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Circulation Research. 2002;90:1044-1054
doi: 10.1161/01.RES.0000020201.44772.67
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(Circulation Research. 2002;90:1044.)
© 2002 American Heart Association, Inc.

Reviews

Cardiomyocyte Cell Cycle Regulation

Kishore B.S. Pasumarthi, Loren J. Field

From the Wells Center for Pediatric Research and Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, Ind.

Correspondence to Loren J. Field, Herman B Wells Center for Pediatric Research, James Whitcomb Riley Hospital for Children, 702 Barnhill Dr, Room 2600, Indianapolis, IN 46202-5225. E-Mail ljfield{at}iupui.edu


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowCardiomyocyte Cell Cycle...
 down arrowCardiomyocyte Cell Cycle...
 down arrowExperimental Manipulation of...
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Although rapid progress is being made in many areas of molecular cardiology, issues pertaining to the origins of heart-forming cells, the mechanisms responsible for cardiogenic induction, and the pathways that regulate cardiomyocyte proliferation during embryonic and adult life remain unanswered. In the present study, we review approaches and studies that have shed some light on cardiomyocyte cell cycle regulation. For reference, an initial description of cardiomyogenic induction and morphogenesis is provided, which is followed by a summary of published cell cycle analyses during these stages of cardiac ontology. A review of studies examining cardiomyocyte cell cycle analysis and de novo cardiomyogenic induction in the adult heart is then presented. Finally, studies in which cardiomyocyte cell cycle activity was experimentally manipulated in vitro and in vivo are reviewed. It is hoped that this compilation will serve to stimulate thought and experimentation in this intriguing area of cardiomyocyte cell biology.


Key Words: cardiomyocyte proliferation • cardiac myocyte apoptosis • heart regeneration • gene targeting • transgenic mice


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowCardiomyocyte Cell Cycle...
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Studies using embryos from different species (fly, chick, zebrafish, xenopus, and mouse) have shed some light on the cardiogenic induction process. During gastrulation of the chick embryo, primitive streak cells migrate to the lateral plate mesoderm. Those cells that migrate to the anterior lateral plate mesoderm are destined to form heart tissue.1–3 On migration, these cells proliferate extensively4 while remaining in close contact with the anterior endoderm3 (a prerequisite for their subsequent cardiomyogenic induction).5 Recent studies have elegantly identified several growth factors that regulate cardiomyogenic induction of the precursor cells in the anterior mesoderm. These include molecules that promote cardiomyogenic induction (BMPs, FGFs, inhibitors of Wnt family of morphogens such as Crescent, Dkk-1, and glycogen synthase kinase-3), as well as molecules that inhibit the process (Wnt family of morphogens, noggin, and chordin).6–9 The failure of these growth factors to promote cardiomyogenic induction in more primitive precursor cells indicates that additional as of yet unidentified factors participate in the process.

In contrast to our somewhat limited understanding of the inductive clues that mediate cardiomyogenic lineage determination, the morphogenetic transformation of the primitive heart into a 4-chambered structure is fairly well characterized. The heart field initially forms as a crescent shaped structure in the anterior part of the embryo that later develops into a linear tube.10 The tubular heart undergoes segmentation along the anterior-posterior axis, followed by rightward looping. This process results in the formation of the right and left ventricles, the atrioventricular canal, the sinoatrial, and the outflow tract segments.11,12 Subsequently, the ventral side of the heart tube rotates and forms the outer curvature of the heart, with the dorsal side becoming the inner curvature.13 These outer and inner curvatures play critical roles in the morphogenesis of the 4-chambered heart, as the individual chambers balloon out from the outer curvature due to the rapid proliferation of resident myocardial cells. These morphogenic changes are accompanied by the expression of chamber-specific genes exclusively in the ventral side and the outer curvature of the heart tube. In contrast, myocardial cells residing in the inner curvature are thought to be relatively undifferentiated, which allows this region to participate in the alignment of the right atrium with the right ventricle and the left ventricle with the outflow tract. This process results in the separation of the pulmonary and systemic circulations.13 Gene-targeting experiments have identified a number of transcription factors that are required for cardiac development, morphogenesis, and/or chamber specification. These include members of Nkx,14 GATA,15,16 MEF2,17 HAND,18,19 Irx,20 Tbx,21 and HRT22 families of transcription factors.


