© 2001 American Heart Association, Inc.
Molecular Medicine |
Cardiac-Specific Overexpression of Cyclin-Dependent Kinase 2 Increases Smaller Mononuclear Cardiomyocytes
From the Cardiovascular Division, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass.
Correspondence to Seigo Izumo, MD, Cardiovascular Division, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. E-mail sizumo{at}caregroup.harvard.edu
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Abstract |
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Abstract—Cyclin-dependent kinase 2 (cdk2) plays a critical role in the G1- to S-phase checkpoint of the cell cycle. Adult cardiomyocytes are believed to withdraw from the cell cycle. To determine whether forced overexpression of cdk2 results in altered cell-cycle regulation in the adult heart, we generated transgenic mice specifically overexpressing cdk2 in hearts. Transgenic hearts expressed high levels of both cdk2 mRNA and catalytically active cdk2 proteins. Cdk2 overexpression significantly increased the levels of cdk4 and cyclins A, D3, and E. There was an increase in both DNA synthesis and proliferating cell nuclear antigen levels in the adult transgenic hearts. The ratio of heart weight to body weight in cdk2 transgenic mice was significantly increased in neonatal day 2 but not in adults compared with that of wild-type mice. Analysis of dispersed individual adult cardiomyocytes showed a 5.6-fold increase in the proportion of smaller mononuclear cardiomyocytes in the transgenic mice. Echocardiography revealed that transgenic heart was functionally normal. However, adult transgenic ventricles expressed ß-myosin heavy chain and atrial natriuretic factor. Surgically induced pressure overload caused an exaggerated maladaptive hypertrophic response in transgenic mice but did not change the proportion of mononuclear cardiomyocytes. The data suggest that overexpression of cdk2 promotes smaller, less-differentiated mononuclear cardiomyocytes in adult hearts that respond in an exaggerated manner to pressure overload.
Key Words: cardiomyocyte regeneration • cell cycle • cyclin-dependent kinase
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Introduction |
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Cardiomyocytes withdraw from the cell cycle after birth, and the postnatal heart grows by cellular hypertrophy rather than by cell division.1 2 3 4 Because loss of cardiomyocytes is the most prominent feature of congestive heart failure, a major cause of morbidity and mortality in the United States, considerable effort has been devoted to examining the proliferative capacity of postnatal cardiomyocytes.5 Several studies suggest that adult cardiomyocytes can be stimulated to synthesize DNA in vitro and in vivo.5 6 7 8 9 10 11 Although the absolute values of the DNA synthesis index vary, they are generally extremely low.5 These studies suggest that proliferative capacity may be retained in adult cardiomyocytes, but it is extremely limited in most adult mammal hearts.
In general, the cell cycle is controlled at various cell-cycle checkpoints by cyclins, cyclin-dependent kinases (cdks), and cdk inhibitors.12 13 14 15 Control of the G1- to S-phase transition is mainly regulated by cdk2,16 17 18 19 whereas cdc2 plays a critical role in the transition from G2 to M phase.20 However, in cardiomyocytes, the regulation of the cell-cycle process is not well characterized. Several groups have shown that overexpression of cell-cycle regulators induces DNA synthesis in cardiomyocytes.21 22 23 24 Transgenic models expressing high levels of c-myc mRNA resulted in a 2-fold increase in cardiomyocyte number in the fetus.25 26 Increased incorporation of bromodeoxyuridine (BrdU), a marker of DNA synthesis, and increased cardiomyocyte numbers were observed in insulin growth factor-1 transgenic mice.27 Furthermore, Soonpaa et al28 reported that overexpression of cyclin D1 promotes cardiomyocyte DNA synthesis and multinucleation. These studies indicate that altered levels of cell-cycle regulators can increase DNA synthesis and possibly increase cardiomyocyte numbers. In this study, we examined the effect of cardiac-specific overexpression of cdk2 on DNA synthesis and cell-cycle regulation in adult cardiomyocytes.
