Therapeutic Delivery of Cyclin A2 Induces Myocardial
Regeneration and Enhances Cardiac Function in
Ischemic Heart Failure
Y. Joseph Woo, MD; Corinna M. Panlilio, BA; Richard K. Cheng, MD; George P. Liao, MB;
Pavan Atluri, MD; Vivian M. Hsu, BA; Jeffrey E. Cohen, BA; Hina W. Chaudhry, MD
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
Background—Heart failure is a global health concern. As a novel therapeutic strategy, the induction of endogenous
myocardial regeneration was investigated by initiating cardiomyocyte mitosis by expressing the cell cycle regulator
cyclin A2.
Methods and Results—Lewis rats underwent left anterior descending coronary artery ligation followed by peri-infarct
intramyocardial delivery of adenoviral vector expressing cyclin A2 (n ⫽32) or empty adeno-null (n ⫽32). Cyclin A2
expression was characterized by Western Blot and immunohistochemistry. Six weeks after surgery, in vivo myocardial
function was analyzed using an ascending aortic flow probe and pressure-volume catheter. DNA synthesis was analyzed
by proliferating cell nuclear antigen (PCNA), Ki-67, and BrdU. Mitosis was analyzed by phosphohistone-H3 expression.
Myofilament density and ventricular geometry were assessed. Cyclin A2 levels peaked at 2 weeks and tapered off by
4 weeks. Borderzone cardiomyocyte cell cycle activation was demonstrated by increased PCNA (40.1⫾2.6 versus
9.3⫾1.1; P⬍0.0001), Ki-67 (46.3⫾7.2 versus 20.4⫾6.0; P⬍0.0001), BrdU (44.2⫾13.7 versus 5.2⫾5.2; P⬍0.05), and
phosphohistone-H3 (12.7⫾1.4 versus 0⫾0; P⬍0.0001) positive cells/hpf. Cyclin A2 hearts demonstrated increased
borderzone myofilament density (39.8⫾1.1 versus 31.8⫾1.0 cells/hpf; P⫽0.0011). Borderzone wall thickness was
greater in cyclin A2 hearts (1.7⫾0.4 versus 1.4⫾0.04 mm; P⬍0.0001). Cyclin A2 animals manifested improved
hemodynamics: Pmax (70.6⫾8.9 versus 60.4⫾11.8 mm Hg; P⫽0.017), max dP/dt (3000⫾588 versus
2500⫾643 mm Hg/sec; P⬍0.05), preload adjusted maximal power (5.75⫾4.40 versus 2.75⫾0.98 mWatts/␮L2;
P⬍0.05), and cardiac output (26.8⫾3.7 versus 22.7⫾2.6 mL/min; P⫽0.004).
Conclusions—A therapeutic strategy of cyclin A2 expression via gene transfer induced cardiomyocyte cell cycle activation
yielded increased borderzone myofilament density and improved myocardial function. This approach of inducing
endogenous myocardial regeneration provides proof-of-concept evidence that cyclin A2 may ultimately serve as an
efficient, alternative therapy for heart failure. (Circulation. 2006;114[suppl I]:I-206–I-213.)
Key Words: cyclin 䡲 heart failure 䡲 myocardial regeneration
H
eart failure has become a major global health concern.
Current therapies ranging from revascularization to
remodeling to replacement are limited and variably effective.
Cellular cardiomyoplasty, with its myriad forms of cell types
and delivery routes, has shown great experimental promise
and some benefit in early clinical application.
