Ischemic Protection and Myofibrillar Cardiomyopathy
Dose-Dependent Effects of In Vivo ␦PKC Inhibition
Harvey S. Hahn, Martin G. Yussman, Tsuyoshi Toyokawa, Yehia Marreez, Thomas J. Barrett,
K. Chad Hilty, Hanna Osinska, Jeffrey Robbins, Gerald W. Dorn II
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Abstract—To delineate the in vivo cardiac functions requiring normal ␦ protein kinase C (PKC) activity, we pursued
loss-of-function through transgenic expression of a ␦PKC-specific translocation inhibitor protein fragment, ␦V1, in
mouse hearts. Initial results using the mouse ␣-myosin heavy chain (␣MHC) promoter resulted in a lethal heart failure
phenotype. Viable ␦V1 mice were therefore obtained using novel attenuated mutant ␣MHC promoters lacking one or
the other thyroid response element (TRE-1 and -2). In transgenic mouse hearts, ␦V1 decorated cytoskeletal elements and
inhibited ischemia-induced ␦PKC translocation. At high levels, ␦V1 expression was uniformly lethal, with depressed
cardiac contractile function, increased expression of fetal cardiac genes, and formation of intracardiomyocyte protein
aggregates. Ultrastructural and immunoconfocal analyses of these aggregates revealed focal cytoskeletal disruptions and
localized concentrations of desmin and ␣B-crystallin. In individual cardiomyocytes, cytoskeletal abnormalities
correlated with impaired contractile function. Whereas desmin and ␣B-crystallin protein were increased ⬇4-fold in ␦V1
hearts, combined overexpression of these proteins at these levels was not sufficient to cause any detectable cardiac
pathology. At low levels, ␦V1 expression conferred striking resistance to postischemic dysfunction, with no measurable
effects on basal cardiac structure, function, or gene expression. Intermediate expression of ␦V1 conferred modest basal
contractile depression with less ischemic protection, associated with abnormal cardiac gene expression, and a
histological picture of infrequent cardiomyocyte cytoskeletal deformities. These results validate an approach of ␦PKC
inhibition to protect against myocardial ischemia, but indicate that there is a threshold level of ␦PKC activation that is
necessary to maintain normal cardiomyocyte cytoskeletal integrity. (Circ Res. 2002;91:741-748.)
Key Words: ischemia/reperfusion 䡲 protein kinase C 䡲 myofibrillar cardiomyopathy 䡲 congestive heart failure
he ␦ and ⑀ isoforms of protein kinase C (PKC) are
implicated in heart failure, myocardial hypertrophy, and
ischemic preconditioning, based largely on patterns of PKC
isoform translocation in human heart disease and experimental animal models.1–5 Accordingly, delineating the in vivo
consequences of PKC isoform activation in myocardial tissue
is a scientific priority. Studying these signaling molecules by
modulating their expression can potentially be confounded by
promiscuous interactions between overexpressed signaling
proteins and their downstream effectors or substrates, or by
opportunistic compensation of related signaling proteins in
gene ablation models. These concerns are especially relevant
to PKC, a group of ubiquitous enzymes composed of at least
11 different, but related, isoforms, many of which are
coexpressed in the same tissue or cell type.6 – 8 To avoid
alterations in enzyme-substrate stoichiometry that are inherent with PKC isoform overexpression or mutational ablation,
we have utilized complementary gain- and loss-of function,
achieved by modulating PKC isoform interactions with their
T
respective membrane anchor proteins, or RACKs.9 –12 In this
manner, in vivo ␦ and ⑀PKC activation revealed opposing
effects on myocardial ischemic protection, but identical
cardiotrophic influences,12 which suggested a novel therapeutic approach for myocardial ischemic salvage by ⑀PKC
activation and ␦PKC inhibition, alone or in combination.
However, growth-modifying effects of chronically activated
␦ and ⑀PKC suggest caution in manipulating their myocardial
activity. Indeed, chronic in vivo inhibition of myocardial
⑀PKC has caused a dilated cardiomyopathy,10,11 and initial
attempts to generate a viable mouse model of chronic ␦PKC
inhibition were unsuccessful due to early development of a
lethal cardiomyopathy.
In the present study, we report the results of chronic
myocardial ␦PKC inhibition through transgenic expression of
␦V1, which encodes the putative ␦PKC RACK binding
domain.12 Viable ␦V1-expressing mice were obtained using
mutant ␣-myosin heavy chain (␣MHC) promoters lacking
one or the other thyroid response element (TRE). Mice
Original received July 1, 2002; revision received August 28, 2002; accepted September 3, 2002.
