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Endocrinology Vol. 145, No. 3 1356-1366
Copyright © 2004 by The Endocrine Society

Targeted Expression of Calmodulin Increases Ventricular Cardiomyocyte Proliferation and Deoxyribonucleic Acid Synthesis during Mouse Development

Josep M. Colomer, Motomu Terasawa and Anthony R. Means

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

Address all correspondence and requests for reprints to: Anthony Means, Department of Pharmacology and Cancer Biology, Box 3813, Durham, North Carolina 27710. E-mail: means001{at}mc.duke.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cell signaling pathways that control ventricular cardiomyocyte proliferation during development are poorly understood. Here we show that increasing levels of the ubiquitous Ca2+ receptor calmodulin (CaM) can regulate cardiomyocyte proliferation in vivo. Targeted overexpression of calmodulin in the heart during embryonic development leads to a 37% or a 79% increase in the number of ventricular myocytes present at embryonic d 17 in mice heterozygous or homozygous for the transgene, respectively. Whereas all homozygous mice die within 10 d after birth, most of the heterozygous mice survive even though they contain 40% more ventricular myocytes relative to the wild-type mice throughout development and into adulthood. The CaM transgene continues to be overexpressed postnatally and, although cell proliferation ceases soon after birth, the elevated levels of CaM lead to an increase in DNA synthesis, which correlates with an increase in the degree of ventricular myocyte polyploidy. Only after proliferation has ceased and polyploidy has become maximal does the continued presence of overexpressed CaM lead to ventricular hypertrophy. However, unlike the case for myocyte number, turning off expression of the CaM transgene results in regression of the hypertrophic response. Together, our results reveal that excess CaM enhances the extent of cell proliferation and DNA synthesis as well as development of hypertrophy of ventricular myocytes in vivo, in a manner consistent with the normal timing of these events during heart development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VENTRICULAR CARDIOMYOCYTE PROLIFERATION during development is a strictly regulated process although the signaling pathways that control this process remain obscure. Analysis of DNA synthesis indicates that nearly 100% of cardiac myocytes are proliferating by d 11 of gestation in mouse embryos [reviewed by P. P. Rumyantsev (1)]. Thereafter, the number of dividing myocytes in rodents decreases progressively until cell division terminates completely by postnatal d 3 (2, 3). As a consequence of the proliferative period, the number of myocytes increases during development from 0.8 x 106 at embryonic d (E) 15 to 9.6 x 106 and then remains constant through adulthood (1). Expression of several cell cycle regulatory genes, such as cyclins D1 and D3, cyclin-dependent kinase (cdk) 4, p107, and p53, closely parallel the relative level of cardiomyocyte DNA synthesis from E12 to postnatal d 15 (2). This extended time in development includes not only the proliferative period but also the period between postnatal d 5–10, during which the last wave of DNA synthesis occurs and results in binucleation of 90–95% of the cardiac myocytes in mice (4) and rats (3). Thus, after the proliferative period the growth of the heart is entirely due to increases in the size of individual cardiomyocytes (3). However, the mechanisms responsible for this switch from proliferation to myocyte growth are poorly understood.

Due to the cessation of myocyte proliferation soon after birth, myocytes that die in the adult heart cannot be replaced. This remarkable block of proliferation has prompted numerous attempts to reinduce cardiomyocyte proliferation in hearts of adult mice by genetic manipulation. Unfortunately, the only protein shown to be successful in this regard when overexpressed in the heart is the large T antigen of simian virus 40 (SV40), which not only reinduces myocyte proliferation but also leads to tumorigenesis (5). Overexpression of other proteins involved in cell cycle regulation, such as cyclin D1, cdk2, and the catalytic subunit of telomerase, delay but do not overcome the cessation of cardiomyocyte proliferation that normally occurs after birth (6, 7, 8). In a similar vein, the absence of the cell cycle inhibitor p27 also results only in a delay in termination of myocyte proliferation (9). These studies indicate that the block of myocyte proliferation that occurs soon after birth is profound and unlikely to be either prevented or reversed without resulting in dire consequences.

Calmodulin (CaM) is a ubiquitous Ca2+ receptor that is critical for cell cycle regulation in cultured mammalian cells as well as in the fungus Aspergillus nidulans. Indeed, CaM levels increase in Chinese hamster ovary cells when they exit the cell cycle (from G1–G0) (10), decrease by 2-fold when cells reenter the cell cycle (from G0–G1), remain low during G1 and then double at the G1/S transition (11, 12). Moreover, CaM overexpression in the mouse cell line C-127 shortens the cell cycle length due to an accelerated G1 phase (13) but is also required for the G2/M transition (14). CaM antagonists such as W13 and W7 block progression of cycling cells in both G1 (11) and G2 (12), as well as the reentry of quiescent cells into the cycle in response to mitogenic stimuli (10, 11). In addition, a 2- to 3-fold decrease in CaM levels, resulting from the expression of antisense RNA, arrests the cycle of mouse C-127 cells in both G1 and G2 (14). The importance of CaM in cell cycle regulation has been confirmed in the genetically tractable fungus Aspergillus nidulans. Strains of this organism engineered to express decreased levels of CaM suffer blocks in both G1 and G2 of the nuclear division cycle, whereas overexpression of CaM shortens the cell cycle due to a decrease in the length of G1 (15). Thus, the roles for CaM in the cell cycle are remarkably similar in Aspergillus and mammalian cells in culture. However, no studies have been reported that assess the ability of excess CaM to drive proliferation of mammalian cells in vivo.

