Novoyatleva, Max-Planck-Institute for Heart and Lung Rsch, Bad
Nauheim, Germany; Paolo Magni, Riccardo Bellazzi, Univ of Pavia, Pavia, Italy; Felix Engel;
Max-Planck-Institute for Heart and Lung Rsch, Bad Nauheim, Germany
Early embryonic development and adult physiology are well studied. In contrast, little is known
about late developmental stages, i.e. organ growth and maturation. For example, it is poorly
understood how the rat heart increases its weight 100fold from embryonic day 13 (chamber
formation) to postnatal day 10. It is known that cardiomyocyte proliferation (hyperplasia) stops
around birth and that subsequent heart growth is mostly achieved by increase in cardiomyocyte
size (hypertrophy). However, it is unclear what regulates cell cycle arrest, cell maturation, and
how the different cell types required for heart formation communicate with each other.
Therefore, we have performed a temporally resolved expression analysis describing rat heart
development from embryonic day 11 to postnatal day 10 at 12 hour intervals. Expression
analysis was performed using Affymetrix GeneChip Rat Expression Set 230. Preprocessing of
the data, i.e. background subtraction, normalization and probe-set summarization, was
performed according to the robust multi-array average (RMA) procedure. Our analysis revealed
that 3.353 out of 30.248 transcripts are differentially expressed (expression change ⱖ 2.5
fold). We found in the literature detailed expression analyses for 43 genes. These genes are
represented in our analysis by 59 probe sets showing the same developmental regulation.
Clustering, aimed at grouping genes with similar temporal expression profiles, allowed
categorizing genes according to their temporal pattern: downregulated, upregulated, transiently
or bi-phasically expressed. Each of these main patterns is represented by more than one
cluster, characterized by a different starting point of down-up regulation, different time of
maximum expression or peak width. The correlation of these data with the current knowledge
of functional and morphological changes during development allows predicting a function for
unknown genes. Our data provide the basis for inferring key genes and regulatory networks
underlying heart growth. These data have the potential to shed light onto congenital heart
disease, cardiac stem cell differentiation and regeneration.
P99
Cytoglobin: A Calcineurin-Dependent Hemoprotein Regulating Cardiac
Growth and Apoptosis
Sarvjeet Singh, Shilpa M Manda, UT Southwestern Med Cntr, Dallas, TX; Pradeep P
Mammen; UT Southwestern Med Cntr, Plano, TX
The redox state within a cardiomyocyte is important in regulating cell growth and apoptosis.
Cytoglobin (Cygb), a stress-responsive cardiac hemoprotein regulated by calcineurindependent transcription factors (AP-1, NFAT), is able to scavenge free radicals/NO. We
hypothesize that Cygb may serve a cardioprotective role in the stressed heart. A transgenic
mouse was engineered with cardiac-specific overexpression of Cygb (TgCygb). Surprisingly,
TgCygb hearts are hypertrophied at baseline compared to non-transgenic mice (NTg) (Hrt/BW
ratio: 6.3⫾0.4 vs 4.9⫾0.2mg/g; ECHO anterior wall: 11⫾0.3 vs 9.1⫾0.3mm; ECHO posterior
wall: 11.2⫾0.3 vs 8.8⫾0.2mm; p⬍0.05; n⫽5). In addition, ECHO revealed a 28% reduction
in LV systolic function in TgCygb mice (%FS: 53⫾2.1% vs 74⫾1.4%, vs NTg mice; p⬍0.05,
n⫽5). qRT-PCR revealed activation of the fetal gene program within TgCygb hearts (2.5, 1.4
and 1.3 fold increases in ␤-MHC, ANP and BNP). In vitro assays were undertaken. By FACS
analysis, overexpression of Cygb (Cygb-OE) in myocytes attenuated apoptosis as compared to
control cells upon exposure to thapsigargin (23⫾2% vs 30⫾1%; p⬍0.05, n⫽6). In addition,
there was a decrease in both caspase activity (3,4,7) and mitochondrial depolarization in
Cygb-OE cells. Site-directed mutagenesis of the conserved histidines in the heme-binding
domain of Cygb abolished the ability of Cygb-OE cells to inhibit apoptosis compared to
wild-type Cygb-OE cells (35⫾1% vs 26⫾1%; p⬍0.05, n⫽3). Knockdown of Cygb (siCygb) in
myocytes led to increased caspase-dependent apoptosis at baseline (30⫾5% vs. 10⫾4%, vs
siRNA control cells; p⬍0.05, n⫽6) as well as increased caspase activity, mitochondrial
membrane depolarization and mitochondrial release of cytochrome c. Pretreatment of siCygb
cells with a pan-caspase inhibitor prevented apoptosis. Finally, more Cygb-OE cells were in the
proliferative phase while more siCygb cells were growth arrested [Cygb-OE cells vs control
cells: FACS S phase: 15⫾0.4% vs. 8⫾0.7%; BrdU (⫹): 56⫾2% vs. 39⫾2%; p⬍0.05; n⫽3;
siCygb cells vs siRNA control cells: FACS G0G/1 phase: 80⫾3% vs 67⫾5%; BrdU (⫹): 18⫾2%
vs 26⫾2.%; p⬍0.05; n⫽3]. Collectively, these new data establish that by effecting cellular
apoptosis and cell cycle, Cygb serves to modulate cardiac growth.
P101
Hyperglycemia Enhances Intracellular Ang II Levels in Rat Heart:
Implications for Cardiac Remodeling and Therapeutic Intervention
Vivek P Singh, Texas A&M Health Science Cntr, College of Medicine, Temple, TX; Bao Le,
Scott & White, Temple, TX; Kenneth M Baker, Rajesh Kumar; Texas A&M Health Science
Cntr, College of Medicine, Temple, TX
Upregulation of the renin-angiotensin system (RAS) has a major role in end-organ damage in
diabetes. Recent reports have suggested that angiotensin converting enzyme inhibitors (ACE-I)
and angiotensin receptor (AT1) blockers (ARBs) do not provide complete benefits in diabetic
BCVS Conference 2008 Abstracts
patients. Recently, we identified a complete and functional intracellular RAS (iRAS) in cardiac
myocytes, which was not inhibited by ACE-I and ARBs. These observations led us to determine
whether the cardiac iRAS is activated in diabetes. Diabetes was produced in adult SD rats by
streptozotocin. Intracellular levels of Ang II (iAng II) were measured in isolated cardiac myocytes
and non-myocytes by ELISA and confirmed by confocal immunohistochemistry. A pathological
role of iAng II was studied by treating diabetic animals with an ARB (candesartan, 1 mg/kg, IP),
ACE-I (benazepril 10 mg/kg, orally), or a renin inhibitor (aliskiren 30 mg/kg, orally). After one
week of diabetes, a significant 9.9-fold increase in Ang II levels in cardiac myocytes of diabetic
rats (0.59 ⫾ 0.01 fmol/mg heart) was observed, compared to controls (0.06 ⫾ 0.01 fmol/mg
heart), which was blocked by insulin treatment. Diabetic rats treated with candesartan
demonstrated increased iAng II levels (7-fold), suggesting intracellular synthesis of Ang II, not
uptake through the AT1 receptor. Aliskiren reduced iAng II to basal levels, while benazepril did
not have any effect. Non-myocyte cells also showed enhanced iAng II levels in diabetic rats
(0.11 ⫾ 0.02 fmol/mg heart), compared to controls (0.04 ⫾ 0.01 fmol/mg heart). Plasma Ang
II levels did not differ significantly among the groups. Diabetic rat hearts showed enhanced
oxidative stress, cardiomyocyte apoptosis, and fibrosis. These pathological parameters were
completely attenuated by aliskiren, while benazeprilat and candesartan were only partially
effective. The latter findings suggested that iAng II has a pathological role in diabetic
cardiomyopathy. In conclusion, this study suggests a novel mechanism of the RAS in cardiac
complications of diabetes and provides an explanation for incomplete blockade of the RAS by
ACE-I and ARBs. Novel interventions, such as renin inhibition, may provide greater benefits in
diabetic conditions.
P102
Resistin Induces Hypertension and Increases Passive Stiffness of the Left
Ventricle in Rats
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Elie R Chemaly, Maeng Jo Kim, Roger J Hajjar, Djamel Lebeche; Mount Sinai Sch of
Medicine, New York, NY
Our group has recently shown that resistin, an adipocytokine involved in insulin resistance, was
highly expressed in left ventricular (LV) tissue in rat models of diabetes mellitus. Adenoviral
overexpression of resistin induced a hypertrophic response in neonatal rat cardiomyocytes and
decreased contractility in adult rat cardiomyocytes. To investigate the in vivo effects of resistin
on left ventricular hypertrophy and hemodynamics, we administered neonatal rats an
intracardiac injection of adenovirus expressing resistin (Ad.Retn, n⫽7) or beta-galactosidase
(Ad.␤-gal, n⫽4) with a CMV promoter, to allow long-term expression of the transgene. Based
on hypertrophy assessment by LV anterior wall thickness measured by echocardiography, the
Retn group showed a mild but statistically significant hypertrophic response at 2, 3 and 4
months compared to the ␤-gal group (0.24 ⫾0.02 cm vs. 0.20 ⫾ 0.005 cm at 3 months,
p⬍0.05). At 5 and 6 month, the ␤-gal group showed a similar, likely age-related, wall
thickening of the left ventricle. The Retn group also displayed a trend to a reduced fraction of
shortening and ejection fraction of the LV, with an increase in the LV end-systolic volume
throughout the study. At end of study (6 months), animals underwent invasive hemodynamic
measurements by pressure-volume loop analysis. The Retn group had a higher maximal LV
pressure than the ␤-gal group (139 ⫾ 13.0 vs. 123 ⫾ 6.3 mmHg, p⬍0.05), higher minimal
LV pressure (12.7 ⫾ 3.0 vs. 2.6 ⫾ 1.4 mmHg, p⬍0.01), and higher LV end-diastolic pressure
(18.0 ⫾ 2.9 vs. 7.7 ⫾ 4.2 mmHg, p⬍0.01). Invasive contractility parameters did not differ
between groups. End-systolic and end-diastolic pressure-volume relationships of the left
ventricle were obtained during inferior vena cava occlusion. A steeper end-diastolic pressurevolume curve was found in the Retn group, indicating a higher passive stiffness of the left
ventricle (P⫽ 0.1). Blood glucose was normal in both groups at the end of the study. There was
a significant increase in the phospholamban/SERCA2a ratio in the Retn group suggesting
abnormal calcium handling associated with myocardial dysfunction. Our data support a role for
resistin in cardiac dysfunction associated with insulin resistance.
Low Levels of TNF␣ Prevent Ceramide Accumulation in Cardiac Cell
Membranes and Lipid-Induced Systolic Dysfunction
P103
Martina Cebova, Catherine J Knowles, Calvin P Vary, Ilka Pinz; Maine Med Cntr,
Scarborough, ME
Obesity increases tumor necrosis factor ␣ (TNF␣) and free fatty acid (FFA) levels, known risk
factors for cardiovascular disease. To test whether TNF␣ and FFA change ceramide (Cer)
content of the cardiomyocyte plasma membrane a high palmitate fed (HPD) mouse model with
low over-expression of TNF␣ (TIA-1-/-) was studied. Wild type (WT) and TIA-1-/- mice were fed
HPD or a medium-chain triglyceride control diet (MCTD), with comparable caloric intake from
fat, for 12 weeks. To determine contractile performance, hearts were perfused in the
isovolumic Langendorff mode in the presence of varying extracellular Ca2⫹ concentrations (1.5
- 4 mM). Total lipid extracts of left ventricular tissue were used to measure C18-Cer content
by mass spectrometry. We found no differences in heart weight or cardiac dimensions between
groups. HPD WT hearts showed a 5– 6 fold increase in C18 Cer within 2 weeks of HPD feeding
(Figure A, n⫽4, * p ⬍ 0.01). In contrast, TIA-1-/- hearts maintained normal Cer content even
after 12 weeks of HPD. At 12 weeks systolic performance in WT hearts decreased in all indices
in the HPD group, in contrast to HPD TIA-1-/- and MCTD WT hearts: systolic pressure -26%, rate
pressure product -27% (Figure B), and ⫹dP/dt -39% (n⫽ 5– 8, p⬍0.01). This difference was
most apparent under conditions of high extracellular [Ca2⫹]. We conclude that low TNF␣
expression preserves the Cer profile of cellular membranes and thus protects the heart from
palmitate induced systolic dysfunction. Thus, we suggest that low TNF␣ expression in obesity
is a cardio-protective mechanism.
e55
P104
Endothelial Cell-Derived Endothelin-1 Promotes Cardiac Dysfunction in
Diabetes Experimental Model
Bambang Widyantoro, Noriaki Emoto, Kazuhiko Nakayama, Dyah W Anggrahini, Kazuya
Miyagawa, Hidemi Nonaka, Vita Y Anggraeni, Kobe Univ Graduate Sch of Medicine, Kobe,
Japan; Takashi Suzuki, Tohoku Univ Graduate Sch of Medicine, Sendai, Japan; Yaz Y
Kisanuki, Masashi Yanagisawa, Howard Hughes Med Institute, Univ of Texas Southwestern
Med Cntr, Dallas, TX; Kenichi Hirata; Kobe Univ Graduate Sch of Medicine, Kobe, Japan
Diabetes may affect cardiac structure and function which may lead to heart failure in the
absence of coronary atherosclerosis and hypertension. However, multifactorial nature behind
the disease remains incompletely understood. The evidence of persistently high plasma
endothelin-1 (ET-1) level in diabetes patients has brought indication of ET-1 involvement in
mediating diabetic complication. Since endothelial dysfunction plays an important role in
pathophysiology of diabetic macroangiopathy, we hypothesized that specific disruption of ET-1
in endothelial cells might be sufficient to attenuate diabetes-induced cardiac injury. To test this
hypothesis, we developed type 1 diabetes in vascular endothelial cell-specific ET-1 knockout
(VEETKO) mice, of which ET-1 expression in major organs including heart were reduced by
60%, and in their wild type (WT) littermates. Early stage of diabetes (8 weeks) increased
cardiac ET-1 expression only in WT mice, revealed stimulation of ET-1 production from
endothelial cells. ET-1 stimulates pro-fibrotic genes expression (TGF-␤, CTGF and Collagen-1)
and promotes cardiac fibrosis in diabetic-WT mice. Interestingly, after 6 months of diabetes we
observed in both genotypes that ET-1 expressed mainly in the fibrotic area, indicates ET-1
release from accumulated fibroblast. In addition, impairment of cardiac angiogenesis by
diabetes was prevented by lack of ET-1, and this is associated with preservation of
HIF1-␣-induced VEGF expression in diabetic-VEETKO mice. Diabetes also increased intra
cardiac accumulation of Triglyceride which further causes disruption of myofibril and
mitochondria in WT mice, but not in VEETKO mice. We further observe heart failure after 9
months of diabetes in WT mice, which is prevented in VEETKO mice (fractional shortening
37.55⫾2.73% vs. 43.63⫾0.91%, respectively, p⬍0.001, n⫽6 each). In conclusion, disruption
of ET-1 in vascular endothelial cell is sufficient to prevent the development of cardiac injury and
heart failure in type 1 diabetic mice model. This result suggests an important role of endothelial
cell-derived ET-1 in mediating diabetic cardiomyopathy and may provide basic rationale of ET-1
blockade as potential therapeutic strategy in preventing diabetic heart disease.
P105
The Mitochondrial ATP-Binding Cassette Protein 2 Is Overexpressed in
Ischemic Heart Disease and Regulates Cellular ROS Levels
Michael A Burke, Rongxue Wu, Tejaswitha Naik, Northwestern Memorial Hosp, Chicago, IL;
Sathyamangla V Prasad, Cleveland Clinic Foundation, Cleveland, OH; Hossein Ardehali;
Northwestern Memorial Hosp, Chicago, IL
ATP-binding cassette (ABC) proteins are a remarkably diverse family of molecules found in all
cells and organelles. Four mammalian mitochondrial ABC proteins have been characterized to
date; however, the functions of most of these proteins are not known. We recently showed that
a mitochondrial ABC protein, mABC1, is protective against oxidant induced cell death. Another
mitochondrial ABC protein, mABC2, is expressed in all human tissues, including the heart, but
its primary function and its role in human disease are not known. To better characterize the
function of mABC2 protein and its role in ischemic heart disease, we sought to study whether
mABC2 levels are altered in response to ischemia in the heart and whether its overexpression
induces an increase in cellular metabolism and production of reactive oxygen species. We first
demonstrated that the levels of mABC2 protein are increased in response to ischemia. This was
confirmed in three different models: explanted hearts of patients with chronic ischemic heart
disease, a dog model of acute low flow coronary ischemia, and a mouse model of acute
myocardial infarction (MI). In the mouse MI model, mABC2 was robustly overexpressed in the
border zone myocardium as well as in the area of infarction as early as 2 days post-MI. These
results strongly suggest that mABC2 levels are increased in response to ischemia. We then
studied whether overexpression of mABC2 leads to an increase in cellular metabolism. HEK293
cells overexpressing a green fluorescent protein (GFP)-tagged mABC2 did not display a change
in the rate of mitochondrial O2 consumption or total ATP levels compared to GFP-transfected
control cells. Interestingly, overexpression of mABC2 resulted in a significant increase in the
levels of reactive oxygen species (ROS) in the presence of H2O2. These data suggest that
mABC2 may play a role in both the myocardial response to ischemia as well as post-infarct
ventricular remodeling, possibly through changes in the levels of cellular ROS production. Thus,
mABC2 may provide a potential therapeutic target in the treatment of ischemic heart disease.
e56
Circulation Research
Vol 103, No 5
August 29, 2008
P106
TAT-Mediated Protein Transduction in Mitochondria: Regulation by Sodium
Channel Blockers
Jayanagendra P Rayapureddi, Wendy Tomamichel, Ronald M Payne; Indiana Univ Sch of
Medicine, Indianapolis, IN
Protein transduction is a process that enables proteins to be directly introduced into cells. TAT
(Transactivator of Transcription) is one of the well known protein transduction domains, which
has been shown to deliver cargo molecules across the plasma membrane. Previously from our
laboratory it was reported that TAT not only can deliver the proteins but also localize them to
mitochondria by using TAT.mMDH.eGFP protein. We are developing this approach of non viral
enzyme replacement therapy for the treatment of mitochondrial diseases like Friedreich’s
ataxia, mitochondrial Trifunctioal protein (TFP␣) deficiency and medium chain acyl coA
dehyrogenase (MCAD) deficiency. It was reported that TAT mediated transduction occurs
through the plasma membrane by macropinocytosis in T cells. However, nothing is known
about TAT mediated protein transduction in mitochondria and we want to address the question
of whether it follows the same mode of endocytosis. So far, we have observed that it is a
receptor independent and energy independent process. None of the endocytosis inhibitors
could inhibit the TAT mediated protein transduction significantly. Interestingly, both flowcytometry and western blot experiments showed that Amiloride, which is a known inhibitor of
macropinocytosis, increased TAT mediated protein transduction in mitochondria. Our further
analyses of Sodium channel inhibitors have shown that of the three Sodium channels in
mitochondria, Na⫹/Ca⫹2 exchanger inhibition had increased the transduction of TAT.mMDH.eGFP into mitochondria.
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
P107
Evidence for a Functional Coupling Between the L-Type Calcium Channel
and the Mitochondria
Helena M Viola, Peter G Arthur, Livia C Hool; The Univ of Western Australia, Perth, Australia
Oxidative stress is a feature of cardiovascular disease and hydrogen peroxide (H2O2) can act as
a signaling molecule to mediate cardiovascular pathology. We have previously demonstrated
that transient exposure of ventricular myocytes to H2O2 leads to a further increase in reactive
oxygen species (ROS) from the mitochondria (supporting the “ROS-induced ROS-release”
hypothesis). This occurs due to a persistent increase in L-type Ca2⫹ channel basal current and
diastolic calcium. The effect persists because a positive feedback exists between increased
basal channel activity, elevated intracellular calcium and superoxide production by the
mitochondria. Enhanced superoxide production occurs with enhanced calcium uptake into the
mitochondria. However superoxide is also produced when mitochondrial membrane potential
(MMP) is increased. We therefore examined the relationship between L-type Ca2⫹ channel
activation and regulation of MMP. Ventricular myocytes were isolated from hearts from
anesthetised guinea-pigs. Activation of the L-type Ca2⫹ channel with BayK(-) or 45 mM KCl
caused a 23.7⫾3.6% increase in MMP assessed with the fluorescent indicator JC-1 (n⫽10;
P⬍0.05). This effect was reproduced when myocytes loaded with JC-1 were patch-clamped
and L-type Ca2⫹ channels were activated by voltage stepping from -40mV to ⫹10mV (n⫽6).
The increase in MMP occurred in the absence of extracellular calcium and presence of Na⫹/H⫹
exchanger inhibitor amiloride and Na⫹ channel inhibitor tetrodotoxin (n⫽5). In addition Ru360,
an inhibitor of the mitochondrial Ca2⫹ uniporter did not prevent the increase in MMP induced
by 45 mM KCl (n⫽6). Activation of the L-type Ca2⫹ channel is modulated by the auxiliary ␤
subunit that is also tethered to the cytoskeleton. Depolymerisation of F actin with latrunculin
A or application of a peptide directed against the alpha interacting domain (AID) of the channel
(inhibiting movement of the ␤ subunit) prevented the increase in MMP by 45 mM KCl (n⫽6).
We conclude that the L-type Ca2⫹ channel can regulate mitochondrial function. A functional
coupling of the channel with the mitochondria may assist with regulating ATP production on a
beat to beat basis. This may be disturbed where there is pathology involving disruption of actin
filaments.
Transcriptional Coactivators PGC-1␣ and PGC-l␤ Control Overlapping
Programs Required for Cardiac Mitochondrial Maturation
P108
Ling Lai, Teresa Leone, Christoph Zechner, Washington Univ in Saint Louis, Saint Louis,
MO; Paul Schaeffer, Miami Univ, Oxford, OH; Sean Kelly, Daniel Flanagan, Washington Univ
in Saint Louis, Saint Louis, MO; Denis Medeiros, Kansas State Univ, Manhattan, KS; Attila
Kovacs, Daniel Kelly; Washington Univ in Saint Louis, Saint Louis, MO
The transcriptional coactivators, PPAR␥ coactivator-1 (PGC-1) ␣ and ␤, have been implicated
in the transcriptional control of myocardial energy metabolism and mitochondrial function. In
animal models of pathologic cardiac hypertrophy and heart failure, the expression of PGC-1␣
is downregulated suggesting that deactivation of this regulatory cascade contributes to
pathologic cardiac remodeling. Surprisingly, however, single PGC-1␣ or ␤ “knockout” mice
exhibit minimal cardiac phenotype under non-stressed conditions. In this study, the functional
roles of PGC-1␣ and PGC-1␤ were investigated in vivo by generating mice with combined
deficiency of PGC-1␣ and PGC-1␤ (PGC-1␣␤-/- mice). In striking contrast to the singly deficient
PGC-1 lines, PGC-1␣␤-/- mice died shortly after birth with small hearts. The cardiac function
of PGC-1␣␤-/- mice at birth was assessed by echocardiography and Doppler analysis, showing
bradycardia, intermittent second-degree heart block, reduced ventricular systolic and diastolic
function, and a markedly diminished cardiac output. Cardiac-specific ablation of the PGC-1␤
gene on a PGC-1␣-deficient background phenocopied the generalized PGC-1␣␤-/- mice.
Investigation of the cellular and molecular phenotype of the PGC-1␣␤-/- heart revealed
signatures of a profound defect in developmental maturation including a late fetal arrest in
mitochondrial biogenesis, cellular derangements, and persistence of a fetal pattern of gene
expression. Specifically, electron microscopy studies revealed a significant diminution in
mitochondrial number and size, with a variety of ultrastructural abnormalities including
vacuoles and reduced cristae density in 0.5 day old PGC-1␣␤-/- mice. The expression of genes
involved in OXPHOS (Cycs, Cox4, and Atp5b) and FAO (Acadm, Acadvl, and Cpt1b) were
significantly reduced and fetal markers (ANP, BNP) abnormally increased in the PGC-1␣␤-/hearts. We conclude that PGC-1␣ and PGC-1␤ share target genes that are necessary for the
perinatal mitochondrial maturation and function of the heart. Given the importance of
mitochondrial dysfunction in the pathogenesis of common myocardial diseases, we propose
that the PGC-1 regulatory cascade is a rational candidate therapeutic target.
P109
In Situ Activation of Constituent Platelets Enhances the Therapeutic
Capacity of Peripheral Blood Mononuclear Cell Implantation
Kaoru Tateno, Junji Moriya, Kentaro Miura, Tohru Minamino, Issei Komuro; Chiba Univ,
Chiba, Japan
Therapeutic neovascularization by cell implantation has become widespread. We have
previously shown that implantation of autologous peripheral blood mononuclear cell preparation
(PBMNC) induced therapeutic neovascularization in ischemic skeletal muscle tissue both in
animals and in patients with critical limb ischemia. We have also revealed that PBMNC
implantation promoted myoblast proliferation, resulting in bulk mass of regenerating myotubes
that produced various angiogenic ligands in rich and continuous manner. The therapeutic effect
of PBMNC was abolished when muscle regeneration was compromised, suggesting a critical
role of tissue myoblasts in therapeutic neovascularization. However, mechanism by which
PBMNC induced myoblast proliferation remained unclear. To elucidate the component within
PBMNC that acts on muscle regeneration, we first separated PBMNC peparation into MNCs and
platelets using density gradient method. PBMNC depleted of coexisting platelets showed less
effect on myoblast proliferation in vitro, as well as on muscle regeneration and neovascularization in vivo. On the contrary, platelets had tremendous ability to enhance proliferation of
co-incubated myoblast cell line C2C12. The supernatant of this co-incubation could still induce
proliferation of another dish of C2C12, suggesting that the effect relies on paracrine
mechanism. These effects were abolished when C2C12 and platelets were incubated in Boyden
chambers, indicating the importance of direct myoblast-platelet contact. Furthermore, we
found that the ability of platelets or its supernatant to induce proliferation of myoblasts was
similarly compromised when platelets were treated with anti-integrin alpha IIb neutralizing
antibody prior to direct co-incubation with C2C12. Finally, pretreatment of PBMNC preparation
with this neutralizing antibody partly attenuated the tissue regeneration property of PBMNC in
vivo. These results indicate that in-situ activation of co-existing platelets within PBMNC
preparation plays a supplemental role on its therapeutic effect when implanted into ischemic
skeletal muscle tissue.