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Materials and Methods |
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Reagents and Chemicals
Anti-human cyclophilin A was purchased from Upstate Biotechnology as an internal control for protein loading and transfer. Other antibodies and chemicals were obtained as described previously.29
Generation and Identification of cdk2
Transgenic Mice
The coding region of the human cdk2 cDNA was
subcloned into a vector (clone 26; a gift of Jeffrey Robbins,
University of Cincinnati, Cincinnati, Ohio) containing 
-myosin heavy
chain (-MHC) promoter and the human growth hormone polyadenylation
site.30 31
Transgenic (Tg) founders were identified by Southern blot analysis.
Total RNA was isolated from ventricular tissue using Tri reagent
(Sigma) according to the manufacturer’s protocol. Total RNA (20
µg/lane) was separated by electrophoresis using 1% denatured gels
and then blotted onto Hybond Nylon+ membrane and probed to GAPDH, cdk2,
ß-MHC, and atrial natriuretic factor
(ANF).32 33 The
hybridization signals were detected by exposing a film
overnight.
Total proteins extracted from ventricles were analyzed for expression of cdk2, cdc2, cdk4, and cyclins by immunoblotting as described previously.29 For the cdk2 kinase assay, immunoprecipitation was performed using cdk2 antibody. The immunocomplex was incubated with histone H1, as described previously.29
Dispersed Cardiomyocyte Preparations, Nuclear
Staining, and Area Analysis
Highly pure adult cardiomyocytes were prepared by
enzymatic dissociation using retrograde perfusion with
collagenase,34 35
and the isolated cardiomyocytes were plated on laminin-coated dishes.
Nuclear staining was done with Hoechst 33258. Nuclei per cell were
counted under a fluorescent microscope. The surface area of individual
cardiomyocytes was analyzed using an National Institutes of Health
program.
Bromodeoxyuridine Incorporation, Nuclear
Density, and DNA Content
Three-month-old wild-type (WT) and cdk2 Tg mice were
injected intraperitoneally once a day with BrdU (100 mg/kg body weight)
for 3 consecutive days. The heart was perfusion-fixed with 4%
paraformaldehyde, paraffin-embedded, and sectioned at standard
locations including both ventricles and the interventricular septum.
The incorporation of BrdU was detected with a mouse monoclonal
anti-BrdU antibody (Sigma) and a fluorescent-labeled donkey anti-mouse
IgG secondary antibody. The sections were counterstained with propidium
iodide dye and troponin T antibody to identify nuclei and
cardiomyocytes, respectively. The results were analyzed by confocal
microscopy. Nuclear density was analyzed as described
previously.36 DNA content
was determined by a procedure reported previously using crude
homogenated heart
tissues.37
Animal Model of Pressure Overload
Ascending aortic constriction was performed as
described
previously.38
Statistics
Data were analyzed by one-way ANOVA followed by
assessment of differences by Duncan’s multiple-range tests.
P<0.05 was considered
statistically significant.
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Results |
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Generation of Cardiac-Specific cdk2 Tg and Baseline Analysis
To create mice in which cdk2 is specifically overexpressed in cardiomyocytes, we used the
-MHC promoter
(Figure 1A
).30 31
Eight lines of animals that exhibited germ-line transmission were
generated, and 3 lines of cdk2 transgenic mice were used for additional
analysis. All 3 lines of cdk2 Tg expressed high levels of cdk2 mRNA and
proteins in adult hearts but not in adult WT hearts
(Figures 1B and 1C). Cdk2 activity could not be detected in
adult WT heart, as determined by cdk2 kinase assay using histone H1 as
a substrate
(Figure 1D). In contrast, adult cdk2 Tg expressed high levels
of cdk2 activity, indicating that overexpressed cdk2 proteins were
catalytically active.