An effective endogenous repair strategy would be a theoretically ideal therapy. However, because of the post-mitotic
state of adult cardiomyocytes, the predominant myocardial
response to injury is cardiomyocyte hypertrophy. Although
recent research has identified a putative population of resident cardiac progenitor stem cells, the native role of these
cells during injury is clearly clinically insufficient for main-
taining cardiac function and the potential role of the manipulation of these cells is still highly uncertain.1–5 A mechanistically more attractive approach is to attempt to induce native
cardiomyocytes to reenter the cell cycle and replicate.6 –9
Multiple potential cell cycle regulators such as cyclins and
cyclin-dependent kinases (cdk) serve as potential therapeutic
targets.10 Manipulation of the restriction point control cyclin
D has shown initial promise.11
Cyclin A2 possesses a unique role in its 2-point control of
the cell cycle, first by interacting with cdk2 in controlling the
G1/S transition into DNA synthesis and then by interacting
with cdks 1 and 2 to control the G2/M entry into mitosis.12
We have previously demonstrated that constitutive expres-
From Division of Cardiothoracic Surgery (Y.J.W., C.M.P., G.P.L., P.A., V.M.H., J.E.C.), Department of Surgery, University of Pennsylvania School
of Medicine, Philadelphia, Pa; Division of Cardiology (R.K.C., H.W.C.), Department of Medicine, Columbia University School of Medicine, New York,
NY.
Presented at the American Heart Association Scientific Sessions, Dallas, Tex, November 13–16, 2005.
Correspondence to Y. Joseph Woo, Division of Cardiothoracic Surgery, University of Pennsylvania, 3400 Spruce St, Philadelphia PA 19104.E-mail:
[email protected]
© 2006 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/CIRCULATIONAHA.105.000455
I-206
Woo et al
sion of the cell cycle regulator cyclin A2 in a transgenic
mouse yields robust postnatal cardiomyocyte mitosis and
hyperplasia.13 To examine the potential role of this regenerative strategy as a therapy for heart failure, a cyclin A2expressing adenoviral vector was constructed and delivered
to rat hearts after left anterior descending coronary artery
infarction and subsequent evidence of cardiomyocyte proliferation, peri-infarct geometric enhancement, and cardiac
functional improvement were observed.
Methods
Animal Care and Biosafety
Adult, male Lewis rats weighing 250 to 300 grams were obtained
from Charles River Laboratories (Boston, Mass). Food and water
were provided ad libitum. This study was performed in accordance
with the standard humane care guidelines of the Guide for the Care
and Use of Laboratory Animals and the Institutional Animal Care
and Use Committee of the University of Pennsylvania, which
conform to federal guidelines.
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Induction of Heart Failure
Male Lewis rats (n ⫽64) were anesthetized with intraperitoneal
ketamine (75 mg/kg) and xylazine (7.5 mg/kg), endotracheally
intubated with a 14-gauge angiocatheter, mechanically ventilated
(Hallowell EMC, Pittsfield, Mass) and supplemented with 1.5%
isoflurane as maintenance anesthesia. A left fourth intercostal space
thoracotomy was performed and the pericardium was incised. The
left anterior descending coronary artery (LAD) was identified and
encircled with a 7-0 prolene suture at the level of the left atrial
appendage. The suture was briefly snared to confirm adequate
consistent ligation, as evidenced by blanching of the arterial region
of distribution, and then permanently ligated. This highly reproducible method causes an infarction of 30% of the left ventricle. The
animals were closed in 3 layers over a temporary thoracostomy tube
and allowed to recover for six weeks. Over the course of 6 weeks, the
animals predictably progressed into ischemic cardiomyopathy.14 –16
A subset of animals received serial intraperitoneal injections of
bromodeoxyuridine (BrdU; BD Pharmingen San Diego, Calif) at a
concentration of 80 ␮g/kg at 4-day intervals to examine DNA
synthesis.
Adenoviral Vector Delivery
Replication-deficient (E1, E3 deleted) adenoviral vectors containing
murine cyclin A2 (Pubmed gene bank ID X75483) driven by the
cytomegalovirus promoter were made by the University of Iowa
Gene Transfer Vector Core (Iowa City, Iowa). Corresponding empty
replication-deficient adenovirus with null content was used as a
control. At the time of coronary ligation, animals were randomized
to either therapy with adeno-cyclin A2 or adeno-null injection.