P110
Granulocyte-Macrophage Colony-Stimulating Factor Antibody Improves Left
Ventricular Function and Limits Maladaptive Remodeling Following
Myocardial Infarction
Jordan J Lancaster, Southern Arizona VA Med Cntr, Tucson, AZ; Robert S Kellar,
Development Engineering Sciences, Flagstaff, AZ; Hoang M Thai, Elizabeth Juneman,
Nicholle M Johnson, Howard G Byrne, Maribeth Stansifer, Southern Arizona VA Med Cntr,
Tucson, AZ; Christopher Beddington, Geoffery Yarranton, Kalobios Pharmaceuticals, Palo
Alto, CA; Steven Goldman; Southern Arizona VA Med Cntr, Tucson, AZ
Background: Recent studies have shown granulocyte-macrophage colony-stimulating factor
(GM-CSF) promotes infarct expansion and inappropriate collagen synthesis in the infarcted
ventricle. Thus suggesting, enhanced proliferation of monocytes and macrophages at the time
of acute myocardial infarction (MI) may have deleterious effects on left ventricular (LV)
remodeling. This study was designed to determine if blocking the effects of GM-CSF alters LV
remodeling and hemodynamics in rats with acute MI. Methods: Acute MI was created by
ligating the left coronary artery of rats; treatment with the GM-CSF antibody (5mg/kg) was
initiated 24 h prior to coronary ligation. Closed chest echocardiography and solid-state
micromanometers were used to measure outcome variables 3 weeks after ligation. N⫽6 –10
in each group. Results: The GM-CSF antibody increased (P⬍0.05) LV ejection fraction (37⫾3
vs 47⫾5%) and decreased (P⬍0.05) LV end-systolic diameter (0.75⫾0.12 vs. 0.59 ⫹0.05 cm)
with no changes in LV systolic pressure (109⫹4 vs 104⫹5 mmHg), LV-end diastolic pressure
(22⫹4 vs 21⫹2 mmHg), LV-end diastolic diameter (0.96⫹0.04 vs. 0.92⫹0.05 cm), or Tau
(25.4⫹2.4 vs. 22.7⫾1.4 msec). Conclusion: We report improvements in LV ejection fraction
and partial reversal of LV remodeling using an antibody against GM-CSF initiated 24 h prior to
MI. These findings suggest that inhibition of monocyte and marcophage migration may be
beneficial in the treatment of heart failure after MI.
P111
Increased Proliferative Capacity of Cardiosphere-Forming Cells Early After
Acute Myocardial Infarction
Jianqin Ye, Andrew Boyle, Henry Shih, Muhammad Khan, Yagai Yang, Junya Takagawa,
Richard Sievers, Hua Su, Yan Zhou, William Grossman, Yerem Yeghiazarians; UC, San
Francisco, San Francisco, CA
Background: Cardiospheres (CSs) represent one type of cardiac stem cell (CSC) which can be
expanded in in vitro culture and may be an excellent cell source for myocardial regeneration.
However, there are limited numbers of CSC in the heart, and whether enough CSs can be
generated from hearts after acute myocardial infarction (MI) remains unknown. In this study,
BCVS Conference 2008 Abstracts
we hypothesized that the number of cardiosphere (CS)-forming cells is altered after acute MI.
Methods: Nine-month old male C57BL/6J mice underwent permanent left anterior descending
artery ligation to induce MI and were sacrificed at 1-, 2- and 4-weeks post-MI (n⫽5– 6/group).
Sham-operated and non-operated mice served as controls. The hearts were collected for CS
generation. The growth rate of CS-forming cells from cardiac explants was analyzed and the
number of CSs from each heart was quantified. The cells of CSs were analyzed by FACS,
immunocytochemistry and RT-PCR. Results: It took 14⫾1, 13⫾2 and 18⫾5 days for
CS-forming cells from hearts 1-, 2- and 4-weeks post-MI to grow to 90% confluence in vitro,
while it took 21⫾1 and 32⫾4 days for sham-operated and non-operated hearts. Analysis
showed that CS-forming cells from hearts 1-, 2-weeks post MI grew faster than both control
groups (P⬍0.004). However, this increase significantly slowed down by 4-week post MI. The
number of CSs formed from hearts harvested 1-week (340⫾22/heart) and 2-weeks
(213⫾16/heart) post-MI were considerably higher than that from sham-operated (80⫾15/
heart) and non-operated hearts (18⫾6/heart) (P⬍0.01). This increase was significantly
attenuated by 4-weeks post-MI (141⫾17/heart). About 11% and 20% of cells of CSs were
Sca1⫹/lineage- and c-Kit⫹ respectively. The cells of CSs expressed early cardiac lineage
markers NKx2.5, GATA4 and endothelial, smooth muscle cells markers. Spontaneous beating
was observed in a few CSs. Conclusion: The proliferative capacity of CS-forming cells following
acute MI increases dramatically 1–2 weeks after injury, resulting in increased number of CSs
in vitro. The time “window” of this increase post-MI has major implications for the design of
clinical trials of CSC therapy. Our data suggest that early acquisition of cardiac tissue post-MI
may facilitate higher yields of CSs for autologous CSC therapy.
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P112
Myocyte-Specific M-CAT Elements Regulate G-Protein ␥ 3 Expression in
Cardiac Myocytes
Charlene McWhinney; NY College of Osteopathic Medicine, Old Westbury, NY
Little is known regarding the mechanisms that control the expression of G-proteins ␣, ␤ and
␥ subtypes. We have previously shown that the G-protein ␥3 gene is expressed in the heart,
brain, lung, spleen, kidney, muscle and testis in mice. We have also reported that the G-protein
␥3 subunit is expressed in rat cardiac myocytes, but not in cardiac fibroblasts. Other studies
have shown that the ␥3 subunit couples to the angiotensin A1A receptor in portal vein
myocytes, and has been shown to mediate ␤-adrenergic desensitization in cardiac myocytes
treated with atorvastatin. In the present study, we evaluated G-protein ␥3 promoter-luciferase
reporter constructs in primary myocytes to identify key regulatory promoter regions. We
identified two important regions of the promoter (UPR and DPR), which are required for
expression in cardiac myocytes. We observed that removal of 48-bp in the UPR diminished
gene transcription by 75%, and that the UPR contains consensus elements for myocyte-specific
M-CAT and MEF-1 elements. The UPR and DPR share transcription factor elements for
myocyte-specific M-CAT element. We observed that cardiac myocyte proteins bind to ␥3
oligonucleotides containing transcription factor elements for myocyte-specific M-CAT and
MEF-1. Myocyte-specific M-CAT proteins were supershifted with TEF-1 antibodies binding to
the ␥3 M-CAT element, which is in agreement with reports showing that the M-CAT element
binds the transcriptional enhancer factor-1 (TEF-1) family of transcription factors. The 150-bp
DPR contains three M-CAT elements, an INR element, an USF1 element and the transcription
start site. We have shown that myocyte ␥3 gene expression is regulated by myocyte-specific
M-CAT and MEF-1 elements.
P113
Hydrogen Peroxide Regulates Proliferation and Extracellular Matrix Proteins
Through Decreased CREB Content in Pulmonary Artery Smooth Muscle
Cells
Chrystelle V Garat, Dwight J Klemm; Univ of Colorado at Denver and Health Sciences Cntr,
Denver, CO
Cyclic AMP response element binding protein (CREB) content is diminished in smooth muscle
cells (SMC) in remodeled pulmonary arteries from animals with pulmonary hypertension.
Several studies have indicated that elevated reactive oxygen species (ROS) production may play
a role in experimental models of hypertension. ROS include superoxide, hydrogen peroxide
(H2O2), hydroxyl anion and reactive nitrogen species. The objective of this study was to
determine whether H2O2 plays a role in pulmonary artery (PA) remodeling by controlling
proliferation and extracellular matrix (ECM) production by PA SMCs through the transcription
factor CREB. For these experiments rat PA SMCs were isolated and cultured from the medial
layer of main PA. Experiments were carried out using quiescent PA SMCs exposed to increasing
concentrations of H2O2. CREB expression was determined by western blot analysis. Results
showed that CREB expression was downregulated in response to H2O2 and that treatment of
PA SMCs with specified concentrations of the antioxidant Tiron prior to exposure to H2O2
restored nuclear CREB levels in H2O2 -treated cells in a dose dependent manner. CREB
depletion with small interfering siRNA induced an increase in SMC proliferation. Furthermore
using the reverse transcriptase polymerase chain reaction and primers for several ECM
proteins, we demonstrated that downregulation of CREB decreased fibronectin and elastin but
not Collagen I mRNA levels. Also, stimulated PA SMCs with H2O2 induced the expression of the
matrix-degrading enzyme gelatinase B (MMP-9) as determined by zymography. In conclusion,
H2O2 induced decrease of CREB expression contributes to PA remodeling through increased
SMC proliferation and altered ECM production.
e57
P114
MURC, A Muscle-Restricted Coiled-Coil Protein, Induces Cardiomyocyte
Hypertrophy Through the Extracellular Signal-Regulated Kinase Pathway
Takehiro Ogata, Univ of Tsukuba, Tsukuba, Japan; Tomomi Ueyama, Kyoto Univ Hosp,
Kyoto, Japan; Koji Isodono, Kyoto Prefectural Univ Sch of Medicine, Kyoto, Japan; Hiroko
Imoto, Kyoto Univ Hosp, Kyoto, Japan; Masashi Tagawa, Kyoto Prefectural Univ Sch of
Medicine, Kyoto, Japan; Norifumi Takehara, Kyoto Univ Hosp, Kyoto, Japan; Satoshi Asada,
Kyoto Prefectural Univ Sch of Medicine, Kyoto, Japan; Atsuo Adachi, Kyoto Univ Hosp,
Kyoto, Japan; Tomosaburo Takahashi, Hiroaki Matsubara, Kyoto Prefectural Univ Sch of
Medicine, Kyoto, Japan; Hidemasa Oh; Kyoto Univ Hosp, Kyoto, Japan
[Backgrounds] Recently, we identified a novel muscle-restricted coiled-coil protein, MURC,
which is evolutionarily conserved from frog to human. MURC is localized to the cytoplasm with
accumulation in the Z-line of the sarcomere and that forced overexpression of MURC in
cardiomyocytes induces activation of the RhoA/ROCK pathway. Transgenic mice expressing
MURC (Tg-MURC) in the heart shows cardiomyocyte hypertrophy and subsequently exhibits
cardiac contractile dysfunction, conduction disturbances, reduced thickness of the ventricular
wall, and interstitial fibrosis in mice. However, the molecular mechanisms responsible for
MURC-induced hypertrophy have not been elucidated. [Methods and Results] To examine the
involvement of MURC in the developmental process of cardiac hypertrophy and heart failure,
we analyzed MURC expression in the heart of Dahl salt-sensitive (DS) rats fed either a low-salt
diet (0.3% NaCl) or a high-salt (8% NaCl) diet. MURC expression was significantly upregulated
both in the hypertrophied and failing heart of DS rats fed the high-salt diet. We then examined
signaling pathways involving MURC in the heart of Tg-MURC mice at 6 weeks of age when they
showed cardiomyocyte hypertrophy. Extracellular signal-regulated kinase (ERK) but not p38
MAPK, JNK, Akt, and PKC␣ was markedly activated in the heart of Tg-MURC mice at 6 weeks
of age. Furthermore, forced overexpression of MURC in cardiomyocytes induced ERK activation,
hypertrophy, and brain natriuretic peptide (BNP) mRNA expression in vitro. RNA interferencemediated knockdown of MURC in cardiomyocytes impaired phenylephrine-induced hypertrophy
and ERK activation. MURC-induced BNP mRNA expression and increase in cell size were
attenuated by a MEK inhibitor in a dose-dependent manner. [Conclusion] These findings
indicate that MURC is upregulated during development of cardiac hypertrophy and heart failure,
and that MURC utilizes the ERK pathway to induce cardiomyocyte hypertrophy.
P115
Targeting the Intrinsic Coagulation Cascade to Protect from Cardiac
Ischemia Reperfusion Injury
Stefan Frantz, Johann Bauersachs, Anna Adamek, Nadja Blömer, Geor Ertl, Medizinische
Universitaetsklinik, Wuerzburg, Germany; Thomas Renne; Institut für Klinische Biochemie
und Pathobiochemie, Wuerzburg, Germany
Formation of fibrin is critical for limiting blood loss at a site of blood vessel injury (hemostasis),
but may also contribute to vascular thrombosis and thus exaggerate myocardial ischemia/
reperfusion injury. Hereditary deficiency of factor XII (FXII), the protease that triggers the
intrinsic pathway of coagulation in vitro, is not associated with spontaneous or excessive
injury-related bleeding, but can reduce thrombus formation. Therefore, we studied the
contribution of FXII for myocardial ischemia reperfusion injury. Following 30 minutes of
ischemia and 24 hours of reperfusion infarct size in FXII deficient mice was significantly
reduced as compared to wild type controls (infarct/area at risk, WT vs. FXII-/-, 73.5%⫾4.4%
vs. 42.3⫾1.6%, p⬍0.001) despite similar area at risk (WT vs. FXII-/-, 28.3%⫾1.7% vs.
29.9⫾1.0%, p⫽n.s.). There was no difference in bleeding complications and bleeding time.
Mice deficient in the FXII substrate factor XI were similarly protected from ischemia reperfusion
injury (infarct/area at risk, FXI-/-, 16.9%⫾4.1%, vs. WT p⬍0.001), as were factor XII/XI double
KO mice. The phenotype of FXII-/-mice could be rescued by intravenous application of human
FXII (infarct/area at risk, FXII-/-⫹FXII, 77.3%⫾8.4%, p⬍0.001 vs. FXIIKO). WT mice treated
with the FXII inhibitor PCK (Pro-Phe-Arg-chloromethylketone) were similarly protected from
myocardial ischemia/reperfusion injury (infarct/area at risk, 46.5% ⫾3.7%, vs. WT p⬍0.001).
Fibrin formation was significantly inhibited in FXII, FXI KO, as well as mice treated with FXII
inhibitor when compared to WT mice. The data suggest that the intrinsic pathway is crucial for
pathological clotting in myocardial ischemia reperfusion injury. FXII inhibition may offer a
selective and safe strategy to reduce ischemic injury.
P116
Tissue Histidine Deprivation Mediates Cardiac Antioxidant Defense via
Activation of GCN2-eIF2␣-ATF4 Pathway
Takaharu Katayama, Motoaki Sano, Jin Endo, Kentaro Hayashida, Keiichi Fukuda; Keio Univ
Sch of Medicine, Tokyo, Japan
[Introduction] We recently report aldehydes accumulation by overexpression of aldehyde
dehydrogenase (Aldh)2*2 paradoxically maintained high levels of glutathione via activation of
amino acid biosynthesis through eIF2␣-ATF4 pathway and conferred upon hearts favorable
tolerance to oxidative stress. This study is designed to investigate how cells sense aldehydes
and transduce signals to eIF2␣-ATF4 pathway. [Method & Result] (1) Among eIF2␣ kinases,
phosphorylation levels of GCN2 kinase, sensor for amino acid depletion, were selectively
increased in the hearts from Aldh2*2-TG mice. (2) Metabolome analysis by capillary
electrophoresis mass spectrometry revealed that tissue histidine concentration in Aldh2*2-TG
hearts was exceptionally reduced by half compared to that in non-TG littermates. (3) To clarify
the functional significance of observed reduction of tissue histidine concentraion, Aldh2*2-Tg
mice were fed high histidine diet. The phosphorylation levels of GCN2/eIF2␣ and the expression
levels of ATF4 and its downstream target genes encode enzymes related to amino acid
biosynthesis were diminished by 50% in the hearts from Aldh2*2-TG mice fed high histidine
e58
Circulation Research
Vol 103, No 5
August 29, 2008
diet, in agreement with the normalization of tissue histidine concentration. Accordingly, both
elevated glutathione concentration and tolerance to ischemia-reperfusion injury were partially
diminished in the hearts from Aldh2*2-TG mice fed high histidine diet compared to Aldh2*2-TG
mice fed normal chew. (4) In cultured cardiomyocytes, aldehyde [4-hyderoxy-2-nonenal,
(4-HNE)] phosphorylated GCN2- eIF2␣ in parallel to a decrease in the intracellular histidine
concentration. (5) GCN2-/- mouse embryonic fibroblasts failed to induce the phosphorylation of
eIF2␣ and the expression of Atf4 in response to 4-HNE. (6) Histidine preferentially formed
4-HNE-adducts, which could explain the selective decrease in intracellular concentration of
histidine by 4HNE. [Conclusions] We identified the novel molecular mechanism of cross-talk
between oxidative stress signaling and eIF2␣-ATF4 pathway. Aldehyde accumulation is being
converted into the signal for amino acid (histidine) starvation and sensed by GCN2 kinase.
P117
Spatial Distribution of Soluble Guanylyl Cyclase in Adult Mouse Ventricular
Cardiac Myocytes Is Altered Under Stress Condition of Pressure Overload
but Is Partially Restored with Sildenafil Treatment
Emily J Tsai, Takahiro Nagayama, Eiki Takamoto, David A Kass; Johns Hopkins Univ,
Baltimore, MD
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Soluble guanylyl cyclase (sGC), a key component of the NO-cGMP signaling pathway, is
conventionally deemed to be cytosolic. Its distribution and NO-sensitivity distinguishes sGC
from particulate GC, an integral protein in the plasma membrane which is stimulated by
natriuretic peptide. The two GC forms partly control the intracellular cGMP pool but do so with
differential functional effects and regulation. Compartmentation of cGMP and its source is also
suggested by the association of sGC with the plasma membrane in neurons, skeletal muscle,
endothelial cells, and platelets. Despite differences in sensitivity to NO and intracellular Ca2⫹
of membrane-associated and cytosolic sGC, the role of membrane-associated sGC in cardiac
myocytes (CMs) is unknown. We hypothesized that sGC translocation plays a role in the CM
response to pressure-overload and that dysregulated sGC translocation is associated with heart
failure. CMs were isolated from adult mice subjected to transaortic constriction (TAC). Some
mice were treated with PDE5-inhibitor sildenafil (SIL) starting 3 weeks post-TAC. Isolated CMs
were cultured, stimulated with an NO donor, and immunostained for sGCa1. In control CMs,
sGC was located predominantly at the plasma membrane at baseline. Upon NO stimulation, sGC
remained largely at the membrane but cytosolic translocation could be detected. In CMs from
mice subjected to TAC for 3 weeks (TAC3wk), sGC was distributed throughout the cytosol. Upon
NO stimulation, sGC distribution mirrored that of NO-stimulated control CMs. In CMs from mice
subjected to long-term TAC (TAC8wk), sGC remained at the membrane. No appreciable
cytosolic translocation was detected upon NO stimulation. In SIL-CMs, sGC distribution at
baseline and upon NO stimulation were similar to that of control CMs. TAC8wk hearts were
more severely dilated and dysfunctional than TAC3wk hearts (LVd 4.9mm vs 4.1mm, FS 11.9%
vs 35.4%). SIL hearts were comparable to TAC3wk (LVd 4.2mm, FS 32.4%). Our findings
suggest that sGC translocation plays a role in cGMP compartmentation in the CM stress
response and that cGMP compartmentation is dysregulated in heart failure. Restoration of
cGMP compartments may in part account for the beneficial effects of sildenafil in pressureoverload heart failure.
P118
Role of Protein Phosphatase 1 Isoforms on Calcium Regulation in the
Sarcoplasmic Reticulum
Yasuhiro Ikeda, Hidekazu Aoyama, Hiroki Aoki, Koichi Yoshmura, Takeshi Yamamoto,
Masafumi Yano, Masunori Matsuzaki; Yamaguchi Univ, Ube, Japan
Increased protein phosphatase 1 activity is associated with dysregulation of Ca2⫹ cycling in
heart failure. We previously reported that the increased content of PP1 alpha and beta in the
sarcoplasmic reticulum is tightly coupled with increased PP1 activity in the sarcoplasmic
reticulum and progressive decrease in cardiac contractility in the UMX7.1 cardiomyopathic
hamster. Specific role of each PP1 isoform in cardiac SR-mediated calcium cycling is not yet
clearly understood. In the present study, we investigated the isoform specific role of PP1 on
SR-mediated calcium cycling and cardiomyocyte contractility. Methods: Content of each PP1
isoforms in the cardiac SR microdomain was determined in the longitudinal and junctional SR
microvesicles which were isolated by differential centrifuge and sucrose gradient fractionation
method from canine hearts. Isoform specific PP1 knockdown was achieved by using adenovirus
shRNA technique, and subcellular distribution pattern was determined by transfecting
adenoviruses encoding enhanced green fluorescent protein (EGFP)-fused PP1 catalytic subunits
in isolated adult rat cardiomyocytes. Results: Each adenoviral PP1 isoform-specific RNAi
(MOI⫽500) induced approximately 80% reduction in protein levels of corresponding PP1
isoform without affecting other isoforms. Among the PP1 isoform specific knockdown, PP1beta
knockdown most effectively enhanced cell shortening and Ca2⫹ transient with and without
beta-adrenergic stimulation. Increased phospholamban phosphorylation at Ser16 was observed
in adenoviral PP1b-RNAi (1.6⫹/- 0.2 folds at baseline), whereas the ryanodine receptor
phosphorylation at Ser 2808 was not affected. PP1beta content was most significantly enriched
in the longitudinal SR fraction in which phospholamban is most abundantly enriched, although
each EGFP-tagged PP1 isoform showed similar lattice like structure and cytosolic staining
pattern. Conclusion: PP1beta is the mostly enriched in the SR and most dominant isoforms
regulating PLN dephosphorylation in cardiomyocytes. Because SR content of PP1beta is tightly
coupled with heart failure progression and increase in SR PP1 activity, PP1beta specific
inhibition may provide novel treatment strategy for heart failure.
P119
The Inositol(1,4,5)trisphosphate, IP3-Receptor Axis in Experimental and
Clinical Atrial Fibrillation
Elizabeth A Woodcock, Julie R McMullen, Xiao-Jun Du, Silvana Marasco, Tiffany
McLeod-Dryden, Jieting Luo, Anthony M Dart; Baker Heart Rsch Institute, Melbourne,
Australia
Atrial fibrillation (AF) is a complex disorder associated with a range of cardiac diseases. AF
requires a combination of triggers and substrates and may be influenced by genetic factors. We
studied the activity of the phospholipase C (PLC), inositol(1,4,5)trisphosphate (IP3), IP3-receptor
(IP3-R) axis in atria from mouse model, as well from patients with mitral valve disease. Mice
with cardiac overexpression of the stress-activated protein kinase, mammalian sterile 20 like
kinase 1 (Mst1) were crossed with mice expressing dominant negative (dn) PI 3-kinase. This
generated a strain with severe dilated cardiomyopathy with conduction block, and AF
developed spontaneously in 40% of the animals. Atria were labeled with [3H]inositol and IP3
was measured by anion-exchange HPLC. Atria from Mst1Xdn-PI3K mice had markedly
increased IP3 responses, which were higher in left atrium (LA (4 fold)) than in right atrium (RA
(3 fold). Atria from Mst1Xdn-PI3K mice also had increased expression of IP3-receptors (IP3-R)
(qRT-PCR), and this involved a change in subtype from the type 2 receptor in WT mice to
primarily type 1. Changes in LA (50 fold) were bigger than in RA (10 fold). LA and RA from
patients with mitral valve disease had increased IP3 responses that were higher in LA (37310 ⫾
11934) than RA (19172 ⫾ 1442 CPM/100 mg tissue, mean ⫾ SEM, n⫽7). Furthermore,
responses were higher in RA from patients with chronic AF than in those presenting in sinus
rhythm (4375 ⫾ 882). In LA, IP3 responses showed a positive correlation with atria size
(r⫽0.75, p⬍0.05). Heightened IP3 responses were associated with a selective increase in
expression of the ‘b’ splice variant of PLC␤1 and the expression of PLC␤1b was higher in LA
than in RA. We conclude that heightened activity of the IP3/IP3-R axis may contribute to the
initiation of AF in some experimental and patient groups.