From the Department of Internal Medicine, Division of Cardiology (H.S.H., M.G.Y., T.T., Y.M., T.J.B., G.W.D.), University of Cincinnati Medical
Center, Cincinnati, Ohio; and the Division of Cardiovascular Molecular Biology (H.O., J.R.), the Children’s Hospital Research Foundation, Cincinnati,
Ohio.
Correspondence to G.W. Dorn II, Division of Cardiology, University of Cincinnati Medical Center, 231 Albert B. Sabin Way, Cincinnati, Ohio
45267-0542. E-mail [email protected]
© 2002 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000037091.64492.69
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October 18, 2002
Figure 1. Schematic depiction of the
three ␦V1-␣MHC promoter constructs.
TRE indicates thyroid responsive
element.
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expressing ␦V1 at the lowest levels exhibited marked resistance to myocardial ischemia but were otherwise normal in
terms of myocardial structure, function, and gene expression.
In contrast, when expressed at higher levels, ␦V1 transgenic
mice developed, in a transgene dose-dependent fashion, a
cardiomyopathy characterized histologically and ultrastructurally by cytoskeletal disorganization and intracellular proteinaceous aggregates resembling those in human myofibrillar cardiomyopathies.13,14 In the context of ␦V1 localization
to cytoskeletal elements within cardiac myocytes, these
results indicate a role for ␦PKC not only in the myocardial
response to ischemia, but also in maintaining normal myofibrillar integrity.
Materials and Methods
Generation of Transgenic ␦V1 Mice
Analogous to the previously described ⑀PKC selective inhibitory
protein fragment, ⑀V1,9,15 ␦V1 was modeled after the first variable
region of rat ␦PKC (amino acids 2 to 144), which encodes the
putative ␦PKC RACK binding domain.12 For transgenic expression,
the cDNA for the ␦V1 peptide, preceded by a FLAG epitope, was
inserted into the SalI-HindIII site of the full-length ␣MHC promoter16 followed by the human growth hormone polyadenylation sequence (␣MHC-␦V1). For attenuated ␦V1 expression, a Bgl-II
cassette of the ␣MHC promoter, with either the first or second
thyroid responsive element individually ablated,17 was inserted into
the analogous portion of the ␣MHC-␦V1 construct, generating
TRE-1 ␣MHC-␦V1 and TRE-2 ␣MHC-␦V1 (Figure 1). All transgene constructs were confirmed by double-stranded sequencing,
microinjected into male pronuclei of fertilized mouse oocytes (FVB/
N), and implanted into pseudopregnant dames by the University of
Cincinnati Transgenic Core (directed by John Neumann). Founders
were identified by genomic Southern analysis of tail clip DNA.
Studies were performed in accordance with protocols approved by
the University of Cincinnati Institutional Animal Care and Use
Committee, and animals were supplied by the UC Transgenic Mouse
Core facility.
5% dry milk, and incubated with primary antibody and secondary
antibodies. Blots were developed with enhanced chemifluorescence
and quantitated using a Storm PhosphorImager. To accurately
quantitate cytosolic PKC␦, it was necessary to load 75 ␮g of protein,
compared with 25 ␮g in the membrane fraction. Antibodies were as
follows: anti–␣B-crystallin (Calbiochem), anti–FLAG-M2 and antidesmin (Sigma), anti–␦ and ⑀PKC, and anti– heat shock protein 60
(Santa Cruz).
Dot-Blot Analysis
mRNA levels were quantified by dot blotting as described.18,19 Total
ventricular RNA was applied (2 ␮g/dot) to nylon membranes,
hybridized with 32P-labeled antisense oligonucleotides specific for
atrial natriuretic factor (ANF), ␣MHC, ␤MHC, sarcoplasmic reticular ATPase (SERCA), ␣ skeletal actin, or glyceraldehyde-3phosphate dehydrogenase (GAPDH), and quantified using a Storm
PhosphorImager (Molecular Dynamics).
Ischemic Injury and Recovery
Isolated hearts from 12- to 20-week-old mice were Langendorffperfused for 20 minutes for equilibration, followed by 40 minutes of
no-flow ischemia and a 20-minute reperfusion period.12 Contractile
function was measured as left ventricular developed pressure (LVP)
and the peak rate of increase of pressure (⫹dP/dt). Creatine kinase
(CK), a marker of myocyte injury, was assayed in the perfusate using
a Sigma kit.
Invasive Hemodynamics
Polyethylene catheters were introduced into the femoral artery and
vein and a 1.4F Millar catheter placed into the left ventricle through
the right carotid artery to monitor real-time heart rate, arterial and
left ventricular pressures, and ⫹dP/dt and ⫺dP/dt. Data were
archived on a G4 computer (Apple Computers) using MacLab
software and interface.19
Myocyte Mechanics
Isolated adult ventricular myocytes were prepared20 and analyzed
according to genotype and presence of intracellular aggregates
determined by phase contrast microscopy. Myocyte ⫹dL/dt (peak
rate of unloaded shortening), ⫺dL/dt (peak rate of relengthening), and
percent shortening were recorded during field stimulation at 0.25 Hz.