Immediately after LAD ligation, a total of 3⫻109 plaque forming
units were injected into 5 predetermined regions in the peri-infarct
borderzone as previously described.17
Confirmation of Protein Expression
In vivo expression of the cyclin A2 protein was confirmed in a subset
of normal noninfarct animals (n ⫽8/group) by direct intramyocardial
injections of adeno-cyclin A2 or adeno-null vector into the cardiac
apex. Myocardial tissue was harvested at 3 days, 1 week, 2 weeks, 3
weeks, and 4 weeks and frozen immediately in liquid nitrogen.
Myocardium was subsequently dounced in homogenization buffer
consisting of 50 mmol/L tris/HCl (pH 7.5), 100 mmol/L NaCl,
5 mmol/L EDTA, 1% v/v Triton X-100, 1 mmol/L NaF, 1 mmol/L
Na2VO4, 0.2 mmol/L phenymethylsulfonyl fluoride, 10 ␮g/mL
leupeptin, and 10 ␮g/mL aprotinin. Lysates were cleared by centrifugation at 12 000 rpm for 10 minutes at 4°C and analyzed for protein
content via the Bradford method (BioRad, Hercules, Calif). Then, 40
␮g of each protein sample was then denatured at 70°C for 10 minutes
Cyclin A2 Induces Myocardial Regeneration
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and electrophoresed onto a 4% to 12% sodium dodecyl sulfate-polyacrylamide gel. Equal loading of protein was verified by Coomassie
blue staining. Proteins were transferred to Immobilon-P PVDF
membranes (Millipore, Bedford, Mass) and immunoblotting was
performed using a mouse anti-cyclin A monoclonal antibody (Abcam, Cambridge, Mass). This antibody has been tested in mice and
has demonstrated cross-reactivity with murine cyclin A. Proteins
were visualized via horseradish peroxidase conjugated anti-mouse
antibody (Amersham Biosciences, Piscataway, NJ) and chemiluminescence detection (Amersham Biosciences).
Immunohistochemisty was used to confirm protein expression
within the harvested hearts; 10-␮m thin myocardial sections were
fixed in 4% paraformaldehyde, permeabilized with Triton X-100,
and blocked with 5% normal goat serum for 2 hours at room
temperature, followed by incubation with rabbit anti-human cyclin A
(1:600; Abcam, Cambridge, Mass) overnight at 4°C. Antibody
cross-reactivity with mouse and rat cyclin A has been demonstrated
by the manufacturer. The slides were washed 3 times in phosphatebuffered saline and incubated with FITC-conjugated goat anti-rabbit
secondary antibody (1:1000; Abcam) for 45 minutes at room
temperature. Cyclin A2 expression was quantitated in 4 fields/
specimen of the region of interest of both cyclin A2 and null hearts
by fluorescence microscopy (40⫻ air magnification; Leica Microsystems, Wetzlar, Germany).
Tissue Section Preparation
Six weeks after infarction and before explantation, the right atrium
was incised and the hearts were perfused and rinsed with 10 mL
phosphate-buffered saline via the aortic root with resultant blanching
of the myocardium. The hearts were arrested in diastole, distended
with Optimum Cutting Temperature embedding compound at a fixed
distending pressure, and snap-frozen in liquid nitrogen. Explanted
hearts were analyzed in a blinded fashion. Sequential transverse
10-␮m tissue sections were made at the level of the papillary
muscles. Sections were immediately fixed in 4% paraformaldehyde,
washed, and blocked in 5% normal goat serum/phosphate-buffered
saline.
Immunohistochemical Assessment of
Cardiomyocyte DNA Synthesis and Proliferation
Myocardial thin sections were fixed, permeabilized, and blocked as
described. Immunohistochemical analysis of cardiomyocyte DNA
synthesis was performed by staining for PCNA (1:600 rabbit
anti-human PCNA; Abcam) and Ki-67 (1:500 goat anti-mouse
Ki-67; Santa Cruz Biotechnology, Santa Cruz, Calif) as indicators of
DNA synthesis and cellular proliferation. Positive nuclei were
co-localized to ␣-sarcomeric actin (1:700 mouse anti-rabbit
␣-sarcomeric actin; Sigma Aldrich, St. Louis, Mo) to confine PCNA
and Ki-67–positive staining specifically to cardiomyocytes. The
␣-isoform of sarcomeric actin will label only ␣-cardiac muscle actins
and ␣-skeletal muscle actins, and will not stain smooth muscle actin.