P121
Differential Modulation of TLR2 and TLR4 Protects the Myocardium Against
Ischemia/Reperfusion Injury
Tuanzhu Ha, Fang Hua, Jim Kelley, Race L Kao, I W Browder, David L Williams, Chuanfu Li;
East Tennessee State Univ, Johnson City, TN
Recent studies have shown that Toll-like receptor 2 (TLR2) and TLR4 play an important role in
myocardial ischemia/reperfusion (I/R) injury. However, the effects of differential modulation of
TLR2 and TLR4 on myocardial ischemic injury have not been investigated entirely. We
examined the effects of peptidoglycan, a specific TLR2 ligand and lipopolysaccharide (LPS), a
specific TLR4 ligand on myocardial I/R injury. Peptidoglycan (PDG, 75 ␮g/25g) was
administered to experimental mice (n⫽8) one hr before the hearts were subjected to ischemia
(1 hr)/reperfusion (4 hrs), while LPS (0.2 mg/kg) was given to the mice (n⫽8) 24 hrs before
myocardial I/R. Untreated mice (n⫽8) were also subjected to I/R. Myocardial infarction was
determined by TTC staining. PDG administration significantly reduced Infarct size (10.5 ⫾
3.03%) compared with untreated mice (30.1 ⫾ 7.59%, p⬍0.01). Pretreatment of mice with
LPS for 24 hrs also significantly reduced infarct size by 66.5% compared to untreated controls
(11.7 ⫾ 1.45% vs 34.9 ⫾ 5.39%, p⬍0.01). Importantly, PDG administration significantly
increased the levels of phospho-Akt (0.80 ⫾ 0.10 vs 0.45 ⫾ 0.09) and phospho-GSK3␤
(0.66 ⫾ 0.14 vs 0.33 ⫾0.10) in the myocardium compared with untreated I/R mice. Similarly,
LPS pretreatment also significantly increased the levels of phospho-Akt and phospho-GSK-3␤
in the myocardium. Pharmacological inhibition of PI3K by LY294002 or genetic modulation
employing kinase defective Akt (kdAkt) transgenic mice abolished the cardioprotection induced
by either PDG or by LPS. The data suggests that TLR2 agonist (PDG)-mediated protection
against I/R injury does not require prolonged pretreatment while TLR4 agonist LPS requires 24
hours of pretreatment to induce a tolerogenic phenotype in the heart. The results suggest that
modulation of TLR2, or TLR 4 will induce cardioprotection through a PI3K/Akt-dependent
signaling pathway although the mechanisms by which TLR2 or TLR4 ligand-induced activation
of the PI3K/Akt signaling pathway may be different.
P122
Plasma Membrane Ca2ⴙ-ATPase Isoform 4 Is Antihypertrophic Possibly Due
to Inhibition of Calcineurin Activity
Xu Wu, Scott N Blair, Michelle Sargent, Allen York, Cincinnati Children’s Hosp, Cincinnati,
OH; Gary E Shull, Univ of Cincinnati, Cincinnati, OH; Jeffery D Molkentin; Cincinnati
Children’s Hosp, Cincinnati, OH
It has been well established that calcineurin induces cardiac hypertrophy, however, the Ca2⫹
sources for calcineurin activation remains elusive. Plasma membrane Ca2⫹-ATPase (PMCA)
extrudes Ca2⫹ out of the cell but in cardiac myocytes it contributes little to excitationcontraction coupling (ECC). In the present study we demonstrated that overexpre ssion of
PMCA4b in the adult heart decreased cardiac hypertrophy (both cell surface areas and heart
weight normalized to body weight ratio) upon 2 or 4 weeks of transaortic constriction (TAC) or
phenylephrine/angiotensin II infusion. Consistent with these observations, Pmca4 genetargeted mice showed increased cardiac hypertrophy after 2 weeks of TAC indicating that
endogenous PMCA4 may have an antihypertrophic function. Overexpression of PMCA4b also
reduced nuclear factor of activated T cell (NFAT) luciferase activity and decreased ANF mRNA
level after stimulation. Interestingly, we also observed that PMCA4b was physically associated
with calcineurin by immunoprecipitation analyses. PMCA4b overexpression in transgenic mice
also reduced cardiac hypertrophy associated with overexpression of transient receptor channel
3 (TRPC3) (which alone has been shown to increase calcineurin activity). Similarly, overexpression of PMCA4b in cultured neonatal rat cardiomyocytes by adenoviral gene transfer
BCVS Conference 2008 Abstracts
inhibited agonist-induced hypertrophy associated with reduced calcineurin-NFAT signaling.
Finally, PMCA4b overexpression also reduced TAC-induced cardiac fibrosis after 8 weeks and
the degree of functional decompensation. However, global Ca2⫹ transients and cell contraction
were not affected by PMCA4b overexpression, collectively indicating that PMCA4b functions in
regulating local sub-sarcolemmal Ca2⫹ signals that might specifically communicate with
calcineurin.
P123
Loss of Bmx Nonreceptor Tyrosine Kinase Ameliorates Pressure
Overload-Induced Hypertrophy in Adult Myocardium
Scherise Mitchell, Shuxun Ren, UCLA, Los Angeles, CA; Kari Alitalo, Molecular/Cancer
Biology Laboratory, Helsinki, Finland; Yibin Wang, Thomas M Vondriska; UCLA, Los Angeles,
CA
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Bmx is a non-receptor tyrosine kinase belonging to the Tec family of protein kinases with an
established role in leukocyte development and angiogenesis in non-cardiac systems. Recent
studies have suggested that Bmx may participate in protective signaling in acute setting in both
cardiac and skeletal muscle; however, the role of this family of kinases in long-term stress to
the myocardium remains completely unexplored. In this study, we examined the response of
genetically modified mice lacking Bmx (Bmx KO mice) to chronic myocardial pressure overload
induced by aortic banding. In comparison to the significant hypertrophy (HW/BW:10.8⫾2.9 TAC
vs. 5.5⫾0.4 sham mg/g, p⬍0.01) observed in WT control mice subjected to banding,
concomitant with deterioration of cardiac function (FS: 19.4% TAC vs. 30.96% sham, p⬍0.01)
as measured by echocardiography, Bmx KO mice demonstrated neither hypertrophic growth
(HW/BW: 6.5⫾1.0 TAC vs. 4.6⫾0.9 sham mg/g, p⫽0.6) nor impaired contractile function (FS:
28.66% TAC vs. 27.95% sham, p⫽0.8) after 8 weeks of pressure overload. Histologic analyses
of cardiac sections (wheat germ agglutinin staining) demonstrate hypertrophic cardiomyocyte
growth in WT but not in Bmx KO animals following 8 weeks of pressure overload. Quantitative
mRNA analyses demonstrate activation of fetal gene program in both WT and Bmx KO banded
mice. Immunoblotting of whole heart lysates was used to detect protein expression and activity
of the mTOR/AKT and AKT/GSK3b pathways in the adult animal. These findings provide the first
evidence of an important role for Bmx mediated signaling and AKT regulation in hypertrophic
myocardial growth in the setting of pressure overload.
P124
Interaction of Scaffolding Adaptor Protein Gab1 with Tyrosine Phosphatase
SHP2 Negatively Regulates IGF-I-Dependent Myogenic Differentiation via
ERK1/2 Signaling Pathway
Yoshikazu Nakaoka, Tatsuya Koyama, Kitaro Okamoto, Wataru Shioyama, Osaka Univ
Graduate Sch of Medicine, Osaka, Japan; Keigo Nishida, RIKEN Rsch Cntr for Allergy and
Immunology, Yokoyama, Japan; Yasushi Fujio, Toshio Hirano, Osaka Univ Graduate Sch of
Medicine, Osaka, Japan; Naoki Mochizuki; National Cardiovascular Cntr Rsch Institute,
Osaka, Japan
Scaffolding adaptor protein Grb2-associated binder 1 (Gab1) coordinates various receptor
tyrosine kinase signaling. Although skeletal muscle differentiation is regulated by various kinds
of growth factors, it remains elusive whether Gab1 coordinates myogenic signals. Here, we
examined the molecular mechanism of insulin-like growth factor-I (IGF-I)-mediated myogenic
differentiation, focusing on Gab1 and its downstream signaling. We found that Gab1 underwent
tyrosine-phosphorylation and subsequent complex formation with protein tyrosine phosphatase
SHP2 upon IGF-I stimulation in C2C12 myoblasts. On the other hand, Gab1 constitutively
associated with phosphatidylinositol 3-kinase regulatory subunit p85. To delineate the role of
Gab1 in IGF-I-dependent signaling, we examined the effect of adenovirus-mediated forced
expression of wild-type Gab1 (Gab1WT), mutated Gab1 unable to bind SHP2 (Gab1⌬SHP2), or
mutated Gab1 unable to bind p85 (Gab1⌬p85), on the differentiation of C2C12 myoblasts.
IGF-I-induced myogenic differentiation was enhanced in myoblasts overexpressing Gab1⌬SHP2,
but inhibited in those overexpressing either Gab1WT or Gab1⌬p85. Conversely, IGF-I-induced
extracellular signal-regulated kinase 1/2 (ERK1/2) activation was significantly repressed in
myoblasts overexpressing Gab1⌬SHP2, but enhanced in those overexpressing either Gab1WT or
Gab1⌬p85. Furthermore, overexpression of catalytic-inactive SHP2 modulated IGF-I-induced
myogenic differentiation and ERK1/2 activation similarly as that of Gab1⌬SHP2, suggesting that
Gab1-SHP2 complex inhibits IGF-I-dependent myogenesis through activation of ERK1/2.
Consistently, the blockade of ERK1/2 pathway reversed the inhibitory effect of Gab1WT
overexpression on IGF-I-dependent myogenic differentiation. In addition, adenovirus-mediated
constitutive activation of ERK1/2 pathway suppressed the enhanced myogenic differentiation by
overexpression of Gab1⌬SHP2. Taken together, these data suggest that Gab1-SHP2-ERK1/2
signaling pathway comprises an inhibitory axis for IGF-I-dependent myogenic differentiation.
P125
Erythropoietin Prevents Left Ventricular Remodeling After Myocardial
Infarction Through Erythropoietin Receptor-Mediated Cytoprotective
Signaling Pathways in Cardiomyocytes
Kazutaka Ueda, Hiroyuki Takano, Raita Uchiyama, Hiroshi Hasegawa, Issei Komuro; Chiba
Univ Graduate Sch of Medicine, Chiba-shi, Japan
Background: Erythropoietin (EPO) has been reported to improve cardiac function after
myocardial infarction (MI) through promoting mobilization of endothelial progenitor cells to the
injured heart and enhancing neovascularization. However, the precise mechanisms of the
beneficial effects of EPO against MI are unknown. In this study, we investigated the molecular
mechanisms of how EPO prevents left ventricular (LV) remodeling after MI. Methods: We
e59
produced MI in wild type (WT) mice and transgene-rescued EPO receptor null mutant (RES)
mice, which lack EPO receptor in nonhematopoietic tissues. We also produced MI in transgenic
mice which express dominant-negative STAT3 in cardiomyocytes (dnSTAT3-tg) and in the mice
of which bone marrow (BM) was replaced by BM cells of enhanced green fluorescent
protein-expressing mice. We examined anti-apoptotic effects of EPO in cultured neonatal rat
cardiomyocytes. Results: EPO significantly prevented LV dysfunction and reduced infarct size
after MI in WT mice but not RES mice (p⬍0.01, n⫽10). EPO decreased the number of apoptotic
cells and increased the capillary density after MI in WT mice but not RES mice (p⬍0.01, n⫽10).
EPO activated Akt and ERK and increased VEGF in post-MI hearts. There were no differences
in LV function and infarct size after MI between EPO-treated dnSTAT3-tg mice and WT mice.
Although the number of circulating CD34⫹/Flk-1⫹ cells was increased by EPO after MI in WT
mice but not RES mice, EPO did not increase the number of BM-derived cells in the post-MI
hearts. EPO was not effective even in RES mice whose BM was replaced by that of WT mice.
In cultured cardiomyocytes, EPO activated Akt and ERK signaling and attenuated H2O2-induced
apoptosis (⬃31%, p⬍0.05, n⫽8) by regulating apoptosis-related proteins such as Bcl-2 family
and caspase3. Furthermore, the cytoprotective effects of EPO were reduced by PI3K and MEK
inhibitors or dominant-negative form of EPO receptor, but not by dominant-negative STAT3.
Conclusions: EPO prevents LV remodeling and dysfunction after MI by directly acting on the
heart rather than recruiting blood cells. EPO-mediated cardiomyocyte survival may be caused
by Akt and ERK activation but not STAT3, which plays a critical role in G-CSF-induced
cardioprotection.
P126
Molecular and Structural Basis for Mechanical Stress-Induced Activation of
Angiotensin II Type 1 Receptor
Noritaka Yasuda, Hiroshi Akazawa, Chiba Univ, Chiba, Japan; Shin-ichiro Miura, Fukuoka
Univ, Fukuoka, Japan; Kaoru Ito, Yingjie Qin, Yoko Kudo, Chien-hui Liao, Chiba Univ, Chiba,
Japan; Yunzeng Zou, Fudan Univ, Shanghai, China; Keijiro Saku, Fukuoka Univ, Fukuoka,
Japan; Issei Komuro; Chiba Univ, Chiba, Japan
Angiotensin II (AngII) type 1 (AT1) receptor is a G protein-coupled receptor (GPCR) that plays a
crucial role in the development of load-induced cardiac hypertrophy. We previously reported
that mechanical stress, the most important stimulus for cardiac hypertrophy, activates the AT1
receptor through an AngII-independent mechanism, and that this activation is inhibited by an
inverse agonist candesartan. However, it remains unclear how mechanical stress activates AT1
receptor and how candesartan exerts inverse agonism. Here we show that mechanical stress
directly activates AT1 receptor by changing the conformation of AT1 receptor. Studies using
substituted cysteine accessibility mapping revealed that transmembrane (TM) 7 of AT1 receptor
showed a counterclockwise rotation and shift into the ligand-binding pocket in response to
mechanical stretch, and candesartan suppressed this helical movement induced by stretch.
Comparison of the inverse agonist activities of several AT1 receptor blockers identified the
carboxyl group at the imidazole ring as a critical chemical pharmacophore that is responsible
for potent inverse agonism. In addition, CV-7H, a candesartan’s derivative lacking the carboxyl
group, showed no inhibitory effect on stretch-induced conformational change of the AT1
receptor. Furthermore, the binding affinities of candesartan were 10-fold reduced in Q257A and
T287A mutants compared with wild-type receptor, and the inverse agonist activity of
candesartan was abolished in these mutants. These results suggest that candesartan
suppressed a stretch-induced helical movement of TM 7 via the bindings of the carboxyl group
of candesartan to Gln257 in TM 6 and Thr287 in TM 7 of AT1 receptor. A molecular modeling
proposes that the tight binding of candesartan to AT1 receptor in an unaligned inactive state [R]
results in stabilization of the receptor in the inactive conformation [R0] and prevents a shift to
the active conformation [Rstretch]. Our results provide a previously unknown basis for the
conformational switch of the AT1 receptor that couples mechanical tress-induced activation and
inverse agonist-induced inactivation.
P127
Inhalation of Hydrogen Gas Reduces Infarct Size in the Rat Model of
Myocardial Ischemia-Reperfusion Injury
Kentaro Hayashida, Motoaki Sano, Keio Univ Sch of Medicine, Tokyo, Japan; Ikuro Ohsawa,
Nippon Med Sch, Kawasaki, Japan; Ken Shinmura, Kayoko Tamaki, Kensuke Kimura, Jin
Endo, Takaharu Katayama, Keio Univ Sch of Medicine, Tokyo, Japan; Shigeo Ohta, Nippon
Med Sch, Kawasaki, Japan; Satoshi Ogawa, Keiichi Fukuda; Keio Univ Sch of Medicine,
Tokyo, Japan
[Background] The extent of ischemia-reperfusion injury determines the final myocardial infarct
volume. Hydrogen (H2) gas selectively reduces the level of hydroxyl radicals, one of the most
highly reactive oxygen species (ROS). The present study investigates whether inhalation of H2
gas confers cardioprotection against ischemia-reperfusion injury. [Methods and Results] (1)
Cardiomyocytes cultured in media with or without dissolved H2 were stimulated with antimycin
A to produce ROS. Media containing dissolved H2 inhibits the dissipation of mitochondrial
membrane potential and improves cell viability. (2) Cardiomyocytes were treated with Cu2⫹ and
ascorbate to induce hydroxyl radical formation by the Fenton reaction. H2 reduces cellular levels
of hydroxyl radicals and restores cell viability. (3) Isolated Langendorff perfused hearts were
subjected to anoxia-reoxygenation. H2 enhances recovery of left ventricular function during
reoxygenation. (4) Tissue H2 levels were monitored with a needle-shaped hydrogen sensor
electrode. Inhaled H2 gas is rapidly transported and reaches ‘at risk’ ischemic myocardium
before reestablishment of coronary blood flow of the occluded infarct-related artery. (5) The
degree of cardioprotection against ischemia-reperfusion injury was evaluated by measuring
oxidative damage and infarct size 30 min after left anterior descending coronary artery
occlusion and reperfusion. Inhalation of an incombustible level of H2 gas (2%) before
reperfusion significantly reduces oxidative stress-induced myocardial injury and infarct size
without affecting hemodynamic parameters, and thereby prevents deleterious LV remodeling.
[Conclusion] Inhalation of H2 gas limits the extent of myocardial infarction in rat models of
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Circulation Research
Vol 103, No 5
August 29, 2008
ischemia-reperfusion injury. The selective anti-oxidant action of H2 provides cardioprotection
against ischemia-reperfusion injury.
P128
Enhancement of Ischemic Angiogenesis by a CXCR4 Antagonist Derived
from Human SDF-1␤ Is Mediated by a Monocyte-Dependent Mechanism
could also attenuate phosphorylated JNK (an important component of MAPK cascade), c-Jun
(downstream molecules of JNK) in ET-1-induced hypertrophied cardiomyocytes. PPAR-␣
expression and PPAR-PPRE binding activity was suppressed in ET-1 administered cardiomyocyte and this suppression was improved by EPA treatment. In conclusion, the present study
showed that ET-1 could induce significant cardiomyocyte hypertrophy with hypertrophic
markers upregulation, and that this remodeling was effectively prevented by EPA-preadministration through the upregulation of PPAR-␣ and the suppression of phosphorylated JNK,
and c-Jun.
Yi Tan, Univ of Louisville, Louisville, KY; Xiaokun Li, Wenzhou Med College, Wenzhou,
China; Lu Cai; Univ of Louisville, Louisville, KY
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Antagonizing CXCR4 with the potent antagonist AMD3100 induced hematopoietic cells
peripheral mobilization and achieved acute blood restoration and angiogenesis, but a long-term
benefit has not been established due to its potentially chronic adverse effects. We developed
a novel antagonist against CXCR4, SDF-1␤P2G, which derived from human SDF-1␤ by genetic
recombinant biotechnology. Treatment with SDF-1␤P2G (5 mg/kg) significantly improved the
limb blood flow and angiogenesis in a hindlimb ischemic mouse model as early as 7 days after
surgical operation, which were measured by a Laser Doppler Image (LDPI) analyzer monitoring
in vivo and CD31⫹ cells/fiber counting on cryo-sections (p⬍0.05 vs PBS). Compared to
AMD3100 (5 mg/kg), SDF-1␤P2G showed a more significant acceleration of revascularization
(p⬍0.01). Markers related to angiogenesis and pro-angiogenic cell migration, including
phosphorylated-Akt, VEGF, SDF-1 and CXCR4, were co-localized with CD31 positive cells.
Endothelial progenitor cells (EPCs, CD31⫹ c-Kit⫹) in the peripheral blood of ischemia mice
were analyzed by flowcytometry at the day 1, 4 and 7 after operation. Compared to PBS group,
unexpectedly, EPCs was not significantly increased in SDF-1␤P2G group (p⬎0.05), but
significantly increased in AMD3100 group (p⬍0.05). In contrast, SDF-1␤P2G was found to
significantly induce a peripheral mobilization of monocytes as early as 1 hour post-injection
(p⬍0.05 vs PBS), and signficantly gastrocnemius accumulation of monocytes/macrophages on
day 3, 7 and 14 after ischemia. These data indicate that the antagonist of CXCR4, SDF-1␤P2G,
stimulates angiogenesis through a distinct mechanism from AMD3100, i.e. continuing
mobilization of monocytes from bone marrow into circulation, and accumulation in the ischemic
sites. Failure of AMD3100 to induce continual blood restoration implies that EPCs may not play
the critical role in ischemic angiogenesis.
P129
C-Reactive Protein Promotes Cardiac Fibrosis in Angiotensin II-Induced
Hypertensive Cardiovascular Diseases
Rongxin Zhang, Yuanyuan Zhang, Xiao Ru Huang, Yin Wu, The Univ of Hong Kong, Hong
Kong, China; Alexander J Szalai, Univ of Alabama at Birmingham, Birmingham, AL; BCY
Wong, C P Lau, Ed Xuekui Wu, Hui Yao Lan; The Univ of Hong Kong, Hong Kong, China
C-reactive protein (CRP) is an acute phase reactant protein considered to be as a prototypic
marker for inflammation and a risk factor for cardiovascular diseases. However, little is known
about the functional role of CRP in cardiac fibrosis in hypertensive cardiovascular diseases. In
this study, we investigated the potential role of CRP on cardiac fibrosis in Angiotensin II (Ang
II) induced hypertension. Transgenic (Tg) CRP and wild type (Wt) mice were infused with Ang
II at a dose of 1.46mg/kg/day via subcutaneous osmotic minipumps for 28 days. The effect of
CRP on heart ejection fraction and blood pressure were examined by Magnetic Resonance
Imaging (MRI) and an automated tail-cuff system, while cardiac fibrosis was determined by
real-time PCR and immunohistochemistry. Results showed that mice with overexpression of
CRP significantly reduced the heart ejection fraction (p⬍0.001), but not affected the blood
pressure and heart rates in response to Ang II. Interestingly, infusion of Ang II significantly also
induced further serum levels of endogenous CRP as measured by ELISA. Cardiac fibrosis as
identified by overexpression of fibrosis markers collagen I and alpha-smooth muscle actin in
both mRNA and protein levels was significantly increased in CRP transgenic mice compared to
the Wt mice (p⬍0.05, respectively). This was associated with a marked upregulation of Ang
II type I receptor, cardiac levels of TGF-␤1, and importantly, activation of the TGF-␤/Smad
signaling pathway (all p⬍0.05). In conclusion, CRP is pathogenic in hypertensive cardiovascular disease in response to Ang II. Upregulation of AT1 receptor and activation of the
TGF-␤/Smad signaling pathway may be a critical mechanism by which CRP enhances Ang
II-mediated cardiac fibrosis. (Correspondence to: H.Y. Lan, [email protected])
P130
Inhibitory Effect of Eicosapentaenoic Acid on Cardiomyocyte Hypertrophy
via PPAR-␣ Upregulation
P131
Hint to Biomarkers of Acute Aortic Dissection by Pathway Analysis
Salah A Mohamed; Univ of SH-Luebeck, Luebeck, Germany
Background: A clinical feature of acute aortic dissection (AAD) is the characteristic tearing
retrosternal pain often confused with that of myocardial infarction. Similar as in myocardial
infarction, a rapid test for the diagnosis of AAD would be vitally helpful. In this study, pathway
analysis of the differentially expressed genes in dissected aortic tissues were used to test the
hypothesis that interaction of important proteins with Marfan syndrome fibrillen 1 may play an
important role in acute rupture of the aorta. Methods: Aortic tissue was collected during
surgery from 19 patients with acute aortic dissection (mean age 61.7⫾13.1 years), from 8
patients with MS (mean age 32.9⫾12.2 years), and from other 6 patients without any known
™
aortic disease (mean age 56.7⫾12.3 years), used further as control. The PIQOR Immunology
microarray with 1076 probes in quadruplicates was used; quantitative RT-PCR (q RT-PCR) and
protein analysis was performed. The differentially expressed genes were subjected to biological
pathway analysis by using a MedScan search. Results: Interactions of MS FBN1 in the
MedScan pathway analysis considered 4 genes, fibulin 1 (FBLN1), fibulin 2 (FBLN2), Decorin
(DCN), and microfibrillar associated protein 5 (MFAP5), which were differentially expressed in
all tissue from AAD. Validation of these genes by q RT-PCR followed by protein analysis
presented at least a 3-fold down-regulation of FBLN1 (0.5⫾0.4 vs 6.1⫾2.3 fold, p ⫽ 0.003),
and of DCN (2.5⫾1.0 vs 8.5⫾4.7 fold, p ⫽ 0.04) in AAD compared to MS and control samples.
Conclusions: We provide first evidence that pathway analysis followed by genomics with
transcriptional profiling may become a powerful diagnostic tool in AAD.
P132
Deletion of Tenascin-C Prevents Left Ventricular Remodeling and Cardiac
Failure After Myocardial Infarction
Kyoko Imanaka-Yoshida, Tomohiro Nishioka, Katsuya Onishi, Mie Univ, Tsu, Japan; Hiroyuki
Tsutsui, Hokkaido Univ, Sapporo, Japan; Toshimichi Yoshida, Mie Univ, Tsu, Japan; Michiaki
Hiroe; International Med Cntr of Japan, Tokyo, Japan
Background: Tenascin-C (TNC) is an extraceulluar matrix glycoprotein, with strong bioactivity,
transiently expressed during embryogenesis, injury, inflammation, or cancer invasion, and
suggested to play an important role during wound healing. TNC is sparsely expressed in healthy
adult heart but markedly upregulated under various pathological condition. Recently, we
reported that the patients with high serum level of TNC after acute myocardial infarction have
a greater incidence of ventricular remodeling. Objective: The main aim of the present study was
to examine whether TNC accelerates adverse ventricular remodeling. Methods and Results:
Myocardial infarction (MI) was produced by ligating the coronary artery in BALB/c background
10- to 12-wk-old male TNC knockout (TNKO) mice and sibling wild type (WT) mice. In WT mice,
TNC was expressed at border zone between intact myocardium and infarct lesion within 24
hours after ligation, peaked around day 3–5, then down-regulated and disappeared by day 28.
