Proliferation Of Mammalian Cardiomyocytes Research Articles

Drug Discovery for Adult Cardiomyocyte Regeneration: Opportunities and Challenges.

Significance: Cardiovascular disease is a major contributor to human mortality and morbidity. The cardiac tissue undergoes fibrotic healing after injury because of the limited regenerative capacity of adult mammalian cardiomyocyte (CM). Extensive research has been performed to identify therapeutic targets for CM regeneration, as the success of promoting adult human CM regeneration to repair the injured heart is considered the Holy Grail in the field. Recent Advances: To date, more than 30 target genes have been shown to regulate adult mammalian CM proliferation. More than 20 targets have been validated in adult mouse myocardial infarction (MI) model in a therapeutic setting. In this review, the translational efficacy readouts from 17 selected pharmaceutical targets are summarized, among which the Hippo-yes-associated protein (Yap) pathway is the most extensively investigated and fits the criteria for a promising target for pro-CM-regeneration therapy development. Critical Issues and Future Directions: As the pro-CM-regeneration potential of current drug treatment for cardiovascular patients is limited, to help identify and fill the gap between basic research and drug discovery in this specific field, details regarding target identification, validation in mouse MI models, high-throughput screening assay development, and preclinical in vivo efficacy model optimization are discussed. Finally, suggestions and recommendations are also provided to help establish a common guideline for in vivo translational studies for drug discovery focusing on CM regeneration. Antioxid. Redox Signal. 39, 1070-1087.

Live cell screening identifies glycosides as enhancers of cardiomyocyte cell cycle activity.

Promoting cardiomyocyte proliferation is a promising strategy to regenerate the heart. Yet, so far, it is poorly understood how cardiomyocyte proliferation is regulated, and no factor identified to promote mammalian cardiomyocyte proliferation has been translated into medical practice. Therefore, finding a novel factor will be vital. Here, we established a live cell screening based on mouse embryonic stem cell-derived cardiomyocytes expressing a non-functional human geminin deletion mutant fused to Azami Green (CM7/1-hgem-derived cardiomyocytes). We screened for a subset of compounds of the small molecule library Spectrum Collection and identified 19 potential inducers of stem cell-derived cardiomyocyte proliferation. Furthermore, the pro-proliferative potential of identified candidate compounds was validated in neonatal and adult rat cardiomyocytes as well as human induced pluripotent stem cell-derived cardiomyocytes. 18 of these compounds promoted mitosis and cytokinesis in neonatal rat cardiomyocytes. Among the top four candidates were two cardiac glycosides, peruvoside and convallatoxin, the flavonoid osajin, and the selective α-adrenoceptor antagonist and imidazoline I1 receptor ligand efaroxan hydrochloride. Inhibition of PTEN and GSK-3β enhanced cell cycle re-entry and progression upon stimulation with cardiac glycosides and osajin, while inhibition of IP3 receptors inhibited the cell cycle-promoting effect of cardiac glycosides. Collectively, we established a screening system and identified potential compounds to promote cardiomyocyte proliferation. Our data suggest that modulation of calcium handling and metabolism promotes cardiomyocyte proliferation, and cardiac glycosides might, besides increasing myocardial contraction force, contribute to cardiac repair by inducing cardiomyocyte proliferation.

GW28-e1093 Features of Adult Mammalian Cardiomyocyte Proliferation with Complete Cytokinesis

GW28-e1093 Features of Adult Mammalian Cardiomyocyte Proliferation with Complete Cytokinesis

Cnot3 enhances human embryonic cardiomyocyte proliferation by promoting cell cycle inhibitor mRNA degradation

Uncovering the molecular basis of mammalian cardiomyocyte proliferation may eventually lead to better approaches for heart regeneration. Compared to extensively-studied transcriptional regulation, the roles of posttranscriptional regulation in cardiac cell fate decisions remain largely unknown. Here, we identified Cnot3 as a critical regulator in cardiomyocyte proliferation at the late stage of cardiac differentiation from human ESCs. Cnot3 was highly expressed in cardiomyocytes with higher proliferation potential in both human and mouse, and its depletion resulted in significant reduction in the proliferative capacity of cells. Furthermore, Cnot3 overexpression greatly enhanced proliferation in both cultured human cardiomyocytes and infarcted murine hearts. Mechanistically, the Ccr4-Not complex preferentially interacted with anti-proliferation gene transcripts in a Cnot3-dependent manner, and promoted their degradation. Together, our study supported the model that Cnot3 enhances cardiomyocyte proliferation by promoting cell cycle inhibitor mRNA degradation. It revealed a previously unrecognized role of mRNA degradation in cardiomyocyte growth, and suggested a potential strategy to control cardiac cell fates in development and diseases.