Dual-positive cells in 4 high-power peri-infarct fields were then
quantitated in a blinded fashion for each specimen and averaged
(40⫻ air magnification; Leica Microsystems, Wetzlar, Germany).
From the subset of animals that had received serial injections of
BrdU, tissue sections were processed as detailed and BrdU staining
was analyzed. After fixation and antigen retrieval, tissue sections
were incubated with a biotinylated mouse anti-BrdU antibody
(1:600; BD Pharmingen, San Diego, Calif). Streptavidin-horseradish
peroxidase was used to detect the presence of antibody via the
Diaminobenzidine (DAB) substrate system. (BrdU detection assay;
BD Pharmingen).
Immunohistochemical Assessment of Mitosis
Sections were analyzed for mitosis using the nuclear mitosis marker,
phosphohistone-H3 (1:600 mouse anti-human phosphohistone-H3;
Upstate Biotechnology, Lake Placid, NY). Dual-positive staining for
both phosphohistone-H3 and cardiomyocytes localized to DAPstained nuclei was performed as described. The number of dual-
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July 4, 2006
positive stained cells was counted in a blinded fashion in 4
high-power fields per specimen.
Measurement of Ventricular Geometry
Tissue sections were obtained at the level of the papillary muscle.
The sections were stained with hematoxylin and eosin to delineate
morphology. Digitized photomicrographs were taken with a Nikon
Coolpix 4300 camera using standardized imaging distances. Geometric measurements were then computed in a blinded fashion using
Scion Image Beta Release 4 (Scion Corporation, Frederick, Mass)
from 2 representative tissue sections for each animal. The left
ventricular diameter was measured in two perpendicular axes and
averaged for each animal. The wall thickness of the left ventricular
borderzone was measured and analyzed from 2 separate areas of each
tissue section and averaged for each animal. The borderzone was
defined as 1 field lateral to myocardial scar.18,19 The wall thickness
index was calculated for each specimen and was defined as the ratio
of borderzone wall thickness to remote normal myocardial wall
thickness ⫻100.
Assessment of Myofilament Density
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Myocardial borderzone hematoxylin and eosin-stained tissue sections were imaged and myofilament density was analyzed per
high-power field. Myofilament density was defined as the total
myocytes per high-power field. Four high-power fields were quantitated and averaged per tissue section in both borderzone and remote
noninfarcted areas. Data analysis was performed in a blinded
fashion, recorded, and analyzed for statistical significance.
Assessment of Hemodynamic Function
Six weeks after initial LAD ligation, a median sternotomy was
performed and hemodynamic measurements were obtained using an
ascending aortic Doppler flow probe and an intraventricular
pressure-volume catheter. A 2.5-mm peri-aortic flow probe (Model
2.5PSL492; Transonic Systems Ithaca, NY) was placed around the
ascending aorta to measure the cardiac output. A 2-French pressure–
volume conductance microcatheter (model SPR838; Millar Instruments Houston, Tex) was volume and pressure calibrated20 and
placed via the left ventricular apex into the left ventricular cavity.
Hemodynamic measurements were analyzed in a blinded fashion
utilizing Chart v4.1.2 software (AD Instruments, Colorado Springs,
Colo) and ARIA1 Pressure Volume Analysis software (Millar
Instruments). In addition to steady-state hemodynamic measurements, contractility was measured from pressure–volume relationships obtained via preload reduction after occlusion of the inferior
vena cava. Volume measurements were calibrated by 2-point linear
interpolation with fixed-volume cuvettes of heparinized rat blood,
and parallel conductance was excluded with the hypertonic saline
injection technique.
Statistical Analysis
Statistical analysis was performed using JMP IN 5.1 software using
2-way ANOVA to test for differences among means. All results were
expressed as means⫾SEM. Statistical significance was determined
as a P⬍0.05.
Statement of Responsibility
The authors had full access to the data and take full responsibility for
their integrity. All authors have read and agree to the manuscript as
written.