The morphometrically determined infarct size and survival rate on day 28 were comparable
between the WT⫹MI and TNKO⫹MI groups. Echocardiograph and hemodynamic analysis
demonstrated no significant differences in LV size and function between sham operated KO and
WT. LV end-diastolic diameter was significantly increased accompanied with reduced ejection
fraction in WT⫹MI mice to compare with WT⫹sham mice. Myocardial stiffness and LV
end-diastolic pressure were also increased in WT⫹MI mice. In KO⫹MI mice, LV end-diastolic
diameter, myocardial stiffness and LV end-diastolic pressure was significantly increased to
compare with KO⫹sham mice. However, end-diastolic pressure, dimension, and myocardial
stiffness in KO⫹MI were lower than those in WT⫹MI. Histological examination showed that
tissue healing appeared to proceed normal and no distinct difference in collagen fibers
formation in scar tissue. However, quantitative analysis demonstrated interstitial fibrosis in the
residual myocardium adjacent to infarcted area was significantly lower in KO⫹MI to compare
with WT⫹MI. Conclusion: TNC may accelerate ventricular remodeling, cardiac failure and
fibrosis in residual myocardium after myocardial infarction.
Nobutake Shimojo, Satoshi Sakai, Seiji Maeda, Univ of Tsukuba, Tsukuba, Japan; Masaaki
Soma, Mochida Pharmaceutical Co, Ltd, Tokyo, Japan; Subrina Jesmin, Takashi Miyauchi,
Kazutaka Aonuma; Univ of Tsukuba, Tsukuba, Japan
Growing body of evidences state the cardiovascular benefit of fish oil including eicosapentaenoic acid (EPA) in humans and experimental animals, but the effect of EPA on endothelin
(ET)-1-induced cardiomyocyte hypertrophy is unknown. Previous study demonstrated that
peroxisomal proliferator-activated receptor (PPAR)-␣ ligand (fenofibrate) prevents ET-1induced cardiomyocyte hypertrophy. Though EPA is a ligand of PPAR-␣, there was no study
linking relationship between EPA and PPAR-␣ in the field of cardiomyocyte hypertrophy. The
present study investigated whether ET-1-induced cardiomyocyte hypertrophy could be
prevented by EPA pre-treatment with possible mechanistic insights. At day 4 of culture,
neonatal rat cardiomyocytes were divided into three groups: control, ET-1 (0.1nM) treated and
EPA-pre-treated (10 ␮M) ET-1 groups. 2-fold increase in cardiomyocyte surface area, 1.8-fold
increase in total protein synthesis rate and an enhanced ␣-actinin expression in cardiomyocyte
were observed after ET-1 administration and these changes were greatly prevented by EPA
pre-treatment. ET-1-induced hypertrophied cardiomyocytes showed increases in ANP and BNP
mRNA expression, which were also suppressed by EPA pre-treatment. Pre-treatment of EPA
P133
Vasomotion Links with Transient Receptor Potential Channels and Its
Response to Angiotensin Receptor Blocker in Hypertension
Xiaoping Chen, Daoyan Liu, Dachun Yang, Xiaoli Feng, Liqun Ma, Zhidan Luo, Zhiming Zhu;
Cntr for Hypertension and Metabolic Diseases, Daping Hosp, Third Military Med Univ,
Chongqing Institute of Hypertension, Chongqing, China
Vasomotion and transient receptor potential channels (TRPCs) participate in the regulation of
vascular reactivity and blood pressure. In clinically, angiotensin II receptor blocker (ARB) is
commonly used to improve the vascular reactivity and lower high blood pressure. However, the
association between vasomotion and TRPCs is unclear. In this study, we investigate the
relationship between vasomotion and TRP channels and effect of ARB on them in mesenteric
artery from spontaneously hypertensive rats (SHR). Endothelium dependent and independent
relaxations of mesenteric arteries were impaired in SHR compared with WKY. The magnitude
of rhythmic oscillatory vasomotion induced by norepinephrine (NE) was significantly greater in
BCVS Conference 2008 Abstracts
mesenteric artery from SHR than WKY (p⬍0.01). In mesenteric artery, TRPC subtype 1 and 5
expressions were significantly higher in SHR compared with WKY. SKF96365, a non-specific
inhibitor of TRPCs, not only inhibited NE induced calcium influx in fura2-loaded cultured VSMCs,
but also significantly reduced the magnitude of the vasomotion from 29⫾3% to 11⫾2%
(P⬍0.01). Administration of candesartan (4mg/kg/day) or telmisartan (5mg/kg/day) for 16
weeks significantly lowered SBP and improved relaxation of mesenteric artery in SHR. In
addition, the magnitudes of vasomotion were significantly lower in mesenteric artery from
treated SHR by candesartan (14⫾3.3% ) and by telmisartan (8⫾1.9%) compared with
untreated SHR (66⫾7% p⬍0.01, n⫽6 per group)). Administration of ARB significantly reduced
TRPC subtypes 1 and 5 expressions of mesenteric arteries from treated SHR compared with
untreated SHR. TRPC 1 and 5 expressions are associated with nitric oxide signaling pathway.
In cultured VSMCs, inhibition of NOS by 1mmol/L L-NMMA resulted in enhancement of the
expressions of TRPC 1/5, which was abolished by NO donor, 1mmol/L sodium nitroprusside.
Furthermore, administration of telmisartan significantly increased eNOS expression in mesenteric artery from SHR. Conclusions: Enhanced vasomotion in mesenteric artery of SHR was
associated with increase expression of TRPC 1/5, which was inhibited by ARB through
promoting the nitric oxide production.
P134
Role of KChIP2 in Cardiac Contraction and Hypertrophy
Hongwei Jin, Cardiovascular Rsch Institute, Mount Sinai Sch of Medicine, New York, NY;
Julieta Palomeque, Cntr de Investigaciones Cardiovasculares, La Plata, Argentina; Roger
Kaprielian, AMGEN Canada-Med Affairs, Ontario, Canada; Roger Hajjar, Djamel Lebeche;
Cardiovascular Rsch Institute, Mount Sinai Sch of Medicine, New York, NY
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
KChIP2 has been hypothesized to regulate the Kv4 channels which encode for Ito in
cardiomyocytes and its deletion in mice decreases Ito density. In hypertrophy and heart failure
KChIP2 expression has been found to be significantly decreased. We sought to examine the role
of KChIP2 in cardiac hypertrophy and the effect of restoring its expression back to normal in
hypertrophied and failing cardiomyocytes on electrical remodeling and cardiac mechanical
function using a combination of molecular, biochemical and gene targeting approaches.
Quantitative RT-PCR and immunoblotting analyses showed that the expression of KChIP2 is
highly restricted to the adult myocardium and it is hardly expressed in the neonatal rat heart.
KChIP2 overexpression through gene transfer of Ad.KChIP2 in neonatal cardiomyocytes for 36
hours resulted in a significant increase in Kv4.2 and Kv4.3 protein levels but had no effect on
the expression patters of Kv1.4, another Ito-channel forming protein. In vivo gene transfer of
KChIP2 in aortically banded adult rats showed that, compared to sham-operated or
Ad.␤-Gal-transduced hearts, KChIP2 has significantly attenuated the left ventricular developed
hypertrophy, robustly increased Ito densities and markedly shortened action potential duration
at 90% repolarization. Interestingly, 1-day old rat pups intracardially transduced with KChIP2
for 2 months then subjected to aortic banding for 4 – 8 weeks (to induce hypertrophy) showed
similar echocardiographic and electrical remodeling parameters as in adult hearts. In addition,
in cultured neonatal cardiomyocytes, KChIP2 overexpression reversed AngII-induced hypertrophic changes in protein synthesis and MAP kinase activation, namely ERK and p38. Associated
with the attenuation of cardiac hypertrophy, overexpression of KChIP2 in adult cultured
cardiomyocytes significantly altered myocyte mechanics by shortening contraction amplitudes
and maximal rates of contraction and relaxation velocities and increasing Ca2⫹ transients.
Altogether, these data show that KChIP2 attenuates cardiac hypertrophy most likely through
restoration of Ito densities and modulation of MAPK pathway.
P135
Intramyocardial Transplantation of Genetically Modified Fibroblasts
Expressing VEGF Induces Angiovasculogenesis and Improves Cardiac
Function Post-Myocardial Infarction in Rats
Giovana A Goncalves, Paula F Vassalo, Leandro Cardoso, Heart Institute (InCor)/Univ Sao
Paulo Med Sch, Sao Paulo SP, Brazil; Leonardo Santos, Paulo J Tucci, Fed Univ Sao Paulo,
Sao Paulo SP, Brazil; Jose E Krieger; Heart Institute (InCor)/Univ Sao Paulo Med Sch, Sao
Paulo SP, Brazil
Preliminary studies in our lab showed that pre-emptive cardiac transplantation of either
genetically modified syngeneic myoblasts or fibroblasts expressing VEGF could increase cardiac
capillary and vascular densities 3 wks following ischemia/reperfusion injury. Here we tested the
therapeutic efficacy of modified cardiac fibroblasts (CF) expressing VEGF (vascular endothelial
growth factor) and/or IGF-1 (insulin-like growth factor) associated to fibrin polymer to induce
angiogenesis, vasculogenesis and improve cardiac function post myocardium infarction. The
groups: VEHICLE⫽ control, POLYMER⫽ fibrin polymer, CELL⫽ 106 CF, NULL, VEGF, IGF-1 and
IGF⫹VEGF⫽ 106 modified CF by AdCMVNULL, AdCMVIGF-1EGFP or AdRSVVEGFP, were directly
injected in the left ventricle muscle of rats 24 h following ligation of the descending coronary
artery. After 4 weeks, VEGF and IGF-1 protein increased in IGF-1, VEGF and IGF-1⫹VEGF
groups, p⬍0.0001. Interestingly, only groups that received VEGF alone or combined with IGF-1
increased capillary and vascular densities and reduced total myocardial collagen area (35,12 ⫾
7,05 vs. 31,28 ⫾ 5,03 vs. 30,07 ⫾ 6,21 vs. 25,89 ⫾ 2,92 vs. 15,43 ⫾ 2,02* vs. 16,07 ⫾
1,83%*, *p⬍0,05, VEHICLE, POLYMER, CELL, NULL, IGF-1, VEGF, and IGF-1⫹VEGF, respectively). Basal morphometric and functional cardiac indices remained unchanged in all groups;
however, pharmacological stress using phenylephrine bolus injection, showed that the VEGF
groups displayed a significant improvement in cardiac work, stroke volume and end-diastolic
pressure while all other groups displayed deterioration of the phenotype. These results
indicated that CF expressing VEGF alone or in combination with IGF-1 can induce angiogenesis
and vasculogenesis in ischemic myocardium decreasing myocardial scar area and improving
cardiac performance 4 weeks after coronary ligation.
e61
P136
Delivery of Superoxide Dismutase with Polyketal Particles for Cardiac
Regeneration
Gokulakrishnan S Iyer, Emory Univ, Atlanta, GA; Stephen C Yang, Georgia Institute of
Technology, Atlanta, GA; Sergey Dikalov, Emory Univ, Atlanta, GA; Niren Murthy, Georgia
Institute of Technology, Atlanta, GA; Michael E Davis; Emory Univ, Atlanta, GA
Oxidative stress is increased in the myocardium following infarction, playing significant roles
in cardiac myocyte death and progression of cardiac dysfunction. Superoxide dismutase (SOD)
protein therapy may have broad clinical implications; however intracellular delivery and poor
delivery kinetics are critical obstacles. Polyketal particles are a new class of biomaterials with
controllable kinetics that degrade into FDA-approved compounds. SOD was encapsulated with
25% efficiency within polyketal microparticles (PK-SOD) using a double-emulsion technique.
Superoxide scavenging capability of PK-SOD was tested by measuring superoxide production
of PMA-stimulated macrophages in the presence of either empty microparticles (PK) or
PK-SOD. Both extracellular and intracellular superoxide were dose-dependently scavenged only
by PK-SOD treatment. This decrease in superoxide levels by PK-SOD resulted in a decrease in
LPS-stimulated TNF␣ production. To determine if PK-SOD could also scavenge reactive oxygen
species in vivo, we used a rat model of ischemia/reperfusion (I/R) followed by particle injection.
Prior studies done in mice and rats demonstrated favorable retention and biocompatibility of the
particles. Rats were injected with either PK or PK-SOD in a randomized and double-blind
manner and total oxidative stress in the infarct zone was measured after three days by
cytochrome C and lipid peroxidation assays. I/R injury resulted in a significant increase in both
reactive oxygen species and lipid peroxidation after three days (p⬍0.05). While there was no
effect of PK, PK-SOD significantly reduced these values to sham levels (p⬍0.05 vs. I/R). These
results demonstrate the potential of polyketal particles to deliver proteins for the treatment of
cardiac dysfunction.
P137
Determining Gene Expression Elucidates the Benefit with Stem Cell
Treatment of Ischemic Myocardium
Suna Wang, Yifu Zhou, Zuxi Yu, Timothy Hunter, Robert F Hoyt, Jr, Keith A Horvath;
National Institutes of Health, Bethesda, MD
Background: We previously reported that transplantation of autologous bone marrow-derived
stem cells (BMC) increased capillary density and improved the function of chronic ischemic
myocardium. However, the knowledge regarding the underlying biological process is limited.
Obtaining a framework of the regulatory genes expressed can help us to evaluate or predict the
outcomes of the cell-based therapy. Therefore, we tested for the genes that link to hypoxia,
angiogenesis and cell differentiation in chronic ischemic myocardium six weeks (wks) post
direct BMC injection. Methods: In a porcine model, adult bone marrow (BM) was harvested at
the time of placing an Ameroid constrictor around LCX to create chronic ischemia zone. After
gradient centrifugation, the mononuclear cells from the BM were placed in DMEM plus 10%
FBS or in EGM2 (growth factor enriched medium) in a density of 106/cm2 cell culture flask.
Four wks later, each swine underwent direct intramyocardial injection of 120 million BMC in
the ischemic zone or saline as a control (11 BMC, 6 saline). Six wks post-injection, 17 paired
ischemic and non-ischemic tissues were collected. Thirty-four total-RNAs were extracted and
cDNA was synthesized. qRT-PCRs were prepared using a SYBR green PCR kit and performed
by iCycler IQ. Results: qRT-PCR revealed HIF-1 was significantly downregulated and VEGF
expression was two times lower in BMC-injected group compared with saline group. However,
IGF1 was upregulated in BMC-injected group and down-modulated in controls. Its receptor,
IGF1R differentially downregulated in controls, while no significant modulation confirmed in the
BMC treated group. One of the IGF regulators, IGFBP5 was over expressed in BMC-injected
ischemia, but no differential expression in saline group. Our preliminary results imply the
modulation of HIF1, VEGF1 and IGF1/IGF1R/IGFBP5 is induced by hypoxic surrounding and
adjusted with the progress provoked by the cell-based therapy. Conclusion: The modulations
of genes, HIF-1a, VEGF and IGF1/IGF1R/IGFBP5 associate with the beneficial effects of the BMC
therapy, and it may lead to the enhancement of angiogenesis and cell differentiation. The gene
regulation patterns may also indicate the outcome of the cell-based therapy.
P138
Importance of Timing in Catalase Overexpression for Cardiac Regeneration
Susan Varghese, Gokulakrishnan S Iyer, Milton Brown, W Robert Taylor, Michael E Davis;
Emory Univ, Atlanta, GA
Increased superoxide following infarction damages the local myocardium, and through
dismutation to hydrogen peroxide (H2O2), increases lipid peroxidation and cardiac fibrosis.
Myocardial levels of the H2O2 scavenger catalase decrease in the weeks following infarction and
its absence may also lead to incomplete regeneration by stem cells. Overexpression of catalase
has been examined, but mice had 90-fold higher catalase levels from birth, two conditions that
are physiologically unlikely. Using a cre-lox system, we created mice that had inducible
catalase overexpression only in myocytes. Following tamoxifen treatment, there was a
significant increase in cardiac catalase activity in transgenic mice (0.9 ⫹ 0.3 vs. 3.6 ⫹ 0.5
U/mg), as well as an increase in catalase protein levels. To test this overexpression in an injury
setting, we examined two situations, either inducing catalase overexpression before myocardial
infarction, or concurrently. Myocyte-specific catalase overexpression decreased H2O2 production 7 days following myocardial infarction (SH ⫽ 1.2 ⫹ 0.2, MI ⫽ 3.3 ⫹ 0.6, CAT SH ⫽ 1.5
⫹ 0.2, CAT MI ⫽ 1.4 ⫹ 0.3 nM/mg/min). Interestingly, if catalase was induced prior to MI,
there was an improvement in function seen at this 7 day time point. However, if catalase was
induced concurrently, despite the reduction in H2O2 levels, there was no effect on function.
When cardiac tissue from the concurrent mice was examined, we found a significant decrease
e62
Circulation Research
Vol 103, No 5
August 29, 2008
in mRNA levels for tumor necrosis factor-␣ and connective tissue growth factor, suggesting a
potential future decrease in fibrosis that is currently being examined. These changes may have
effects not only on myocytes, but on cardiac stem cells (CSCs) as well. We examined c-kit⫹,
lin- CSCs and found not only did they express less catalase than cardiac myocytes, but H2O2
had significant effects on apoptosis. Our data also demonstrate that CSCs themselves produce
H2O2 in response to TNF-␣ treatment. In summary our data suggest that inducible,
cardiac-specific overexpression of physiological levels of catalase has differential effects
depending on when it is induced in the disease process. These studies give valuable insight in
to the ideal timing for catalase protein therapy, and may have effects on stem cell therapy.
P139
Cytokines Secreted from a 3-Dimensional Fibroblast Construct (3DFC)
Potentially Mediate Improvements in Cardiac Function and Angiogenesis:
The Potential Role of Biomechanical Strain
Michael R Hicks, Arizona State Univ, Tempe, AZ; Kate Meltzer, Univ of Arizona, Phoenix, AZ;
Jordan J Lancaster, Howard Byrne, Nicholle M Johnson, Hoang M Thai, Steven Goldman,
Southern Arizona VA Med Cntr, Tucson, AZ; Paul Standley; Univ of Arizona, Phoenix, AZ
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Initial studies utilizing 3DFC in a rat model of chronic ischemic heart failure and acute
myocardial infarction show improvements in left ventricular function, myocardial blood flow,
and microvessel density. The 3DFC is currently being evaluated in Phase I clinical trials in
patients with end stage heart failure and in patients undergoing elective coronary artery bypass
grafting. While data from these studies are promising, the mechanisms of action of 3DFC are
unclear. To investigate the potential roles of fibroblast-derived cytokines in mediating the
effects of 3DFC, we developed an in vitro strain model to mechanically strain 3DFC. Subsequent
to strain, the following were analyzed: dsDNA, total protein, and fibroblast secretion of twenty
cytokines with known roles in angiogenesis and inflammation. 3-Dimensional Fibroblast
Constructs (N⫽3) were attached to BioFlex® collagen-coated membranes and incubated in
media containing 2% FBS. 3-Dimensional Fibroblast Constructs were then strained sinusoidally
at 110% of their initial resting length at 1Hz for 48 hours. Control 3DFCs were not strained.
There were no significant changes in dsDNA concentration between static and strained groups
(0.0035 ⫾ 0.0003; 0.0037 ⫾ 0.0003 ␮g/ml cytokine per mg 3DFC; respectively). Protein
content was significantly elevated with strain (2.32 ⫾ 0.3 vs. 3.44 ⫾ 0.3 ␮g/ml; p⬍0.05)
suggesting a hypertrophic response. Conditioned media samples from control and strained
3DFC contained measurable MCP-1*, HGF*, Leptin*, IL-8*, VEGF*, IFN␥†, Angiogenin*, TNF␣†,
IL-1␣†, HB-EGF*, bFGF*, ANG-2*, IL-1␤†, IL-4†, IL-6†, IL-13†, PIGF*, PDGF-BB*, IL-10†, and
EGF† (in decreasing order of concentration). The † indicates a significant decrease (p⬍0.05)
with strain; while * indicates no significant change with strain. Occurrences of decreased
cytokine concentrations may be due to reduced fibroblast secretion, cytokine degradation, or
cell uptake. Thus with sinusoidal strain, the 3DFC undergoes a hypertrophic response with
increases in protein content and altered activation in cytokine expression. These data suggest
that this altered cytokine expression may be in part responsible for the beneficial effects of this
fibroblast construct.
P140
Enrichment of Cardiomyocyte Progenitor Cells from Human Embryonic
Stem Cells
Reza Ardehali, Irving L Weissman, Micha Drukker; Stanford, Stanford, CA
It is widely accepted that the regenerative capacity of human myocardium is limited and
inadequate to compensate for cellular loss after myocardial injury. Recent studies have
indicated that cellular-based therapy may improve cardiac function. Human embryonic stem
cells (hESC) have the capacity to renew and differentiate into mature cardiomyocytes (CM).
Using standard immunization techniques in rats, over 400 monoclonal antibodies were raised
against surface markers expressed in the early hESC differentiation. These generated
antibodies along with a panel of commercially available antibodies, were tested on partially
differentiated hESCs and several novel surface markers that are expressed in early mesodermal
differentiation were identified. We next tested the potential application of these antibodies to
enrich for cardiomyocyte progenitors in a clonogenic assay. Human ESCs, carrying green
fluorescent protein (GFP) under regulation of cardiac specific-gene promoters (myosin heavy
chain or troponin), were differentiated in the presence of bone morphogenic protein- (BMP) 4
or retinoic acid (RA) and were screened by fluorescence-activated cell sorting (FACS) using the
candidate antibodies. The sorted cells were then placed in non-labeled embryoid bodies and
the beating sectors containing GFP label were identified. Identification of cardiac progenitors is
an iterative process that requires multiple rounds of sub-fractionation until unit efficiency is
reached. The regenerative potential of the enriched population of cardiomyocyte progenitors,
while eliminating teratogenic sub-population, are currently under investigation in in vivo
models. This approach will allow us to assess clinical application of enriched cardiomyocyte
progenitors from human embryonic stem cells to restore injured myocardial function.
P141
Injection of Self-Assembling Nanopeptides with Clonal Cardiac Sca-1
Positive Cells Improves Cardiac Function After Myocardial Infarction
Through Angiogenesis and Antiapoptosis
Masakuni Tokunaga, Toshio Nagai, Koji Iwanaga, Chiba Univ, Chiba, Japan; Katsuhisa
Matsuura, Tokyo Women’s Med Univ, Tokyo, Japan; Toshinao Takahashi, Mei Lan Liu,
Tomomi Oyama, Masato Kanda, Atsuhiko Naito, Issei Komuro; Chiba Univ, Chiba, Japan
™
Self-assembling nanopeptides, PuraMatrix (PM), have been reported to offer microenvironment for the transplanted cells and support the cell survival. To examine whether cell
transplantation with PM can restore the cardiac function of C57BL/6 mice myocardial infarction
(MI) models , we injected the 0.1% (v/v) cell/PM into the peri-MI area and then patched 0.5%
(v/v) cell/PM on MI area. The mice were divided into following groups; skeletal myoblasts with
PM (SM/PM), bone marrow mononuclear cells with PM (BM/PM), clonal Sca-1 positive cardiac
progenitors with PM (cSca-1/PM) and PM alone (PM). Non-treated MI models (Cont) were
prepared as controls. Infarct area was examined by Masson’s trichrome staining, and cardiac
dimension and function were examined by echocardiography 2 weeks after transplantation.
cSca-1/PM attenuated ventricular enlargement in comparison with SM/PM, BM/PM, PM and
Cont (left ventricular diastolic dimension: cSca-1/PM 4.9⫾0.7mm, SM/PM 5.3⫾1.1mm,
BM/PM 5.8⫾0.6 mm, PM 5.5⫾0.8mm, Cont 5.9⫾0.6mm, p⬍0.01 vs Cont). Fractional
shortening of cSca-1/PM was better than that of SM/PM, BM/PM, PM and Cont (cSca-1/PM
15⫾9%, SM/PM 14⫾8%, BM/PM 10⫾5%, PM 12⫾7%, Cont 7⫾2%, p⬍0.01 vs Cont). Infarct
area of cSca-1/PM (38⫾14%, p⬍0.05 vs SM/PM, BM/PM, PM, p⬍0.01 vs Cont) was
significantly smaller than that of SM/PM, BM/PM, PM and Cont (58⫾7%, 55⫾6%, 54⫾14%,
57⫾10%, respectively). To elucidate the mechanisms of the beneficial effects of cSca-1/PM,
the number of von Willebrand factor (vWF), smooth muscle actin (SMA) 2 weeks after
transplantation and TUNEL positive cells 1 day after transplantation were examined by
enzymatic immunostaining. Both vWF positive capillary density and SMA positive arteriole
density in infarct area of cSca-1/PM were significantly higher than that of SM/PM, BM/PM, PM,
and Cont. The percentage of TUNEL positive cells in border and infarct area of cSca-1/PM
(13.0%, p⬍0.01 vs PM, Cont) was significantly lower than that of PM and Cont (18.9%, 26.8%,
respectively). In summary, transplantation of cSca-1/PM prevents cardiac remodeling, improves cardiac contraction and reduces infarct area through angiogenesis and anti-apoptosis.