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No cdk2 Tg mice were prone to early lethality over 1 year of
observation. Gross cardiac defects and histological evidence of
ventricular hypertrophy or fibrosis were absent in adult cdk2 Tg
hearts. The ratio of heart weight to body weight (HW/BW) of cdk2 Tg did
not differ from that of WT in adults
(Figure 2A). However, the HW/BW ratio in cdk2 Tg mice was
significantly increased on neonatal day 2 but not on neonatal day 5 or
10
(Figure 2A). To investigate whether the cardiac-specific
overexpression of cdk2 would affect in vivo cardiac function, we
performed echocardiography on 3-month-old
mice.39 There was no
difference in posterior wall thickness in diastole, anterior wall
thickness in diastole, left ventricular dimension in diastole, and left
ventricular dimension in systole and endocardial fractional shortening
between WT and cdk2 Tg at baseline
(Table 1). Therefore, global cardiac structure and cardiac
function were normal in adult cdk2 Tg.
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Increases of DNA Synthesis in Adult cdk2
Tg Heart
To determine whether overexpression of cdk2 promotes
DNA synthesis in adult heart, we first examined the DNA content by
staining a crude homogenated adult heart tissue with Hoechst
dye.37 Cdk2 Tg and WT mice
did not differ in the DNA content of heart tissues (WT=3.97±0.27; cdk2
Tg=3.76±0.39 µg DNA/mg heart tissues, n=6,
P=NS). Next, we measured the
nuclear density using adult heart section. There was also no
significant difference in nuclear density between adult WT and cdk2 Tg
(WT=2350±557; cdk2 Tg=2257±432 nuclei/mm2,
n=6, P=NS). Finally, we
analyzed DNA synthesis using the incorporation of BrdU into newly
synthesized DNA.36 We found
that the ventricular labeling index was 0.06±0.007% in cdk2 Tg, which
was >100 times higher than that of age-matched WT mice (cdk2 Tg=23±3
BrdU-positive cardiac nuclei/4x104 total
nuclei versus WT=2±4.2 BrdU-positive cardiac
nuclei/4x105 total nuclei, n=10,
P<0.001). These findings
indicate that overexpression of cdk2 increased DNA synthesis in adult
hearts by a very limited amount.
Because proliferating cell nuclear antigen (PCNA) is a
component of the DNA replication fork that is required for both DNA
synthesis and repair, we measured the expression of PCNA at different
developmental stages. WT and cdk2 Tg mice expressed equally high levels
of PCNA proteins in the heart at embryonic day 18 (E18)
(Figure 2B
). In contrast, at neonatal day 2, there was
significantly higher expression of PCNA in cdk2 Tg than in WT heart
(Figures 2B and 2C). Thus, the stage-specific increase in
cardiac mass
(Figure 2A) is consistent with the differential expression of
PCNA in neonatal cdk2 Tg (see Discussion). More modest but also
significant increases of PCNA were found at neonatal days 5 and 10 and
in the adult
(Figures 2B and 2C).
Increases in the Proportion of Smaller
Mononuclear Cardiomyocytes in Adult cdk2 Tg
In the murine fetus, almost all cardiomyocytes are
mononuclear, whereas in the adult, >90% are
binuclear,3 40
suggesting that binucleation of the cardiomyocyte is associated with
cardiomyocyte maturation. Therefore, we asked whether overexpression of
cdk2 affects nucleation of individual cardiomyocytes. We counted the
nuclei of isolated adult cardiomyocytes by staining with Hoechst. In
agreement with the previous reports, the large majority of
cardiomyocytes in the adult WT heart was binuclear; only 8% of
cardiomyocytes were
mononuclear3 40
(Figures 3A and 3B). Interestingly, in cdk2 Tg mice, there was
a marked increase in the relative proportion of mononuclear
cardiomyocytes (45%) with a concomitant decrease in the binuclear
cardiomyocytes
(Figures 3A and 3B). There was no change in the number of
cardiomyocytes with >2 nuclei
(Figure 3B).