Results
Adenoviral Expression
Western blot analysis of cyclin A2 protein levels demonstrated statistically significant cyclin A2 levels in the adenocyclin A2 samples as compared with adeno-null controls. As
expected, adeno-null control samples did not contain measurable amounts of cyclin A2 protein. In the experimental
Figure 1. Adenoviral expression. A, Western blot showing
adenoviral mediated expression of cyclin A2 up to 21 days
compared with null adenovirus. 3⫻109 plaque forming units of
adenovirus were injected into 5 distinct areas around area of
infarct created by LAD ligation during initial surgery. B, Animals
were recovered and euthanized at each time point to generate a
corresponding adenoviral expression curve. C, Immunohistochemical analysis yielded statistically significant values of cyclin
molecule in only the cyclin A2 adenovirally treated tissue
sections.
animals, cyclin A2 protein reached peak levels at 14 days and
tapered off by 28 days (Figure 1). Fluorescent immunocytochemical labeling confirmed cyclin A2 protein levels as early
as 3 days after adenoviral injection. Immunocytochemical
labeling for cyclin A2 was absent in the adeno-null animals at
all time points, and absent in the adeno-cyclin A2 animals at
28 days.
Cardiomyocyte Proliferation
PCNA expression colocalized to ␣-sarcomeric actin stained
cells was significantly upregulated in cyclin A2 animals
compared with null controls, suggesting borderzone cardiomyocyte cell cycle activation. Ki-67 expression colocalized
to ␣-sarcomeric actin-stained cells was likewise significantly
upregulated in adeno-cyclin A2 animals compared with null
controls, also suggesting borderzone cardiomyocyte cell cycle activation. Furthermore, BrdU uptake studies demonstrated a significant increase in the proportion of BrdUpositive cells per 1000 cardiomyocytes with adeno-cyclin A2
therapy, thereby confirming active DNA synthesis (Table 1).
Colocalization of phosphohistone-H3 expression to ␣
-sarcomeric actin stained cells in cyclin A2 animals compared
with null controls was significantly elevated, confirming
cardiomyocyte mitosis (Figure 2). These cellular proliferation
studies provided multiple independent measures of cell cycle
activation and cardiomyocyte mitosis with adeno-cyclin A2
therapy. These data demonstrate convincing evidence for
cardiomyocyte proliferation with cyclin A2 activation.
Woo et al
Cyclin A2 Induces Myocardial Regeneration
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TABLE 1. Immunohistochemical Analysis of Proliferation in Cardiac
Tissue Samples
PCNA (positive cells/1000 cells) n⫽12/subset
Ki-67 (positive cells/1000 cells) n⫽12/subset
BrdU (positive cells/1000 cells) n⫽5/subset
Myofilament Density
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Myofilament density analysis demonstrated a statistically
significant increase in peri-infarct borderzone myofilaments
in the adeno-cyclin A2 treatment animals as compared with
null control animals. Quantitation of myofilament density in
the remote, normal myocardium demonstrated no change in
myofilament density or structure between the treatment and
control groups. The similarity in remote myofilament composition and structure validated the assay and denoted similar
baseline myocardial structure (Figure 3).
Ventricular Geometry
Computerized planimetric analysis of ventricular geometry
demonstrated a statistically significant increase in borderzone
Null
Cyclin A2
P
Borderzone
9.3⫾1.1
40.1⫾2.6
⬍0.0001
Remote
8.4⫾1.4
10.3⫾1.6
0.38
Borderzone
20.4⫾6.0
46.3⫾7.2
⬍0.0001
Remote
16.9⫾4.8
19.2⫾6.1
0.24
Borderzone
5.2⫾5.2
44.2⫾13.7
0.0287
Remote
0⫾0
5.8⫾5.8
0.36
wall thickness in the adeno-cyclin A2 treated animals as
compared with the adeno-null treated animals (1.8⫾0.04
versus 1.4⫾0.04 mm; P⬍0.001). The calculated wall thickness index (defined as the percent ratio of borderzone wall
thickness to remote normal myocardial wall) was significantly increased in cyclin A2 experimental tissue sections as
compared with controls (73.9⫾6.7 versus 62.4⫾5.4;
P⬍0.001). Enhanced preservation of left ventricular geometry and diameter (8.8⫾0.2 versus 9.9⫾0.1 mm; P⬍0.001)
was noted in the adeno-cyclin A2 group compared with
control animals (Figure 4).
