Cardiac Regeneration and Repair: Mechanisms and Therapy Cardiomyocyte proliferation in cardiac development and regeneration: a guide to methodologies and interpretations

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 prom- ising approach for improving adult human cardiac function after injury, but it is important to elucidate the mechanisms arresting mammalian cardiomyocyte pro- liferation after birth and to utilize better assays to determine In the literature, 2 in vivo systems have been described to visualize clonal growth of cardiomyocytes. A : the mosaic analysis with double markers (MADM) mouse model is based on split genes encoding for NH 2 - and COOH-terminal parts of the ﬂuorescent proteins red ﬂuorescent protein (RFP) and green ﬂuorescent protein (GFP). The system is designed such that, upon Cre recombination during mitosis, the split genes are recombined to allow the expression of RFP in 1 daughter cell and GFP in the other (G2-X). If cell division/cytokinesis is not completed, cells will be binucleated, expressing both RFP and GFP and appearing yellow. This model has been used to determine homeostasis in the heart of the Myh6CreERT2 mouse. B : the 2nd model is based on the Brainbow mouse model containing several genes encoding different ﬂuorescent proteins under the control of a general promoter. Cre recombination results in the random expression of individual ﬂuorescent proteins. Thus, the use of a cardiac-speciﬁc promoter (Myh6) driving Cre will result in the labeling of individual cardiomyocytes. Activation of proliferation in such an individual labeled cardiomyocyte will then result in single color-labeled clones. Yet this system works well only if few cardiomyocytes are labeled, several different ﬂuorescent proteins are utilized, and cardiomyocytes are induced to undergo several cell division. However, there is always an uncertainty, especially in the case of small-sized clones, whether a single color-labeled clone arises from proliferation or by the same recombination event in neighboring cells. This system was utilized as a dual-color system (RFP and GFP) to determine the effect of Yes-associated protein (YAP) on cardiomyocyte proliferation. YAP was expressed under the control of a doxycycline-dependent reverse tetracycline activator protein.