Towards regenerating the mammalian heart: challenges in evaluating experimentally induced adult mammalian cardiomyocyte proliferation

In recent years, there has been a dramatic increase in research aimed at regenerating the mammalian heart by promoting endogenous cardiomyocyte proliferation. Despite many encouraging successes, it remains unclear if we are any closer to achieving levels of mammalian cardiomyocyte proliferation for regeneration as seen during zebrafish regeneration. Furthermore, current cardiac regenerative approaches do not clarify whether the induced cardiomyocyte proliferation is an epiphenomena or responsible for the observed improvement in cardiac function. Moreover, due to the lack of standardized protocols to determine cardiomyocyte proliferation in vivo, it remains unclear if one mammalian regenerative factor is more effective than another. Here, we discuss current methods to identify and evaluate factors for the induction of cardiomyocyte proliferation and challenges therein. Addressing challenges in evaluating adult cardiomyocyte proliferation will assist in determining 1) which regenerative factors should be pursued in large animal studies; 2) if a particular level of cell cycle regulation presents a better therapeutic target than another (e.g., mitogenic receptors vs. cyclins); and 3) which combinatorial approaches offer the greatest likelihood of success. As more and more regenerative studies come to pass, progress will require a system that not only can evaluate efficacy in an objective manner but can also consolidate observations in a meaningful way.

Cardiomyocyte proliferation in cardiac development and regeneration: a guide to methodologies and interpretations.

The newt and the zebrafish have the ability to regenerate many of their tissues and organs including the heart. Thus, a major goal in experimental medicine is to elucidate the molecular mechanisms underlying the regenerative capacity of these species. A wide variety of experiments have demonstrated that naturally occurring heart regeneration relies on cardiomyocyte proliferation. Thus, major efforts have been invested to induce proliferation of mammalian cardiomyocytes in order to improve cardiac function after injury or to protect the heart from further functional deterioration. In this review, we describe and analyze methods currently used to evaluate cardiomyocyte proliferation. In addition, we summarize the literature on naturally occurring heart regeneration. Our analysis highlights that newt and zebrafish heart regeneration relies on factors that are also utilized in cardiomyocyte proliferation during mammalian fetal development. Most of these factors have, however, failed to induce adult mammalian cardiomyocyte proliferation. Finally, our analysis of mammalian neonatal heart regeneration indicates experiments that could resolve conflicting results in the literature, such as binucleation assays and clonal analysis. Collectively, cardiac regeneration based on cardiomyocyte proliferation is a promising approach for improving adult human cardiac function after injury, but it is important to elucidate the mechanisms arresting mammalian cardiomyocyte proliferation after birth and to utilize better assays to determine formation of new muscle mass.

Epigenetic modification at Notch responsive promoters blunts efficacy of inducing notch pathway reactivation after myocardial infarction.

The Notch pathway plays a key role in stimulating mammalian cardiomyocyte proliferation during development and in the early postnatal life; in adult zebrafish, reactivation of this pathway is also essential to drive cardiac regeneration after injury. We wanted to assess efficacy of Notch pathway stimulation in neonatal and adult hearts as a means to induce cardiac regeneration after myocardial infarction in mice. In early postnatal life, cardiomyocyte exit from the cell cycle was paralleled by decreased Notch signaling and the establishment of a repressive chromatin environment at Notch-responsive genes, characterized by recruitment of the polycomb group enhancer of zeste homolog 2 methyltransferase and the acquisition of the histone 3 Lysine 27 trimethylation histone mark, as detected by chromatin immunoprecipitation. Forced Notch pathway activation by adenoassociated virus gene transfer of activated Notch1 or its ligand Jagged1 expanded the proliferative capacity of neonatal cardiomyocytes; this correlated with increased transcription of Notch target genes and maintenance of an open chromatin conformation at their promoters. The same adenoassociated virus vectors, however, were largely ineffective in stimulating cardiac repair after myocardial infarction in adult mice, despite optimal and long-lasting transgene expression. Analysis of Notch-responsive promoters in adult cardiomyocytes showed marks of repressed chromatin and irreversible CpG DNA methylation. Induction of adult cardiomyocyte re-entry into the cell cycle with microRNAs was independent from Notch pathway reactivation. Notch pathway activation is crucial in regulating cardiomyocyte proliferation during the early postnatal life, but it is largely ineffective in driving cardiac regeneration in adults, because of permanent epigenetic modification at Notch-responsive promoters.