This combination of enhanced borderzone wall thickness
and preservation of ventricular geometry increases the potential for enhanced myocardial function.
Cardiac Function
Myocardial functional analysis 6 weeks after LAD ligation
indicated a statistically significant increase in cardiac output
as measured by Doppler analysis of ascending aortic blood
flow in the adeno-cyclin A2 animals as compared with
adeno-null animals. Dynamic pressure-volume analysis
showed a statistically significant improvement in maximum
generated ventricular pressure (Pmax), maximum dP/dT,
elastance, and preload-adjusted maximal power with adenocyclin A2 therapy. Also, the ventricular volumes appear to be
smaller in the cyclin hearts. There was no difference in the
baseline heart rate between the treatment and control groups.
Therefore, it appears that the adeno-cyclin A2-treated animals
had statistically significant preservation of cardiac function
compared with control animals (Table 2). Cyclin A2-treated
animals also had significantly improved cardiac contractility,
as indicated by an increased slope of the pressure–volume
relationship during caval occlusion as compared with
controls (0.75⫾0.19 versus 0.57⫾0.18; P⫽0.048). Representative pressure–volume loops from each group are
shown in Figure 5.
Figure 2. Immunohistochemical analysis of mitosis in tissue
samples by phosphohistone-H3 staining. Phosphohistone-H3
was stained for as an indicator of active mitosis. A, Corresponding null-treated tissue sections did not stain positive for
phosphohistone-H3. B, Representative cyclin-treated tissue section showing active intranuclear mitosis. Phosphohistone-H3
appears green, alpha-sarcomeric actin appears red, and DAPIstained nuclei appear blue. C, Graphical representation of
phosphohistone-H3 ratios in cardiomyocyte nuclei. Cyclin tissue
sections (n ⫽13) had significantly greater phosphohistone-H3
positive cells localized to cardiomyocyte nuclei than null controls (n ⫽12) in the borderzone (12.7⫾1.4 vs 0⫾0; P⬍0.0001)
Discussion
In infarcted hearts, targeted expression of the cell cycle
regulator cyclin A2 induced cardiomyocyte mitotic activity,
increased borderzone myofilament density, and improved
ventricular function.
Controlling myocardial cell cycle regulation is a complex
interaction between cyclins, cyclin-dependent kinases, cdk
inhibitors such as p57, p21, p27, and other regulators including retinoblastoma protein, PCNA, and E2F.21–23 The cyclins,
which play a central role in cell cycle control, form an
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Figure 3. Myofilament density. Myofilament density was analyzed from respective tissue sections. A, Cyclin-treated
tissue sections showed significantly
greater myofilament density in the borderzone as compared with null-treated
sections (n ⫽12/subset). Cyclin and nulltreated sections did not differ in myofilament density in the remote right ventricular areas that were not treated with
adenovirus. B, Representative photomicrograph of remote myofilament density
in null tissue sections. C. Representative
photomicrograph of remote myofilament
density in cyclin-treated tissue sections.
D, Representative photomicrograph borderzone myofilament density in null tissue sections. E, Representative photomicrographs borderzone myofilament
density in cyclin-treated tissue sections.
appealing therapeutic target for inducing cardiomyocyte replication. Subsequent endogenous myocardial regeneration
would seem an intuitively well-matched therapy to address
necrotic and apoptotic cardiomyocyte loss in heart failure.
Figure 4. Ventricular geometry. Ventricular geometry was measured from hematoxylin and eosin-stained sections of cyclintreated and control tissue samples and displayed graphically. A,
Quantitative analysis of ventricular wall thinning in tissue sections revealed that cyclin-treated tissue sections had greater left
ventricular wall thickness. B, Quantitative analysis of intracavitary dimension in tissue sections revealed that cyclin-treated
tissue sections had diminished left ventricular dilatation.