We previously generated lines of transgenic mice in which CaM was overexpressed in the heart using a 500-bp fragment of the atrial-natriuretic factor (ANF) promoter (16). We have shown that this overexpression leads to hypertrophy of both atrial and ventricular cardiomyocytes and that at least one CaM target involved in the hypertrophy response is CaM kinase II (16, 17). In addition, hypertrophy induced by pressure overload also correlates with increases in both the amount of specific CaM-dependent protein kinase (CaMK) II isoforms and CaMKII autonomous activity in the heart (18), and overexpression of CaMKII {delta} in the heart results in cardiac hypertrophy (19, 20, 21).

Because the CaM transgene in mice is regulated by the ANF promoter and this promoter is functional in the ventricles during the developmental period in which myocytes proliferate, we questioned whether the overexpression of CaM could increase the number of myocytes in the ventricles during this proliferative period, and, if so, whether the duration of this proliferative period was altered. We report here that the presence of excess CaM during embryogenesis does indeed result in an increase in the number of ventricular myocytes, and that a positive correlation exists between the degree of CaM overexpression and the number of myocytes per ventricle. In fact, at the highest levels of CaM we could obtain in mice homozygous for the transgene, all the mice die before the postnatal d 10. Although we find that CaM overexpression does not extend the duration of the proliferative period, it does result in an increase in the number of myocytes engaged in postnatal DNA synthesis, which leads to a 3-fold enhancement in endoreduplication (polyploidy). Finally, only after the proliferative and DNA synthesis effects of CaM have ceased does CaM-induced hypertrophy occur. Thus, overexpression of CaM can drive the cell cycle in vivo, and CaM is the first protein shown to affect all three stages of heart growth when overexpressed in ventricular myocytes during mouse development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
The transgenic mice used in this study were originally generated by Gruver et al. (16). Mice were housed in the Duke University Levine Science Research Center Vivarium under a 12-h light, 12-h dark cycle. Food and water were provided ad libitum, and care was given in compliance with National Institutes of Health guidelines on the use of laboratory and experimental animals. Animal experiments were carried out in compliance with a protocol approved by the Duke University Animal Care and Use Committee.

Genotyping was performed by dot blot analysis after isolation of tail DNA after proteinase-K digestion, according to the method of Hogan et al. (22). Ten micrograms of DNA were applied in duplicate onto a zeta-probe membrane (Bio-Rad, Hercules, CA), and hybridized to a restriction fragment (SphI/HindIII) overlapping the 3' end of the chicken CaM cDNA radiolabeled with 32P by random priming using the method of Feinberg and Vogelstein (23). To distinguish heterozygous from homozygous transgenic mice, a parallel dot blot was done using a probe specific for glyceraldehyde 3-phosphate dehydrogenase as described before (17). Quantification of the dot blots was done by PhosphorImager (Molecular Dynamics Inc., Piscataway, NJ) and the ratio CaM/glyceraldehyde 3-phosphate dehydrogenase was used to determine the CaM transgene dosage.

CaM quantification
Ventricles of WT, heterozygous, and homozygous CaM mice were dissected at E17 and homogenized in RIPA buffer [50 mM Tris-Cl (pH 8), 200 mM CaCl2, 0.4% sodium dodecyl sulfate, 1% Nonidet P-40, 1 mM dithiothreitol, 0.5% deocycholate, and 1 mM benzamadine, with the following protease inhibitors: 5 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM pefablock). The protein content was assayed using the DC Protein Assay kid (Bio-Rad), and equivalent amounts of extracts from each genotype were analyzed by Western blot, along with 10, 50, and 150 ng of purified bovine testes CaM as standard, using a CaM-specific antibody (Upstate Biotechnology, Lake Placid, NY). The bands were scanned (Personal Densitometer SI, Molecular Dynamics) and quantified using IQMac software (Molecular Dynamics).

Histology
Hearts were dissected, processed and stained with hematoxylin and eosin as described before (16).

PCNA (proliferating cell nuclear antigen) immunostaining was performed on tissue sections prepared as indicated above followed by blocking the endogenous peroxidase with 0.5% H2O2. An antibody specific for PCNA was used (Calbiochem Novabiochem Corp., San Diego, CA) and the signal amplified using an ABC kit (Vector Laboratories Inc., Burlingame, CA).

Analysis of DNA synthesis in ventricular myocytes was determined using a kit for 5-bromo-2'-deoxy-uridine (BrdU) labeling and detection (Roche Molecular Biochemicals Corp., Indianapolis, IN). Mice received 10 µmol of BrdU per 100 g of body weight as a single ip injection following the manufacturer’s procedure. After 1 h. the animals were killed, the hearts removed and processed as indicated above. An antibody against BrdU and a second antibody against mouse IgG labeled with fluorescein isothiocyanate, both provided in the kit, were used to detect nuclei incorporating BrdU. Positively labeled nuclei obtained by PCNA or BrdU labeling were quantified and expressed as a percentage of total myocyte nuclei.

DNA content
The amount of DNA per ventricle in 17 d embryos was determined by a fluorometric method (24) exactly as described before (16), except that only ventricles were used in the present study.

To measure the DNA content per nucleus the Feulgen technique was used as described previously (25). Briefly, mice were killed at 10 d of age, ventricles dissected and fixed in 10% formaldehyde phosphate buffer (pH 7.0). After fixation for 10 d or more, ventricles were incubated in 50% KOH for 16 h, rinsed in saline, and left in saline for 4 h. Thereafter, the saline was replaced and the ventricles mixed vigorously until a homogenous cell suspension was obtained. Aliquots of this suspension were placed on a microscope slide and dried. The slides were stained by the Feulgen method (26). Hydrolysis in 5 N HCl continued for 10 min at 37 C, and incubation in Schiff reagent for 1 h at room temperature. Nuclear staining was analyzed at x1000 using a Zeiss Axioskop microscope (Zeiss, Thornwood, NY) equipped with epifluorescent optics. Images were captured using a Pentamax-cooled charge-coupled device camera (Princeton Instruments, Princeton, NJ) interfaced with Metamorph software (Universal Imaging Corp., West Chester, PA) and analyzed with Metamorph.

