Two decades of heart regeneration research: Cardiomyocyte proliferation and beyond.

Interest in vertebrate cardiac regeneration has exploded over the past two decades since the discovery that adult zebrafish are capable of complete heart regeneration, contrasting the limited regenerative potential typically observed in adult mammalian hearts. Undercovering the mechanisms that both support and limit cardiac regeneration across the animal kingdom may provide unique insights in how we may unlock this capacity in adult humans. In this review, we discuss key discoveries in the heart regeneration field over the last 20 years. Initially, seminal findings revealed that pre-existing cardiomyocytes are the major source of regenerated cardiac muscle, drawing interest into the intrinsic mechanisms regulating cardiomyocyte proliferation. Moreover, recent studies have identified the importance of intercellular interactions and physiological adaptations, which highlight the vast complexity of the cardiac regenerative process. Finally, we compare strategies that have been tested to increase the regenerative capacity of the adult mammalian heart. This article is categorized under: Cardiovascular Diseases > Stem Cells and Development.

Quantitative Three-dimensional Label-free Digital Holographic Imaging of Cardiomyocyte Size, Ploidy, and Cell Division.

Cardiac regeneration in newborn rodents depends on the ability of pre-existing cardiomyocytes to proliferate and divide. This capacity is lost within the first week of postnatal development when these cells rapidly switch from hyperplasia to hypertrophy, withdraw from the cell cycle, become binucleated, and increase in size. How these dynamic changes in size and ploidy impact cardiomyocyte proliferative potential is not well understood. In this study, we innovate the application of a commercially available digital holographic imaging microscope, the Holomonitor M4, to evaluate the proliferative responses of mononucleated diploid and binucleated tetraploid cardiomyocytes. This instrument coupled with the powerful Holomonitor App Suite software enables long-term label-free quantitative three-dimensional tracking of primary cardiomyocyte dynamics in real-time with single-cell resolution. Our digital holographic imaging results provide direct evidence that mononucleated cardiomyocytes retain significant proliferative potential as most can successfully divide with high frequency. In contrast, binucleated cardiomyocytes exhibit a blunted response to a proliferative stimulus with the majority not attempting to divide at all. Nevertheless, some binucleated cardiomyocytes were capable of complete division, suggesting that these cells still do retain limited proliferative capacity. By quantitatively tracking cardiomyocyte volume dynamics during these proliferative responses, we reveal that both mononucleated and binucleated cells reach a unique size threshold prior to attempted cell division. The absolute threshold is increased by binucleation, which may limit the ability of binucleated cardiomyocytes to divide. By defining the interrelationship between cardiomyocyte size, ploidy, and cell cycle control, we will better understand the cellular mechanisms that drive the loss of mammalian cardiac regenerative capacity after birth.

An injury-responsive mmp14b enhancer is required for heart regeneration.

Mammals have limited capacity for heart regeneration, whereas zebrafish have extraordinary regeneration abilities. During zebrafish heart regeneration, endothelial cells promote cardiomyocyte cell cycle reentry and myocardial repair, but the mechanisms responsible for promoting an injury microenvironment conducive to regeneration remain incompletely defined. Here, we identify the matrix metalloproteinase Mmp14b as an essential regulator of heart regeneration. We identify a TEAD-dependent mmp14b endothelial enhancer induced by heart injury in zebrafish and mice, and we show that the enhancer is required for regeneration, supporting a role for Hippo signaling upstream of mmp14b. Last, we show that MMP-14 function in mice is important for the accumulation of Agrin, an essential regulator of neonatal mouse heart regeneration. These findings reveal mechanisms for extracellular matrix remodeling that promote heart regeneration.

Unlocking cardiomyocyte renewal potential for myocardial regeneration therapy.

Cardiovascular disease remains the leading cause of mortality worldwide. Cardiomyocytes are irreversibly lost due to cardiac ischemia secondary to disease. This leads to increased cardiac fibrosis, poor contractility, cardiac hypertrophy, and subsequent life-threatening heart failure. Adult mammalian hearts exhibit notoriously low regenerative potential, further compounding the calamities described above. Neonatal mammalian hearts, on the other hand, display robust regenerative capacities. Lower vertebrates such as zebrafish and salamanders retain the ability to replenish lost cardiomyocytes throughout life. It is critical to understand the varying mechanisms that are responsible for these differences in cardiac regeneration across phylogeny and ontogeny. Adult mammalian cardiomyocyte cell cycle arrest and polyploidization have been proposed as major barriers to heart regeneration. Here we review current models about why adult mammalian cardiac regenerative potential is lost including changes in environmental oxygen levels, acquisition of endothermy, complex immune system development, and possible cancer risk tradeoffs. We also discuss recent progress and highlight conflicting reports pertaining to extrinsic and intrinsic signaling pathways that control cardiomyocyte proliferation and polyploidization in growth and regeneration. Uncovering the physiological brakes of cardiac regeneration could illuminate novel molecular targets and offer promising therapeutic strategies to treat heart failure.