P143
Interleukin-6 Mediates Concentric Hypertrophy and Myocardial Fibrosis in
Rats
Giselle C Melendez, Yan Du, Joseph S Janicki, Gregory L Brower; Univ of South Carolina,
Columbia, SC
Induction of inflammatory cytokines has been implicated in the progression of myocardial
remodeling and heart failure. In previous studies we and others have shown that TNF-␣ is an
important contributor to the pathologic myocardial remodeling. Increased levels of circulating
TNF-␣ and interleukin-6 (IL-6) in patients with congestive heart failure suggest these cytokines
may be involved in the pathogenesis. TNF-␣ is known to mediate collagen degradation as well
as in-series sarcomeric addition contributing to ventricular dilatation. However, the effects of
IL-6 on cardiac remodeling in vivo have not been investigated. Accordingly, in this study we
explore the hypothesis that up-regulation of IL-6 mediates adverse myocardial remodeling. To
this end, a group of adult male Sprague Dawley rats was infused with IL-6 (2.5 ␮g/kg/hr, IP)
for 7 days via osmotic minipump and compared to aged-matched shams. Cardiac size and
function were evaluated by echocardiogram at baseline and after IL-6 infusion. Hearts were
removed, weighed and analyzed for collagen volume fraction (CVF) and isolated cardiomyocyte
size. IL-6 induced concentric LV hypertrophy (749 mg versus 660 mg in Control hearts;
p⬍0.05), whereas, RV mass was not different. IL-6 infusion produced a marked increase in
interstitial collagen relative to that in control hearts (CVF of 6.2% vs. 1.7%, respectively;
p⬍0.001). The cardiomyocyte hypertrophy at the cellular level also reflected a concentric
phenotype, with cells being significantly longer and thicker (18% and 32%, respectively;
p⬍0.01). These novel observations demonstrate a direct effect of IL-6 on cardiac remodeling
in vivo, which in contrast to TNF-␣, induces a dramatic myocardial fibrosis together with
concentric cardiac hypertrophy. This suggests that IL-6 may contribute to the development of
diastolic dysfunction, and as such could drive the transition to heart failure.
P144
Erythropoietin Promotes Angiogenesis and Prevents Heart Failure After
Myocardial Infarction
Raita Uchiyama, Hiroyuki Takano, Kazutaka Ueda, Yuriko Niitsuma, Hiroshi Hasegawa, Issei
Komuro; Chiba Univ Graduate Sch of Medicine, Chiba, Japan
Background: Erythropoietin (EPO) has been reported to increase endothelial cells and exert
cardioprotective effects through mobilizing bone marrow (BM)-derived cells in the heart after
myocardial infarction (MI), but the precise mechanism is unknown. Methods and Results: MI
was produced in C57BL/6 male mice and subcutaneous injection of EPO was started
immediately after MI and continued daily for 5 days. EPO inhibited left ventricular(LV)
dysfunction and reduced infarct size at 2 weeks after MI (p⬍0.01, LV end-diastolic dimension
(LVEDD); Control 5.8⫾0.2 mm, EPO 5.2⫾0.1 mm, %fractional shortening (%FS); Control
8.8⫾0.2%, EPO 16.2⫾1.7%). In border area of infarcted hearts, EPO markedly increased the
ratio of CD31-positive microvessels per cardiomyocytes (p⬍0.01, Control 1.235⫾0.069, EPO
1.385⫾0.036). In the mice whose BM was replaced by that of GFP-expressing mice, there was
no statistical difference in the number of GFP/CD31-double positive cells between salinetreated and EPO-treated mice.Western blot analysis revealed that EPO significantly increased
Akt and ERK activation and the expression levels of vascular endothelial growth factor (VEGF)
in the hearts after MI and in cultured cardiomyocytes. In vitro study, the EPO-induced increase
in VEGF was abolished by a PI3K inhibitor LY294002, but not a MEK inhibitor PD98059. In
HUVECs, EPO-treated cardiomyocyte-conditioned media enhanced BrdU incorporation, and this
effect was inhibited by a VEGF inhibitor CBO-P11, and adenoviral infection of dominantnegative EPO receptor or dominant-negative Akt. In vivo study, the injection of a soluble form
of Flt-1 (sFlt-1), an inhibitor of VEGF, into the thigh muscle markedly reduced EPO-induced
cardioprotective and angiogenic effects after MI (p⬍0.05, %FS; EPO 11.1⫾0.83%,
EPO⫹sFlt-1 7.56⫾1.50%, p⬍0.01, the ratio of microvessels per cardiomyocytes; EPO
1.385⫾0.036, EPO⫹sFlt-1 1.186⫾0.063). Conclusions: These results suggest that EPO
promotes angiogenesis in the post-MI hearts through the Akt/VEGF pathway rather than
recruiting blood cells, and at least in part, this mechanism contributes to prevention of left
ventricular remodeling and heart failure after MI.
BCVS Conference 2008 Abstracts
P145
Resolvin E1 Has a Direct Protective Effect Against Hypoxia-Reoxygenation
Injury: An In Vitro Study in Rat Cardiomyocytes
Yumei Ye, Yu Lin, Regino J Perez-Polo, Univ of Texas Med Branch, Galveston, TX; Per
Gjostrup, Resolvyx Pharmaceuticals, Inc, Bedford, MA; Yochai Birnbaum; Univ of Texas Med
Branch, Galveston, TX
Background: Resolvin E1 (RvE1) is a naturally occurring omega-3 polyunsaturated fatty-acidderived compound, generated by Cyclooxygenase-2 and 5-lipoygenase. RvE1 has strong
anti-inflammatory properties that induce resolution of acute inflammation. In a previous study
we have shown that RvE1, administered before reperfusion, limit myocardial infarct size in the
rat. We asked whether RvE1 has a direct effect on cardiomyocytes, or the protective effect is
mediated by attenuation of the inflammatory cells. Methods: H9C2 cells (derived from
embryonic rat heart tissue) were incubated with RvE1 (0, 1, 10, or 100 nM). Cells were
subjected to 16h of hypoxia⫹ 2h of reoxygenation. Cell death was assessed by trypan blue (TB)
uptake and cell viability by MTT assay. As a measure of apoptotic cell death, the presence of
cytosolic oligonucleosomes was detected using the Cell Death Detection ELISA® (Roche
Applied Sciences, Indianapolis, IN)). Values are represented as fold change relative to controls
not exposed to hypoxia-oxygenation. Results: RvE1 decreased the percentage of TB positive
cells, increased viability and decreased apoptosis. The protective effect was dose dependent.
Conclusions: RvE1 has a direct effect in protecting cardiomyocytes against hypoxiareoxygenation injury that is independent of leukocytes.
TB (%)
MTT (%)
Apoptosis
Vehicle
RvE1 1.0 nM
ReV1 10 nM
RvE1 100 nM
P value
41.7⫾0.4
49.0⫾0.6
3.34⫹0.04
35.5⫾0.4
50.1⫾0.8
3.36⫹0.02
30.3⫾0.4
62.6⫾1.2
2.54⫹0.01
23.8⫾0.9
74.6⫾0.9
2.23⫹0.01
⬍0.001
⬍0.001
⬍0.001
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P146
Pitavastatin Attenuates Doxorubicin-Induced Cardiotoxicity Through
Antioxidant Effect
e63
role in ER stress-induced apoptosis. Thus, PARM-1 expression could be involved in cardiac
remodeling in hypertensive heart diseases.
P148
Foxo1-Induced Stimulation of Autophagy Contributes to Cardiac Atrophy
Nirmala Hariharan, Junichi Sadoshima; Univ of Medicine and Dentistry of New Jersey,
Newark, NJ
The Forkhead box-O (Foxo) family transcription factors are known to regulate diverse cellular
functions, including muscle atrophy, apoptosis, and longevity. Foxo1 and Foxo3 are upregulated
by mechanical unloading and negatively regulate cardiac growth. Autophagy is a bulk
degradation process, in which the cytosolic proteins are sequestrated by double membrane
vesicles called autophagosomes and degraded through lysosomes. We hypothesize that Foxo1
stimulates autophagy in vitro thereby contributing to cardiomyocyte atrophy. In order to reveal
the function of Foxo1 in the heart, we transduced adenovirus harboring Foxo1-WT (AdFoxo1WT) into neonatal rat ventricular myocytes (NRVM). Overexpression of Foxo1 significantly
reduced the cell size at baseline (-54%, p⬍0.01) and in response to phenylephrine stimulation
(-61%, p⬍0.01) in NRVM. Ad-Foxo1WT transduced NRVM upregulated expression of autophagy
genes, including Beclin1 (2.6 fold) and Vps34 (2.4 fold) relative to control Ad-LacZ transduced
cells. Foxo1 increased formation of GFP-LC3 dots, with the number of dots/cell significantly
more in control LacZ (2.5 fold, p⬍0.05). Furthermore, Foxo1 significantly reduced protein
expression of p62/SQSTM1 (-45 %, p⬍0.05), a protein degraded through autophagy. These
results suggest that Foxo1 stimulates autophagy in cardiac myocytes. Treatment of cardiac
myocytes with inhibitors of autophagy, such as 3-Methyladenine (3-MA) and adenovirus
harboring shRNA Beclin1 (Ad-shBeclin1), decreased Foxo1-induced LC3-II formation (Relative
LC3-II/LC3-I ratio: LacZ 1.0, Foxo1 3.6, Foxo1⫹3-MA 1.7, Foxo1⫹shBeclin1 1.3) and reduction
in p62/SQSTM1. 3-MA and Ad-shBeclin1 also attenuated Foxo1-induced reduction in cardiac
myocyte cell size (LacZ 100%; Foxo1 66%; Foxo1⫹3-MA 81%, p⬍0.05; Foxo1⫹shBeclin1
89%, p⬍0.05) and increased the relative protein content significantly (Foxo1⫹3MA vs Foxo1
p⬍0.05; Foxo1⫹shBeclin1 vs Foxo1 p⬍0.01). These results suggest that Foxo1 stimulates
autophagy in cardiac myocytes and that the stimulation of the autophagy-lysosomal pathway
may play an important role in mediating Foxo1 induced cardiac atrophy.
Masashi Yoshida, Ichiro Shiojima, Issei Komuro; Chiba Univ Graduate Sch of Medicine,
Chiba, Japan
Doxorubicin (Dox)-induced cardiotoxicity leads to myocyte cell death and cardiomyopathy. We
tested whether HMG-CoA reductase inhibitors (statins) exert cardioprotective effects against
Dox-induced cardiotoxicity. In neonatal rat ventricular myocytes, Pitavastatin attenuated
oxidative stress, ATM kinase activation, p53 accumulation, and myocyte cell death induced by
Dox treatment. Dox-induced oxidative stress was also decreased by N-acetylcysteine (NAC) but
not by ATM kinase inhibitor wortmannin, whereas Dox-induced p53 accumulation was reduced
both by NAC and wortmannin. Since statins mainly inhibit mevalonate synthesis pathway, we
tested whether treatment with intermediate products of mevalonate pathway and their enzymes
abolish the reduction of Dox-induced oxidative stress and p53 accumulation. Mevalonate,
farnesyl pyrophosphate and geranylgeranyl pyrophosphate reversed the inhibitory effects of
pitavastatin on Dox-induced oxidative stress and p53 accumulation, whereas Rac1 inhibitor and
geranylgeranyl transferase inhibitor, but not farnrsyl transferase inhibitor, reduced Dox-induced
oxidative stress and p53 accumulation. In wild-type mice, treatment with Dox resulted in a
decrease in left ventricular contractile function and an increase in apoptotic cell death, and
pitavastatin treatment reduced the Dox-induced contractile dysfunction and apoptosis.
Moreover, in heterozygous p53 knockout mice, Dox-induced cardiotoxicity was significantly
attenuated when compared to wild type mice. These data collectively suggest that Dox-induced
cardiotoxicity is mediated by oxidative stress-ATM-p53 pathway, and is attenuated by
pitavastatin through anti-oxidant effect involving Rac1 inhibition.
P147
PARM-1, a Novel Transmembrane Molecule Involved in Endoplasmic
Reticulum Stress-Induced Apoptosis in Cardiac Myocytes
Tomosaburo Takahashi, Koji Isodono, Kyoto Prefectural Univ of Medicine, Kyoto, Japan;
Takehiro Ogata, Kyoto Univ Hosp, Kyoto, Japan; Satoshi Asada, Hiroko Imoto, Atsuo Adachi,
Kyoto Prefectural Univ of Medicine, Kyoto, Japan; Tomomi Ueyama, Hidemasa Oh, Kyoto
Univ Hosp, Kyoto, Japan; Hiroaki Matsubara; Kyoto Prefectural Univ of Medicine, Kyoto,
Japan
To identify novel transmembrane and secretory molecules expressed in cardiac myocytes,
signal sequence trap screening was performed in rat neonatal cardiac myocytes. One of the
molecules identified was a transmembrane protein, prostatic androgen repressed massage-1
(PARM-1). While PARM-1 has been identified as a gene induced in prostate in response to
castration, its function is largely unknown. Our expression analysis revealed that PARM-1 was
specifically expressed in hearts and skeletal muscles, and in the heart, cardiac myocytes, but
not non-myocytes expressed PARM-1. Immunostaining of PAMR-1 in cardiac myocytes showed
that PARM-1 localized in endoplasmic reticulum (ER). In Dahl-salt sensitive rats, high salt diet
resulted in hypertension, cardiac hypertrophy and subsequent heart failure, and significantly
stimulated PARM-1 expression in the hearts with concomitant increase in atrial natriuretic
factor expression. In cultured neonatal rat cardiac myocytes, PARM-1 expression was
stimulated by proinflammatory cytokines such as TGF-b, TNF-a and IL-1b, but not by
hypertrophic stimuli such as phenylephrine, isoproterenol and leukemia inhibitory factor. A
marked increase in PARM-1 expression was observed in response to ER stress inducers such
as thapsigargin and tunicamycin. Silencing PARM-1 expression by adenoviral expression of
shRNAs against PARM-1 resulted in increased susceptibility of cardiac myocytes to apoptosis
induced by ER stresses. These results indicated that PARM-1 is an ER protein specifically
expressed in cardiac myocytes, and PARM-1 expression is increased in the heart of
hypertensive heart failure, and by ER stresses. Further, expression of PARM-1 has a protective
P149
Hypoxia-Induced Expression of E2F-1 Activates Bnip3 and Apoptosis in
Ventricular Myocytes
James Shaw, Natalia Yurkova, Lorrie Kirshenbaum; Univ of Manitoba, Winnipeg, Canada
Previously we have shown that E2F-1 provokes cell death of post-mitotic adult and neonatal
ventricular myocytes, however, the underlying mechanism was undetermined. In this report we
show that E2F-1 triggers cell death of ventricular myocytes through a mechanism that directly
impinges upon the intrinsic apoptotic pathway and involves the activation of the death factor
Bnip3. Overexpression of E2F-1 in cells caused a 4.9 fold increase (p⬍0.01) in myocyte cell
death compared to control cells. E2F-1 provoked mitochondrial perturbations consistent with
hypoxic injury and the intrinsic death pathway including permeability transition pore opening,
Smac/Dibalo release and caspase-3 activation. Bnip3 gene transcription was increased by 2.1
fold in cells expressing wild type E2F-1 but not in cells expressing a transactivation defective
mutant. During hypoxia, Rb was proteolytically cleaved and inactivated. ChIP analysis verified
increased binding of E2F-1 to the Bnip3 promoter during hypoxia - a finding consistent with the
induction of Bnip3 transcription. The Bnip3 homologue, Nix/Bnip3L was unaffected in
ventricular myocytes by either E2F-1 or hypoxia. Genetic ablation of E2F-1 or expression of a
caspase resistant form of Rb suppressed basal and hypoxia-inducible Bnip3 gene transcription.
Loss of function mutations of Bnip3 abrogated mitochondrial defects and cell death elicited by
E2F-1. To our knowledge the data provide the first direct evidence that de-regulated E2F-1
activity during hypoxia impinges upon the intrinsic death pathway through the transcriptional
activation of Bnip3 in the heart.
Nampt Is a Critical Regulator of NADⴙ Synthesis in Cardiomyocytes
P150
Chiao P Hsu, Junichi Sadoshima; UMDNJ, Newark, NJ
Cellular NAD⫹ content is regulated by both de novo and salvage pathways. Despite the
presence of the de novo pathway, the salvage reactions are essential in humans. Nicotinamide
phosphoribosyltransferase (Nampt) is a rate-limiting enzyme in the salvage pathway. NAD⫹ is
not only required for cellular respiration and oxidative phosphorylation, but is also used as a
substrate for NAD⫹-dependent enzymes, such as Sirt1. At present, little is known about the
function of Nampt in the heart. To study the role of Nampt in the heart, we examined expression
levels of Nampt in heart samples. Adenovirus constructs harboring Nampt and shRNA-Nampt
were transduced into neonatal rat cardiomyocytes. The expression of Nampt in the heart was
decreased in pathological conditions, including ischemia (56%), ischemia/reperfusion (60%),
pressure overload (42%) and heart failure (37%). Transduction of adenovirus harboring Nampt
and shRNA-Nampt respectively caused increases and decreases in NAD⫹ and ATP concentrations in cardiomyocytes, suggesting that Nampt critically regulates NAD⫹ and ATP levels in
them. Overexpression of Nampt in cardiomyocytes increased survival against the stress
imposed by methylmethane sulfonate (MMS, 1.2 mM), an agent known to hyperactivate
PARP-1 and reduce the cellular NAD⫹ content, and against glucose deprivation. Knockdown of
Nampt alone induced death of cardiomyocytes and made them more sensitive to cell death
induced by MMS (0.6 mM). Death of cardiomyocytes due to Nampt knockdown was not
accompanied by increases in caspase 3 cleavage or cytoplasmic accumulation of mono and
oligo-nucleosomes, suggesting that the death may not be apoptotic. Although Nampt
knockdown increased LC3-II and accumulation of autophagosomes, it was also accompanied
e64
Circulation Research
Vol 103, No 5
August 29, 2008
by accumulation of p62 and impaired autophagic flux. In summary, Nampt critically regulates
NAD⫹ and ATP content in cardiomyocytes. Expression of Nampt is decreased in many cardiac
pathogenesis. Nampt is protective against NAD⫹/ATP deprivation, whereas reduced expression
of Nampt causes non-apoptotic cell death and impairment of autophagic flux in cardiomyocytes. These results suggest that Nampt is an essential gatekeeper of energy status,
autophagic flux and survival in cardiomyocytes.
nucleotide-releasing factor 1. Conclusions: While there are numerous gene modules that are
unique to fetal from normal adult myocardium, only a small proportion are recapitulated in heart
failure. The identification of these modules provides novel targets for functional genomic
analysis and therapetic abrogation of maladaptive responses to myocardial injury.
P154
P151
Polymorphisms of MMP-2 Gene Are Associated with Systolic Heart Failure
Risk
Withdrawn
Yihong Hua, Li Song, Naqiong Wu, Xiangfeng Lu, Xianmin Meng, Dongfeng Gu, Yuejin Yang;
Cardiovascular Institute and Fuwai Hosp, Beijing, China
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Background Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes responsible
for myocardial extracellular protein degradation. MMP-2 is an important member of MMPs.
MMP-2 has been demonstrated to play a pivotal role in myocardial remodeling process that
occurs in congestive heart failure. We hypothesized MMP-2 genetic variations could confer high
risk of systolic heart failure. Methods To test the association of single nucleotide polymorphisms of MMP-2 with systolic heart failure risk, we performed a hospital-based, case-control
study of 605 patients with systolic heart failure and 689 controls without heart failure. Three
single nucleotide polymorphisms of MMP-2 (rs243864, rs243866, rs17859821) were analyzed
by restriction fragment length polymorphism (RFLP) methods. Results We found that the
genotype frequencies of MMP-2 rs243866 AA and AG in the control group were much higher
than that in the patients group (24.7% vs. 17.9%,P⬍0.01). After adjustment for age, body mass
index, smoke and uric acid by using multiple logistic regression analysis, MMP-2 A allele
carriers had lower risk of systolic heart failure than did GG genotype carriers (OR
0.693;95%CI:0.492– 0.975;P⫽0.035). After adjustment for above traditional cardiovascular
risk factors in haplotype analysis, haplotype GGG was associated with higher systolic heart
failure risk. MMP-2 rs17859821, rs243864 had no association with systolic heart failure.
Conclusion The findings of the present study suggest that MMP-2 rs243866 A allele confers
lower risk of systolic heart failure.
Activation of CaMKII␦ Is Critically Involved in Mutated Cardiac
␣-Actin-Induced Dilated Cardiomyopathy
P152
Haruhiro Toko, Tohru Minamino, Issei Komuro; Chiba Univ Graduate Sch of Medicine, Chiba,
Japan
[Purpose] Dilated cardiomyopathy (DCM) is characterized by dilatation and impaired contraction
of the left ventricle, and patients with DCM often develop heart failure. Various mutations in the
genes encoding the cytoskeletal proteins have been identified in DCM patients, but the
mechanisms of how such mutations lead to DCM remain unknown. [Methods and Results] To
examine how gene mutations cause DCM, we generated transgenic mice (Tg) that overexpress
the mutated cardiac alpha-actin in the heart. This mutation has been reported in patients with
DCM. Echocardiography revealed that left ventricular dimension was gradually increased and
that fractional shortening was reduced in the Tg mice compared with the wild-type mice. The
Tg mice died of heart failure at ⬃30 weeks of age. The activity of CaMKIIdelta was increased
in Tg mice and this increase was associated with cardiac dysfunction. Inhibition of this
activation by KN-93 prevented an increase in left ventricular dimension and preserved cardiac
function. [Conclusion] We established a murine model of DCM that has the mutation identical
to that of human DCM. The activation of CaMKIIdelta plays a critical role in the development
of heart failure in this model. Further studies will provide novel insights into the treatment of
DCM patients.
P153
Reevaluating Dogma: Recapitulation of Fetal Gene Expression Programs in
Heart Failure
Frederick E Dewey, Marco V Perez, Matthew T Wheeler, Euan A Ashley; Stanford Univ Sch
of Medicine, Stanford, CA
Background: Myocardial insult leading to the clinical syndrome of heart failure is thought to
invoke a recapitulation of fetal gene expression programs. It remains unclear,however, what
modules of coexpression comprise the fetal gene program and to what extent they are
recapitulated in heart failure. We aimed to identify coexpression programs unique to fetal
myocardium from normal adult myocardium and evaluate their degree of preservation in failing
myocardium. Methods: All gene expression microarray data available in the Gene Expression
Omnibus database from human fetal (n ⫽ 17), normal adult (n ⫽ 213) and failing (n⫽340)
myocardium were combined. Weighted gene co-expression network analysis was used to
identify fetal gene modules based on topological overlap. Marginal module analysis was
employed to identify fetal gene modules that are not present in normal adult myocardium or
exhibited significant modular differential expression. Functional enrichment analysis was
performed on fetal modules for identification of over-represented gene ontologies in each
module. Preservation of these fetal modules was then assessed in failing myocardium to
identify fetal gene programs that are re-capitulated in heart failure. Results: We identified 58
gene modules comprised of between 20 and 443 genes unique to fetal myocardium. The
overall network preservation for fetal gene modules in failing myocardium was 16%, but of the
58 gene modules that were unique to fetal myocardium from adult myocardium, only 3 (5%)
were reproduced in failing myocardium. Significantly over-represented pathways included
neuronal system development (p ⫽ 0.00023), cell-cell adhesion (p ⫽ 0.00063), and sodium
ion transport (p ⫽ 0.0008). Hub genes identified in fetal modules re-capitulated in heart failure
included splicing factor 1, Ras-like without CAAX 1, and Ras protein-specific guanine
P155
Transcriptional Upregulation of Glutathione Metabolic Pathways: An Early
Biosignature of R120GCryAB Cardiomyopathy in Mice
Rajasekaran Namakkal Soorappan, Univ of Utah Med Sch, Salt Lake City, UT; Matthew A
Firpo, Univ of Utah Med Sch, Salt Lake City, UT; Brett Milash, Univ of Utah Huntsman
Cancer Institute, Salt Lake City, UT; Ivor J Benjamin; Univ of Utah Med Sch, Salt Lake City,
UT
Background: Protein aggregation cardiomyopathy is a life-threatening manifestation of a
multisystem disease (also termed desmin-related myopathy) caused by the missense R120G
mutation in the gene encoding the human small HSP ␣B-crystallin (hR120GCryAB). Recent
genetic studies in mice have shown that selective hR120GCryAB expression in the heart
induces a novel toxic gain-of-function mechanism, termed reductive stress, caused by G6PD
overexpression, the major source of reducing equivalents and GSH/GSSG redox couples.
Methods and Results: To gain insights into the cellular pathways affected by hR120GCryAB,
we used DNA micro arrays to assess the transcriptional events in 3 month old transgenic mice:
wild-type transgenic (hCryABWT) and mutant (hR120GCryAB Tg) mouse lines. Total CryAB
levels were increased in hCryABWT Tg hearts (1.5 fold) but considerably greater for
hR120GCryAB Tg mice (6.0 fold), the latter associated with overt heart failure and 100%
premature death. The earliest upregulated biological pathways were belonging to stress
response activation (e.g., Hsp70, Hsp 90) and the glutathione metabolism (Gpx1, Gpx3,
glutathione S-transferase, G6pdx) in hR120GCryAB Tg hearts compared with hCryABWT
transgenic lines. Upregulation of these pathways persisted only in hR120GCryAB Tg mice at 6
months. Using RT-PCR with these selected candidate genes as a ‘training’ set, we
independently confirmed both sensitivity and specificity in ‘test’ samples from hR120GCryAB Tg
and control mice at 3 months. Conclusions: Our findings establish the utility of a novel
biosignature linked to dysregulation of glutathione metabolism whose reproducibility and
reliability might predict the onset and rate of progression of protein aggregation R120G CryAB
cardiomyopathy in humans.