*    Cardiomyocyte Cell Cycle Regulation During Development
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Cell cycle activity is an intrinsic component of cardiac differentiation and morphogenesis, as evidenced by traditional tritiated thymidine incorporation studies. An exceedingly high level of DNA synthesis (labeling indices approaching 70%) is seen in the precardiac mesoderm of the myoepicardial plate in E8 mouse embryos.23–25 The onset of cardiomyogenic differentiation (ie, the first appearance of a cardiomyocyte phenotype) is accompanied by transient reduction in the tritiated thymidine labeling index that recovers to approximately 45% by E11 during mouse ontology.23–25 A similar modulation of cell cycle activity is observed during early cardiac development in the chick.23 The high rate of cell cycle activity during the early stages of cardiomyogenic differentiation undoubtedly contributes to the "ballooning" of ventricular cardiomyocytes from the tubular heart as described in the morphological studies of Moorman and colleagues.13 As morphogenesis of the ventricle progresses, the tritiated thymidine labeling index is approximately 2-fold greater in cardiomyocytes of the compact layer of the myocardium as compared with those in the inner trabeculae.23–25 The overall rate of cardiomyocyte proliferation gradually declines during later stages of embryogenesis.23,26–28

Retroviral tagging experiments using ß-galactosidase–expressing viruses have provided insight into the interplay between cardiac cell cycle and morphogenic formation of the heart.29,30 These studies indicated that on myogenic differentiation, proliferation of a single infected myocyte in the tubular heart, gives rise to transmural, cone-shaped growth units. It was hypothesized that assembly of these cone-shaped units contributes to the 3-dimensional ovoid structure of the ventricular walls. The gradient in the rate of cardiomyocyte proliferation observed between the compact layer and the inner trabeculae23–25 would account for the formation of the cone-shaped growth units. Recent analysis of the proliferative capacity of individual embryonic cardiomyocytes cultured at clonal density supports the general concept that coordinated cell cycle regulation contributes to the formation of cone-shaped growth units.31 These studies revealed that the absolute proliferative capacity of individual cardiomyocytes isolated from E15 rats varied greatly. Such variation of growth potential between individual cells would be anticipated given the intrinsic differences in cell cycle activity observed between cells from the compact zone versus the trabeculae at any given time of cardiac development. These studies also suggested that daughter cells from a given progenitor had a propensity to undergo a similar number of cell divisions prior to terminal differentiation. The symmetrical structure of the cone-shaped growth units would be dependent on similar growth potential in daughter cells.

Shortly after birth, there is a transition from hyperplastic to hypertrophic myocardial growth. At the morphological level, this transition is characterized by a marked increase in myofibril density, the appearance of mature intercalated discs, and the formation of binucleated cardiomyocytes. Numerous studies have documented a gradual decrease in radiolabeled thymidine incorporation that is coincident with the appearance of binucleated cardiomyocytes, 23,26–28 leading to the suggestion that binucleation results from a round of genomic duplication and karyokinesis in the absence of cytokinesis. Additional studies demonstrated that in mice there are two temporally distinct phases of cardiomyocyte DNA synthesis.26 The first phase of DNA synthesis occurred in fetal life and was associated exclusively with cardiomyocyte proliferation, whereas the second phase occurred in immediate postnatal life and was associated largely with binucleation. This suggested that cardiomyocyte proliferation ceases before birth in mice, and that the key gene products regulating proliferation (as opposed to nuclear endoreduplication) would likely be expressed differentially during fetal versus neonatal life.

Many regulatory molecules that control the mammalian cell cycle have recently been identified (a simplified schematic diagram of the cell cycle is depicted in the Figure). A number of descriptive studies examining expression of known cell cycle regulatory genes during cardiac ontology have been reported. In general, positive cell cycle regulators (as for example, cyclins, the cyclin-dependent kinases [Cdks], and protooncogenes) are highly expressed in the embryonic hearts where cardiomyocyte cell cycle activity is relatively high. Most of these positive cell cycle regulators are downregulated in the adult heart. In general, expression patterns of negative cell cycle regulatory genes (as for example, Cdk inhibitors) are frequently increased in adult hearts, where cardiomyocyte cell cycle activity is largely absent. A review of the expression patterns of these proteins is provided (Table 1).