Size and number of myocytes
Ventricles from mice of different ages (E17; and 0, 4, 7, 10 and 14 d) were dissected and weighed. Isolated myocytes were prepared by digestion of minced ventricles with collagenase in Joklik media as described before (27), slightly modified. Briefly, a digesting solution composed of Joklik’s MEM (Life Technologies, Inc., Rockville, MD) containing in (mM) 113 NaCl, 4.7 KCl, 12 NaHCO3, 10 KHCO3, 0.6 NaH2PO4, 0.5 MgCl2, 10 HEPES, 20 D-glucose, 30 taurine, 2 carnitine, 2 creatine, and 0.020 Ca2+ at pH 7.4, with 150 U/ml of type 2 collagenase (Worthington Biochemical Corp., Freehold, NJ), was incubated with 1-mm pieces of ventricular tissue at 37 C for 5 min. The first digestion was discarded and the tissue was incubated again with the digesting solution for 5 min. and centrifuged at 50 x g for 2 min. The pellet, containing free cells and tissue, was resuspended in washing solution (Joklik’s modified, with 1% of BSA, without collagenase) and filtered through a 250-µm nylon mesh. The remaining tissue was digested again with digesting solution for 5 min at 37 C, washed in washing solution, and filtered. The two filtered solutions were combined and contained a population of cells consisting primarily of myocytes. Immediately after filtering, the cells were fixed in glutaraldehyde (final concentration 2%), which has been shown not to alter myocyte size (28).

A Coulter counter (Coulter Electronics Inc., Hialeah, FL) was used to determine the volume of fixed myocytes obtained from the ventricles (29) (30). Latex microspheres of 10.28 µm of diameter were used to calibrate the machine. After counting the number of myocytes per milliliter at the microscope, the same cell suspension was run through the Coulter counter and the minimal threshold volume was adjusted to count half the amount of cells per milliliter. The resulting value represents the median size.

The number (N) of myocytes per ventricle (right and left ventricles together) was calculated according the following equation: N = (myocyte volume fraction x ventricular volume)/median myocyte size. The myocyte volume fraction used was 75%, and ventricular volume was calculated as the ventricular weight divided by the ventricular specific gravity (1.06) (30) for both normal and CaM transgenic mice because no differences in fibrosis or water content were observed between these mouse strains (see results section).

Cell diameter was measured microscopically in cross-sectioned myocytes microscopically. The myocytes were prepared as described in Histology.

Statistical analysis
ANOVA (two-factor ANOVA in Fig. 1Go) and the Student’s t test analysis were applied in every comparison except to compare the observed to the expected number of embryos based on mendelian segregation, where a square-Ji test was applied. All the statistical analysis was done using Statview (Abacus Concepts Inc., Berkeley, CA). Statistical significance was accepted when P < 0.05.



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  		FIG. 1. Effect of CaM overexpression on ventricular growth. Ventricular growth is represented as ventricular weight corrected by body weight (V/B) from birth to 28 d of age for mice with the indicated genotypes; mutant CaM (CaM-8) and WT-CaM transgenes are similarly expressed up to postnatal d 5. Mean ± SE; n >= 10 for each point. Two-factor ANOVA indicates that both genotype (P < 0.0001) and the interaction (genotype x age, P < 0.0001) contribute significantly to the V/B ratio. Also included are the individual V/B ratios of pups found dead.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overexpression of CaM results in reduced survival
We previously generated transgenic mice in which the ANF gene promoter targeted CaM overexpression to the heart. Because the level of CaM has been shown to regulate the rate of cell proliferation in mammalian cell lines, we used the ANF-CaM mice to determine whether this effect occurs in vivo during organogenesis of the heart. Male and female mice expressing the transgene (called CaM heterozygous) were mated to produce mice with extra high (homozygous), high (heterozygous), or normal (wild-type, WT) levels of CaM in the ventricular myocytes. Figure 1Go shows the phenotypic consequences of elevated CaM levels on ventricular size (expressed as ventricle/body weight ratio, V/B) as a function of postnatal development. As expected, WT mice had a constant V/B throughout postnatal development. However, mice transgenic for CaM showed a significant increase in their V/B ratio, indicating that increased CaM levels in the ventricles result in larger ventricles. Specifically, there is a 50% increase in the V/B in CaM mice compared with WT at birth, which eventually increases to 100% at d 14 postnatal, then decreases to 20% by d 28 (see Fig. 1Go), and this difference is maintained in adults (V/B ratio at 3 months of age is 3.86 ± 0.07 and 5.29 ± 0.45 in WT and CaM mice respectively, P <= 0.026). Moreover, these effects are specific for the Ca2+ transducing action of CaM because mice expressing a mutant form of CaM (CaM-8) that binds Ca2+ normally but cannot bind to CaM-target proteins (31) show a V/B ratio identical with WT during the time that both transgenes are expressed (Fig. 1Go). Furthermore, two-factor ANOVA shows that the interaction of genotype with age has a significant effect on the V/B, so that CaM mice at 14 d of age have a significantly higher V/B compared with the average V/B for CaM mice whereas CaM mice at 28 d of age or during adulthood have a V/B lower than the average V/B for CaM mice. The decrease of the V/B with age parallels the down-regulation of the ANF-CaM transgene in the ventricles as the mice age, which reduces the amount of CaM produced by the transgene as shown previously (17). These results indicate that the size of the ventricles is positively correlated with the CaM levels.