SDS-PAGE and Immunoblot Analysis
Western blotting was performed as described.9 Briefly, myocardial
homogenates were fractionated by differential centrifugation into
cytosolic (100 000g supernatant) and Triton X-100 – extracted particulate (100 000g pellet) fractions, size-separated on 10% SDSPAGE gels, transferred to polyvinylidene membranes, blocked with
Microscopy
Routine histological examination was performed on formalin-fixed
Masson’s trichrome-stained sections. Samples were preserved in
glutaraldehyde for ultrastructural analysis. Immunoconfocal studies
used desmin, ␣B-crystallin, vimentin, and ␦PKC antibodies (the
Hahn et al
␦PKC Cardiomyopathy
743
latter a gift from Dr Daria Mochly-Rosen, Stanford University,
Stanford, Calif), all at a 1:200 dilution, with Texas red- or fluorescein-conjugated secondary antibodies (1:200). Imaging was performed with a dual laser Nikon PCM2000 system and a Nikon
Eclipse E800 microscope using emission wavelengths of 515⫾30
nm (fluorescein) and 605⫾32 nm (Texas Red).
Statistical Analysis
Results are presented as mean⫾SEM. Experimental groups were
compared using Student’s t test or 1-way ANOVA, as appropriate.
The Bonferroni test was applied to all significant ANOVA results
using SigmaStat software. A value of P⬍0.05 was considered
significant.
Results
Premature Lethality and Myofibrillar
Cardiomyopathy of ␣MHC-␦V1 Transgenic Mice
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Our prior studies of transgenic mice expressing a ␦PKC
activating peptide revealed exaggerated postischemic injury.12 We therefore considered whether inhibition of ␦PKC
could be cardioprotective. To test this possibility, transgenic
mice were created expressing the ␦PKC inhibitory protein
fragment, ␦V1, in the heart. However, the response of these
␦PKC-inhibited hearts to ischemia could not be assessed due
to early lethality. Of three original ␣MHC-␦V1 founders, two
died before breeding, whereas the third sired only 9 first
generation ␦V1 transgenic mice (out of 245 pups; 3.7%)
before he also died. No ␣MHC-␦V1 first generation mice
survived long enough to breed; all were dead by the age of 41
days (mean 27⫾3 days). Before death, ␣MHC-␦V1 transgenic mice appeared lethargic, with rapid respirations and
tissue edema, suggesting heart failure. Indeed, although only
one ␣MHC-␦V1 mouse survived to an age and size sufficient
for invasive hemodynamic evaluation, this mouse exhibited a
64% reduction in peak dobutamine-stimulated ⫹dP/dt compared with its control (NTG⫽19 877 versus ␣MHC␦V1⫽6720 mm Hg/s), and pathological examination of all 9
␣MHC-␦V1 mice revealed pleural effusions, ascites, and
enlarged, thick-walled hearts with intramural thrombi (Figure
2A). Compared with NTG siblings, whole-heart weights of
␣MHC-␦V1 mice were increased by 89% (93⫾38 mg NTG
versus 176⫾13 mg ␣MHC-␦V1; P⬍0.001), and lung weights
were increased, indicating pulmonary congestion (158⫾20
mg NTG versus 215⫾21 mg ␣MHC-␦V1; P⫽0.024). The
thick-walled ␦V1 phenotype contrasts with dilated cardiomyopathy in the analogous ⑀PKC inhibitor (⑀V1) transgenic
mice (Figure 2A) (which is, however, equally lethal10,11).
Immunoconfocal microscopy of ␦V1 using the incorporated
FLAG-epitope (Figure 2G) revealed decoration of cytoskeletal elements, similar to anti-␦PKC (Figure 2F).21 This
pattern of subcellular localization contrasts with localization
of ⑀PKC-derived ⑀V1 to cross-striated elements.10
A unique feature of the ␦V1 phenotype, and one that
could explain the development of lethal heart failure
without ventricular dilation, was frequent intramyocyte
blue-staining areas on Masson’s trichrome-stained myocardial sections that did not specifically label for mucin,
collagen, glycogen, or mucopolysaccharides (Figure 2B).
On ultrastructural examination these areas corresponded to
Figure 2. ␦V1 expression induces myofibrillar cardiomyopathy.