Cyclin A2 is unique in its control of both major transitions
of the cell cycle at G1/S and G2/M. Cyclin A2 is the only
cyclin that is completely silenced after birth in mice,13 rats,24
and humans.24 This disappearance of cyclin A2 occurs at a
rate consistent with the rate of withdrawal of cardiomyocytes
from the cell cycle.24 A targeted deletion of cyclin A2 in the
mouse exhibited embryonic lethality at embryonic day 5.5.25
A recent study has demonstrated that the development of the
majority of mouse tissues can occur independently of all 3
D-type cyclins.26 It has also been shown with a double
knockout that cdk4 and cdk6 are not essential for cellular
proliferation.27 It has also been reported that E-type cyclins
are largely dispensable for normal development in mice.28
Both E1 and E2 knockout mice (⫺/⫺) developed normally.
The D-type cyclins control restriction point movement into
DNA synthesis and have been studied as a potential therapeutic target; however, the potential for cell cycle arrest
before mitosis may be a limitation.29 Thus, cyclin A2 expression offers enhanced potential for inducing cell cycle re-entry
given its control of the G2/M transition in addition to G1/S
and makes cyclin A2 a particularly attractive therapeutic
target.
We have previously demonstrated biologic marker evidence of cardiomyocyte mitosis and histomorphologic evi-
Woo et al
TABLE 2.
Cyclin A2 Induces Myocardial Regeneration
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In Vivo Hemodynamic Measurements
Null
(n⫽18)
Cyclin A2
(n⫽18)
Heart rate, bpm
187.0⫾17.9
198.3⫾30.0
NS
Maximum volume, ␮L
263.6⫾56.9
213.0⫾61.1
0.035
Maximum pressure, mm Hg
60.4⫾11.8
70.6⫾8.9
0.017
Elastance, mm Hg/␮L
0.67⫾0.33
0.97⫾0.41
0.036
P
dP/dt Max, mm Hg/sec
2500⫾643
3000⫾588
0.048
Preload adjusted maximal power, mWatts/␮L2
2.75⫾0.98
5.75⫾4.40
0.048
Cardiac output via flow probe, mL/min
22.7⫾2.6
26.8⫾3.7
0.004
N⫽18 for each subset.
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dence of cardiomyocyte hyperplasia in a transgenic mouse
expressing cyclin A2.13 Up to 70% increase in the calculated
number of cardiomyocytes was noted, as well as a corresponding increase in heart weight-to-body weight ratio. These
encouraging findings have prompted us to develop a therapeutic strategy based on inducing cardiomyocyte cyclin A2
expression.
In our model, cyclin A2 adenoviral expression peaked at 2
weeks and tapered off by 4 weeks. This limited duration of
adenoviral expression may be of utility; providing cyclin A2
expression during an active period of adverse post-infarction
ventricular remodeling when the presence of additional functional cardiomyocytes may be of particular benefit. Potential
detrimental hypertrophy from excessive hyperplasia or perhaps neoplasia from prolonged cyclin A2 expression consequently may be avoided, because we did not observe any
nests of aberrant appearing cells in any of the specimens.
Furthermore, our examinations of transgenic animals constitutively expressing cyclin A2 for as long as 1.5 years did not
reveal any neoplastic transformation.13
We examined multiple biologic markers of cellular proliferation in this study. While PCNA and Ki-67 are wellaccepted
indicators
of
cellular
proliferation,
phosphohistone-H3 expression is a very precise assay of
mitosis and thus serves as the ultimate marker of the desired
product of cell cycle re-entry.30 The complete absence of
phosphohistone-H3 in borderzone and remote myocardium in
our control animals confirmed the post-mitotic state of the
adult rat heart. The expression levels in the remote uninjured
myocardium were equivalent between null and cyclin A2
groups, thereby excluding any systemic factors to which
differences could potentially be attributed. Additionally, each
Figure 5. Pressure–volume loops. Improved contractility was measured in cyclin A2-treated animals as compared with control animals
(P-V slope 0.75⫾0.19 mm Hg/␮L vs 0.57⫾0.18 mm Hg/␮L). Representative pressure–volume loops are shown.