We noticed that a number of pups resulting from heterozygous matings died within the first 10 d after birth. Although we could not do biochemical measurements on the hearts from these mice, we did genotype them and measured the V/B ratio. As shown in Fig. 1Go, the V/B ratio of the dead pups was greater in every case than that of age-matched transgenics that lived to adulthood. The genotype analysis suggested the possibility that the mice that died might have been homozygous for the transgene, indicating that survival might be influenced by the CaM content. Indeed, the content of CaM in the ventricles is dependent on the number of transgenes, as shown in Fig. 2AGo. Semiquantitative Western blot analysis, using different amounts of purified bovine testis CaM as a standard, indicates that at E17 WT ventricles contain between 1 and 1.7 ng of CaM/µg protein, whereas heterozygous transgenic mice contain between 3 and 6.5 ng of CaM/µg protein and homozygous contain between 5.5 and 9.2 ng of CaM/µg of protein. Therefore, the number of transgenes determined by genotyping accurately reflects changes in the CaM content of the ventricular myocytes.



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  		FIG. 2. Effect of cardiac CaM dose on mouse survival. A, CaM content in the ventricles of WT, heterozygous and homozygous CaM transgenic mice at E17. Representative quantification of a CaM Western blot analyzing 15 µg of protein from ventricular extracts of the indicated genotypes using 10, 50, and 150 ng of purified CaM as standard. B, The survival was monitored from birth to postnatal d 21 in WT mice (n = 76), heterozygous (n = 102), and homozygous (n = 62) CaM mice and expressed as a percentage of the number of initially born mice of each genotype.

 
To test the hypothesis that survival might be influenced by the CaM content, we analyzed the correlation between survival and genotype of mice at different ages, from late embryogenesis to postnatal d 21. Indeed, the ratio of WT:heterozygous:homozygous mice was 1.0:1.8:0.5, based on the analysis of 66 embryos at E17, compared with the expected 1.0:2.0:1.0 (P <= 0.015 for observed vs. expected homozygous, using the square-Ji test), indicating that only half of the expected CaM homozygous mice are found at that early age. In a separate experiment in which 164 transgenic mice were analyzed, all 62 of the homozygous and 25 of the heterozygous (of 102) had died by postnatal d 21 (Fig. 2BGo). On the other hand, no deaths occurred among the WT (n = 160) or CaM-8 (n = 123, data not shown) mice, indicating that the degree of CaM overexpression correlates with decreased survival (Fig. 2BGo). We conclude that once a threshold of CaM is achieved in ventricular cardiomyocytes, further increases result in death. Based on the mendelian segregation at different ages, we estimate that the pups died during late embryogenesis as well as soon after birth.

CaM induces myocyte proliferation before birth in a dose-dependent manner
In an attempt to correlate the CaM dose-dependent mortality with an abnormal ventricular morphology, cross sections of ventricular tissue were stained with hematoxylin and eosin and analyzed microscopically for gross structural anomalies. At E17, none of the ventricles from either homozygous or heterozygous CaM mouse embryos showed any scars, necrosis, or fibrosis (Fig. 3Go, A–C). Similarly, CaM overexpression did not result in any changes in vacuolization (which is due to deposits of glycogen) or Aristchow nuclei (which are due to longitudinally oriented chromatin). The number of cardiomycytes per unit area was quantified as an indicator of cardiomyocyte size but was also found to be very similar in all three genotypes (37.7 ± 3 myocytes/104 µm2, 38.4 ± 1.5 myocytes/104 µm2, 33.1 ± 1.3 myocytes/104 µm2, in WT, heterozygous and homozygous respectively, n >= 3). In fact, the only structural difference we noted was that the thickness of the ventricular wall increased in parallel with the number of CaM transgenes and the ventricles became more rounded in appearance. Clearly, as shown in Fig. 3DGo, E17 CaM mice appear to have enlarged ventricles relative to WT. To confirm this finding quantitatively and establish whether the ventricular size is dependent on dose of the CaM transgene, we quantified the V/B ratio in embryos at E17. As shown in Fig. 4AGo, there is a 50% increase in the size of the ventricles of heterozygous CaM mice and a 77% increase in the ventricles of homozygous CaM mice compared with WT (P <= 0.0001 in both cases). The increase in the V/B ratio between heterozygous and homozygous CaM mice was also significant (P <= 0.0006), supporting the contention that CaM overexpression enlarges the ventricles in a dose-dependent way.



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  		FIG. 3. Effect of the CaM dose on the morphology of the hearts at E17. Representative microphotograph of WT (A), heterozygous (B), and homozygous (C) embryonic ventricular tissue. Bar, 10 µm. D, Representative microphotographs of the whole heart of WT (left), heterozygous (middle), and homozygous (right) E17 embryos (low magnification).

 


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  		FIG. 4. Quantification of the effect of the CaM dose on ventricles of mice at E17. A, The V/B ratio was calculated for WT, heterozygous and homozygous E17 embryos. Mean ± SE; n >= 10 for each genotype; *, P <= 0.0001; {dagger}, P <= 0.0006. B, The DNA content in ventricles was measured and expressed as the total amount of DNA per ventricle of E17 embryos. Mean ± SE; n >= 4 for each genotype; *, P <= 0.03; {dagger}, P <= 0.0003; {ddagger}, P <= 0.013. C, The median volume of ventricular myocytes was estimated from WT, heterozygous and homozygous E17 embryos. Mean ± SE; n >= 5; *, P <= 0.05. D, The number of myocytes per ventricle was estimated from WT, heterozygous, and homozygous E17 embryos. Mean ± SE; n >= 5; *, P <= 0.025; {dagger}, P <= 0.0002.