A, Representative 30-day-old NTG, ␦V1, and ⑀V1 hearts. B, Histological section (Masson’s trichrome) from a ␦V1 heart demonstrating protein aggregates (*). C, Electron micrograph of ␦V1
heart demonstrating amorphous regions (*) and sarcomere
streaming. D, Aggregate labeled with desmin. E, Aggregate
stained for ␣B-crystallin. F, Anti-␦PKC and (G) anti-FLAG-␦V1
demonstrating an identical cytoskeletal pattern, but not staining
within the aggregates (*).
electron-dense regions of amorphous protein within focal
subcellular cytoskeletal distortions (Figure 2C). Mitochondria, other cellular organelles, and streaming fragments of
sarcomeres were visualized within these areas (see online
Figure in the online data supplement available at
http://www.circresaha.org).
The distortions of cardiomyocyte subcellular architecture
in ␣MHC-␦V1 hearts closely resemble those seen in human
myofibrillar myopathies13,14,22 and their genetically modified
mouse cardiomyopathy analogs.23,24 In these myopathies,
blue-staining intramyocyte protein aggregates contain high
levels of desmin and ␣B-crystallin, due to mutations affecting
one or the other protein.13,14 We therefore examined ␦V1expressing cardiomyocytes for similar immunohistological
features. As shown in Figures 2D and 2E, the intracellular
aggregates in ␦V1 myocardium stained positively for both
desmin and ␣B-crystallin, the latter of which, as in human
myofibrillar myopathies,14,22 was preferentially localized to
the periphery (Figure 2E). In contrast, ␦V1 staining was not
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October 18, 2002
Figure 3. Increased expression of
desmin and ␣B-crystallin is not sufficient
to cause cardiomyopathy. A, Representative Western blots demonstrating
increased desmin and ␣B-crystallin in
␦V1 mice. B, Representative Western
blots for desmin and ␣B-crystallin in
desmin (Des), ␣B-crystallin (␣B), NTG,
and dual transgenic (Des/␣B) hearts. C,
Normal in vivo contractile function at
baseline and with peak dobutamine. D,
Masson’s trichrome section demonstrating absence of myocyte aggregates.
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more prominent within the aggregates (Figure 2G), and there
was no qualitative change in desmin or ␣B-crystallin in
normal-appearing ␦V1 cardiomyocytes (not shown). Not
surprisingly, given the abnormal accumulation of these proteins into intramyocyte aggregates, immunoblot analysis
showed that expression of both desmin and ␣B-crystallin was
increased approximately 4-fold in ␣MHC-␦V1 myocardium,
compared with nontransgenic (Figure 3A). In contrast, expression of a related chaperone, HSP60, was not altered
(Figure 3A). A direct phosphorylation effect of ␦PKC on
desmin and ␣B-crystallin was excluded by 2-dimensional
immunoblotting, which showed no differences in phosphorylation states between hearts from ␣MHC-␦V1, nontransgenics, and the recently described ␦PKC activator mice12 (not
shown).
Given the previously established roles of mutant desmin
and ␣B-crystallin in human myofibrillar myopathies, their
upregulation and localization within protein aggregates in
cardiomyopathic ␦V1 hearts raised the possibility that increased expression of these two proteins in combination
might contribute to the cardiomyopathy. To test this notion,
we crossbred recently developed mouse models of desmin
and ␣B-crystallin overexpression, neither of which are reported to exhibit myocardial pathology.23,24 Immunoblot
analysis demonstrated that the double transgenics maintained
the ⬇4-fold levels of desmin and ␣B-crystallin overexpression of the parent lines (Figure 3B). Confirming the previous
characterization of these mice, single transgenic animals were
normal in terms of myocardial pathology and cardiac contractile function (Figure 3C and data not shown). The desmin/
␣B-crystallin double transgenic mice also were normal in all
respects, including heart weight, ventricular contractility at
baseline and in response to dobutamine (Figure 3C and data
not shown), and myocardial histological appearance (Figure
3D). From this, we conclude that simple upregulation of
myocardial desmin and ␣B-crystallin is not sufficient to
cause the ␦V1 myofibrillar cardiomyopathy phenotype, and
infer that ␦PKC activation and targeting to the cardiomyocyte
cytoskeleton is necessary for normal cardiomyocyte structure
and contractile function.
To confirm that the observed cardiomyocyte cytoskeletal
abnormalities impaired ␣MHC-␦V1 myocardial performance,
contractile function of isolated ventricular myocytes was
assessed as a function of the presence or absence of cytoskeletal distortions. Using phase contrast optics, cardiomyocytes
with normal cellular architecture were easily distinguished
from those with intracellular aggregates. Accordingly, compared with normal-appearing ␣MHC-␦V1 cells, the unloaded
fractional shortening of abnormal ␣MHC-␦V1 cardiomyocytes was depressed by 63%, and the peak rate of unloaded
shortening was decreased by 59% (Table 1). Normalappearing ␣MHC-␦V1 myocytes had unloaded fractional
shortening similar to NTG (Table 1).