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  		Simplified schematic of the mammalian cell cycle.


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  		Table 1. Expression Patterns of Cell Cycle Regulatory Proteins During Cardiac Development


*    Cardiomyocyte Cell Cycle Activity and De Novo Cardiomyogenesis in Adult Life
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The capacity of adult cardiomyocytes to reenter the cell cycle has received considerable attention recently. It is generally accepted that adult cardiomyocytes retain some capacity to synthesize DNA. However, there is considerable debate regarding the frequency at which this occurs and if re-initiation of DNA synthesis necessarily leads to cell division. Indeed, classic studies documented that DNA synthesis contributes to increased nuclear ploidy in severely hypertrophic hearts (reviewed in Rumyantsev23). Key experimental issues relevant to this debate include (1) the fidelity of the assay used to monitor cell cycle activity, (2) the fidelity of the assay used to distinguish cardiomyocyte nuclei from nonmyocyte nuclei, (3) the degree to which myocardial injury may enhance cardiomyocyte cell cycle activity, and (4) the degree to which this response varies between species. The level of subjectivity involved in the assessment of cardiomyocyte cell cycle progression in the adult heart is reflected by the fact that different laboratories, despite using similar techniques, have reported markedly dissimilar rates for a given species.32–39 Issues relevant to the assessment of cardiomyocyte cell cycle activity in the adult heart have recently been reviewed in detail38,39 and are not discussed in detail here. However, it is noteworthy that a relatively nonsubjective tritiated thymidine incorporation assay revealed only exceedingly low levels of DNA synthesis in normal (0.0005%) and injured (0.008%) adult mouse cardiomyocytes (which were identified by expression of a nuclear localized ß-galactosidase reporter gene under the transcriptional regulation of the {alpha}-cardiac myosin heavy chain promoter37). It is important that the levels of cardiomyocyte cell cycle activity be accurately determined, as the magnitude of the activity will likely reflect the degree to which it is therapeutically exploitable.

Until recently, the notion of de novo cardiomyogenic induction and subsequent cardiomyocyte proliferation in the adult heart was not viewed as a likely prospect. However, the identification of multipotent adult stem cells in several tissues raises the possibility that some degree of neocardiomyogenesis might occur in the adult heart. To directly test this, the Goodell group produced myocardial infarcts in mice that had previously undergone bone marrow reconstitution with genetically tagged cells.40 A limited number of myocytes expressing the genetic tag were observed in the infarcted heart at later time points. The proliferative capacity of these myocytes has not yet been characterized; however, the appearance of lone stem cell–derived myocytes in sections prepared from injured hearts (as opposed to clusters of myocytes) suggests that cell cycle activity might be limited. Although it would be of considerable interest to recapitulate this result using lineage-restricted reporter genes, these data nonetheless strongly suggest that de novo myogenesis from bone marrow derived precursors can occur at low levels in the adult heart.

Subsequent studies have shown that stem cells with myogenic potential can be derived from neuronal,41 hepatic,42 mesenchymal,43 and endothelial44 sources. It has also been suggested that direct transplantation of bone marrow–derived stem cells into infarcted mouse hearts can result in substantial neocardiogenesis45; however, preliminary efforts entailing transplantation of similar stem cells carrying a lineage-specific reporter gene have failed to demonstrate cardiomyogenic differentiation.46 Finally, a remarkable degree of stem cell–based neocardiomyogenic induction (approaching 10% of the myocardium) was recently reported in transplanted human hearts.47 It should be noted that the significance of the marker used to identify cardiomyogenic stem cells in this study (Sca1 immune reactivity) was confusing, given the absence of any reported examples of Sca1 immune reactivity in humans. Other studies suggested that the rate of de novo myogenic induction in transplanted human hearts is more on par with that seen in the Goodell bone marrow transplant studies.48 Yet another study failed to identify any host-derived cardiomyocytes in transplanted hearts (although some endothelial cells were detected).49

Thus, even though the field of neocardiomyogenesis in the adult heart is relatively new, there already appears to be some debate regarding the rate at which it occurs. Many of the experimental issues that impact on the assessment of cardiomyocyte DNA synthesis are also relevant to the assessment of neocardiomyogenesis. This point is further illustrated by the recent observations that genetically tagged bone marrow stem cells50 and neuronal stem cells51 have the capacity to fuse with embryonic stem cells, and in so doing, generate multipotent hybrid cells that retain the expression of reporter gene. These results further underscore the need of rigorous genetic analyses in these types of studies. With respect to the myocardium, it will be very interesting indeed to see how research in this area progresses over the next several years and, in particular, to see how results from these newer model systems are reconciled with experimental and clinical experiences that do not support high levels of intrinsic regenerative cardiac growth.