 
Because the histological analysis shows no increase in the amount of extracellular matrix, the enlargement of the ventricles must be due to either an increase in the size of the myocytes or to an increase in the number of myocytes. If the number of myocytes is increased in a CaM dose-dependent manner, the increase in the number of CaM transgenes should result in a parallel increase in the amount of DNA in the ventricles. Therefore, the total DNA content in the ventricles of embryos at E17 was quantified for the three genotypes. Indeed, heterozygous CaM mice contained 13% more DNA/ventricle than WT (P <= 0.03), as shown in Fig. 4BGo; and homozygous CaM mice contained 15% more DNA/ventricle than heterozygous CaM mice (P <= 0.013). Note that DNA of nonmyocyte cells is also quantified in this assay although these cells do not overexpress the CaM transgene. Therefore, the increases in DNA content due to CaM overexpression in myocytes is an underestimation of what occurs in individual myocytes. Still, these results confirm that ventricular DNA content is increased in a CaM gene number-dependent way and are compatible with an increased rate of myocyte proliferation during embryonic development of the heart.

To address directly the hypothesis that the number of myocytes accounts for the ventricular size differences observed among the different genotypes, we dissected the ventricles from E17 mice, weighed them, digested them with collagenase, and fixed the myoyctes in gluteraldehyde, which preserves the size of the cells (28). Then we counted the number of myocytes in suspension by microscopy to distinguish myocytes from fragmented cells and small nonmyocyte cells. Then the samples were analyzed by a Coulter counter. The size threshold was adjusted to count half the number of cells. This measurement provides the median size of the cells (30). The results indicate that the size of ventricular myocytes from WT (618 ± 43 fl), heterozygous (596 ± 65 fl), and homozygous (550 ± 50 fl) mice is very similar (Fig. 4CGo). If anything, higher CaM levels result in rather smaller myocytes, as the difference becomes significant between WT and homozygous (P = 0.047). This result is consistent with the similar number of moycytes per unit area as seen by microscopy in Fig. 3Go, and indicates that CaM overexpression does not result in increased growth (hypertrophy) of individual myocytes during development.

Based on the median size of the myocytes and the known size of the ventricles, we can estimate the approximate number of myocytes in the ventricles (29). Figure 4DGo shows that the number of myocytes significantly increases when CaM levels are higher: WT ventricles contain fewer myocytes (5.4 ± 0.8 million) than heterozygous mice (7.4 ± 1.5 million, P < 0.025), which contain fewer myocytes than homozygous mice (9.7 ± 2.1 million, P < 0.025). These results show that CaM results in increased proliferation of ventricular myocytes during development. They also indicate that the proliferative effect of CaM is dose dependent, consistent with the observations of bigger ventricles and more DNA/ventricle as a function of the CaM transgene copy number. Collectively our results confirm that the increased ventricular mass during embryogenesis is due exclusively to increased myocyte proliferation and is positively correlated with the number of CaM transgenes. Our data are the first to show that CaM overexpression induces hyperplasia in an animal tissue in vivo.

Overexpression of CaM results in increased DNA synthesis and polyploidy after birth
Although cell proliferation in the ventricles is largely restricted to embryonic development, DNA synthesis continues in these cells for the first few days after birth (2). This afforded us the opportunity to examine whether overexpression of CaM resulted in increased DNA synthesis after birth. We monitored DNA synthesis in postnatal mice by two independent immunocytochemical methods: BrdU incorporation as shown in Fig. 5AGo and PCNA content as shown in Fig. 5BGo. Both methods produced nearly identical results. In WT mice, only a small number of cardiomyocytes was engaged in DNA synthesis at birth. This number gradually increased until d 7 and then began to decrease. DNA synthesis had ceased in cardiomyocytes of WT mice by d 17. On the other hand, the CaM mice showed a 5-fold increase in the number of myocytes engaged in DNA synthesis at birth relative to WT, as seen by both methods. With some fluctuation, this large number of cells incorporating DNA was maintained through d 7 before beginning to decline. DNA synthesis was undetectable in CaM mice by postnatal d 17. These results show that, whereas overexpression of CaM does not change the timing of postnatal DNA synthesis, the number of ventricular myocytes engaged in DNA synthesis is proportional to the number of CaM transgenes.



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  		FIG. 5. Effect of CaM overexpression on postnatal DNA synthesis in cardiomyocytes. A, BrdU incorporation was quantified in ventricular sections as the percentage of positive cardiomyocyte nuclei. B, PCNA labeling was also quantified as a percentage of positive cardiomyocyte nuclei. Mean ± SE; n = 3 for each time point and strain; *, P <= 0.0001 comparing CaM to WT mice of the same age; {dagger}, P <= 0.006 comparing CaM with WT mice of the same age.

 
Because DNA synthesis is required for cell proliferation, and DNA synthesis during postnatal cardiac development depends on the number of CaM transgenes, we investigated whether postnatal ventricular myocyte proliferation (hyperplasia) was dependent on CaM overexpression. However, to accurately estimate the number of myocytes by the Coulter counter method as was done in the embryos, both WT and transgenic CaM ventricles must have a similar nonmyocyte content.

The nonmyocyte content consists of two main components: fibroblasts and collagen deposits (excess of which is called fibrosis), and accumulation of water (excess of which is called edema). Microanalysis of cross sections of hearts from different ages stained with the collagen-specific dye picrosirius red showed no signs of collagen accumulation or differences in the rearrangement of cardiomyocytes in the ventricles (results not shown). Therefore, excess CaM does not induce fibrosis. Similarly, analysis of the water content (differences between wet and dry weight) of the ventricles showed no cardiac edema. Because edema due to congestive heart failure is more evident in other tissues such as lung and liver, the water content of those tissues was also analyzed, and we found no difference in CaM mice compared with WT (results not shown). Therefore overexpression of CaM does not change the nonmyocyte content of the ventricles.