Bone morphogenetic protein-10 induces cardiomyocyte proliferation and improves cardiac function after myocardial infarction.

Heart disease is among the leading causes of death worldwide, and the limited proliferation of mammalian cardiomyocytes prevents heart regeneration in response to injury. Bone morphogenetic protein-10 (BMP10) exerts multiple roles in various developmental events; however, the effect of BMP10 and the underlying mechanism involved in cardiac repair remains unclear. After stimulation with the recombinant BMP10, an obvious dose-dependent cardiomyocyte proliferation and reentry of differentiated mammalian cardiomyocytes into the cell cycle was observed. Furthermore, BMP10 stimulation strikingly enhanced Tbx20 expression. Further analysis demonstrated that T-box 20 (Tbx20) was involved in BMP10-induced proliferation of differentiated cardiomyocytes as preconditioning with Tbx20 siRNA significantly attenuated BMP10-induced DNA synthesis. In vivo, BMP10 induced rat cardiomyocyte DNA synthesis and cytokinesis. After myocardial infarction (MI), BMP10 stimulated cardiomyocyte cell-cycle reentry and mitosis, resulting in the decrease of infarct size and improvement of cardiac repair. Taken together, these data indicated that BMP10 stimulated cardiomyocyte proliferation and repaired cardiac function after heart injury. Consequently, BMP10 may be a potential target for innovative strategies against heart failure.

Knockdown of Cyclin-dependent Kinase Inhibitors Induces Cardiomyocyte Re-entry in the Cell Cycle

Proliferation of mammalian cardiomyocytes stops rapidly after birth and injured hearts do not regenerate adequately. High cyclin-dependent kinase inhibitor (CKI) levels have been observed in cardiomyocytes, but their role in maintaining cardiomyocytes in a post-mitotic state is still unknown. In this report, it was investigated whether CKI knockdown by RNA interference induced cardiomyocyte proliferation. We found that triple transfection with p21(Waf1), p27(Kip1), and p57(Kip2) siRNAs induced both neonatal and adult cardiomyocyte to enter S phase and increased the nuclei/cardiomyocyte ratio; furthermore, a subpopulation of cardiomyocytes progressed beyond karyokynesis, as assessed by the detection of mid-body structures and by straight cardiomyocyte counting. Intriguingly, cardiomyocyte proliferation occurred in the absence of overt DNA damage and aberrant mitotic figures. Finally, CKI knockdown and DNA synthesis reactivation correlated with a dramatic change in adult cardiomyocyte morphology that may be a prerequisite for cell division. In conclusion, CKI expression plays an active role in maintaining cardiomyocyte withdrawal from the cell cycle.

Dedifferentiation and Proliferation of Mammalian Cardiomyocytes

BackgroundIt has long been thought that mammalian cardiomyocytes are terminally-differentiated and unable to proliferate. However, myocytes in more primitive animals such as zebrafish are able to dedifferentiate and proliferate to regenerate amputated cardiac muscle.Methodology/Principal FindingsHere we test the hypothesis that mature mammalian cardiomyocytes retain substantial cellular plasticity, including the ability to dedifferentiate, proliferate, and acquire progenitor cell phenotypes. Two complementary methods were used: 1) cardiomyocyte purification from rat hearts, and 2) genetic fate mapping in cardiac explants from bi-transgenic mice. Cardiomyocytes isolated from rodent hearts were purified by multiple centrifugation and Percoll gradient separation steps, and the purity verified by immunostaining and RT-PCR. Within days in culture, purified cardiomyocytes lost their characteristic electrophysiological properties and striations, flattened and began to divide, as confirmed by proliferation markers and BrdU incorporation. Many dedifferentiated cardiomyocytes went on to express the stem cell antigen c-kit, and the early cardiac transcription factors GATA4 and Nkx2.5. Underlying these changes, inhibitory cell cycle molecules were suppressed in myocyte-derived cells (MDCs), while microRNAs known to orchestrate proliferation and pluripotency increased dramatically. Some, but not all, MDCs self-organized into spheres and re-differentiated into myocytes and endothelial cells in vitro. Cell fate tracking of cardiomyocytes from 4-OH-Tamoxifen-treated double-transgenic MerCreMer/ZEG mouse hearts revealed that green fluorescent protein (GFP) continues to be expressed in dedifferentiated cardiomyocytes, two-thirds of which were also c-kit+.Conclusions/SignificanceContradicting the prevailing view that they are terminally-differentiated, postnatal mammalian cardiomyocytes are instead capable of substantial plasticity. Dedifferentiation of myocytes facilitates proliferation and confers a degree of stemness, including the expression of c-kit and the capacity for multipotency.