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heart was matched to an internal control to confirm that the
therapy was localizable and contained within the region of
administration. BrdU labeling further verified that DNA
synthesis actively occurred within cardiomyocytes.
This rationale was extended to the comparison of myofilament density. The remote myocardium revealed equivalent
myofilament density among groups. Ischemic injury resulted
in a notable decrement in myofilament density which was
partially rescued with cyclin A2 therapy, yielding a 25%
increase in density. Interestingly, the observed increase in
borderzone wall thickness in cyclin A2 hearts equaled a 23%
improvement. Although the absolute values may appear less
than significant, on a relative scale, a 25% increase in
myocytes is substantive. Additionally, in a heart which at
baseline is only millimeters thick, any increase may be
sufficient to yield considerable improvement in functional
capacity. These histologic improvements were mirrored in
multiple functional parameters.
Two theoretical limitations of this study warrant discussion. In using an adenoviral expression system to effect a
nuclear process, a question arises as to whether an adenoviral
gene product will undergo appropriate processing and targeting to attain appropriate intranuclear localization. One study
reported that the fusion of a nuclear localization signal to an
adenoviral expressed cyclin D1 was necessary to target the
cyclin D1 to the nucleus and prevent cytoplasmic accumulation, which precluded cell cycle activation.31 A more recent
study11 questioned the necessity of such a nuclear localization
signal and postulated that the observation of cytoplasmic
accumulation had more to do with the adenoviral delivery
protocol and an overwhelming multiplicity of infection with
the in vitro cardiomyocyte model used in the TamamoriAdachi study.31 Furthermore, 3 other studies of adenoviralmediated expression of indirect cell-cycle regulators E2F,21
E1A,32 and FGF-533 have all demonstrated cell cycle re-entry
to some degree. These studies imply that adenovirally transcribed and translated proteins can be appropriately processed
by intrinsic post-translational modification and subcellular
localizing machinery and thus can target and impact nuclear
events.
The second limitation of this study pertains to the proposed
existence of a putative resident cardiac progenitor stem cell
population. Although the data in this study are consistent with
the induction of native cardiomyocyte cell cycle activity and
replication, there exists the theoretical possibility that it is in
fact this stem cell population that may have been activated,
thus providing a second potential mechanism to explain the
degree of cardiac repair noted as a result of cyclin A2
administration. This putative mechanism is being investigated
further. If such a population of stem cells is in fact being
stimulated with this cyclin expression strategy, this may
provide an unexpected but added benefit.
In this report, the therapeutic strategy of myocardial gene
transfection to express cyclin A2 induced cardiomyocyte cell
cycle activation. This activation yielded increased borderzone
myofilament density and improved myocardial function. This
approach details proof-of-concept evidence that can be further used to design a novel therapeutic strategy to administer
or activate cyclin A2 and, hence, endogenous myocardial
regeneration may be an attainable goal in the near future to
limit the morbidity and mortality of human heart failure.
Sources of Funding
This work was supported in part by grants from the NIH National
Heart Lung and Blood Institute/Thoracic Surgery Foundation for
Research and Education HL072812 (Y.J.W.), NIH National Heart
Lung and Blood Institute HL067048-03 (H.W.C.), National Heart
Lung and Blood Institute, Ruth L. Kirschstein National Research
Service Award, Individual Fellowship 1 F32 HL79769-01 (P.A.) and
AHA Heritage Affiliate Medical Student Research Fellowship
(R.K.C. while in medical school at Columbia University).
Disclosures
None.
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Therapeutic Delivery of Cyclin A2 Induces Myocardial Regeneration and Enhances
Cardiac Function in Ischemic Heart Failure
Y. Joseph Woo, Corinna M. Panlilio, Richard K. Cheng, George P. Liao, Pavan Atluri, Vivian
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Circulation. 2006;114:I-206-I-213
doi: 10.1161/CIRCULATIONAHA.105.000455
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