Thus, the number of cardiomyocytes in the ventricles was estimated using the Coulter counter method. Figure 6Go shows the number of total ventricular myocytes as a function of postnatal development. WT mice show a slight increase in myocyte number between postnatal d 1 and 4, after which time the number of myocytes stabilizes coincident with the cessation of proliferation known to occur soon after birth (3). However, CaM mice show an increased number of myocytes per ventricle at all ages examined (Fig. 6Go), consistent with the hyperplasia observed in E17 embryos. Specifically, at postnatal d 1 there was a 75% increase in total ventricular cardiomyocyte number in the CaM mice relative to WT (8.36 ± 0.42 x 106 cardiomyocytes per ventricle and 14.65 ± 0.791 x 106 cardiomyocytes per ventricle in WT and CaM heterozygous mice, respectively). The increase in the number of cardiomyocytes in newborn mice is specific for functional CaM because mice of the same age expressing the mutant form of CaM, CaM-8, contained 8.52 ± 0.67 x 106 cardiomycoytes per ventricle, similar to WT newborns. Although the number of cardiomyocytes per ventricle increased progressively between E17 and postnatal d 4 in both WT and CaM heterozygous mice, no further increase was measurable between postnatal d 4 and 14 in CaM heterozygous mice; CaM mice retain 40% more myocytes than WT mice at all time points (Fig. 6Go). These results reveal that the presence of additional CaM does not have a demonstrable effect on proliferation after birth. In adult mice, although CaM levels have normalized and the difference in ventricular size has diminished relative to WT as seen by the V/B ratio, the number of cardiomyocytes remained 34% higher in CaM mice (9.28 ± 1.21 x 106 vs. 12.45 ± 0.89 x 106 cardiomyocytes per ventricle in WT and CaM mice, respectively, n = 3, P <= 0.05). This result demonstrates that the increase in cardiomyocyte number that occurs exclusively during embryogenesis in the CaM mice persists throughout the life of the mouse.



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  		FIG. 6. Effect of CaM overexpression on postnatal ventricular myocyte number. The estimated numbers of myocytes per ventricle were plotted from birth to postnatal d 14. Mean ± SE; n >= 5 for each time point and strain; P <= 0.002 comparing CaM to WT at all ages.

 
Because CaM overexpression induces an increase in postnatal DNA synthesis but does not result in further proliferation, the accumulated DNA could result in two outcomes: the cells could undergo nuclear division without cytokinesis (polynucleation) or the extra DNA could remain in the nucleus and increase the ploidy (polyploidy). To distinguish between these two possibilities we quantified polynucleation and polyploidy in both WT and CaM ventricles at postnatal d 10, as previous studies on WT mice had established that binucleation occurs primarily between postnatal d 5 and 10 (2). Consistent with these previous studies, we found a binucleation index of almost 80% in WT mice (Table 1Go). However, CaM mice contained significantly less binucleated cardiomyocytes than WT mice (P <= 0.025), and no polynucleated (more than two nuclei) cardiomyocytes were detected at this age in mice of either genotype. Therefore, the increased amount of DNA in cardiomyocytes in response to increased CaM levels does not result in an increase in binucleation.


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  		TABLE 1. Binucleation and DNA content per nucleus in ventricular myocytes of normal and CaM transgenic mice at postnatal d 10

 
Nuclear polyploidy was assessed in cardiomyocytes by quantifying the DNA content per nucleus using the Feulgen method. Table 1Go shows that, as expected by postnatal d 10, WT mice contained a low percentage of polyploid nuclei. However, overexpression of CaM increased the percentage of nuclei with polyploidy by 3-fold (P <= 0.008, Table 1Go). The fact that some of the polyploid nuclei seem to be aneuploid together with the presence of some degree of DNA synthesis at postnatal d 10, suggest that overreplication, although slowing down, is still proceeding at d 10. Thus, the 3-fold increase in DNA synthesis that occurs during postnatal development in the CaM mice results exclusively in a 3-fold increase in polyploidy.

Overexpression of CaM results in postnatal enhanced cardiac hypertrophy
We have previously shown that overexpression of CaM in cardiomyocytes results in hypertrophy in 14-d-old mice (17). Because our results show that the number of myocytes per ventricle remains constant after postnatal d 4, but CaM overexpression continues to increase the V/B ratio up to d 14, we analyzed the contribution of CaM overexpression to hypertrophy at different postnatal time points. For this purpose, cardiomyocyte volume was quantified as a function of postnatal age using the Coulter counter method, as shown in Fig. 7Go. The median volume of ventricular myocytes increased severalfold between postnatal d 1–7, but this volume increase was similar in WT and CaM mice. Thus, the increased V/B ratio of the hearts of CaM mice during the first week of postnatal life did not include a hypertrophy component. Whereas the cardiomyocyte volume remained relatively constant between postnatal d 10 and 14 in WT mice, the volume of these cells in the CaM mice continued to increase. By postnatal d 14, the volume of the cardiomyocytes was 46% greater in the CaM mice than in WT mice and this difference was highly significant (Fig. 7Go, P <= 0.0001). These results were confirmed by an independent assessment of myocyte volume, namely measuring cardiomyocyte diameter. Indeed, analysis of cross sections of the ventricles of 14-d-old mice revealed that cardiomyocyte diameter in CaM mice is significantly higher than in WT mice (15.2 ± 1.0 µm in WT and 18.6 ± 1.3 µm in CaM mice; n = 6; P <= 0.002). Finally, we also monitored the myocyte volume in adult mice and found the volume to be similar in both genotypes (10200 ± 2364 fl in WT and 9640 ± 425 fl in CaM mice; n = 3; not significant). Together, our results show that hypertrophy of ventricular myocytes in CaM mice occurs as a function of age and is only apparent after CaM-induced hyperplasia and polyploidy have ceased. Moreover, hypertrophy regresses after expression of the transgene is silenced and CaM levels have returned to normal.



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  		FIG. 7. Effect of CaM overexpression on ventricular myocyte size. The median volume of ventricular myocytes was plotted from birth to postnatal d 14. Mean ± SE; n >= 3 for each time point and strain; *, P <= 0.0001 comparing WT to CaM at postnatal d 14.