Visualization of regenerating and repairing hearts.

With heart failure continuing to become more prevalent, investigating the mechanisms of heart injury and repair holds much incentive. In contrast with adult mammals, other organisms such as teleost fish, urodele amphibians, and even neonatal mammals are capable of robust cardiac regeneration to replenish lost or damaged myocardial tissue. Long-term high-resolution intravital imaging of the behaviors and interactions of different cardiac cell types in their native environment could yield unprecedented insights into heart regeneration and repair. However, this task remains challenging for the heart due to its rhythmic contraction and anatomical location. Here, we summarize recent advances in live imaging of heart regeneration and repair, discuss the advantages and limitations of current systems, and suggest future directions for novel imaging technology development.

Mending a broken heart with novel cardiogenic small molecules.

Adult mammalian cardiomyocytes are unable to proliferate to regenerate lost tissue after heart injury. Du et al., reporting in Cell Stem Cell, employ a FUCCI- and MADM-based system to screen for small molecules combinations that produced a collaborative effect on cardiomyocyte cycling and cytokinesis. The authors generate a cocktail of five small molecules that increase cardiomyocyte proliferation and regeneration in vitro and in vivo with high efficiency, and explore its potential in cardiac regenerative repair after myocardial infarction through a new potential pathway for cardiomyocyte cell-cycle re-entry.

Measuring cardiomyocyte cell-cycle activity and proliferation in the age of heart regeneration.

During the past two decades, the field of mammalian myocardial regeneration has grown dramatically, and with this expanded interest comes increasing claims of experimental manipulations that mediate bona fide proliferation of cardiomyocytes. Too often, however, insufficient evidence or improper controls are provided to support claims that cardiomyocytes have definitively proliferated, a process that should be strictly defined as the generation of two de novo functional cardiomyocytes from one original cardiomyocyte. Throughout the literature, one finds inconsistent levels of experimental rigor applied, and frequently the specific data supplied as evidence of cardiomyocyte proliferation simply indicate cell-cycle activation or DNA synthesis, which do not necessarily lead to the generation of new cardiomyocytes. In this review, we highlight potential problems and limitations faced when characterizing cardiomyocyte proliferation in the mammalian heart, and summarize tools and experimental standards, which should be used to support claims of proliferation-based remuscularization. In the end, definitive establishment of de novo cardiomyogenesis can be difficult to prove; therefore, rigorous experimental strategies should be used for such claims.

Thyroid hormone-dependent regulation of metabolism and heart regeneration.

While adult zebrafish and newborn mice possess a robust capacity to regenerate their hearts, this ability is generally lost in adult mammals. The logic behind the diversity of cardiac regenerative capacity across the animal kingdom is not well understood. We have recently reported that animal metabolism is inversely correlated to the abundance of mononucleated diploid cardiomyocytes in the heart, which retain proliferative and regenerative potential. Thyroid hormones are classical regulators of animal metabolism, mitochondrial function, and thermogenesis, and a growing body of scientific evidence demonstrates that these hormonal regulators also have direct effects on cardiomyocyte proliferation and maturation. We propose that thyroid hormones dually control animal metabolism and cardiac regenerative potential through distinct mechanisms, which may represent an evolutionary tradeoff for the acquisition of endothermy and loss of heart regenerative capacity. In this review, we describe the effects of thyroid hormones on animal metabolism and cardiomyocyte regeneration and highlight recent reports linking the loss of mammalian cardiac regenerative capacity to metabolic shifts occurring after birth.

Genetic, Epigenetic, and Post-Transcriptional Basis of Divergent Tissue Regenerative Capacities Among Vertebrates.

Regeneration is widespread across the animal kingdom but varies vastly across phylogeny and even ontogeny. Adult mammalian regeneration in most organs and appendages is limited, while vertebrates such as zebrafish and salamanders are able to regenerate various organs and body parts. Here, we focus on the regeneration of appendages, spinal cord, and heart - organs and body parts that are highly regenerative among fish and amphibian species but limited in adult mammals. We then describe potential genetic, epigenetic, and post-transcriptional similarities among these different forms of regeneration across vertebrates and discuss several theories for diminished regenerative capacity throughout evolution.