P156
Evaluation of Polymorphisms in Candidate Genes in the Dramatic Response
to Pharmacologic Therapy of Heart Failure
Matthew T Wheeler, Aleksandra Pavlovic, Frederick Dewey, Marco Perez, Devin Absher,
Michael Y Ho, Elizabeth Cretti, Audrey Southwick, David N Rosenthal, Daniel Bernstein,
Richard M Myers, Paul A Heidenreich, Michael B Fowler, Robert C Robbins, Euan A Ashley;
Stanford Univ, Stanford, CA
Background Heart failure patients exhibit differential responses to conventional pharmacologic
therapy. Studies have evaluated single nucleotide polymorphisms (SNPs) as possible determinants of these responses. We sought to comprehensively evaluate SNPs in candidate genes in
two groups of patients with differential response to therapy. Methods By screening more than
1200 heart failure patients, 136 patients were identified with an absolute ejection fraction
BCVS Conference 2008 Abstracts
increase of greater than 20 percentage points. After exclusion of those with coincident surgical
or resynchronization therapy, 50 patients were matched with age- race- and sex-matched
controls who failed optimum medical therapy and required orthotopic heart transplantation.
Whole blood DNA obtained from each individual was hybridized to Illumina HapMap 550K
BeadChips. Data were subjected to additive logistic regression analysis. Candidate genes were
identified by literature review in three groups: those previously tested, those in downstream
signaling pathways, and those known to be involved in pathophysiology of heart failure. SNPs
in these genes or SNPs in high LD to previously studied SNPs were evaluated for between group
significance. Results We found 3065 SNPs scored on the 550K chip associated with candidate
genes. Of these, SNPs representing polymorphisms in the candidate genes encoding dystrophin
(DMD), transforming growth factor beta-1 (TGFB1), neuregulin-1 (NRG1), protein kinase C,
alpha (PRKCA), and adenosine A1 receptor (ADORA1) were most significantly different (all p less
than 0.005). SNPs previously identified as modulators of pharmacologic response were not
found to be significantly different (all p greater than 0.05). In this study, no SNP reached
statistical significance using a conservative multiple testing correction algorithm, with an alpha
of 1.6 x 10^-5. Conclusions Evaluation of candidate gene polymorphisms in a study of dramatic
response to therapy identified polymorphisms in several genes involved in the pathogenesis of
heart failure. No SNP in a drug target gene was identified as being significant. This small phase
I study identifies coding and non-coding modulators of ‘extreme phenotype’ which will be
tested in a larger cohort.
P157
Mechanism of HDL Revealed by Electron Microscopy
e65
P159
High Molecular Weight Fibroblast Growth Factor-2 Can Be Released by
Human Cardiac Fibroblasts and Can Promote Cardiomyocyte Hypertrophy
Jon-Jon Santiago, Xin Ma, Robert Fandrich, Rakesh Arora, Elissavet Kardami; Institute of
Cardiovascular Sciences, Winnipeg, Canada
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Background: Fibroblast growth factor-2 (FGF-2) is translated as high molecular weight (hi-,
⬎20 kDa) or low molecular weight (lo-, 18 kDa) isoforms. Previously, we showed that
administration of rat recombinant hi-FGF-2, but not lo-FGF-2, resulted in cardiomyocyte
hypertrophy in vitro and in vivo. Here we investigated whether endogenously made hi-FGF-2,
a predominantly nuclear protein, is a credible candidate for promoting cardiomyocyte
hypertrophy. Specifically we asked if human cardiac fibroblasts can export hi-FGF-2, and if so,
whether exported hi-FGF-2 can stimulate hypertrophy. Methods: Primary cultures of human
cardiac (myo)fibroblasts were obtained from atrial tissue explants; atrial tissue was collected
from patients undergoing coronary artery bypass grafting (CABG) or valve replacement surgery.
Angiotensin II (Ang II) was used to stimulate release of pro-hypertrophic cytokines from these
cells. Results: Firstly, we established that hi-FGF-2 is indeed expressed in human atrial tissue
and tissue-derived fibroblasts: Western blotting showed that while about 50% of tissue FGF-2
was composed of hi-FGF-2 (22–25 kDa), this isoform predominated (over 90% of total) in
lysates of human fibroblasts. Cardiac fibroblasts released only hi-FGF-2 to the conditioned
medium, in response to Ang II. Conditioned media from stimulated fibroblasts (but not from
non-stimulated cells) promoted hypertrophy (increased size) of neonatal rat cardiomyocytes in
culture. Pilot studies indicated that the pro-hypertrophic effect of fibroblast-conditioned media
was decreased by anti-FGF-2 neutralizing antibodies. Conclusion: We have shown, for the first
time in human, that adult-heart derived fibroblasts express and release hi-FGF-2 which can
then act in a paracrine fashion to promote cardiac myocyte hypertrophy. Targeting chronic
release, and/or preferential translation, of hi-FGF-2 by cardiac fibroblasts has the potential of
preventing cardiac hypertrophy and thus improve cardiac prognosis.
Gang Ren; Univ of California, San Francisco, San Francisco, CA
During reverse cholesterol transport (RCT), high-density lipoprotein (HDL) conveys excess
cholesterol from peripheral tissues to the liver and steroidogenic organs. It is through HDL’s
mediation of RCT that a majority of HDL’s ability to reverse or prevent the onset of
cardiovascular disease is derived. As HDL proceeds through RCT, it forms a variety of
subclasses, dependent on the HDL particle’s lipid cargo status. To drive the process of RCT,
each subclass of HDL has a unique set of biological properties (receptor affinity, enzyme
activation capabilities, etc.), most of which are derived from apolipoprotein A-I (apoA-I), the
main protein constituent of HDL. Despite many years of investigation into the structure of
apoA-I, very little is known of apoA-I’s HDL subclass-specific structure and the role of apoA-I
structure in HDL subclass-specific properties. Here, we use electron microscopy, electron
cryo-microscopy and advanced image processing to examine reconstituted HDL subclasses
(7.8, 8.4, 9.6nm diameter discoidal and 9.4nm diameter spherical HDL) on negatively stained
and frozen-hydrated native state specimens. Three-dimensional reconstruction of particles
revealed several structural features unique to each subclass. For instance, apoA-I, the high
density component, appears as a figure “6” on particles of 7.8 and 8.4nm diameter, and as a
“ring” on 9.6nm particles, in contrast, the protein appears as large “6” shape in spherical HDL.
These results bring up with a hypothesis on how apoA-I mechanistically regulate and organize
its lipids pocket during RCT.
P158
Bone Marrow Stromal Cells but Not Hematopoietic Stem Cells of the
Smooth Muscle Myosin Heavy Chain Promoter-Driven LacZ Transgenic
Mice Give Rise into Vascular Smooth Muscle Cells in Vitro
P160
Heat Shock Protein 27 Induced Cardiac Hypertrophy in a Dose-Dependent
Manner in Mice
Li Liu, Xiaoyan Min, Bo Qian, Xiaojin Zhang, Youwei Shi, Sisi Pang, Liqing Bi, Guoxian Ding,
First Affiliated Hosp with Nanjing Med Univ, Nanjing, Nanjing, China; Xiang Gao, National
Resource Cntr of Mutant Mice, Nanjing Univ, Nanjing, China; Chuanfu Li, East Tennessee
State Univ, Johnson City, TN; Yunlin Cheng; First Affiliated Hosp with Nanjing Med Univ,
Nanjing, Nanjing, China
Increased expression of heat shock protein27 (Hsp27) has been observed in hypertrophic
hearts induced by isoproteronal or pressure overload. However, little is known about the role
of Hsp27 in the development of cardiac hypertrophy. In the present study, we examined the
biological role of Hsp27 in cardiac hypertrophy. Transgenic mice with cardiac-specific
expression of Hsp27 at 4-month old were employed and the gender-matched wild type
littermates (WT) served as controls. In Tg mice with moderate expression of Hsp27, the ratio
of heart weight/Tibia length (HW/TL) was increased by 8.1% compared with WT controls
(5.90⫾0.34, n⫽9 vs 5.46⫾0.21, n⫽8, p⬎0.05). However, the ratio of HW/TL in Tg mice with
high expression of Hsp27 (HTG) was increased by 29.6% compared with WT controls
(7.08⫾0.16, n⫽ 9 vs 5.46⫾0.21, n⫽8, p⬍0.01). Cardiac myocyte sizes in HTG mice were
increased by 57.7% and mRNA expression of ANP and BNP increased by 93.9% and 43.0% in
HTG mice compared with WT controls. Left ventricle (LV) function measured by echocardiography showed that FS% and EF% decreased by 22% (30.79⫾1.98 vs. 39.43⫾2.70, P⬍0.05)
and by 14% (63.55⫾2.71 vs. 74.15⫾2.65, P⬍0.05) in HTG mice (n⫽12) compared with WT
mice (n⫽14). Histological examination exhibited significant interstitial fibrosis in the myocardium of HTG mice. In addition, the levels of phosphorylated ERKs were increased by 116.7%
(2.17⫾0.03 vs 1.00⫾0.08, P⬍0.001) in HTG mice (n⫽4) compared with WT controls (n⫽4).
Our findings suggest that overexpression of HSP27 in the myocardium will induce cardiac
hypertrophy in a dose-dependent manner and that the mechanism may involve activation of
ERK signaling pathway.
Youichi Katoh, Juntendo Univ Sch of Med, Urayasu Hosp, Chiba, Japan; Ryota Hashimoto,
Juntendo Univ Sch of Med, Tokyo, Japan; Yuji Nakazato; Juntendo Univ Sch of Med,
Urayasu Hosp, Chiba, Japan
Background — Bone marrow contains hematopoietic stem cells (HSCs) and mesenchymal
stem cells. While some studies have reported that HSCs may give rise to vascular smooth
muscle cells (SMCs) in response to acute injury, others indicate that transdifferentiation would
have to be an extremely rare event. Methods and Results — To examine the plasticity of
HSCs without acute mechanical injury, we created a transgenic smooth muscle myosin heavy
chain (SM-MHC)-LacZ mouse that carries a LacZ gene coupled with SM-MHC regulatory
element and performed in vitro study. Bone marrow cells obtained from SM-MHC-LacZ mice
were cultured in DMEM media supplemented with 10% FCS usually used for the culture of
vascular SMCs. Although no obvious LacZ positive cells were observed at the initiation of
culture, cells with high LacZ intensity became conspicuous among the cells attached to the
culture plate within 1 to 2 weeks. During the culture, LacZ positive cells gradually dominated
among the attached cells. The bone marrow cells were also cultured in Dexter media that can
support in long-term the growth of hematopoietic cells as well as stromal cells. LacZ
expression was also detected only in the stromal cells that were strongly attached to culture
plates. On the other hand, no floating cells including HSCs grown in the same culture showed
LacZ expression. Consistent with our previous report, these results indicate that bone marrow
contains stromal progenitors that differentiate into SMCs expressing SM-MHC. The progenitor
for these SM-MHC-expressing SMCs is distinct from HSCs. Conclusions — These experiments
suggest that developmental plasticity of HSCs may be strictly limited, and the transdifferentiation of HSCs into mesenchymal cells unlikely occurs, at least without acute mechanical injury
in vitro.
P161
Resistin Is Expressed in the Heart and Promotes Cardiac Hypertrophy
Maengjo Kim; Mt Sinai Sch of Medicine, New York, NY
Heart failure is the major cause of death in diabetic patients. A contributing factor to heart
failure in these patients is the development of diabetic cardiomyopathy. Although several
factors may contribute to the development of cardiac dysfunction the underlying pathogenesis
is still partially understood. Recently, a novel paradigm for the role of the adipocytokine resistin
in diabetes has emerged. Resistin has been proposed to be a link between obesity, insulin
resistance and diabetes. Using microarray analysis, we have recently found that cardiomyocytes isolated from type 1 and 2 diabetes expressed high levels of resistin. However, the
functionality of resistin in respect to cardiac function is largely unknown. In this report we show
that resistin, not only is expressed in the heart, but also promotes cardiac hypertrophy.
Adenovirus-mediated overexpression of resistin in cultured neonatal rat ventricular myocytes
(NRVM) significantly increased sarcomere organization and cell size, increased protein
synthesis and increased the expression of atrial natriuretic factor and ␤-myosin heavy chain.
Overexpression of resistin in NRVM was also associated with activation of the mitogenactivated protein (MAP) kinases, ERK1/2 and p38 as well as increased Ser-636 phosphorylation
of insulin receptor substrate-1 (IRS-1), indicating that IRS-1/MAPK pathway may be involved in
the observed hypertrophic response. Associated with the development of cardiac hypertrophy,
overexpression of resistin in adult cultured cardiomyocytes significantly altered myocyte
mechanics by depressing cell contractility, as well as contraction and relaxation velocities.
e66
Circulation Research
Vol 103, No 5
August 29, 2008
Intracellular Ca2⫹ measurements showed slower Ca2⫹ transients decays in resistin-transduced
myocytes compared to controls, suggesting impaired cytoplasmic Ca2⫹ clearing. We conclude
that resistin overexpression confers to primary cardiomyocytes all the features of the
hypertrophic phenotype and promote cardiac hypertrophy possibly through the IRS-1/MAPK
pathway.
P162
Cardiac Lipotoxicity Is Associated with Defective ␤-Adrenergic Response
Kalyani G Bharadwaj, Shota Ikeda, Rajiv Agarwal, Tae-Sik Park, Ni-Huiping Son, Ira J
Goldberg; Columbia Univ, New York, NY
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Diabetic cardiomyopathy has been modeled in mice by altering myocyte lipid metabolism.
Cardiac lipid accumulation causes mechanical dysfunction, metabolic dysregulation, and
myocyte death. Transgenic mouse models of cardiac lipotoxicity include LpL GPI
(glycosylphosphatidylinositol-anchored human lipoprotein lipase), MHC-PPAR␥1 (cardiacspecific overexpression of Peroxisome Proliferator Activated Receptor ␥1), and MHC-ACS
(cardiac-specific overexpression of acyl CoA synthase). These transgenic mice have echocardiographic demonstrable dilated cardiomyopathy. In an attempt to further characterize heart
malfunction in these models, we employed a sensitive technique involving in vivo cardiac
catheterization whereby we assessed basal heart function and its response to adrenergic
stimulus. In order to assess this, the right carotid artery was cannulated with a catheter and
advanced through the ascending aorta into the left ventricle. Following baseline measurements,
responses to increasing doses of dobutamine (1, 3, 10, and 30 ␮g/kg) administered via the
femoral vein were determined to evaluate heart function. At baseline, 10 month old LpLGPI mice
showed reduced contractile function measured as LVdP/dtmax compared to wildtype (wildtype:
11058⫾1543, LpLGPI: 6957⫾518 mmHg/s; P⬍0.05). Furthermore, the LpLGPI mice showed
marked reductions in inotropic response to dobutamine (wildtype: 21631⫾1283, LpLGPI:
9879⫾1498 mmHg/s; P⬍0.05). As early as 4 months of age, LpLGPI mice showed significantly
reduced response to dobutamine in comparison to wild type (wildtype: 19684⫾1041, LpLGPI:
10236⫾2545 mmHg/s; P⬍0.05). This effect was also observed in the MHC-PPAR␥1 and
MHC-ACS mice. Membrane associated fraction of PKC␣ protein was increased 150% in the
LpLGPI hearts at 4 and 10 months compared to wild type (P⬍0.05). In addition, there was a 43%
increase in total cellular PKC␣ in the LpLGPI compared to wildtype (P⬍0.05). MHC-PPAR␥1
hearts showed a 25% and 21% increase in the membrane and total PKC␣ protein respectively
compared to the wildtype (P⬍0.05). These data suggest that cardiac lipotoxicity is associated
with defective ␤ adrenergic response and elevated PKC␣ function, which may partially
contribute to the ensuing cardiac dysfunction.
P163
All-trans Retinoic Acid Prevents Hyperglycemia-Induced Cardiac
Remodeling Through Inhibition of Expression of Renin-Angiotensin System
Components in Cardiomyocytes
Jing Pan, Rakeshwar Guleria, Texas A&M Univ, Health Science Cntr, Cardiovascular Rsch
Institute, Temple, TX; Rashmi Choudhary, Univ of Colorado Health Sciences Cntr, Denver,
CO; Kenneth M Baker; Texas A&M Univ, Health Science Cntr, Cardiovascular Rsch Institute,
Temple, TX
We have previously reported that all-trans retinoic acid (RA), the metabolite of vitamin A, has
an important role in preventing mechanical stimuli-induced cardiac remodeling, through
inhibition of expression of the renin-angiotensin system (RAS), in vitro and in vivo. The
metabolic availability of vitamin A is impaired and associated with insulin resistance in diabetic
patients and animal models. Diabetes mellitus (DM) is a well known risk factor for the
development of congestive heart failure. However, the cellular mechanisms that relate to the
development of cardiac remodeling in DM have not been fully elucidated. Using neonatal
cultured cardiomyocytes, we determined the effect of high glucose on expression of RA
receptors (RAR and RXR) and the mechanisms involved in RA mediated regulation of
hyperglycemia-induced cardiac remodeling. Cardiomyocytes were exposed to normal or high
glucose (25 mM), for 24 h, in the presence or absence of RA (0.01–10 ı̀M). High glucose down
regulated nuclear expression of RAR␣, RXR␣ and RXR␥, which was partially reversed by RA.
Cell apoptosis was analyzed by Annexin V staining, using flow cytometry. High glucose induced
an increase in Annexin V positive cells, which was dose-dependently inhibited by RA. RA also
prevented high glucose induced cleavage of caspase 3. Flow cytometric analysis, along with
DCF staining, demonstrated significant inhibition of high glucose-induced reactive oxygen
species generation in RA treated cells. The protective effects of RA on cardiomyocytes were
abrogated, by pretreatment with either AGN193109 (RAR antagonist) or HX531 (RXR
antagonist), indicating the participation of both RA receptor types in the cardioprotective effect.
High glucose induced gene expression of renin, angiotensinogen and AT1 receptor, as well as
the production of angiotensin II (Ang II), were all dose-dependently inhibited by RA. Ang
II-induced cell apoptosis and oxidative stress were prevented by RA. In summary, downregulation of RA receptors is involved in hyperglycemia-induced cardiac remodeling. Activation of
RAR/RXR-mediated signaling protects cardiomyocytes from hyperglycemia-induced apoptosis
and intracellular oxidative stress, through inhibition of expression of intracardiac RAS
components.
P164
Induction of Lipotoxicity in a Cardiomyocyte-Derived Cell Line Reduces Cell
Responsiveness to ␤-Adrenergic Stimulation
Konstantinos Drosatos, Joshua D Brown, Tae-Sik Park, Ira J Goldberg; Columbia Univ, New
York, NY
Lipotoxicity in tissues including the heart leads to cellular dysfunction and cell death. Here we
report that induction of lipotoxicity in a cardiomyocyte-derived cell line (AC-16) by treatment
with either saturated (palmitic acid - PA) or unsaturated (oleic acid - OA) long chain fatty acids
resulted in intracellular lipid droplet formation, and affected cellular gene expression and
␤-adrenergic receptor responsiveness. PA treatment increased intracellular lipid droplets and
increased expression of genes associated with heart failure (BNP), apoptosis (Bax, caspase-1),
endoplasmic reticulum stress (CHOP, ATF6) and inflammation (TNF-␣). OA led to less
pronounced effects. PA increased the expression of the fatty acid transporter CD36 by 2.6 fold,
as compared with non-treated cells, while it did not modulate the expression of Carnitine
Palmitoyl Transferase-1 (Cpt1) and Peroxisome Proliferator Activated Receptor (PPAR)-␣,
indicating an imbalance between fatty acid “input” and “output” that might result in triglyceride
accumulation. Stimulation of fatty acid-treated AC-16 cells with 100 nM isoproterenol
(␤-adrenergic receptor agonist) followed by assessment of cyclic AMP (cAMP) levels showed
that PA treatment reduced cAMP production by 40%. Oleic acid treatment led to a smaller
reduction of cAMP levels. In contrast, cAMP generation after forskolin stimulation was not
altered by PA. In transgenic mice, cardiac overexpression of PPAR␥ leads to lipotoxic
cardiomyopathy. Based on this observation, we attempted to reproduce lipotoxicity in vitro by
adenovirus-mediated expression of PPAR␥ in AC-16 cells. PPAR␥ increased CD36, CPT1 and
Pyruvate Dehydrogenase Kinase-4 (PDK4) gene expression, as well as BNP and Bax. Treatment
of PPAR␥-overexpressing AC-16 cells with PA, but not OA increased BNP mRNA levels 4.2 fold.
PA-treatment, but not OA treatment of PPAR␥-expressing AC-16 cells reduced the cAMP
response by 25% as compared with PPAR␥-expressing cells that were not treated with fatty
acids. Thus, as has been found in in vivo models of lipotoxicity, increased cardiomyocyte lipid
accumulation by treatment with PA or overexpression of PPAR␥ stimulates BNP production and
leads to defective adrenergic responsiveness.
P165
p53 Plays a Critical Role in Angiotensin II-Induced Mitochondrial
Cytochrome C Release-Dependent Cardiac Apoptosis
Guihua Zhou, Lu Cai; Univ of Louisville, Louisville, KY
Our previous studies showed that angiotensin II (Ang II)-induced cardiac apoptotic cell death
and nitrosative damage plays a critical role in the development of diabetic cardiomyopathy.
Although tumor suppressor gene p53 was implicated in the pathogenesis of Ang II-induced
cardiac cell death, the direct causative role of p53 in Ang II-induced cardiac apoptosis and its
signaling pathways have not been defined. In the present study, therefore, cardiac H9C2 cells
were exposed to Ang II at 100 nM for 24 hr in vitro, which increased the total and
phosphorylated p53 expressions. Interestingly, Ang II induced a time-dependent translocation
of p53 from cytosol to mitochondria and then to nucleus. We found that exposure of H9C2 cells
to Ang II also induced significant increases in protein nitration, measured by 3-nitrotyrosine
(3-NT), and apoptotic cell death, determined by DNA fragmentation. Furthermore, mitochondrial
permeability, detected by fluorescent staining and flow cytometry, was significantly increased
along with an increased mitochondrial cytochrome c release, determined by Western blotting
assay. All these effects of Ang II were inhibited by p53 inhibitor pifithrin-␣ (PFT-␣). To validate
the in vitro finding into in vivo condition, FVB mice were given subcutaneously injection of Ang
II at the dose of 1 mg/kg body weight with and without twice I.P. injections of PFT-␣ at 2.2
mg/kg body weight half of an hour before and 1 hour after Ang II treatment. Expressions of both
total and phosphorylated p53 were increased in Ang II-treated WT mice, but not in Ang
II/PFT-␣-treated WT mice. Ang II-induced capase-3 activation and 3-NT accumulation in the
hearts were also prevented in Ang II/PFT-␣-treated WT mice. These results suggest that Ang
II-induced cardiac apoptosis is mediated predominantly by p53 activation-dependent, mitochondrial cytochrome c release pathway (Supported by ADA and AHA grants).
P166
Strain Activation of Calcineurin-Mediated Mitochondrial Apoptotic Pathway
After Myocardial Infarction in Ovine Model
Tieluo Li, Kinjal Savai, Changfu Wu, Kwame G Yankey, Guangming Cheng, Christopher
DeFilippi, Meredith Bond, Zhongjun J Wu, Bartley P Griffith; UMB Sch of Medicine,
Baltimore, MD
Objective: Pathological remodeling of the left ventricle (LV) after myocardial infarction (MI) is
a major cause of heart failure. Calcineurin (CaN) has been reported to lead to apoptosis through
BAD. The goal of this study was to evaluate the relationship between the CaN/BAD
mitochondrial apoptotic pathway activation and the degree of myocardial remodeling, as well
as the regional myocardium contractile function. Methods: An MI with 25% free wall mass was
created in 9 Dorsett hybrid sheep. The in vivo deformation of the LV was monitored by 16
sonomicrometric crystals implanted into the myocardium free wall. Sonomicrometric data were
collected pre-infarction, and weekly until 11 weeks post-infarction. The regional contractile
function (systolic strain) at each time point and the regional dilatation (remodeling strain) over
time in the non-infarcted region (adjacent and remote) as well as the infarct were quantified
by analyzing the sonomicrometry data. The myocardial tissue collected at termination and
isolated mitochondria from the tissue samples underwent Western blotting and phosphorimager quantification for the expression of major regulatory proteins in the CaN/BAD pathway.
Positive apoptotic cells were determined by TUNEL. Results: The relative abundance of
apoptotic proteins correlated well with the remodeling strain: CaN (R2 ⫽0.44 P⬍0.05), BAD (R2
BCVS Conference 2008 Abstracts
⫽0.19 P⬍0.05), mitochondrial BAD (R2 ⫽0.36 P⬍0.05), cytosol cytochrome c (R2 ⫽0.31
P⬍0.05), cleaved caspase-3 (R2 ⫽0.43 P⬍0.05). TUNEL positive cells were 1.56 ⫾ 0.16 and
6.44 ⫾ 0.58* (cells per high power field, *p⬍0.05 as compared to normal myocardium) in the
remote and adjacent zones, respectively. The contractile function at termination negatively
correlated with these apoptotic activities: CaN (R2 ⫽0.34 P⬍0.05), BAD (R2 ⫽0.36 P⬍0.05),
mitochondrial BAD (R2 ⫽0.16 P⬎0.05), cytosol cytochrome c (R2 ⫽0.29 P⬍0.05), cleaved
caspase-3 (R2 ⫽0.31 P⬍0.05). Conclusions: The differences in regional strain are related to
regional alterations in protein expression in the CaN/BAD mitochondrial apoptotic pathway.