Phenotypic ␦V1 Dose-Response With Attenuated
␣MHC Promoters and Cardiac Protection With
␦PKC Inhibition
The unexpected phenotype of myofibrillar cardiomyopathy
and lethality in the ␣MHC-␦V1 mice precluded an analysis of
the effects of ␦PKC inhibition on the cardiac response to
ischemia/reperfusion injury. To obtain viable animals suitable
for testing our original hypothesis, two approaches were
undertaken. First, the original ␣MHC-␦V1 transgene construct was reinjected at lower concentrations of DNA (⬇2
versus ⬇6 ng/␮L) to obtain additional founders with fewer
TABLE 1.
Isolated Myocyte Mechanics
NTG
␣MHC-␦V1
Normal
␣MHC-␦V1
Aggregates
⫹dL/dt
117.4⫾7.2
147.4⫾14.8
48.7⫾4.8*
⫺dL/dt
⫺103.3⫾6.7
⫺125.2⫾15.2
⫺33.2⫾4.4*
Shortening, %
*P⬍0.05 vs NTG.
10.6⫾0.5
11.9⫾1.4
4.5⫾0.6*
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␦PKC Cardiomyopathy
745
Figure 4. Variable ␦V1 expression and phenotype achieved with mutant TRE ␣MHC
promoters. A, Top, Western blot analysis of
FLAG-␦V1 expression. Bottom, mRNA
expression. B, Selective inhibition of ischemia/reperfusion-induced ␦PKC translocation
in ␦V1 expressing hearts. Top, ␦PKC partitioning in NTG at baseline, and NTG, TRE-1,
and TRE-2 hearts after ischemia/reperfusion.
Bottom, Parallel studies of ⑀PKC.
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integrated copies of the transgene. Second, the ␣MHC promoter was modified in order to attenuate its expression while
retaining its characteristic cardiomyocyte-specificity. Toward
this end, we mutated the ␦V1 construct to individually ablate
the two thyroid responsive elements (TRE), which have
previously been reported to be necessary for full activity of
this promoter.16,17 Herein, the mutant promoters are designated TRE-1 when the first thyroid response element is
ablated, and TRE-2 when the second site is ablated (see
Figure 1).
Multiple founders were identified for each transgene construct, ␣MHC-␦V1, TRE-1, and TRE-2. Because results were
similar between lines for each promoter, the data have been
combined. Expression of ␦V1, measured by immunoblot blot
analysis of the incorporated amino terminal FLAG epitope,
corresponded with the anticipated activities of the promoters;
TRE-1 mice expressed 72% and TRE-2 mice 40% of the level
of the reinjected, lower copy number ␣MHC-␦V1 mouse
(Figure 4A). At levels of ␦V1 expression achieved in the
␣MHC-␦V1 and TRE-1 mice, heart weight increased in
proportion with ␦V1 expression, whereas TRE-2 hearts were
normal in size (Table 2). Likewise, the increase in fetal
cardiac genes that characterizes cardiomyopathic syndromes25 also showed a relationship to ␦V1 expression level
(Figure 4A). Compared with NTG (and TRE-2) mice,
␣MHC-␦V1 mouse ventricles had a 3-fold increase in ␤MHC
and a 15-fold increase in ANF mRNA expression, whereas
TRE-1 mice similarly demonstrated a 3-fold increase in
␤MHC, but an 8-fold increase in ANF mRNA. In addition,
␣MHC-␦V1 mice had a 50% reduction in SERCA expression, compared with a 30% reduction for TRE-1; again,
TRE-2 mice were similar to NTG. TRE-1 and TRE-2 mice
exhibited no increase in mortality up to 12 months of age.
However, like those of the original ␦V1 line, first generation
mice from the reinjected ␣MHC-␦V1 founder died prematurely from apparent heart failure, with a mean survival time
of 18⫾2 weeks (n⫽44).
TABLE 2.
The effects of different levels of ␦V1 expression on
myocardial ␦PKC activation, assessed as translocation to
subcellular membrane particulates, were assessed in isolated
perfused mouse hearts. As shown in Figure 4B, a 40-minute
period of global ischemia followed by 20 minutes of reperfusion stimulated a marked redistribution of ␦PKC from the
cytosolic to membrane subcellular fraction (compare control
NTG to ischemia/reperfused NTG). In contrast, translocation
was significantly inhibited in TRE-2 hearts (76⫾2.3% of
NTG), and TRE-1 hearts (42⫾4.4% of NTG; Pⱕ0.05 versus
NTG and TRE-2). No effect of ␦V1 expression was seen on
subcellular partitioning of related ⑀PKC (Figure 4B). Thus,
variable myocardial expression of ␦V1 has specific, doserelated inhibitory effects on ischemia/reperfusion-stimulated
␦PKC translocation.