Endocrine Influence on Cardiac Metabolism in Development and Regeneration.

Mammalian cardiomyocytes mostly utilize oxidation of fatty acids to generate ATP. The fetal heart, in stark contrast, mostly uses anaerobic glycolysis. During perinatal development, thyroid hormone drives extensive metabolic remodeling in the heart for adaptation to extrauterine life. These changes coincide with critical functional maturation and exit of the cell cycle, making the heart a post-mitotic organ. Here, we review the current understanding on the perinatal shift in metabolism, hormonal status, and proliferative potential in cardiomyocytes. Thyroid hormone and glucocorticoids have roles in adult cardiac metabolism, and both pathways have been implicated as regulators of myocardial regeneration. We discuss the evidence that suggests these processes could be interrelated and how this can help explain variation in cardiac regeneration across ontogeny and phylogeny, and we note what breakthroughs are still to be made.

Vertebrate cardiac regeneration: evolutionary and developmental perspectives.

Cardiac regeneration is an ancestral trait in vertebrates that is lost both as more recent vertebrate lineages evolved to adapt to new environments and selective pressures, and as members of certain species developmentally progress towards their adult forms. While higher vertebrates like humans and rodents resolve cardiac injury with permanent fibrosis and loss of cardiac output as adults, neonates of these same species can fully regenerate heart structure and function after injury - as can adult lower vertebrates like many teleost fish and urodele amphibians. Recent research has elucidated several broad factors hypothesized to contribute to this loss of cardiac regenerative potential both evolutionarily and developmentally: an oxygen-rich environment, vertebrate thermogenesis, a complex adaptive immune system, and cancer risk trade-offs. In this review, we discuss the evidence for these hypotheses as well as the cellular participators and molecular regulators by which they act to govern heart regeneration in vertebrates.

Hormonal control of cardiac regenerative potential.

Research conducted across phylogeny on cardiac regenerative responses following heart injury implicates endocrine signaling as a pivotal regulator of both cardiomyocyte proliferation and heart regeneration. Three prominently studied endocrine factors are thyroid hormone, vitamin D, and glucocorticoids, which canonically regulate gene expression through their respective nuclear receptors thyroid hormone receptor, vitamin D receptor, and glucocorticoid receptor. The main animal model systems of interest include humans, mice, and zebrafish, which vary in cardiac regenerative responses possibly due to the differential onsets and intensities of endocrine signaling levels throughout their embryonic to postnatal organismal development. Zebrafish and lower vertebrates tend to retain robust cardiac regenerative capacity into adulthood while mice and other higher vertebrates experience greatly diminished cardiac regenerative potential in their initial postnatal period that is sustained throughout adulthood. Here, we review recent progress in understanding how these three endocrine signaling pathways regulate cardiomyocyte proliferation and heart regeneration with a particular focus on the controversial findings that may arise from different assays, cellular-context, age, and species. Further investigating the role of each endocrine nuclear receptor in cardiac regeneration from an evolutionary perspective enables comparative studies between species in hopes of extrapolating the findings to novel therapies for human cardiovascular disease.

KrasP34R and KrasT58I mutations induce distinct RASopathy phenotypes in mice.

Somatic KRAS mutations are highly prevalent in many cancers. In addition, a distinct spectrum of germline KRAS mutations causes developmental disorders called RASopathies. The mutant proteins encoded by these germline KRAS mutations are less biochemically and functionally activated than those in cancer. We generated mice harboring conditional KrasLSL-P34Rand KrasLSL-T58I knock-in alleles and characterized the consequences of each mutation in vivo. Embryonic expression of KrasT58I resulted in craniofacial abnormalities reminiscent of those seen in RASopathy disorders, and these mice exhibited hyperplastic growth of multiple organs, modest alterations in cardiac valvulogenesis, myocardial hypertrophy, and myeloproliferation. By contrast, embryonic KrasP34R expression resulted in early perinatal lethality from respiratory failure due to defective lung sacculation, which was associated with aberrant ERK activity in lung epithelial cells. Somatic Mx1-Cre-mediated activation in the hematopoietic compartment showed that KrasP34R and KrasT58I expression had distinct signaling effects, despite causing a similar spectrum of hematologic diseases. These potentially novel strains are robust models for investigating the consequences of expressing endogenous levels of hyperactive K-Ras in different developing and adult tissues, for comparing how oncogenic and germline K-Ras proteins perturb signaling networks and cell fate decisions, and for performing preclinical therapeutic trials.

In vitro and in vivo roles of glucocorticoid and vitamin D receptors in the control of neonatal cardiomyocyte proliferative potential.