*    Experimental Manipulation of Cardiomyocyte Cell Cycle Activity
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 up arrowCardiomyocyte Cell Cycle...
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Primary cardiomyocyte cultures from enzymatically dispersed fetal, neonatal, or adult hearts have been widely used for cell cycle studies. Initial applications of this approach entailed testing the effect(s) of exogenous agents on cultured cells. Numerous molecules that promote or repress cardiomyocyte DNA synthesis and/or proliferation have been identified in this manner (Table 2). An interesting report recently suggested that the absence of a 3-dimensional matrix in primary monolayer cultures may render cardiomyocytes less responsive to exogenous growth factors,52 although it should be noted some factors that failed to elicit a cell cycle effect with monolayer cultures in this study had previously been shown to be effective.53 In the 1980s, the advent of gene transfer technology permitted direct cause/effect relation for cell cycle activation to be established. Initial experiments utilized traditional calcium phosphate or lipofection techniques; however, the low efficiency of gene transfer with these techniques limited their utility. Subsequently, the development of viral transduction approaches with recombinant retro- and adenoviruses have been used to effect highly efficient cardiomyocyte gene transfer in vitro.


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  		Table 2. Effects of Exogenously Added Molecules on Cell Cycle Activity in Primary Cardiomyocyte Cultures

Despite the large number of studies utilizing gene transfer in primary cardiomyocyte cultures, this approach is not without its limitations. For example, cultures are frequently subject to strict temporal constraints due to the rapid proliferation of noncardiomyocytes. It has also been argued that the marked physiological and molecular differences between fetal and neonatal cardiomyocytes as compared with terminally differentiated adult cells renders these primary cultures unsuitable for studies aimed at cell cycle reactivation in quiescent adult cells. Difficulties encountered in the generation of large-scale cultures of differentiated adult cardiomyocytes further limits in vitro cell cycle analyses. These limitations can be partially circumvented by the use of pure, terminally differentiated cardiomyocyte cultures derived from embryonic stem (ES) cells54; however, exploitation of this approach has been limited to date.55,56 This latter approach is also readily amenable to cotransfection with multiple expression constructs.55,56 In vitro gene transfer studies with positive effects on cardiomyocyte cell cycle activity are summarized (Table 3).


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  		Table 3. Genetic Manipulations That Enhance Cardiomyocyte Proliferation

The ability to perform gene transfer in vivo bypasses a number of the limitations encountered with primary cardiomyocyte cultures. This approach encompasses the direct delivery of viral vectors into the myocardium, as well as the generation of genetically modified animals (that is, traditional gain of function transgenic animals produced by pronuclear injection, as well as knockout mice produced by homologous recombination in ES cells). Further refinements of these approaches, as for example, the development of regulated transgenes that permit conditional (ie, temporal and spatial) expression of a given gene,57,58 or alternatively conditional elimination of a gene using cre/lox technology,59 provide powerful tools to further characterize cardiomyocyte cell cycle regulation. Care must be exercised in ascertaining if a given cardiac phenotype is a primary versus secondary effect of the genetic manipulation. This is particularly true for knockout animals, because all cells that express a given gene are effected (as opposed to transgenic models, which frequently use tissue-restricted promoters to target transgene expression). A priori, phenotypes resulting from secondary effects are likely to be more prevalent if a gene expression is globally altered as opposed to a tissue-restricted alteration. Combinatorial approaches, as for example the use of viral gene transfer into the hearts of genetically modified animals, provide still more experimental options.60 The ability to determine if genetic background can influence the effect of a known genetic modification of cell cycle activity provides a novel way to identify modifying genes.61,62 Collectively, these approaches constitute an extremely versatile arsenal of technologies with which to study cardiomyocyte cell cycle regulation, which is only now beginning to realize its full potential. Gene transfer studies with positive and negative effects on cardiomyocyte cell cycle activity are summarized (Tables 3 and 4, respectively).