Because a hallmark feature of the original ␦V1 cardiomyopathy was the presence of intracellular desmin/crystallincontaining aggregates, we performed histological examination to establish that the phenotypic continuum achieved with
variable ␦V1 expression extended to these characteristic
structural abnormalities. Although (as hoped) none of the new
␦V1 transgenic lines developed a phenotype as severe as the
original, the proportion of cardiomyocytes (percent of total)
with aggregates that stained blue with Masson’s Trichrome
correlated with ␦V1 expression (7.7⫾0.6% ␣MHC-␦V1,
1.1⫾0.4% TRE-1, 0% TRE-2) (Figure 5). Furthermore,
cardiac functional abnormalities paralleled the alterations in
cardiomyocyte/myocardial structure. Invasive hemodynamic
Morphometric Analysis of ␦V1 Mice
NTG
Body wt, g
24.9⫾0.7
␦V1
22.8⫾0.4
TRE-1
24.2⫾2
TRE-2
21.6⫾0.8
Heart/bd, mg/g
5.8⫾0.3
9.5⫾0.9*
6.42⫾0.8
5.1⫾0.2
Ventricle/bd, mg/g
4.1⫾0.1
5.4⫾0.2*
4.4⫾0.2
3.9⫾0.1
Lung/bd, mg/g
6.5⫾0.01
8.4⫾0.3
7.3⫾0.6
7.8⫾0.6
49.5⫾1.5
55.4⫾1.8
49.6⫾1.1
Liver/bd, mg/g
54.6⫾2
Wt indicates weight; bd, body. n⫽6 to 9 animals per group.
*P⬍0.05 vs NTG.
Figure 5. Pathological aggregate formation parallels ␦V1 transgene expression. Top left, ␣MHC-␦V1 (Masson’s trichrome). Top
right, TRE-1. Bottom left, TRE-2. Bottom right, NTG. Arrows
denote representative aggregates.
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October 18, 2002
Figure 6. Cardiac protection from ischemia/reperfusion injury. A, Enhanced
recovery of left ventricular dP/dt after 40
minutes of ischemia in TRE-2 mice. B,
Postischemic function normalized to
baseline values (error bars omitted for
clarity). C, Attenuated postischemic CK
release from TRE-1 and TRE-2 hearts.
n⫽6 to 10.
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analysis revealed that both the ␣MHC-␦V1 and TRE-1 mice
had significantly depressed basal and stimulated ⫹dP/dt. At
the maximal dobutamine dose, ␣MHC-␦V1 and TRE-1 mice
exhibited 43% and 28% reductions in ⫹dP/dt, respectively
(12265⫾740 mm Hg/s ␣MHC-␦V1, 15418⫾69 TRE-1, and
21217⫾720 NTG; Pⱕ0.05 versus NTG). As with histological appearance, TRE-2 mice were functionally normal (peak
⫹dP/dt 18802⫾2102 mm Hg/s; P⫽NS).
The varying “dose” of ␦V1 expression afforded by the
mutant promoters permitted a correlative analysis of biochemical ␦PKC inhibition and functional response to ischemia/reperfusion. TRE-2 mouse hearts, with only modest
␦PKC inhibition and no detectable abnormalities in structure,
function, or gene expression were nevertheless markedly
resistant to global ischemia, with rapid recovery of contractile
function (Figures 6A and 6B) and strikingly less myocyte
damage (assessed as CK release) compared with NTG (Figure 6C). TRE-1 mouse hearts, with significantly decreased
␦PKC translocation inhibition (see Figure 4B), appeared at
first glance to have increased ischemic injury (Figure 6A).
However, the basal function of TRE-1 hearts was depressed
due to the modest myofibrillar cardiomyopathy (Figures 5
and 6A), and when the postischemic data are corrected for the
baseline functional abnormality, there is no difference in
post-ischemic cardiac functional recovery compared with
NTG controls (Figure 6B). However, as with TRE-2, a
striking cytoprotective effect was evident (Figure 6C). It was
not possible to assess the effects of ischemia/reperfusion on
the ␣ MHC- ␦ V1 mice, due to the severity of the
cardiomyopathy.
Discussion
These studies establish dose-dependent effects of a ␦PKC
translocation inhibitor in the in vivo mouse heart. Lower
levels of ␦V1 expression, which inhibited ischemiastimulated ␦PKC translocation by 24%, protected hearts from
ischemia/reperfusion injury. Importantly, these benefits occurred in the absence of any detectable structural or functional alterations, including highly sensitive assays of myocardial gene expression. With progressively more ␦PKC
inhibition however, cardiomyopathies developed exhibiting
pathological and histological characteristics of myofibrillar
myopathies. Cytoskeletal abnormalities in these ␦V1 cardiomyopathies, together with targeting of ␦V1 (and hence
activated ␦PKC) to the cardiomyocyte cytoskeleton, identify
a critical function for ␦PKC in maintaining myocyte cytoskeletal integrity.
