Mechanisms of cardiomyocyte cell cycle arrest and maturation in postnatal rodents and swine

Heart failure causes millions of deaths annually and presents a large global healthcare burden. A regenerative cure for the damaged myocardium, where new muscle forms by proliferation of pre-existing cardiomyocytes, is an attractive therapeutic goal. Adult mammalian cardiomyocytes are terminally-differentiated, only capable of proliferating at a very low rate that is insufficient for a regenerative response after myocardial infarction. However, over the past decade, a transient innate capacity for cardiac regeneration during the early neonatal period has been described in both rodents and swine. Whether such a capacity exists in newborn human infants is unknown. Studying the mechanisms of regenerative potential in neonatal rodents and swine could offer greater insight into heart development in human neonates, and also facilitate discovery of novel targets for human heart disease therapy.Cardiomyocyte maturational processes occur concurrent with loss of heart regenerative potential in early neonatal mice. These maturational processes, and the transcriptional mechanisms regulating them, have been successfully manipulated to induce cardiac regenerative repair in adult mouse hearts after injury. Pigs also possess a similar period of early neonatal heart regenerative capacity as mice. However, the maturational dynamics of cardiomyocyte growth in the postnatal pig heart are not well-defined, despite popularity of swine as large mammal models for cardiac preclinical studies. In Chapter 2 of this dissertation, we describe cardiac maturation in postnatal swine from newborn to adolescent ages. Our results show discordance between time of terminal cardiomyocyte maturation and loss of heart regenerative potential in postnatal swine, dissimilar to rodents. Further, postnatal pig cardiomyocytes are distinct from rodents and humans, exhibiting extensive multinucleation of up to 16 nuclei per cardiomyocyte by 6 postnatal months. These differences hold importance for preclinical cardiac studies in swine, and present opportunities to investigate unique cardiomyocyte maturational processes in a mammalian model.Cardiomyocyte proliferative arrest occurs within the first postnatal week in rodents, coincident with loss of regenerative potential. Downregulation of developmental signaling alongside activation of various cell cycle inhibitory factors occurs during this time. Neonatal cardiomyocyte maturation is thus a complex process with many compensatory mechanisms, characterization of which is essential for targeted proliferative repair without uncontrolled growth in adult diseased hearts. Previous studies identified a potential inhibitory role in cardiomyocyte proliferation for transcriptional co-regulator Btg2 (B-cell translocation gene 2). Btg2, and its close family member Btg1, are well-known in non-cardiac cell lineages for their anti-proliferative functions, but are virtually unstudied in the heart. In Chapter 3 of this dissertation, utilizing in vivo and in vitro rodent models of Btg1 and Btg2 (Btg1/2) depletion, we show that Btg1/2 loss significantly increases neonatal cardiomyocyte mitotic activity. Also, by transcriptome analysis, a pleiotropic role for Btg1/2 in cardiomyocyte cell cycle regulation is implicated. These data suggest a novel regulatory role for Btg1/2 in neonatal rodent cardiomyocyte cell cycle arrest.Together, the studies described in this dissertation promote understanding of cardiomyocyte maturation in postnatal rodents and swine. Our results highlight distinctive differences in cardiomyocyte biology among various mammals, and also enhance overall understanding of mammalian cardiomyocyte cell cycle regulation.