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  		Table 4. Genetic Manipulations That Decrease Cardiomyocyte Proliferation

Several interesting lessons can be gleaned from gene transfer experiments wherein cardiomyocyte cell cycle activity has been manipulated. For example, a number of studies have shown that forced expression of fundamental cell cycle regulators (ie, D-type cyclins, E2F-1, or c-myc) can drive cardiomyocyte DNA synthesis, which in some instances results in complete genomic replication and karyokinesis in the adult heart. However, it would appear from these studies (and in particular the study utilizing a conditionally active myc transgene58) that induction of DNA synthesis and karyokinesis per se is insufficient to drive adult cardiomyocytes through cell division (at least under the experimental conditions reported to date). This in turn has suggested to some investigators that additional cell cycle checkpoints, as for example at G2/M, must be circumvented for cytokinesis to occur.60,63–66 An alternative explanation is that there is intrinsically a limited number of cell cycles through which cardiomyocytes can progress. This view is supported by the modest effect on total cell number seen in the myc transgenic model wherein transgene was constitutive,67,68 as well as by the developmental "intrinsic timer" studies described earlier in this review.31 Yet another view is that the presence of highly organized myofibers permits karyokinesis but not cytokinesis.23 However, the suggestion that adult cardiomyocytes retain the capacity to undergo cytokinesis39 is inconsistent with the notion that myofiber density imparts an insurmountable cell cycle checkpoint, and this is certainly not the case in newt cardiomyocytes.69

Under certain conditions, the targeted expression of the multifunctional DNA tumor virus oncoproteins appears to bypass all cell cycle checkpoints in cardiomyocytes. For example, targeted expression of the SV40 large T antigen oncoprotein was sufficient to induce sustained cycling of cardiomyocytes in transgenic mice. The observation that tumor cell lines from these animals have continuously been propagated since 1987,70 and that a derivative cell line is capable of continuous in vitro propagation,71 clearly indicated that the all downstream cell cycle checkpoints in cardiomyocytes can be bypassed (although it is not clear if T antigen alone is sufficient for sustained ex vivo proliferation, or if additional as of yet unidentified stochastic genetic modifications are required). The observation that DNA tumor virus oncoproteins subjugate cell cycle activity by binding to, and thereby altering the activity of, endogenous cell cycle regulators has prompted a series of experiments to identify the cardiomyocyte T antigen binding partners.72,73 These studies revealed that 2 of T antigen binding proteins (namely p53 and p193) encode proapoptotic activity and suggested that minimally these 2 pathways must be circumvented to promote sustained cardiomyocyte proliferation. This notion was recently confirmed using ES-derived cardiomyocytes: E1A expression was capable of inducing sustained cell cycle activity only when the activity of p53 and p193 is compromised (accomplished by coexpression of mutant p193 and p53 cDNAs, which encode dominant interfering activity).55 By analogy, analysis of cell lines generated by targeted expression of T antigen under the regulation of promoters that are active during the early phases of cardiomyogenic induction74 may result in the identification of additional cell cycle regulatory proteins.


*    Summary
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 up arrowAbstract
 up arrowIntroduction
 up arrowCardiomyocyte Cell Cycle...
 up arrowCardiomyocyte Cell Cycle...
 up arrowExperimental Manipulation of...
 *Summary
down arrowReferences
 
The future promises to yield new discoveries and advances in our understanding of cardiomyocyte cell cycle regulation that will hopefully give rise to the ability to promote regenerative myocardial growth. With respect to the intrinsic proliferative and de novo cardiomyogenic potential of the adult heart, it is abundantly clear from studies cited herein that the published values for the magnitude of both processes vary dramatically. It is very important to rigorously determine the extent to which these processes do occur. If the intrinsic rates for cardiomyocyte proliferation and/or de novo cardiomyogenic differentiation are exceedingly low, then the ability to exploit these processes for clinical benefit would likely be quite limited. Increasing the frequency of these events would require the existence, identification, and ultimately the successful delivery of cytokines that normally regulate the process. Conversely, if these processes do occur at the frequency that some studies suggest, then their clinical utility could be invaluable. These issues will ultimately be resolved as multiple groups attempt to confirm and extend the current observations.