An unexpected finding of these studies was the degree of
cardiomyocyte protection (as assessed by CK release) af-
forded by ␦V1 expression in both the TRE-1 and TRE-2 lines
(Figure 6C). We considered that the presence of protein
aggregates and depressed basal contractile function of TRE-1
mice would compromise functional recovery after ischemia/
reperfusion. Indeed, absolute recovery of contractile function
was severely depressed in TRE-1 mice, although when
normalized to basal function, TRE-1 mice had exactly the
same degree of functional recovery as NTG controls
(55.5⫾8.6% NTG versus 55.5⫾15.3% TRE-1). Nevertheless,
a dramatic cytoprotective effect was observed, exceeding
even that of the functionally superior TRE-2 mice. A possible
explanation relates to increased expression of ␣B-crystallin in
the TRE-1 mice, which was not seen in TRE-2. ␣B-crystallin,
aka, heat shock protein 22, is a chaperone for the intermediate
filament desmin and is known to exert a cardioprotective
effect in ischemic preconditioning.26 Thus, ␦V1-mediated
upregulation of ␣B-crystallin could contribute to cytoprotection in these hearts. It is therefore interesting that activation of
⑀PKC, which also protects from postischemic cardiac dysfunction, is rather less cytoprotective than ␦PKC inhibition,
and is not associated with regulated expression of ␣Bcrystallin or desmin9 (unpublished results). Another difference between cardiac protection afforded by transgenically
expressed ␺⑀RACK versus ␦V1 was delayed development of
mild, albeit normally functioning ventricular hypertrophy,
with induction of ␤MHC gene expression in ␺⑀RACK
mice.9,10 Absence of any such physical or molecular modifications by the chronically expressed ␦PKC inhibitor (at low
expression levels) may be advantageous in terms of a potential therapeutic approach.
When ␦V1 was expressed at incrementally higher levels in
the heart, a cardiomyopathy developed with severity in
proportion to ␦V1 expression level. The most surprising
aspect of higher-level ␦V1 expression was that this cardiomyopathy had the histopathological and functional characteristics of specific human genetic disorders, the myofibrillar
myopathies. These skeletal and cardiac myopathies are a
heterogeneous group of diseases that share a common histopathological picture of intramyocyte proteinaceous aggregates with sarcomere disorganization.13,14,22 The best characterized of this group are the desmin-related myopathies
caused by mutant forms of desmin or ␣B-crystallin. Missense
mutations in the carboxy-terminal part of the desmin rod
domain cause myopathies by interfering with desmin assembly.13 The ␣B-crystallin R120G mutation leads to myopathy
by impairing its ability to act as a molecular chaperone for
desmin, and interfering with proper desmin integration into
intermediate filaments.14,22 Thus, pathological mutations of
either of these proteins result in abnormal accumulation of
Hahn et al
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protein into characteristic intracellular aggregates, accompanied by cytoskeletal and sarcomere disruption. Interestingly,
a similar phenotype has been observed in the desmin knockout mouse model,27,28 which implies that the pathology is
associated with loss of desmin function, not increased desmin
expression as observed in the ␦V1 mouse model and other
hypertrophy conditions.29 Indeed, our present studies demonstrate that neither overexpression of normal desmin alone, nor
its overexpression in combination with ␣B-crystallin, causes
any measurable functional or structural myocardial abnormalities. Rather, the observation that activated ␦PKC in cardiac
myocyte translocates to the cytoskeleton establishes a mechanistic link between ␦PKC function and myofibrils. Studies in
cultured rat neonatal cardiomyocytes and in ␦V1 mice (data
not shown) demonstrate that activated ␦PKC colocalizes with
cytoskeletal vimentin,30,31 and that vimentin and ␦PKC coimmunoprecipitate.30 Thus, ␦PKC substrates exist at the
cytoskeleton, and our results indicate that attenuated ␦PKC
activation causes loss of cytoskeletal organization. The identities of critical cytoskeletal ␦PKC substrates, and of the
cytoskeletal ␦PKC binding protein, ie, ␦PKC RACK, are
currently unknown.