Significant up-regulation of apoptotic proteins were present at the adjacent zone where
increased regional remodeling strain and decreased contractile function were observed.
P167
Inhibition of Mitochondrial Naⴙ/Ca2ⴙ Exchanger Improves Energy Supply in
Heart Failure
Ting Liu, Brian O’Rourke; The Johns Hopkins Univ, Baltimore, MD
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Heart failure is associated with increased intracellular Na⫹ ([Na⫹]i) level. Our previous work
demonstrated that elevated [Na⫹]i impaired mitochondrial NADH production in cardiomyocyte
by inhibiting mitochondrial Ca2⫹ ([Ca2⫹]m) accumulation, which suggested detrimental effects
of increased [Na⫹]i on mitochondrial bioenergetics in failing heart. In the present work, we
investigated i) whether NADH/NAD⫹ balance is impaired in cardiomyocytes from failing heart,
ii) whether decrease of [Na⫹]i or treatment of CGP-37157, an inhibitor of mitochondrial
Na⫹/Ca2⫹ exchanger, improves NADH production in cardiomyocytes from failing heart by using
a heart failure model. Ascending aortic constriction was applied to 250 –300g male guinea pigs.
Cardiomyocytes were isolated at 6 – 8weeks when decreases of ejection fraction (EF) were
observed. Compared to age-matched non-surgical controls, heart weight/tibia length of aortic
banding animals was increased by ⬃85%. EF of banding hearts was decreased by 20%. [Na⫹]i,
indicated by SBFI, was increased in failing hearts compared to controls (from 5.22⫾1.36 to
16.81⫾3.13 mM). Cardiomyocytes were superfused with Tyrode⬘s solution containing 2mM
Ca2⫹ and 100nM isoproterenol with or without 10mM CGP-37157. Mitochondrial NADH,
expressed as percentage of reduced NADH pool, was recorded at rest and during 4Hz
stimulation, followed by a return to the resting state. In normal cardiomyocytes, NADH level was
maintained during 4Hz stimulation. However, in most cardiomyocytes from failing heart, NADH
levels decreased dramatically (⬃35%) during stimulation. CGP-37157 recovered NADH
production of cells from failing heart during stimulation and decrease of [Na⫹]i by patch clamp
with 5mM Na⫹ in pipette solution also prevented the impaired NADH response in heart failure.
Decrease of [Na⫹]i to 5mM also enhanced [Ca2⫹]m accumulation while compared to that of cells
patch clamped with 15mM Na⫹. We conclude that high intracellular Na⫹ level in heart failure
impairs mitochondrial bioenergetics, and this impairment can be prevented by inhibiting the
mitochondrial Na⫹/Ca2⫹ exchanger. The findings support our hypothesis that mitochondrial
Na⫹/Ca2⫹ exchanger is a potential therapeutic target to improve the function of failing heart.
P168
Age-Associated Mitochondrial Dysfunction and Cardiac Hypertrophy in
Cardiac Targeted Transgenic Models of Mutations in TK2 or Pol ␥ with
NRTI Treatment
James J Kohler, Seyed Hosseini, Ioan Cucoranu, Amy Hoying, Elgin Green, Earl Fields,
David Johnson, Rodney Russ, William Lewis; Emory Sch of Medicine, Atlanta, GA
Background: Mitochondrial biogenesis is critical to normal cardiac performance. DNA
polymerase gamma (Pol ␥) and thymidine kinase 2 (TK2) are two enzymes involved in mtDNA
replication. Point mutations in Pol ␥ and TK2 identified in clinical diseases are associated with
mtDNA depletion syndromes and organ dysfunction. Because nucleoside reverse transcriptase
inhibitors (NRTIs) can also disrupt mtDNA replication, we examined the impact of mutant Pol
␥ (Y955C) or TK2 (H121N and I212N) in combination with NRTI-based HAART on cardiac
function over time. Methods: Targeted transgenic mice (TG) overexpressed Y955C, H121N or
I212N in cardiac tissues driven by the ␣-MyHC promoter. Changes in cardiac and mitochondrial
structure and function were examined by real-time PCR for mitochondrial (mt) DNA abundance,
TEM and ECHO. TGs and wild-type (WT) littermates were treated with vehicle or NRTI-based
HAART (ziduvodine, lamivudine, and indinavir) for 3 and 10 wks. Results: NRTI treatment of
WTs had no impact on mtDNA abundance. In Y955C TGs, mtDNA abundance decreased
independent of NRTI treatment or age. In contrast, H121N TGs temporally increased mtDNA
abundance early (3 wks), returning to comparable WT levels at 10 wks. mtDNA abundance in
I212N TGs increased with or without NRTI treatment at 3 and 10 wks compared to WT
littermates. Ultrastructural changes occured in all TGs and WTs with 10 wks of NRTI treatment
and also in untreated Y955C TGs. LV Mass increased in WTs after 10 wks NRTI treatment. In
Y955C TGs, LV Mass and LVEDD increased early (3 wks) with and without NRTI treatment
compared to WT littermates. I212N TGs had increased LV Mass with or without treatment (3
wks), with additional increase in LV Mass and increased LVEDD at 10 wks NRTI treatment.
Untreated H121N TGs had no change in LV Mass at 3 wks, but increased LV Mass and LVEDD
at 10 wks with vehicle or NRTI treatment. Conclusions: Disparate changes in mtDNA occur
early in Y955C and TK2 mutant TGs and persist (exception of H121N). Cardiac hypertrophy and
dilation progresses in all three TGs over time, although more quickly in Y955C TGs. These
pathogenic point mutations in genes involved in mtDNA replication appear to increase risk for
cardiac hypertrophy and are compounded by NRTI treatment.
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P169
The Expression Pattern of T-Type Ca2ⴙ Channels Changes During Bone
Marrow-Derived Smooth Muscle Progenitor Cells Differentiating into
Smooth Muscle Cells
Ryota Hashimoto, Juntendo Univ Sch of Medicine, Tokyo, Japan; Youichi Katoh, Juntendo
Univ Sch of Medicine Urayasu Hosp, Urayasu, Japan; Seigo Itoh, Juntendo Univ Sch of
Medicine Shizuoka Hosp, Shizuoka, Japan; Hiroyuki Daida, Juntendo Univ Sch of Medicine,
Tokyo, Japan; Yuji Nakazato, Juntendo Univ Sch of Medicine Urayasu Hosp, Urayasu, Japan;
Takao Okada; Juntendo Univ Sch of Medicine, Tokyo, Japan
Background — Functional expression of L- and T-type Ca2⫹ channels is developmentally
regulated in native vascular smooth muscle cell (VSMC)s. We and others have recently been
published the presence of putative smooth muscle progenitor cells in the bone marrow and that
smooth muscle-like cells may be differentiated from bone marrow stromal cell (BMSC)s.
However, few studies have addressed whether the differentiated cells also possess the
functional properties of smooth muscle cell (SMC)s, including functional expression of L- and
T-type Ca2⫹ channels. Methods and Results — To investigate whether BMSC-derived SMCs
exhibit functional vascular SMC properties, voltage-gated Ca2⫹ currents were measured using
whole-cell patch clamp recordings in BMSC-derived SMCs during development. Whereas only
10% (1 of 10) of BMSC-derived SM progenitor cell (BMSC-SMPC)s showed T-type Ca2⫹ channel
current on 0 week, almost all BMSC-SMPCs showed T-type Ca2⫹ channel current after one
week. After two weeks, 40% (5 of 12) of BMSC-SMPCs showed T-type Ca2⫹ channel current
and other 60% showed L-type Ca2⫹ channel current. Conclusions — BMSC-derived SMCs
exhibit functional and physiological properties of vascular SMCs, including voltage-dependent
Ca2⫹ channel currents. Like native vascular SMCs, functional expression of L- and T-type Ca2⫹
channels is developmentally regulated in BMSC-derived SMCs. Since BMSC-SMPCs have the
potential to differentiate into functional vascular SMCs, they can be reliable and expandable
SMC sources for the construction of tissue-engineered vascular grafts.
P170
Priming with Angiopoietin-1 Augments Neovasculogenic Potential of the
Peripheral Blood Stem Cells Mobilized with G-CSF
Jun-Bean Park, Hyun-Jai Cho, Wonjae Lee, Min-Seok Kim, Choon-Soo Lee, Seoul National
Univ Hosp, Seoul, Republic of Korea; Gou-Young Koh, Korea Advanced Institute of Science
and Technology, Daejeon, Republic of Korea; Dong-Soo Lee, Kyung-Woo Park, Hae-Young
Lee, Hyun-Jae Kang, Byung-Hee Oh, Young-Bae Park, Hyo-Soo Kim; Seoul National Univ
Hosp, Seoul, Republic of Korea
Background: The low engraftment rate of stem/progenitor cells which are infused via the
intracoronary route to the ischemic myocardium is the one of the most important factors
limiting the efficacy of cell therapy. We investigated the concept of priming the peripheral blood
stem cells enriched by G-CSF mobilization and apheresis (mobPBSCs) with angiopoietin-1
(Ang1), to enhance the engraftment into the ischemic tissue and neovasculogenic potential.
Methods and Results: In the comparative analysis of several biologic markers between
mob
PBSCs and naı̈ve peripheral blood mononuclear cells (PBMCs), the expression of Tie2, the
Ang1 receptor, was significantly higher in mobPBSCs than PBMCs. (19.2⫾3.0 % vs. 1.2⫾0.8 vs.
1.2⫾0.2, p⬍0.001 for mobPBSCs vs. PBMC AMI vs. PBMC SA).( mobPBSCs from 33 patients
of AMI with G-CSF treatment for 3 days vs. PBMCs from 15 patients with AMI vs. PBMCs from
20 patients with stable angina). After 4 hours of COMP-Ang1 (400 ng/ml) stimulation, mobPBSCs
committed to the endothelial lineage with induction of CD31 and VE-Cadherin expression,
which was mediated by newly-found Tie2/Ets-1 pathway. Priming of mobPBSCs with COMPAng-1 induced the expressions of ␣4␤1 and ␣5␤1 integrins that are also est-1 downstream
molecules, leading to enhanced adhesion to endothelial cells or fibronectin. In rabbit ear
ischemia-reperfusion model, priming of mobPBSCs with COMP-Ang1 resulted in significantly
improved first pass engraftment to distal vascular bed after intra-arterial delivery. In a murine
ischemic hindlimb model, intravascular delivery of primed mobPBSCs enhanced both engraftment and neovascularization. Conclusions: These results indicate that short-term priming of
stem/progenitor cells using COMP-Ang1 may be a feasible and promising option to functionally
augment mobPBSCs by enhancing differentiation and adhesiveness, and to improve the efficacy
of intracoronary cell therapy for patients with myocardial infarction.
P171
Treatment of Acute Myocardial Infarction versus Heart Failure with a
Viable 3-Dimensional Fibroblast Patch
Jordan J Lancaster, Southern Arizona VA Med Cntr, Tucson, AZ; Robert S Kellar, Theregen
Inc, San Francisco, CA; Hoang M Thai, Elizabeth Juneman, Howard G Byrne, Nicholle M
Johnson, Maribeth Stansifer, Monika Schmelz, Steven Goldman; Southern Arizona VA Med
Cntr, Tucson, AZ
Background: Recent clinical trials utilizing cell-based therapies for acute myocardial infarction
(MI) have reported disappointing results. Because of this, we are proposing the use of a viable
3-dimensional fibroblast construct (3DFC) on the infarcted heart. The 3DFC patch provides a
matrix support structure and growth factor stimulation for new cell and blood vessel growth.
This study was designed to compare implanting the 3DFC at the time of the acute MI in rats
versus 3 weeks after coronary ligation when the rat is in heart failure. Methods: Acute MI is
created by ligating the left coronary artery of rats; the 3DFC is implanted at the time of the MI
versus 3 weeks later. N⫽8 –12 in each group. Results: With implanting the 3DFC at the time
of the MI vs. 3 weeks later, there are increases (P⬍0.05) in LV ejection fraction (EF): 40 and
21%, systolic displacement of the infarcted anterior wall (64 and 43%), and myocardial blood
flow (37 and 116% ml/kg/min). While implanting 3DFC at the time of the MI improves LV
remodeling by deceasing (P⬍0.05) LV end-diastolic dimension 19%, implanting the 3DFC with
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Circulation Research
Vol 103, No 5
August 29, 2008
the rat in heart failure does not alter LV remodeling; LV end-diastolic dimension does not
change. Implanting the 3DFC at either time does not alter LV hemodynamics. Conclusion: We
report improvements in LV function and myocardial blood flow in both acute MI and chronic
heart failure treated with a 3DFC patch. This patch partially reverses LV remodeling only if
implanted during an acute MI. Our conclusion is that improving matrix support and myocardial
blood flow prevents adverse LV remodeling only in an acute MI; it does not reverse LV
remodeling in chronic heart failure.
P172
Tumor Necrosis Factor-␣ p75 Receptor Is Required in Post-MI Recovery in
Adult Heart
David A Goukassian, Tkebuchava Tengiz, Marcy Silver, Sharath Sasi, Hui-Ya Gilbert,
Huin-Jai Chow, Tufts Univ, Boston, MA; Young-sup Yoon, Emory Univ, Atlanta, GA; Raj
Kishore; Northwestern Univ, Chicago, IL
(MuRF-1), increase protein degradation in vivo and in vitro. The UPS breaks down proteins in
an ATP-dependent manner. Because the central determinant of the levels of ATP in the cell is
5’ Adenosine Monophosphate Kinase (AMPK), we propose that AMPK plays a role in the
regulation of Mafbx and MuRF-1, thereby reversing cardiac hypertrophy through the UPS. In
order to investigate the role of AMPK in the activation of Mafbx and MuRF-1 in vitro and in vivo,
we used two model systems: neonatal rat ventricular myocytes (NRVM) and WT and Mafbx and
MuRF-1 knockout mice. In both models, AMPK was activated by treatment with the
anti-diabetic drug Metformin or AICAR (5’-phosphoribosyl-5-aminoimidazole-4-carboxamide).
Our data show that Metformin and AICAR both increase transcript and protein levels of
Mafbx/Atrogin 1 and MuRF-1 in vitro. We conclude that AMP kinase potentially activates the
UPS in cardiomyocytes. One of the consequences may be enhanced availability of amino acids
for energy provision.
P176
Evidence for Direct Glutathionylation of the Cardiac L-Type Ca2ⴙ Channel
Protein Responsible for Altered Calcium Signaling During Oxidative Stress
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Aging is a risk factor for coronary and peripheral artery disease. The role of TNF-␣ receptors
(TNFR1/p55 and TNFR2/p75) in post-Acute Myocardial Infarction(post-AMI) recovery is not well
understood. We hypothesized that signaling through TNFR2/p75 may be essential in post-AMI
regeneration in adult heart. To test our hypothesis we evaluated post-AMI survival, cardiac
function and angiogenesis in AMI model (LAD ligation) in age-matched young- and old-WT,
-p75KO and -p55KOs. In old-WTs there was significant 4-fold age-associated increase in
post-AMI mortality. In young-p75KOs post-AMI mortality was approaching mortality in old-WTs
(35% vs. 45%) whereas ⬎1/2 of old-p75KOs (60%) died within 7 days post-AMI. No post-AMI
mortality was observed in young-p55KOs and in old-p55KOs was lower than in young-WTs
(10% vs. 15%). Compared to young-WT, between days 7 and 28 post-AMI functional recovery
(% LV fractional shortening-FS) was 10% lower in old-WTs and young-p75KOs (p⬍0.05) and
more than 20% lower in old-p75KOs (p⬍0.001). To evaluate ongoing MI injury, we
immunostained hearts with cardiac troponin I (cTnI). Compared to non-infarcted tissue, cTnI
expression was significantly increased in infarct border-zone of young-p75KOs, old-WTs and
old-p75KOs 7 days post-AMI and cTnI was the highest in old-p75KOs. Compared to young-WTs
there was significant decrease in capillary density (BS1-lectin staining) and the number of
functional vessels (BS1-lectin perfusion) in old-WT and young-p75KOs in infarct/infarct
border-zone. There was further 3-fold post-AMI decrease (p⬍0.001) in functional vessels in
old-p75KOs compared to old-WTs. To corroborate our findings to human condition peripheral
blood (PB) endothelial progenitor cells (EPCs) from young volunteers and adult CAT-lab patients
were evaluated for expression of angiogenic, pro-survival (VEGF, ANG-2, HIF-1alpha, ET-1) and
stem cell-derived factors, receptors (SDF1, CXCR4 and GCSFR) by qRT-PCR. There was
significant 2–5-fold (p⬍0.02) decreased in EPCs from adult patents. Our data strongly suggests
a critical role of signaling through TNFR2/p75 in post-AMI recovery in adult tissue through
impairment of several ischemia-induced repair and regeneration processes.
William A Macdonald, Livia C Hool; The Univ of Western Australia, Crawley, Australia
Increased generation of reactive oxygen species and elevated intracellular calcium are a feature
of cardiovascular disease. In isolated cardiac myocytes current through the L-type Ca2⫹
channel is increased during mild oxidative stress induced by 30ı̀M hydrogen peroxide (H2O2).
This results in an increase in diastolic calcium that further enhances generation of superoxide
from the mitochondria (supporting the “ROS-induced ROS-release” hypothesis). Of the various
cellular modifications that occur with oxidative stress, there is increasing evidence that
glutathionylation predominates in cells because glutathione is the most abundant low molecular
mass thiol. The aim of this study was to determine whether the function of the L-type Ca2⫹
channel is altered during oxidative stress as a result of direct glutathionylation of thiol groups
on the channel protein. In isolated cardiac myocytes, the thiol-specific reducing agent
dithiothreitol decreases macroscopic current while the thiol-specific oxidizing agent 5,5’-dithiobis[2-nitrobenzoic acid] (DTNB, 200␮M) reverses these effects. To determine whether this is
due to a direct effect on thiol groups on the channel protein, we purified the long N terminal
isoform of the human ␣1C subunit of the L-type Ca2⫹ channel protein and reconstituted the
channel protein into liposomes. Exposure of liposomes to the thiol-specific reducing agent
dithiothreitol (1mM) decreased Po by 48% (P⫽0.003, n⫽7) while DTNB (200␮M) increased
channel open probability (Po) by 98% (P⫽0.033, n⫽9). In addition 5mM GSSG increased Po 3
fold (P⬍0.001, n⫽5), while 0.5mM GSH resulted in a 5 fold decrease in Po (P⬍0.001, n⫽4).
Mild oxidative stress induced by addition of 30ı̀M H2O2 significantly increased Po by 67%
(P⫽0.049, n⫽6). This effect was reversed with washout of H2O2 indicating that lipid
peroxidation does not occur. We treated lysate from guinea pig heart with 30ı̀M H2O2. Western
blot analysis using an anti-GSH antibody and an anti-L-type Ca2⫹ channel antibody confirmed
that glutathionylation of the L-type Ca2⫹ channel occurred following exposure to 30ı̀M H2O2.
This study indicates that changes in the glutathionylation state of the channel account for
altered L-type Ca2⫹ channel function during oxidative stress.
P173
Connexin43-Induced Inhibition of Cardiomyocyte DNA Synthesis Is Not
Dependent on Downstream Activation of TGF␤ Signal Transduction
P177
Antibody Microarray Analysis of Failing Human Heart Ventricles Reveals
Novel Signaling Protein Abnormalities
Madhumathy Jeyaraman, Barbara Nickel, Elissavet Kardami; Institute of Cardiovascular
Sciences, St Boniface Rsch Cntr, Univ of Manitoba, Winnipeg, Canada
We have shown that the cytokine Transforming growth factor ␤ (TGF␤), as well as the
membrane phosphoprotein Connexin43 (Cx43), in its ’inhibitory’ state (lacking phosphorylation
at the mitogen-sensitive site serine 262), can both inhibit mitogenic stimulation of cardiomyocytes. There is some evidence of potential cross-talk between the two inhibitory pathways: (a)
Cx43 was recently reported to activate intracellular signals and genes traditionally associated
with TGF␤, (b), pilot data in our laboratory showed that the C-terminal of Cx43 was capable of
interacting with cardiac Smad2. We thus investigated the hypothesis that Cx43 inhibition of
primary neonatal cardiomyocyte DNA synthesis is dependent on components of the TGF␤
pathway. Conversely, we examined whether TGF␤ would promote the growth ’inhibitory’ state
of Cx43. Pharmacological inhibition of TGF␤ receptor TGF␤RI by SB431542, while effective in
preventing Smad2 phosphorylation (and thus its ability to signal by translocating to the nucleus)
had no effect on Cx43-mediated growth inhibition. Similarly, neither the overexpression of
dominant-negative TGF␤RII nor dominant-negative Smad2 (or Smad3) could restore DNA
synthesis in Cx43-inhibited cardiomyocytes. On the other hand, TGF␤ prevented the
mitogen-induced and protein kinase C-mediated phosphorylation of Cx43 at serine 262,
detected by phospho-specific antibodies. Taken together our data suggest a cardiomyocyte
growth inhibition scenario in which TGF␤ acts upstream of Cx43, promoting its inhibitory
potential by preventing its phosphorylation at S262. Subsequently, Cx43-mediated growth
inhibition does not require downstream engagement of TGF␤ signals.
P174
Myocardial Regulation of Ubiquitin Ligases by AMP-Kinase
Kedryn K Baskin, Peter Razeghi, Heinrich Taegtmeyer; Univ of Texas Med Sch, Houston, TX
Left ventricular hypertrophy (LVH) is an independent risk factor for cardiovascular morbidity and
mortality and the regression of LVH improves cardiac function. Conventional strategies used to
reverse cardiac hypertrophy currently focus on decreasing pro-hypertrophic signaling. However, this approach has often been unsuccessful because of the vast redundancy within the
pro-hypertrophic signaling network. Here we propose a new approach to reverse cardiac
hypertrophy: Through the activation of pro-atrophic signaling pathways. Cardiomyocyte size is
determined by the balance of protein synthesis and degradation. Studies in skeletal muscle
have already shown that the Ubiquitin Proteasome System (UPS) is the major signaling pathway
responsible for skeletal muscle atrophy. The activation of the two muscle-specific ubiquitin
ligases, Muscle atrophy F-box protein (Mafbx/Atrogin-1) and Muscle Ring Finger protein 1
Lori A Walker, Andrea M Vitello, Peter M Buttrick; Univ of Colorado HSC, Denver, CO
Several biochemical signaling pathways contribute to heart failure including those involved in
inflammation, apoptosis, and proliferation. However, it is unclear what signaling pathways are
chamber and/or disease specific. The purpose of this study was to elucidate novel protein
changes that might distinguish right (RV) and left (LV) ventricular failure using a protein
microarray strategy. Human tissue samples were obtained from control and failing right and left
ventricles and samples were homogenized, protein concentration was measured, and an equal
amount of protein sample was labeled with either cyanine 3 or cyanine 5 fluorophores. Samples
were mixed to provide the following comparisons: control RV vs. control LV, control RV vs.
failing RV, control LV vs. failing LV, failing RV vs. failing LV. Protein mixtures were incubated
with an antibody microarray (Clontech). Microarrays were imaged using a Perkin Elmer
microarray scanner and relative cyanine 5 to cyanine 3 fluorescence was determined. Changes
in the cyanine 5 to cyanine 3 ratios were analyzed for 505 different proteins. Interestingly, our
antibody microarray findings indicate that there were no significant protein differences between
control RV and control LV. However, IL-1b and DMPK were upregulated in failing RV compared
with control RV; Caspase-9/ICE - LAP6/Apaf-3 and FADD/ Mort-1 were downregulated in failing
LV compared to control LV; NF-kB, doublecortin, PARP, and nexilin were upregulated in failing
LV compared to control LV; p96 and flotillin2/ ESA were upregulated in failing RV compared to
failing LV; and CSK and cytochrome c/ Apaf were upregulated in failing LV compared to failing
RV. Preliminary western blot data confirm several of the microarray findings. Together, these
data provide the foundation for future studies aimed at the elucidation of novel signaling
pathways that are both chamber and disease specific. By elucidating these mechanisms it may
be possible to design chamber specific therapies that might ameliorate the pathologic changes
observed in heart failure.
