Investigating the pro-regenerative potential of Pontin in the heart.

Project Summary Ischemic heart disease is the leading cause of death worldwide, in part because human heart muscle fails to regenerate after ischemic injury. A primary barrier to human heart regeneration is the near-complete absence of cardiomyocyte cell division following a myocardial infarction (MI). Fortuitously, injured hearts of adult zebrafish and neonatal mice are capable of robust cardiac regeneration achieved through cardiomyocyte proliferation. However, neonatal mice lose their innate cardiac regenerative capacity between P1 and P7 due to cardiomyocyte cell cycle exit, the regulatory details of which remain incompletely understood. Deciphering the pro- and anti-proliferative pathways regulating cardiomyocyte proliferation and cell cycle exit in these highly tractable model organisms will identify druggable pathways for stimulating heart regeneration as a permanent remedy for myocardial infarction. Herein, I propose to expand our fundamental knowledge of the gene regulatory networks controlling cardiomyocyte proliferation through mechanistic studies on the AAA+ ATPase Pontin and high-quality candidate downstream targets. We present preliminary data identifying Pontin as a novel regulator of cardiomyocyte proliferation in zebrafish and mice. Zebrafish pontin mutants are devoid of cardiomyocyte proliferation, and cardiomyocyte-specific overexpression of Pontin (pontinOECM) hyperactivates cardiomyocyte proliferation during cardiac development, growth, and regeneration. Remarkably, Pontin overexpression also improves the efficiency of heart regeneration, as evidenced by reduced scar size. We also demonstrate that myocardial-specific deletion of Pontin in embryonic mice suppresses cardiomyocyte proliferation, leading to embryonic lethality. Interestingly, Pontin levels decrease naturally in cardiomyocytes soon after birth, consistent with the possibility that Pontin downregulation creates a barrier to regeneration by facilitating cardiomyocyte cell cycle exit. Accordingly, cardiomyocyte-specific overexpression of Pontin delays cell cycle exit and, potentially, expands the regenerative window. Accordingly, myocardial infarction of PontinOECM hearts at P8 resulted in improved ejection fraction at P29, consistent with a regenerative response. Here, I propose to test the central hypothesis that Pontin regulates cardiomyocyte proliferation by controlling the chromatin accessibility of high-quality candidate genes identified through ‘omics efforts detailed in preliminary data. In Aim I, we will corroborate the candidate Pontin targets regulating CM proliferation during zebrafish heart development. In Aim II, we will corroborate the direct Pontin targets regulating CM proliferation during zebrafish heart regeneration. In Aim III, we will determine if Pontin is a pro-regenerative factor in mice and whether the zebrafish targets are conserved in mammals. Overall, the proposed studies will produce novel biological insights and positively impact ongoing efforts to stimulate cardiomyocyte proliferation in the context of human heart disease.