The ␦V1 transgenic approach interrogates ␦PKC function
in the in vivo mouse heart through targeted expression of a
protein fragment designed to competitively inhibit ␦PKC
binding with its membrane anchor protein, or RACK.12,32 We
have previously used the approach of translocation inhibition
to characterize in vivo functions of myocardial ⑀PKC.9 –11 In
those studies, ⑀PKC inhibition by transgenic expression of its
first variable region, ⑀V1, had little effect at lower expression
levels, but at the highest expression level caused a lethal
dilated cardiomyopathy.10 In both the current ␦V1 model, and
the previous ⑀V1 model, expression of PKC V1 peptides at
lower levels was well tolerated, but higher expression levels
resulted in development of lethal cardiomyopathies. However, the cardiomyopathies themselves are strikingly different. Whereas ⑀V1 expressors developed profound ventricular
wall thinning and chamber dilation, ␦V1 expressors had thick
ventricular walls and normal chamber dimensions, associated
with the aforementioned histological and ultrastructural abnormalities. Likewise, the antithetic experiments, ie, facilitated translocation of ␦PKC and ⑀PKC through transgenic
expression of their respective ␺RACK peptides, resulted in
similar hypertrophic phenotypes, but opposite effects on
myocardial recovery from ischemia.10,12 Taken together,
these observations indicate that ␦PKC and ⑀PKC have distinct roles in myocardial cytoskeletal maintenance and the
response to ischemic injury, but an overlapping function in
myocardial growth. This latter functional redundancy may
explain why mutational ablation of the ⑀PKC gene has no
overt cardiac phenotype,33 because ␦PKC may opportunistically compensate for the absent ⑀PKC.
In the present studies, a great deal of effort went into
generating mouse models with varying levels of transgene
expression. The need was to generate ␦V1-expressing animals that survived long enough to study, and to assess the
dose-dependent effects of the transgene while retaining the
favorable characteristics of ␣MHC promoter, ie, robust,
cardiomyocyte-specific ventricular transgene expression that
␦PKC Cardiomyopathy
747
largely begins after birth. Thus, we chose to mutationally
attenuate the ␣MHC promoter. Attenuated versions of the
␣MHC promoter were obtained by individually mutating the
thyroid response elements, as originally conceived by Rindt and
Robbins,16,17 and the resulting TRE-1 and TRE-2 ␦V1 phenotypes represent form frustes of the original ␣MHC-␦V1 phenotype, permitting a detailed analysis. Importantly, multiple independent lines of TRE-2 mice did not develop a cardiomyopathy
when followed up to one year, indicating that a threshold
expression level exists for pathological effects of ␦PKC inhibition. We have observed similar threshold effects when overexpressing either intact signaling molecules, such as G␣q,19 or
catalytically inactive peptide fragments, such as the ⑀PKC
inhibitor, ⑀V1.10 G␣q overexpression at twice endogenous levels
had no effect on cardiac structure, function, or gene expression,
whereas 4- and 5-fold G␣q overexpressors developed the characteristic hypertrophy, contractile dysfunction, and abnormalities in gene expression.19 Likewise, in the lowest expressing line
(⬇8 copies) cardiac-specific expression of the ⑀PKC inhibitory
peptide, ⑀V1, had no detectable basal phenotype (although a
phenotype was revealed by superimposed expression with
G␣q11), whereas the highest expressing line (⬎200 copies) died
prematurely of dilated cardiomyopathy, and an intermediate
expressing line (⬇40 copies) exhibited modest but significant
alterations in contractile function and increased fetal cardiac
gene expression.10 In the context of the present and previous
studies where transgenesis has been utilized as a drug delivery
system for catalytically inactive peptides (Gq-mini gene,34
␤ARK-ct35), delineation of dose-dependent effects should be an
essential component of the phenotypic characterization, and the
TRE mutant ␣MHC promoter constructs have demonstrated
utility for this purpose.
In summary, we have delineated essential roles for ␦PKC
in the in vivo myocardial response to ischemic injury and for
normal cardiomyocyte cytoskeletal integrity. A critical function for ␦PKC in maintenance of normal cardiomyocyte
cytoskeletal structure was indicated not only by the histopathological/ultrastructural findings and associated contractile dysfunction in mice expressing ␦V1, but also by decoration of intermediate filaments by ␦V1, thus identifying the
cardiomyocyte cytoskeleton as a subcellular target for activated ␦PKC. Despite the deleterious effects of high-level ␦V1
expression, the potential therapeutic utility of ␦PKC inhibition for postischemic myocardial protection is supported by
identification of a therapeutic window, where low-level ␦V1
expression is protective, without adverse consequences.
Acknowledgments
This work is supported by P50 HL52318 and R01 HL58010. We
wish to thank Gary Lin for his technical contributions to this project.
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Ischemic Protection and Myofibrillar Cardiomyopathy: Dose-Dependent Effects of In Vivo
δPKC Inhibition
Harvey S. Hahn, Martin G. Yussman, Tsuyoshi Toyokawa, Yehia Marreez, Thomas J. Barrett,
K. Chad Hilty, Hanna Osinska, Jeffrey Robbins and Gerald W. Dorn II
Circ Res. 2002;91:741-748; originally published online September 12, 2002;
doi: 10.1161/01.RES.0000037091.64492.69
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
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Online methods
for MS # 4458-R1
Fig 1