P178
Neuregulin-1 Alleviated Doxorubicin-Induced Downregulation of Cardiac
Troponin I and Cardiac Troponin T in the Heart
Yun Bian, Maoyun Sun, Marcy Silver, Caritas St Elizabeth’s Med Cntr, Boston, MA; Kalon K
Ho, Beth Israel Deaconess Med Cntr, Boston, MA; Mark A Marchionni, NRG Biotech,
Arlington, MA; Anthony Caggiano, Acroda Therapeutics, Inc, Hawthorne, NY; Peter M Kang,
Beth Israel Deaconess Med Cntr, Boston, MA; James P Morgan, Xinhua Yan; Caritas St
Elizabeth’s Med Cntr, Boston, MA
Our previous studies showed that heterozygous knock out of the Neuregulin-1 (NRG1) gene
aggravated doxorubicin cardiotoxicity. Here, we tested whether NRG1 injection improved
BCVS Conference 2008 Abstracts
cardiac function in doxorubicin injured mice and the underlying mechanisms. Methods:
C57BL/6 mice were injected with a single dose of doxorubicin (Dox, 20mg/kg, i.p.) with or
without concomitant treatment of NRG1. Cardiac function was analyzed five days after Dox
injection by hemodynamic measurements. In vitro studies were performed in neonatal rat
cardiomyocytes (NRCM). Results: Cardiac function was improved in Dox-NRG1 vs. Dox-Placebo
mice. The protein levels of cardiac troponin I (cTnI) and troponin T (cTnT) were decreased in
Dox-Placebo vs. control mice, but were maintained in Dox-NRG1 hearts. Studies in RNCM
showed that Dox decreased both mRNA and protein levels of cTnI and cTnT. NRG1 abolished
these effects. NRG1’s effects on maintaining cTnI and cTnT proteins were blocked by PI3K
inhibitor LY294002, an Akt inhibitor or inhibitor for translation cycloheximide. The downregulation of cTnI and cTnT was also abolished by caspase inhibitor Z-VAD and proteasome
inhibitor MG132, but not by lysosome inhibitor Bafilomycin A1. Further, inhibitors for
caspase-3, 6, 9 and 13 abolished both down-regulations of cTnI and cTnT. Inhibitors for
caspase-2, 5 and 10 abolished down-regulation of cTnI but not cTnT. Dox increased the activity
of caspase- 3, 6, 9 and10; NRG1 inhibited these effects of Dox, while LY294002 abolished the
effects of NRG1. In addition, Dox increased the ubiquitinylation of cTnI and NRG1 abolished this
effect. Dox decreased the phosphorylation of P70S6K, S6, 4EBP and EIF4G, while NRG1
maintained the phosphorylation level of these proteins. Using transgenic mice with cardiac
specific overexpression of a constitutively active PI3K (CaPI3K) and a dominant negative PI3K
(dnPI3K), we further showed that both survival and cardiac function were significantly improved
in Dox-CaPI3K vs. Dox-WT mice, but were lower in Dox-dnPI3K vs. Dox-WT mice. Conclusion:
NRG1 alleviated Dox induced down-regulation of cTnI and cTnT by inhibiting caspase and
proteasome degradation and increasing transcription and translation. PI3K plays an important
role in mediating NRG1’s effects.
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P179
Subinflammatory Amount of Interleukin-1 Is Constitutively Produced by
Cyclically Stretched Cardiac Cells and Induces IGF-1 Through JAK/STAT
Pathway, Which Is Required for Compensated Hypertrophy in
Pressure-Overloaded Heart
Mitsuhiko Okigaki, Shoken Honsho, Susumu Nishikawa, Hiroaki Matsubara; Kyoto
Prefectural Univ Sch of Medicine, Kyoto, Japan
Background: Heart are consitutively exposed to cyclically stretch. In pressure-overload, this
stress is aggravated and to prevent cardiac damage, compensated cardiomyocyte hypertrophy
occurs. This process is induced by various growth factors such as insulin-like growth factor-1
(IGF-1). We here demonstrated the novel role of interleukin-1 (IL-1) in IGF-1-mediated
compensated cardiac hypertrophy. Methods: Cardiac pressure-overload was performed by
banding abdominal aorta in Interleukin-1-beta-deficient-mice (IL-1-KO). Primary-cultured
cardiac fibroblasts (CF) and cardiomyocytes (CM) were exposed to cyclic stretch (12%;
60cycle/min). Results: 1) At 80-days after -pressure-overload, in WT-mice, heart-to-body
weight or size of cadiomyocyte increased to 52% (p⬍0.01) or 51% (p⬍0.01) higher than the
basal levels (each n⫽15). In IL-1-KO, these parameters were 20⫹/-1.3% or 24.8% lower
(each p⬍0.05 vs. Day-80-WT, each n⫽15), respectively, than the WT-group. Cardiac function
was 25.2% lower than WT (p⬍0.01, n⫽15) by echocardiography. 2) In the basal condition,
IGF-1-mRNA in IL-1-KO-heart was 58% (p⬍0.05) lower than WT-group. At Day-14, in
WT-heart, IGF-1-mRNA increased to 2.0-fold, whereas, in IL-1-KO-heart, it did not increase
(p⬍0.01 vs. WT-heart at day-14). In WT-CFs, 24-hours after exposure of cyclic stretch,
IGF-1-mRNA-levels increased to 3.8-fold over basal level, which was abolished by treatment
with neutralizing anti-IL-1␤-antibody. Treatment with 10ng/mL of IL-1␤ to WT-CFs increased
IGF-1-mRNA to maximum level, whereas higher dose of IL-1␤ rather inhibited IGF-1-induction.
3) Activation of JAK2/STAT5 is required for IL-1␤-induced IGF-1-induction. 4) In IL-KO-CF or
CM, phosphorylation of JNK was remarkably increased and inhibition of JNK restored
stretch-induced IGF-1-induction. 5) Stretch-induced IL-1␤-induction was mediated by stressresponsive tyrosine kinases, PYK2 or Src, and ROS. The amount of stretch-induced IL-1␤ in CF
was just 10% of LPS-induced IL-1␤ and did not induce cytokine induction to CF. Conclusion:
Sub-inflammatory amount of interleukin-1 is constitutively produced in the beating heart, and
induce IGF-1, which is required for compensated hypertrophy in the pressure-overloaded heart.
P180
MicroRNA-210 Is Upregulated in Hypoxic Cardiomyocytes
Raja K Mutharasan, Louis C Dore, Betty Kong, Tejaswitha Naik, Hossein Ardehali;
Northwestern Univ, Chicago, IL
Background: MicroRNAs (miRNAs) are a class of small RNA molecules which regulate
protein-encoding mRNA translation. They play critical roles in cardiac development and
function, yet their role in the response of cardiomyocytes to hypoxia remains unknown.
Methods: Primary cultures of neonatal rat cardiomyocytes (NRCMs) were exposed to normoxia,
1.5% O2, and 0.5% O2 for 48h. At the end of 48h the cardiomyocytes were lysed and the total
RNA fraction was collected. These samples were then hybridized to miRNA microarrays. In
addition, quantitative real-time PCR (RT-PCR) was performed to assess miR-210 levels using
the Taqman assay. Results: The microarray data from 1.5% O2 for 48h versus normoxia (n⫽3
for both groups) demonstrated that miR-210, -322, -503, -190, -126, and -21 were more than
20% upregulated, with miR-210 upregulated 2.3-fold. The microarray data from 0.5% O2 for
48h versus normoxia (n⫽3 for both groups) demonstrated that miR-210, -326, and -542–5p
were more than 20% upregulated, with miR-210 upregulated 6.6-fold. These data suggested
a robust, specific, and apparently dose-responsive upregulation of miR-210 in response to
hypoxia. We then confirmed this observation by RT-PCR, which demonstrated that miR-210 is
7.6-fold and 25-fold upregulated in 1.5% and 0.5% O2, respectively. Time course experiments
performed at 0, 1, 2, 4, 8, and 24h after exposure to 1.5% O2 showed a statistically significant
3.1-fold increase at 8h (p⫽0.015, n⫽3). Finally we assessed the length of miR-210
upregulation in NRCMs post hypoxia exposure. NRCMs were cultured in 1.5% O2 for 48h and
subsequently cultured under normoxic conditions for a further 48 and 120h. Compared to
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normoxic control, miR-210 levels at 48 and 120h post hypoxia exposure were 11.8 and
13.7-fold increased respectively (p⬍0.01 for each comparison, n⫽3). Conclusions: MiR-210
is profoundly upregulated by hypoxia in NRCMs, and may play a role in modulating the cellular
response to myocardial ischemia. Targeting miR-210 may provide a novel therapeutic option
for ischemic heart disease.
P181
Differential Roles of Rac1 and RhoA in Regulating Mechanical
Stretch-Induced Angiotensinogen Gene Expression in Cardiac Fibroblasts
Suresh K Verma, Hind Lal, Cardiovascular Rsch Institute, Temple, TX; Honey B Golden,
TAMUHSC CVRI, Temple, TX; Manuela Smith, Greg Lu, Rakeshwar Guleria, Cardiovascular
Rsch Institute, Temple, TX; Donald M Foster, Central Veterans Health Care System, Temple,
TX; David E Dostal; Cardiovascular Rsch Institute, Temple, TX
The cardiac renin-angiotensin system (RAS) has been shown to be activated in the
hemodynamically overloaded left ventricle. Angiotensin II (Ang II) is an important regulator of
cardiac remodeling and fibrosis in the mechanically overloaded myocardium. Small GTPases
regulate a wide variety of cellular processes including myocardial remodeling; however, the
role of small GTPases in mediating stretch-induced angiotensinogen (Ao) gene regulation is
poorly understood in cardiac fibroblasts (FB). The objective of the present work was to identify
downstream signaling targets responsible for mediating effects of Rac1 and RhoA on Ao gene
expression in FB. Left ventricular FB were isolated from neonatal rat hearts, plated on
deformable membranes coated with collagen IV and were exposed to 20% static-stretch. The
levels of active and GTP-bound GTPase was determined using pull-down assays, in which
GST-PBD (Pak1-binding domain) was used to assay Rac1, whereas RhoA activity was
determined using GST-Rhotekin-RBD beads. Stretch (ST) significantly activated both Rac1 and
RhoA activity and increased Ao gene expression (8h, 1.89⫾0.23; 16h, 2.96⫾0.69; 24h,
5.23⫾0.63 fold) as compared to no stretch (NS). ␤1-integrin receptor blockade, using tac-␤1
adenovirus, impaired stretch-induced Rac1 activation (2 min, 0.80⫾0.03 p⬍0.05; 15 min,
0.59⫾0.02 p⬍0.05 fold), but increased RhoA activity (2 min, 3.0⫾0.08; 15 min, 1.8⫾0.01
fold). Rac1 blockade using dominant-negative (DN) adenovirus significantly enhanced stretchinduced Ao gene expression (NS, 1.29⫾0.02, p⬍0.001; ST, 3.6⫾0.3, p⬍0.001 fold,
compared to GFP virus control), whereas Ao gene expression was significantly suppressed with
RhoA-DN treatment (NS, 2.2⫾0.1, p⬍0.01; ST, 1.48⫾0.2, p⬍0.01 fold, compared to GFP
virus control). Rac1-DN adenovirus inhibited stretch-induced JNK and AKT phosphorylation and
activated p38 phosphorylation, whereas RhoA-DN adenovirus induced JNK and AKT phosphorylation and inhibited p38 phosphorylation. In summary, this is the first study to demonstrate
that mechanical stretch-induced activation of Rac1 and RhoA have opposing roles in the
regulation Ao gene expression in FBs. Thus, Rac1 or RhoA may be viable targets for the
treatment of cardiac remodeling and hypertrophy.
P182
Regulation of T-Type Calcium Channel in Cardiomyocytes
Florentina Pluteanu, Loyola Univ Med Ctr, Maywood, IL; Christopher Hill, Leanne L Cribbs;
Loyola Univ of Chicago, Maywood, IL
The expression of low voltage activated T-type calcium channels (T-VDCC) is regulated in the
cardiovascular system during development and in pathological states such as hypertension and
cardiac hypertrophy, but the relation between T-type channel expression and the onset or
severity of the disease is not known. Therefore we used neonatal rat ventricular myocytes
(NRVM) to measure the regulation of the two cardiovascular T-type calcium channel
alpha-subunits, Cav3.1 and Cav3.2. We have used two approaches to alter the levels of
T-VDCC; (1) conditions of hypoxic injury; and (2) adenoviral knockdown or overexpression of
T-VDCC subunits. In the aforementioned conditions, we measured mRNA levels by RT-PCR in
parallel with measurements of calcium currents, action potentials and calcium transients, to
evaluate the impact of Cav3 regulation on NRVM function. In preliminary experiments, a robust
and reproducible effect on T-VDCC levels was noted in hypoxia/reoxygenation injury conditions.
After 24 hours of 1% oxygen, Cav3.1 mRNA levels decreased 4.6 fold, and Cav3.2 increased
1.9 fold (n⫽11). After 24, 48 and 96 hours fo reoxygenation, mRNA levels ov Cav3.1 were 12.8
(n⫽9), 18.7 and 11 fold (n⫽2) increased, while Cav3.2 increased 1.4, 2 and 0.9 fold compared
to hypoxic conditions, and VEGF mRNA levels as an indicator of hypoxic injury returned to
normal. Down regulation of Cav3.1 using adenoviral shRNA showed that 2 days after infection,
spontaneous beating increased from an average of 45 to 100 beats per mintue (bpm),
accompanied by a compensatory 3 fold up regulation of the Cav3.2 mRNA. High beating
frequency of calcium transients (up to 200 bpm) was also recorded when Cav3.2 was
overexpressed in NRVM. Our results suggest that both Cav3.1 and Cav3.2 alpha-subunits of
T-VDCC are differentially regulated unde pathological conditions, and their impact on
cardiomyocyte function may be subtype specific.
P183
Simulated Ischemia Activates the ATF6 Branch of the Endoplasmic
Reticulum Stress Response in Cultured Cardiac Myocytes
Shirin Doroudgar, Donna J Thuerauf, Marie M Marcinko, Christopher C Glembotski; San
Diego State Univ, San Diego, CA
Stresses that perturb the folding of nascent proteins in the endoplasmic reticulum (ER) or
cytosol can lead to the accumulation of misfolded proteins and to potential cellular dysfunction
and pathological consequences, including vascular and cardiac diseases. One of the pathways
activated by misfolded proteins in the ER is the ER stress response (ERSR), which features the
transcriptional induction of numerous genes encoding proteins that increase ER protein folding
capacity. Although activation of the ERSR program could provide protection from ischemic
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Vol 103, No 5
August 29, 2008
damage, little is known about the mechanisms of ischemia-activated ER stress in cardiac
myocytes. Accordingly, the objective of this study was to determine the mechanism by which
potentially protective ERSR genes are activated by simulated ischemia in cultured cardiac
myocytes. The ER transmembrane protein, ATF6, a primary sensor of misfolded proteins, is
cleaved during ER stress and translocates to the nucleus, serving as a potent transcription
factor that induces numerous ER-targeted proteins that enhance ER protein folding, such as
glucose-regulated protein 78 (GRP78). In this study, we determined that GRP78 levels increase
in neonatal rat ventricular myocyte cultures (NRVMC) during simulated ischemia, and that this
increase is due to transcriptional induction of the GRP78 gene. Moreover, simulated ischemia,
which induced the cleavage of ATF6 in NRVMC, also activated the GRP78 promoter and
increased GRP78 mRNA. Infection with a recombinant adenovirus encoding dominant-negative
ATF6 blocked GRP78 promoter activation by simulated ischemia. Transfection of NRVMC with
siRNA targeted to ATF6 also blocked promoter activation by simulated ischemia. The element
responsible for GRP78 promoter activation by simulated ischemia was identified by mutation
analyses, and it was shown to specifically bind ATF6 in electrophoretic mobility shift assays.
This is the first study to demonstrate ischemia-mediated activation of ATF6 and an ERSR gene
promoter in any cell type. Moreover, these results indicate that during ischemia, a potentially
novel gene program, activated by the ATF6 branch of the ERSR, may lead to the expression of
proteins that could contribute to a cardioprotective stress response.
Exposure to LPS led to downregulation of PGC-1␣ (79%) and ␤ (49%) mRNA expression and
downstream metabolic gene targets (MCPT1, PDK4, GLUT4). LPS repressed the activity of a
PGC-1␣ promoter-luciferase reporter in NRCM indicating that the regulation occurred at the
transcriptional level. Conversely, forced expression of either PGC-1␣ or ␤ in NRCM led to a
reciprocal downregulation of TNF-␣, IL-6, and MCP-1 mRNA by ⬃ 60%. Consistent with these
findings, myocardial expression of TNF-␣, IL-6, and MCP-1 was increased 2–3 fold in PGC-1␤
null mice compared to age- and strain-matched control mice. Surprisingly, LPS led to a
significant diminution in LV fractional shortening in WT but not PGC-1␤ null mice (11% vs.
36%, respectively) suggesting that at least in the short-term, LPS-mediated repression of
PGC-1␤ is adaptive. Taken together, these data suggest that the PGC-1 family of coactivators
can facilitate reciprocal regulatory “cross-talk” between metabolic and inflammatory stimuli.
P184
Stretch-Induced Regulation of Angiotensinogen Gene Expression in Cardiac
Myocytes and Fibroblasts: Opposing Roles of JNK1 and p38␣ MAP Kinases
Background: The incidence of MI rises sharply at the time of menopause, implicating loss of
estrogen (E2) in age-related increases in ischemia/reperfusion (I/R) injury. Failure of chronic E2
replacement to reduce cardiovascular mortality indicates that E2 alone is not sufficient to offset
diminished ischemic tolerance of the aged heart. Purpose: To identify new protein targets for
the treatment of acute MI in aged women using a state-of-the-art proteomics approach to
characterize cardiac mitochondrial proteins related to protein kinase C⑀ (PKC⑀), a critical
mediator of cardioprotection. We hypothesized that E2 deficiency exacerbates age-dependent
disruptions in mitochondrial responses to ischemic stress, and acute activation of PKC⑀ is
sufficient to reduce MI size in female rat heart. Methods: Langendorff-perfused hearts from
adult (5 mo, n⫽32) and aged (23 mo, n⫽32) F344 ovary-intact or ovariectomized (OVX) rats
were administered the PKC⑀-activator peptide, ␺⑀RACK conjugated to Tat (500 nM) prior to 47
min I. MI size was measured by TTC staining, and LV protein fractions were obtained by
differential centrifugation. Targeted proteomic analyses were conducted in mitochondria
isolated from hearts following control perfusions ⫾ ␺⑀RACK, using the newly released isobaric
tags for relative and absolute quantification (iTRAQ) 8plex labeling and tandem MS/MS. Results:
Greater MI size in aged OVX was associated with decreased mitochondrial targeting of PKC⑀.
Administration of ␺⑀RACK in aged OVX reduced MI size vs non-treated controls (33% vs. 78%),
partially restored mitochondrial PKC⑀ and reduced total eNOS levels. Initial MudPIT analyses
revealed n⫽638 distinct mitochondrial proteins. With iTRAQ, significant group differences were
observed in proteins related to cellular metabolism, ion transport, oxidative stress and cell
death regulation. Acute effects of ␺⑀RACK in aged were primarily limited to proteins which
regulate oxidative stress. Conclusions: Selective PKC⑀ activation reduced I/R injury in the aged
female rat heart. Proteomic analysis through iTRAQ was effective in identifying new candidate
proteins for further in-depth study of age- and E2-dependent alterations in PKC⑀-mediated
cardioprotective signaling in mitochondria.
Hind Lal, Suresh K Verma, Honey B Golden, Cardiovascular Rsch Institute, Temple, TX;
Donald M Foster, Central Texas Veterans Health Care System, Temple, TX; Manuela Smith,
David E Dostal; Cardiovascular Rsch Institute, Temple, TX
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The cardiac renin-angiotensin system (RAS) has been implicated in mediating myocyte
hypertrophy, remodeling and fibroblast proliferation in the hemodynamically overloaded heart.
The intracellular signaling mechanisms responsible for regulation of angiotensinogen (Ao), a
substrate of the RAS system, are largely unknown. Here we report the identification of JNK1
as a negative, and p38␣ as a major positive regulator of Ao gene expression in cardiac myocyte
(CM) and fibroblast (FB). Blockade of JNK1/2 with SP600125 increased Ao gene expression in
CM (10.52 ⫾ 1.98 fold, P⬍0.001) and FB (13.32 ⫾ 2.07 fold, P⬍0.001); however specific
JNK2/3 inhibitor treatment had no effect, indicating the role of JNK1. Adenovirus-mediated
expression of wild-type JNK1 inhibited stretch-mediated Ao gene expression, whereas
expression of dominant-negative JNK1 increased CM basal (3.25 ⫾ 0.32 fold, P⬍0.001) and
stretch-mediated (4.32⫾0.44 fold, P⬍0.001) and FB, basal (2.40 ⫾ 0.26 fold, P⬍0.01) and
stretch- mediated (3.98 ⫾ 0.34 fold, P⬍0.001) Ao gene expression, confirming the role of
JNK1. Blockade of p38␣/␤ by SB202190 or p38␣ by SB203580 inhibited stretch-induced Ao
gene expression in both CM (2.22 ⫾0.29 fold, P⬍0.001) and FB (2.54 ⫾ 0.21 fold, P⬍0.001).
Expression of wild type p38␣ increased the stretch-induced Ao gene expression in both CM
(8.41 ⫾ 1.50, P⬍0.001) and FB (3.39 ⫾ 0.74 fold, P⬍0.001). Conversely, expression of
dominant-negative p38␣ inhibited stretch-mediated Ao gene expression in CM (1.88 ⫾ 0.22,
P⬍0.01) and FB (2.44 ⫾ 1.30, P⬍0.01). Moreover, expression of MKK6b (E) stimulated Ao
gene expression in both CM (9.32 ⫾ 2.01 fold, p⬍0.001), and FB (3.36 ⫾ 0.56 fold, p⬍0.001),
indicating that activation of p38 by itself is sufficient to induce Ao gene expression. Prolonged
stretch diminished JNK1 activation, which was accompanied by a reciprocal increase in p38
activation and Ao gene expression. In summary, these studies indicate that p38␣ and JNK1
have opposing roles in the regulation of Ao expression. p38␣ was demonstrated to be a positive
regulator, whereas JNK1 was found to be a negative regulator of Ao. This suggests that a
balance in JNK1 and p38␣ activation determines the level of Ao gene expression in myocardial
cells.
P185
Convergence of Myocardial Inflammatory and Metabolic Signaling on the
Nuclear Receptor Coactivators PGC-1␣ and ␤
Joel D Schilling, Teresa C Leone, Ling Lai, Daniel P Kelly; Washington Univ Sch of
Medicine, St Louis, MO
Increasing evidence implicates inflammation in the pathogenesis of heart failure (HF) due to
diabetes or hypertension. PGC-1␣ and ␤ are inducible, cardiac-enriched, transcriptional
coactivators that regulate fatty acid metabolism and mitochondrial biogenesis by interacting
with transcription factors including the nuclear receptors PPAR␣ and ERR␣. Reduced
expression of PGC-1␣ has been associated with cardiac hypertrophy and HF. To investigate the
impact of inflammation on cardiac PGC-1 ␣/␤ signaling, we performed intraperitoneal LPS
injections in C57BL/6 mice. LPS triggered a rapid wave of inflammatory cytokine expression
followed by a coordinate decrease in PGC-1␣ and PGC-1␤ mRNA levels of 60% and 90%,
respectively (p ⬍ 0.05). Several known PGC-1 target genes were also downregulated by LPS
including direct targets (PPAR␣, ERR␣) and downstream genes involved in fatty acid oxidation
(MCPT1, MCAD) and glucose uptake (GLUT4). Electron microscopic analysis of heart sections
from LPS-injected mice demonstrated increased myocyte lipid accumulation, consistent with a
decrease in myocardial fatty acid oxidation. To further explore the LPS-mediated regulation of
PGC-1 gene expression, neonatal rat cardiac myocytes (NRCM) in culture were exposed to LPS.
P186
iTRAQ Proteomic Approaches Reveal Novel Mitochondrial PKC⑀-Mediated
Targets in Aged Female Rat Heart
Timothy Lancaster, Nanette Tomicek, Christopher LynchPennsylvania State Univ, Univ Park,
PA; Pennsylvania State Univ, Hershey, PA; Bruce Stanley, Donna H Korzick; Pennsylvania
State Univ, Univ Park, PA
P187
Characterization and Role of the Akt Phosphatase PHLPP in the Heart
Shigeki Miyamoto, Nicole H Purcell, Joan Heller Brown; UCSD, San Diego, CA
Akt provides strong survival signals in many cell types including the heart. The PH domain
leucine-rich repeat protein phosphatase, PHLPP, was recently discovered to dephosphorylate
Akt and turn off Akt signaling in cancer cell lines. Two closely related isoforms, PHLPP-1 and
PHLPP-2 have been identified. We have found that both PHLPP-1 and PHLPP-2 isoforms are
expressed in the heart and immunofluorescence analysis indicates that PHLPP distributes
throughout the cardiomyocyte, including the nucleus. To determine whether PHLPP suppresses
Akt activation in cardiomyocytes, PHLPP expression in neonatal rat ventricular myocytes
(NRVMs) was knocked-down by siRNA. Akt activation in response to leukemia inhibitory factor
(LIF) was potentiated and its duration was prolonged. Concomitantly the protective effect of LIF
was increased in NRVMs. We recently generated PHLPP-1 knock-out mice in collaboration with
the Newton laboratory. There is no basal cardiac phenotype (normal heart/body weight ratio
and cell size in KO mice), although the KO mice are somewhat smaller than wild-type mice.
When isolated perfused hearts from PHLPP KO mice are subject to ischemia/reperfusion (I/R),
Akt activation is increased relative to WT and TTC staining reveals smaller infarctions. These
data suggest that potentiation of Akt activation by deletion of PHLPP is protective against I/R
injury in the heart. Interestingly, hypoxia and glucose starvation both reduce PHLPP expression
in NRVMs, suggesting that PHLPP levels are regulated under stress conditions, possibly as a
protective cellular response. Conversely, treatment with LIF for 48 hrs significantly upregulates
PHLPP, a response that is dependent on Akt activity as it is inhibited PI3K or Akt inhibitors.
Upregulation of PHLPP was also observed in IGF-1 transgenic mice in which Akt is chronically
active. Taken together these results suggest that the PHLPP expression level could be an
important regulator of Akt activity and cardiomyocyte viability. We are currently characterizing
PHLPP KO mice subjected to the transverse aortic constriction to determine whether PHLPP
contributes to development of pressure overload induced hypertrophy and development of heart
failure.
BCVS Conference 2008 Abstracts
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Circ Res. 2008;103:e35-e70
doi: 10.1161/01.RES.0000335003.35117.c3
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Copyright © 2008 American Heart Association, Inc. All rights reserved.
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