Cardiomyocyte (CM) proliferative potential varies considerably across species. While lower vertebrates and neonatal mammals retain robust capacities for CM proliferation, adult mammalian CMs lose proliferative potential due to cell-cycle withdrawal and polyploidization, failing to mount a proliferative response to regenerate lost CMs after cardiac injury. The decline of murine CM proliferative potential occurs in the neonatal period when the endocrine system undergoes drastic changes for adaptation to extrauterine life. We recently demonstrated that thyroid hormone (TH) signaling functions as a primary factor driving CM proliferative potential loss in vertebrates. Whether other hormonal pathways govern this process remains largely unexplored. Here we showed that agonists of glucocorticoid receptor (GR) and vitamin D receptor (VDR) suppressed neonatal CM proliferation. We next examined CM nucleation and proliferation in neonatal mutant mice lacking GR or VDR specifically in CMs, but we observed no difference between mutant and control littermates at postnatal day 14. Additionally, we generated compound mutant mice that lack GR or VDR and express dominant-negative TH receptor alpha in their CMs, and similarly observed no increase in CM proliferative potential compared to dominant-negative TH receptor alpha mice alone. Thus, although GR and VDR activation is sufficient to inhibit CM proliferation, they seem to be dispensable for neonatal CM cell-cycle exit and polyploidization in vivo. In addition, given the recent report that VDR activation in zebrafish promotes CM proliferation and tissue regeneration, our results suggest distinct roles of VDR in zebrafish and rodent CM cell-cycle regulation.

Lamin B2, Guardian of Cardiomyocyte Nuclear Division.

Regenerative capacity is robust in the neonatal mouse heart but is lost during postnatal development when cardiomyocytes undergo cell-cycle arrest and polyploidization. In this issue of Developmental Cell, Han et al. (2020) show that Lamin B2, a nuclear lamina filament supporting cardiomyocyte karyokinesis, also facilitates cell division and cardiac regeneration.

Defined factors to reactivate cell cycle activity in adult mouse cardiomyocytes.

Adult mammalian cardiomyocytes exit the cell cycle during the neonatal period, commensurate with the loss of regenerative capacity in adult mammalian hearts. We established conditions for long-term culture of adult mouse cardiomyocytes that are genetically labeled with fluorescence. This technique permits reliable analyses of proliferation of pre-existing cardiomyocytes without complications from cardiomyocyte marker expression loss due to dedifferentiation or significant contribution from cardiac progenitor cell expansion and differentiation in culture. Using this system, we took a candidate gene approach to screen for fetal-specific proliferative gene programs that can induce proliferation of adult mouse cardiomyocytes. Using pooled gene delivery and subtractive gene elimination, we identified a novel functional interaction between E2f Transcription Factor 2 (E2f2) and Brain Expressed X-Linked (Bex)/Transcription elongation factor A-like (Tceal) superfamily members Bex1 and Tceal8. Specifically, Bex1 and Tceal8 both preserved cell viability during E2f2-induced cell cycle re-entry. Although Tceal8 inhibited E2f2-induced S-phase re-entry, Bex1 facilitated DNA synthesis while inhibiting cell death. In sum, our study provides a valuable method for adult cardiomyocyte proliferation research and suggests that Bex family proteins may function in modulating cell proliferation and death decisions during cardiomyocyte development and maturation.

Evidence for hormonal control of heart regenerative capacity during endothermy acquisition.

Tissue regenerative potential displays striking divergence across phylogeny and ontogeny, but the underlying mechanisms remain enigmatic. Loss of mammalian cardiac regenerative potential correlates with cardiomyocyte cell-cycle arrest and polyploidization as well as the development of postnatal endothermy. We reveal that diploid cardiomyocyte abundance across 41 species conforms to Kleiber's law-the ¾-power law scaling of metabolism with bodyweight-and inversely correlates with standard metabolic rate, body temperature, and serum thyroxine level. Inactivation of thyroid hormone signaling reduces mouse cardiomyocyte polyploidization, delays cell-cycle exit, and retains cardiac regenerative potential in adults. Conversely, exogenous thyroid hormones inhibit zebrafish heart regeneration. Thus, our findings suggest that loss of heart regenerative capacity in adult mammals is triggered by increasing thyroid hormones and may be a trade-off for the acquisition of endothermy.

Unconventional Functions of Muscles in Planarian Regeneration.

Muscles are traditionally considered in the context of force generation. Scimone et al. (2017), reporting in Nature, now examine muscles in a developmental setting and find unexpected roles for distinct planarian muscle fibers. The authors show that muscles provide patterning signals to promote regeneration and guide tissue growth after injury.