When considering the intrinsic proliferative capacity of adult cardiomyocytes, it is important to reiterate that DNA synthesis does not necessarily result in genome duplication, that genome duplication does not necessarily result in karyokinesis, and that karyokinesis does not necessarily result in cytokinesis. Although such interpretive restrictions are rather obvious, one must keep them in mind when comparing the results from different laboratories particularly when different assays are utilized. The long-term viability of any new cells, regardless of the origin, is also critical with respect to their clinical utility. When considering the potential for de novo cardiomyogenic induction in the adult, recent studies demonstrating the propensity for stem cell fusion raise the very distinct possibility that several previous studies suggesting stem cell plasticity or transdifferentiation might have been incorrectly interpreted (reviewed in Wurmser and Gage75). Given this, rigorous confirmation with nonambiguous molecular genetic markers should be requisite for any studies assessing de novo adult cardiomyogenesis. Exploitation of novel model systems exhibiting favorable outcomes after myocardial injury may also provide new insights into mechanisms with which to enhance any intrinsic regenerative capacity in the heart. For example, myocardial damage was markedly reduced in MRL mice after trans-diaphragmatic cryoinjury.76 Although it was not clear if this outcome reflected bona fide cardiomyocyte regeneration or simply a global improvement of the wound healing process (which is observed in other tissues of these animals), elucidation of the mechanism responsible for the phenotype could nonetheless give rise to new interventional approaches.

It is also abundantly clear from studies cited herein that genetic interventions are able to promote cardiomyocyte DNA synthesis, karyokinesis, and in some cases, cytokinesis. These existing models are currently being further characterized and developed by many groups. Moreover, additional genes or gene combinations that are able to promote cardiomyocyte proliferation will undoubtedly be identified. It is important to note that in most instances these genetic interventions have been performed in either fetal or neonatal cell culture, or alternatively in transgenic animals that utilized promoters that are transcriptionally active before cardiomyocyte terminal differentiation. Thus, in many cases it remains to be demonstrated that a given intervention will have an analogous effect when introduced into an adult cardiomyocyte. The development of regulated transgenic models and the improvement of viral delivery systems will greatly facilitate these important analyses. Once the potential utility of a genetic pathway is validated in experimental models, clinical exploitation will ultimately require further development. For example, safe and efficient gene transfer in human myocardium has not yet been achieved; this would be a prerequisite if a gene-based regenerative mechanism paralleling current experimental systems is to be developed. Perhaps a more realistic approach (particularly over the short-term) would rely on the identification and/or development of pharmacological agents that can modulate expression of the endogenous genes within the myocardium. Such approaches, if viable, would have certain advantages over interventions that rely on permanent gene transfer in vivo. The wedding of gene transfer and cell transplantation77 technologies provides a particularly attractive alternative approach to rapidly exploit the potential benefit of cell cycle–promoting genes.

In light of our initial progress in the elucidation of cardiomyocyte cell cycle regulatory mechanisms, and the identification of genes that are able to promote cell cycle activity in the myocardium, researchers in this area should be quite enthusiastic that the field will continue to grow and evolve. The realization that identification of genes and/or pathways that are able to promote cardiomyocyte proliferation is only the first step for potential clinical exploitation should not temper our enthusiasm. Rather, this should serve to motivate us to rapidly validate our models, as well as to keep an open mind with respect to the use and development of novel approaches that might permit the exploitation of these genes and/or pathways. With a lot of hard work (and a little luck), perhaps we will be able to see the endgame resulting from such efforts.


*    Acknowledgments
 
We thank NHLBI for support, and Drs Mark Soonpaa, Michael Rubart, Hidehiro Nakajima, and Hisako Nakajima for comments on the manuscript. We also thank our many colleagues working in the field. It is hoped that this review provides an evenhanded and (somewhat) unbiased compilation of cardiomyocyte cell cycle studies. We apologize in advance for any relevant views/studies that were inadvertently not included.


*    Footnotes
 
L.J.F. consults for Cardion-AG, Erkrath, Germany.

Received January 22, 2002; revision received April 16, 2002; accepted April 16, 2002.


*    References
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 *References
 

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