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Other investigators showed that overexpression of G1- to
S-phase cell-cycle regulators decreased cell size in
vitro.41 42 43
Therefore, to examine whether overexpression of cdk2 would also affect
cardiomyocyte size in our model, we measured a 2-dimensional surface
area of cardiomyocytes isolated from adult hearts. Quantitative
analysis indicated that the surface areas of adult cdk2 cardiomyocytes
were significantly smaller compared with those of WT cardiomyocytes
(Figure 3C). The size distribution of cardiomyocytes in cdk2
Tg was shifted to the left compared with WT, additionally supporting
this finding
(Figure 3D). In general, the mononuclear cardiomyocytes were
smaller than binuclear cardiomyocytes
(Figure 3A). The quantitative analysis showed that cell size
of mononuclear cardiomyocytes was 60% of binuclear cardiomyocytes in
cdk2 Tg, suggesting that overall decrease in cell size in cdk2 Tg may
be predominately attributable to increase in the proportion of
mononuclear cardiomyocytes.
To asses the maturation of cardiomyocytes in adult cdk2 Tg,
we measured the expression of cardiac fetal markers, such as ANF and
ß-MHC, by Northern blot analysis using whole ventricle tissues. In
adult WT hearts, the expression of ANF and ß-MHC genes was not
detectable
(Figure 4). However, despite the fact that there was no
evidence of hypertrophy or heart failure, the expression of both genes
was increased in adult cdk2 Tg hearts, suggesting that the
overexpression of cdk2 may disturb maturation of adult
cardiomyocytes.
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Expression of Cell-Cycle Regulators in
Adult cdk2 Tg
To additionally explore how cdk2 overexpression affects
cell-cycle regulation in cardiomyocytes on a molecular level, we
examined the expression of several cell-cycle regulators. Although
neonatal WT hearts expressed substantial levels of positive cell-cycle
regulators, such as cyclins A, D3, and E, cdk4, and cdc2, the adult
hearts expressed undetectable levels, consistent with the cell-cycle
withdrawal in adult hearts
(Figure 5). In contrast, adult cdk2 Tg hearts were
characterized by marked increases of cyclin D3 and cdk4 and modest
increases in cyclin A and E expression
(Figure 5). This suggests that cdk2 activates the expression
of G1- to S-phase cell-cycle regulators. As in WT hearts, cdc2
(Figure 5, top right) and cyclin B1 (data not shown) were
also undetectable in adult cdk2 Tg hearts, indicating that cdk2
overexpression is insufficient in upregulating the cell-cycle
regulators of the G2 to M phase. The expression of p21 and p27 cdk2
inhibitors was unchanged in cdk2 Tg hearts compared with the WT hearts
(Figure 5).
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Effect of Pressure Overload on cdk2 Tg
Mice
To determine the effects of overexpressed cdk2 in
response to the biomechanical stress, we induced cardiac hypertrophy by
pressure overload using an ascending aortic constriction
(AAC).38 After 7 weeks of
AAC, the HW/BW ratio was increased in both cdk2 Tg and WT compared with
sham-operated mice hearts
(Table 2). However, cdk2 Tg showed a significantly greater
increase in heart weight (cdk2 Tg=174.1±19.6 versus WT=146.6±21.4 mg,
n=10, P<0.05). The lung weight
in cdk2 Tg was also significantly increased compared with WT in
response to pressure overload (cdk2 Tg=175.3±13.3 versus WT=156.8±7.1
mg, n=10, P<0.05).