TWEAK is a positive regulator of cardiomyocyte proliferation

Proliferation of mammalian cardiomyocytes stops during the first weeks after birth, preventing the heart from regenerating after injury. Recently, several studies have indicated that induction of cardiomyocyte proliferation can be utilized to regenerate the mammalian heart. Thus, it is important to identify novel factors that can induce proliferation of cardiomyocytes. Here, we determine the effect of TNF-related weak inducer of apoptosis (TWEAK) on cardiomyocytes, a cytokine known to regulate proliferation in several other cell types. Stimulation of neonatal rat cardiomyocytes with TWEAK resulted in increased DNA synthesis, increased expression of the proliferative markers Cyclin D2 and Ki67, and downregulation of the cell cycle inhibitor p27KIP1. Importantly, TWEAK stimulation resulted also in mitosis (H3P), cytokinesis (Aurora B), and increased cardiomyocyte numbers. Loss of function experiments revealed that re-induction of proliferation was dependent on tumour necrosis factor receptor superfamily member 12A (FN14) signalling. Downstream signalling was mediated through activation of extracellular signal-regulated kinases and phosphatidylinositol 3-kinase as well as inhibition of glycogen synthase kinase-3beta. In contrast to neonatal cardiomyocytes, TWEAK had no effect on adult rat cardiomyocytes due to developmental downregulation of its receptor FN14. However, adenoviral expression of FN14 enabled efficient induction of cell cycle re-entry in adult cardiomyocytes after TWEAK stimulation. Our data establish TWEAK as a positive regulator of cardiomyocyte proliferation.

Features of cardiomyocyte proliferation and its potential for cardiac regeneration

The human heart does not regenerate. Instead, following injury, human hearts scar. The loss of contractile tissue contributes significantly to morbidity and mortality. In contrast to humans, zebrafish and newts faithfully regenerate their hearts. Interestingly, regeneration is in both cases based on cardiomyocyte proliferation. In addition, mammalian cardiomyocytes proliferate during foetal development. Their proliferation reaches its maximum around chamber formation, stops shortly after birth, and subsequent heart growth is mostly achieved by an increase in cardiomyocyte size (hypertrophy). The underlying mechanisms that regulate cell cycle arrest and the switch from proliferation to hypertrophy are unclear. In this review, we highlight features of dividing cardiomyocytes, summarize the attempts to induce mammalian cardiomyocyte proliferation, critically discuss methods commonly used for its detection, and explore the potential and problems of inducing cardiomyocyte proliferation to improve function in diseased hearts.

Divergent Siblings

Proliferation of mammalian cardiomyocytes ceases around birth when a transition from hyperplastic to hypertrophic myocardial growth occurs. Previous studies demonstrated that directed expression of the transcription factor E2F1 induces S-phase entry in cardiomyocytes along with stimulation of programmed cell death. Here, we show that directed expression of E2F2 and E2F4 by adenovirus mediated gene transfer in neonatal cardiomyocytes induced S-phase entry but did not result in an onset of apoptosis whereas directed expression of E2F1 and E2F3 strongly evoked programmed cell death concomitant with cell cycle progression. Although both E2F2 and E2F4 induced S-phase entry only directed expression of E2F2 resulted in mitotic cell division of cardiomyocytes. Expression of E2F5 or a control LacZ-Adenovirus had no effects on cell cycle progression. Quantitative real time PCR revealed that E2F1, E2F2, E2F3, and E2F4 alleviate G0 arrest by induction of cyclinA and E cyclins. Furthermore, directed expression of E2F1, E2F3, and E2F5 led to a transcriptional activation of several proapoptotic genes, which were mitigated by E2F2 and E2F4. Our finding that expression of E2F2 induces cell division of cardiomyocytes along with a suppression of proapoptotic genes might open a new access to improve the regenerative capacity of cardiomyocytes.