Reptin is Required to Maintain Cardiomyocytes in a Proliferative Diploid State

Abstract

Cardiomyocyte proliferation drives the addition of new myocardium during heart development and regeneration. We previously reported that the ATPase Reptin is a suppressor of cardiomyocyte proliferation in zebrafish as reptin mutant hearts show increased BrdU incorporation at 3 days post fertilization (dpf). In chapter 2, we determine that the ATPase function of Reptin is essential for its ability to repress cardiomyocyte proliferation at 3 dpf before making the paradigm shifting discovery that despite being hyperproliferative at 3 dpf, reptin-/- cardiomyocytes exhibit an anti-proliferative transcriptional profile. We then demonstrate that reptin-/- cardiomyocyte proliferation rates significantly decrease by 5 dpf and that reptin-/- cardiomyocytes accumulate in a polyploid state. The loss of Reptin drives cellular senescence but not an accumulation of DNA damage or apoptosis in cardiomyocytes. Through RNA-sequencing we identify several pathways and cellular processes which are dysregulated in reptin mutants including AP-1, tp53, cbx7a, and midbody/cytokinesis. We investigate the role of these mechanisms and their relationship to Reptin’s ability to regulate cardiomyocyte proliferation. We show that AP-1 signaling plays a role in controlling ventricular area in reptin mutants, but its effects on proliferation in reptin mutant hearts remain unclear. We further demonstrate that tp53 and cbx7a are not major drivers of the later stage cell cycle exit observed in reptin-/- cardiomyocytes. We further observe using human iPSC derived cardiomyocytes that Reptin localizes to the midbody of cytokinetic cardiomyocytes. We show that in mice, like zebrafish, Reptin is required for healthy heart development and the maintenance of cardiomyocyte proliferation. In the mouse, we show that the regulation of cardiomyocyte cell cycle exit by Reptin is cell autonomous and like in zebrafish loss of Reptin does not result in the significant induction of apoptosis. Our work has led to a new model whereby Reptin initially represses cardiomyocyte cell cycle entry in early heart development, but that it is required to maintain cardiomyocytes in a proliferative diploid state over the course of cardiac development. Since the major barrier to heart regeneration is cardiomyocyte cell cycle exit, therapeutic manipulation of Reptin could be used to stimulate renewal of the diseased heart.