Echocardiography showed that pressure overload caused a greater
increase in the posterior and anterior wall thickness of cdk2 Tg mice
than that of WT mice. However, there was a significant decrease in the
fraction shortening after 7 weeks of ascending aortic constriction in
cdk2 Tg mice, suggesting a left ventricle failure. There was no
significant difference in body weight or liver weight, suggesting the
absence of significant right ventricular failure. Nuclear staining
analysis demonstrated that pressure overload did not change the
relative distribution of individual cardiomyocyte nuclei (compare
Figures 3B and 6B). Pressure overload also had no effect on
PCNA and cdc2 expression in either WT or cdk2 Tg
(Figure 6C). Moreover, pressure overload did not
significantly increase the BrdU incorporation in cdk2 Tg compared with
nonbanding cdk2 Tg (23±3 BrdU-positive cardiac
nuclei/4x104 total nuclei in cdk2 Tg at the
baseline vs 25±2 BrdU-positive cardiac
nuclei/4x104 total nuclei in cdk2 Tg after
AAC, n=10, P=0.12). The
expression of ß-MHC and ANF in cdk2 Tg mice hearts increased
additionally in response to pressure overload
(Figure 4). Interestingly, there was greater relative
hypertrophic growth in mononuclear than binuclear cardiomyocytes in
response to pressure overload compared with sham-operated cdk2 Tg
hearts
(Figures 6A and 6D). The results indicate that pressure
overload in cdk2 Tg mice did not alter the proportion of mononuclear
cardiomyocytes. Compared with binuclear cardiomyocytes, pressure
overload did cause greater hypertrophy of the mononuclear
cardiomyocytes. Cdk2 mice were also more susceptible to left
ventricular dysfunction in response to hypertrophic
stimuli.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, cardiac-specific overexpression of cdk2 resulted in increased numbers of smaller mononuclear cardiomyocytes and suggestion of a less-differentiated adult heart. There was also evidence of increased DNA synthesis and altered cell-cycle regulators favoring the G1 to S phase. Because the adult cdk2 Tg heart weight was unchanged but the cells were smaller compared with WT mice, there was most likely a total increase in the numbers of cardiomyocytes in cdk2 Tg hearts. In fact, hemocytometer cell counts of dispersed cell preparation showed that there was a 42% increase in the total number of cardiomyocytes in cdk2 Tg mice compared with age-matched WT mice (1.7x107±1.3x 105 in cdk2 Tg versus 1.2x107±1x105 in WT, n=6, P<0.001). This finding is in agreement with previous reports that overexpression of G1-phase cell-cycle regulators shorten the G1 phase and decreases cell size41 42 43 and that loss of p27, a major cdk2 inhibitor, increases cell numbers but decreases the cell size in hearts.44 45 46 47
Potential increase in cell numbers in cdk2 Tg hearts could
occur in 3 developmental periods. First, cell number could increase in
the embryonic stage. Although the notion that -MHC promoter is only
activated in the ventricles after birth has been
challenged,30 31 48
the expression of cdk2 in the cdk2 Tg at E18 was not significantly
different than the WT littermates (data not shown). Thus, cdk2 is not
overexpressed at this stage in cdk2 Tg hearts. Second, the cell
division may occur in adulthood. The BrdU labeling index of
cardiomyocytes was 0.00005% in 3-month-old control littermates.
However, the index in cdk2 Tg was increased to 0.06%, which is
consistent with previous reports that overexpression of cyclin D1
promotes DNA synthesis in adult
hearts.28 The increased
expression of PCNA produces independent molecular evidence of DNA
synthesis. However, the absolute value of the BrdU index was still very
low compared with that of dividing
cells.49 50 Thus,
the proliferative capacity of adult cdk2 Tg cardiomyocytes still seems
to be extremely limited. The most likely scenario is that
overexpression of cdk2 causes additional rounds of cardiomyocyte
replication in the neonatal period, when -MHC becomes activated.
This hypothesis is supported by the significantly increased HW/BW ratio
and PCNA expression at neonatal day 2
(Figure 2). At neonatal day 2, most cardiomyocytes are
mononuclear1 3 ;
therefore, cdk2 Tg heart weights would be greater than WT, because cdk2
Tg may have more cells. In the p27 knockout, cardiomyocytes undergo 1
or 2 additional cell divisions before they withdraw from the cell
cycle.51 52 Thus,
the phenotypes of cdk2 Tg mice resemble those of p27 knockout
mice.