 
Taken together, our results indicate that increased levels of CaM cause ventricular enlargement during embryogenesis solely due to hyperplasia, and then contribute to a further postnatal enlargement of the ventricles solely due to hypertrophy. Whereas the increased cell number remains in the adult heart, the hypertrophic response is abated once CaM levels have returned to normal. In addition, increased levels of CaM result in increased polyploidy in ventricular myocytes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Calmodulin plays a pivotal role in regulation of the cell cycle based on studies in mammalian cells in culture and genetically tractable organisms such as Aspergillus nidulans (32). Calcium and calmodulin are required for G1 progression, S phase entry, and the G2/M transition. Here we show that overexpression of CaM in ventricular cardiomyocytes also induces hyperplasia in vivo. This effect is restricted to the embryonic heart and occurs only during the period in heart development known to support cardiomyocyte proliferation. This induction of hyperplasia in vivo by CaM is consistent with studies in culture that show that increases in CaM shorten the duration of G1 and result in a more rapid progression through the cell cycle (13, 14). Moreover, in the postnatal mouse, excess CaM increases the number of cardiomyocytes engaged in DNA synthesis in a manner that leads to polyploidy even after cell division has ceased. These results are consistent with observations that the level of CaM must double at the G1/S boundary in order for cells to enter and progress through S phase of the cell cycle (10, 12). Thus, even without nuclear division, CaM appears to play an important role in endoreduplication of DNA. Our results suggest a role for CaM in regulating the G1/S transition in ventricular cardiomyocytes in vivo.

The mechanisms by which CaM regulates G1 progression and entry into DNA synthesis are not clear. However, the evidence indicates that the hypertrophic pathways involving activation of transcription factors such as myocyte enhancer factor-2 (MEF-2) and nuclear factor of activated T cells (NFAT) by CaM-dependent kinases and phosphatase (calcineurin), may be distinct from the CaM-induced hyperplastic pathways, which involve phosphorylation of several cdks and other cell cycle proteins as well as the accumulation of some proteins such as cyclin D and cdk4. Figure 8Go provides a summary of what is known from the literature and the present report.



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  		FIG. 8. Diagram of CaM growth promoting activities in cardiac myocytes. The diagram shows CaM targets involved in cell proliferation regulation as well as regulation of hypertrophy, which occur at different developmental stages. (SRE) indicates that the target protein of CaMKII is unknown, but it interacts with the serum response element of the ANF promoter. See text for details.

 
In order for quiescent cells to proceed into DNA synthesis in response to a mitogen, CaM is required upstream of the accumulation of the G1 cyclins (33). In mammalian cells, a second role has been proposed in the activation of the cyclin D1/cdk4 complex (34) and phosphorylation of proteins of the retinoblastoma family (35). CaM target proteins have been suggested to include protein phosphatase 2B (calcineurin) and CaMK (32, 33, 36). The evidence indicates that calcineurin regulates the synthesis of cdk4 and cyclin D, which occurs in early G1 of the cell cycle (32) and increased levels of cyclin D1 have been shown to shorten G1 (37). Whether such regulations are mediated by NFAT is not clear, but cAMP response element binding protein has been implicated in this process (33). It is tempting to speculate that overexpression of CaM in the cardiomyocytes would lead to the premature activation of calcineurin and CaMK, which would recapitulate the results in mammalian cells in culture by decreasing the length of G1 (see Fig. 8Go). In support of this possibility, we observed smaller ventricular myocytes in the ventricles of CaM homozygous mice compared with WT, which is indicative of a shorter G1 phase. For example, overexpression of cyclin E or cyclin D1 in fibroblasts, which decreases the length of G1, also results in smaller cells (37, 38). However, because CaM is required for other cell cycle transitions, its overexpression would also shorten the duration of the cell cycle (13, 14, 32). Unfortunately, we were unable to confirm this attractive hypothesis in the CaM mice because ventricles contain several cell types, and these cells proliferate in an asynchronous manner.

Even if CaM-dependent increases in cyclin D1 and cyclin D/cdk4 activity are important for the CaM-induced proliferation of cardiomyocytes, this pathway alone cannot explain the stimulation of endoreduplication that occurs in response to overexpression of CaM. Indeed, whereas CaM overexpression results in a slight decrease in binucleation by d 10, cyclin D1 overexpression results in an increase in the number of binucleated and polynucleated cardiomyocytes as well as larger cardiomyocytes (6). Thus, another CaM-dependent target must be involved in the increased DNA synthesis, we observe once cell division has ceased. One potential target that might be involved in regulation of DNA synthesis is the cyclin E/cdk2 complex (see Fig. 8Go). Interestingly, similar to our observations in mice that contain elevated CaM, overexpression of cdk2 in the heart results in a decreased percentage of binucleated myocytes, an increase in myocyte proliferation around birth and increased DNA synthesis postnatally (7). These remarkable similarities in the phenotypes resulting from overexpression of the two proteins raise the possibility that CaM might regulate cdk2 activity. One level of regulation could be at the level of cyclin accumulation as both cyclin E (which is required for S phase entry) and cyclin A (which is required for S phase progression) bind to and regulate the activity of cdk2 (39). Indeed, cyclin E levels are reduced by treatment of Swiss mouse 3T3 fibroblasts with the calcineurin inhibitor cyclosporin A, and both cyclosporin A and KN-93 (an inhibitor of CaM-dependent kinases) decrease the level of cyclin A (40). Alternatively, CaM might not act directly on cyclin E/cdk2 but rather might participate in regulation of a downstream target of cyclin E/cdk2. Regardless of the precise mechanism involved, our results would be consistent with the presence of a CaM-dependent, cdk2-dependent pathway in ventricular myocytes.