Overexpression of cdk2 revealed a significant accumulation of mononucleated cardiomyocytes in adult hearts, which contrasts with the increase in multinucleated cardiomyocytes associated with cyclin D1 overexpression.28 Two possibilities for this phenomenon can be postulated. First, overexpression of cdk2 may delay cardiomyocyte terminal differentiation and subsequently increase cell numbers in adult cdk2 Tg hearts. In cdk2 Tg mice, hypertrophy growth may be limited by an organ size control.34 53 Thus, the smaller mononuclear cardiomyocytes are prevented from completely differentiating to become binuclear cardiomyocytes and persist in a less-differentiated state. This is consistent with the finding that constitutive overexpression of cdk2 inhibits differentiation of rat pheochromocytoma PC12 cells.54 This scenario is also supported by the fact that the expression of fetal genes, such as ANF and ß-MHC, is significantly increased in adult cdk2 Tg hearts. This result contrasts the previous finding that cyclin D1 Tg had no fetal gene activation.28 The second possibility is that overexpression of cdk2 may inhibit the formation of binucleate cells, because the decline in cdk2 abundance and activity coincides with binucleation during murine development.55
To determine whether hypertrophic growth can stimulate DNA synthesis in cdk2 Tg cardiomyocytes, we tested the effect of pressure overload on cdk2 Tg. Pressure overload did not change the proportion of mononuclear and multinuclear cardiomyocytes and BrdU incorporation. This result is consistent with a previous report that there was no significant difference between the ratio of mononucleate and binucleate cardiomyocytes in control and hypertrophic hearts; in addition, no DNA synthesis occurred in response to hypertrophic stimuli.40 Previous studies demonstrated that hypertrophic growth stimulated PCNA expression in adult hearts and may be attributable to fibroblast proliferation.5 However, in the present study, we did not find a significant increased expression of PCNA after 7 weeks of AAC. One possibility for this is that the window of reactive PCNA or DNA synthesis may have been missed, because most hypertrophic growth occurred within 3 weeks after pressure overload.56 Acute pressure overload resulted in an exaggerated hypertrophic response, mainly from smaller, mononuclear cardiomyocytes. This hypertrophic response, compared with normal WT cardiomyocytes, seemed maladaptive and resulted in increased left ventricular dysfunction.
Transgenic models have been widely applied in the field of cardiovascular research.57 It is generally accepted that experimental and control animals differ only by the presence or absence of the transgene in transgenic mice.57 Any phenotypic difference observed in the 2 groups are attributable, either directly or indirectly, to the transgene expression. Consequently, nontransgenic littermates are generally used as an internal control. However, we have recently demonstrated that cardiac-specific overexpression of green fluorescence protein, a molecule presently believed to be biologically inert, resulted in a dilated cardiomyopathy when the levels of green fluorescence protein expression is very high.58 Therefore, using cdk2 kinase-mutant transgenic mice as a control would make the conclusion much stronger than using a nontransgenic littermate control. This remains to be done. It should be noted, however, that the phenotype of cdk2 Tg mice, an increase in mononuclear myocytes, is unique and thus less likely to be a nonspecific response to a foreign gene in the heart.
Overexpression of cdk2 significantly increased the D-type
cyclin 3 and cdk4
(Figure 5), which may phosphorylate pocket proteins and
disrupt the complex of pocket proteins and E2F transcription factors.
The expression of cyclins A and E was significantly increased in cdk2
Tg compared with WT mice
(Figure 5). This finding is important, because cyclins A and
E are the partners of cdk2 in the cell-cycle process. However, the
expression of G2- to M-phase cell-cycle regulators, such as cdc2 and
cyclin B1, was not observed in adult cdk2 Tg, suggesting that
overexpression of cdk2 was not able to overcome the G2- to M-phase
block.
The mechanism of cell-cycle withdrawal in cardiomyocytes is complicated and poorly understood. The present study suggests a possible role of cdk2 in overcoming G1- to S-phase block in cardiomyocytes. However, additional studies are needed to show whether G2- to M-phase block can be overcome.
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Acknowledgments |
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This work was supported in part by National Institutes of Health grant AG 44976 (to S.I.). H.-S.L. is supported by postdoctoral fellowship from the American Heart Association (Massachusetts affiliate). We thank Dr William Pu for his critical reading of the manuscript.
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Footnotes |
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Original received August 31, 2000; revision received January 4, 2001; accepted January 4, 2001.
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