Although high levels of CaM correlate with increased proliferation, increased DNA synthesis and increased polyploidy of ventricular myocytes, the timing and sequence of these events are identical in CaM mice and WT mice. In fact, although altered levels of a number of cell cycle regulators in the heart such as cyclin D1, cdk2, p27, c-Myc, and telomerase reverse transcriptase result in increased myocyte proliferation and DNA synthesis, none of these changes overcome the block to proliferation that occurs in cardiomyocytes (6, 7, 8, 9, 41). In fact, no mammalian protein has been found to overcome this block when overexpressed in the heart. The nature of the block to proliferation in the heart remains enigmatic. However, the proliferation block does not depend on the number of cell divisions because myocytes cultured at 33 C divide a fewer number of times than at 37 C but still withdraw from proliferation at the same time (42). The independence of cell cycle exit from the number of cell cycles is consistent with the cessation of myocyte proliferation at the same age in both WT and CaM mice.

In addition to affecting proliferation and DNA synthesis, overexpression of CaM also results in hypertrophy, but this latter effect occurs only after the former ones have ceased. In fact, CaM is unique in its ability to induce both hyperplasia and hypertrophy of ventricular myocytes in a sequential manner. Overexpression of other single proteins involved in cell cycle progression or growth control is unable to induce both types of growth. Several proteins such as cyclin D1 (6), cdk2 (7), IGF-1 (43), and SV40 T antigen (5) induce only hyperplasia. Other proteins such as ras (44), akt (45, 46) or a dominant-negative phosphatase and tensin homologue on chromosome ten (PTEN) (47) induce only hypertrophy. These results reveal that pathways distinct than those directly involved in cell cycle are required for hypertrophy. Thus, it is significant that ectopic expression of CaM, which has so many cellular targets in so many signaling pathways induces hyperplasia followed temporally by hypertrophy. How can this single Ca2+ receptor protein exert such pleotypic effects in the heart? The akt/PTEN pathway is not differentially activated in the myocardium of CaM transgenic mice compared with WT because there are no differences in the degree of phosphorylation of akt, S6K, or eIF4 binding protein-1 (EBP-1), as monitored with antibodies specific for the corresponding phosphoproteins (data not shown). However, other pathways that involve CaM target proteins involved in hyperplasia such as calcineurin (48), or CaMK (19, 20, 49) seem also to be sufficient to induce hypertrophy when Ca2+/CaM-independent forms of these proteins are expressed in the heart. In these cases, transcriptional activation has been proposed to be the underlying mechanism by which the hypertrophic response is generated. Thus, as depicted in Fig. 8Go, calcineurin is proposed to dephosphorylate and thereby promote the nuclear translocation of NFAT, whereas CaMK activates other transcription factors such as MEF-2 and an unknown protein that functions through the serum response element in the ANF promoter (50).

The extent of ventricular enlargement that occurs in CaM transgenic mice also correlates with diminished survival. Heterozygous CaM mice do not show any cardiac fibrosis or scars, even in cases where the ventricles are three times the size of those in WT mice of the same age. On the other hand, none of the CaM homozygous mice survive longer than 1 wk after birth. The hearts of these latter animals invariably contain markedly thicker ventricular walls, although we never observed complete collapse of the lumen. Apparently, the appropriate size of the ventricles is important for cardiac function, because ventricular enlargement is associated with heart failure and increased death in humans (51) and both myocyte proliferation and growth are tightly regulated in the heart (30, 42). As an example, overexpression of SV40 T-antigen in the heart, which increases proliferation and eventually leads to tumor formation, also induces a large increase in heart size, which correlates with increased incidence of cardiac arrhythmias and mortality (5, 52). The mechanism by which such increases in heart size lead to morbidity is not clear, but because the death occurs suddenly in CaM transgenic mice, arrhythmias could be responsible for the reduced survival. Indeed, arrhythmias have been linked to mutations in several cardiac cation channels, including K+ channels and the cardiac ryanodine receptor 2 (RyR2) that functions as a Ca2+ channel [reviewed by Keating (53)]. Interestingly, RyR2 binds CaM (54) and is phosphorylated by a CaMK (55). Moreover, the CaM inhibitor W7 inhibits ventricular arrhythmia in rabbits (56). Although through a different mechanism, the CaM inhibitor calmidazolium as well as CaMK inhibitors, such as KN-93 and the autocamtide-2-related peptide that specifically inhibits CaMKII, inhibit atrial arrhythmias (57). Because CaM mice show increased CaMKII activity in the ventricles (17), and CaM levels are very high in the atria (16), these mice are clearly at risk for generation of both ventricular and atrial arrhythmias.

In summary, we show that targeted overexpression of CaM in the heart induces an increase in myocyte proliferation in vivo, which occurs solely during embryonic development and ceases soon after birth. Subsequently, CaM promotes a significant increase in postnatal DNA synthesis, which results in a 3-fold increase in the number of postmitotic cells that exhibit polyploidy. Finally, only after these cell cycle-related events have subsided does the presence of excess CaM induce hypertrophy. Thus, CaM is the first protein shown to result in sequential but temporally distinct hyperplasia and hypertrophy of cardiomyocytes when overexpressed in the heart.


   Footnotes
 
This work was supported by NIH Grants HD-07503 and GM-33976 (to A.R.M.).

Current address for M.T.: Iwakura Hospital Kawai-cho, Iwakura, Aichi 482-0015, Japan.

Abbreviations: ANF, Atrial-natriuretic factor; BrdU, 5-bromo-2'- deoxy-uridine; CaM, calmodulin; CaMK, CaM-dependent protein kinases; cdk, cyclin-dependent kinase; E, embryonic day; MEF-2, myocyte enhancer factor-2; NFAT, nuclear factor of activated T cells; PCNA, proliferating cell nuclear antigen; SV40, simian virus 40; V/B, ventricle/body weight; WT, wild-type.

Received August 27, 2003.

Accepted for publication December 1, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
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