Regulation of cardiomyocyte proliferation by the Reptin ATPase

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ABSTRACT: Cardiomyocyte proliferation is an important source of new myocardium during heart development and regeneration. After birth cardiomyocytes are highly resistant to proliferation. The lack of adult cardiomyocyte proliferation precludes cardiac repair and underlies the high morbidity and mortality rates associated with congenital heart defects and adult cardiovascular disease. Therefore, identifying novel regulators of cardiomyocyte proliferation is key to the development of regenerative heart therapies. Zebrafish are a well- established model to study cardiac development and regeneration because their cardiomyocytes maintain a remarkable capacity to proliferate allowing the heart to regenerate after adult injury. Our lab recently reported that the AAA+ ATPase Reptin is a potent suppressor of cardiomyocyte proliferation. Reptin is a known component of the Tip60 and INO80 complexes which have roles in DNA damage repair and chromatin remodeling. Our lab demonstrated in zebrafish that reptin loss of function mutations cause myocardial hyperplasia at 3 days post fertilization (dpf). We showed that cardiomyocyte-specific overexpression of reptin rescues the cardiomyocyte hyperproliferation phenotype. We further demonstrated that inducible overexpression of reptin after adult cardiac injury resulted in decreased cardiomyocyte proliferation and failure to regenerate. Expanding on our published data, I propose to study the molecular mechanisms by which Reptin suppresses cardiomyocyte proliferation. Preliminary data suggests that the ATPase function of Reptin is essential to dampen proliferation at 3 dpf. RNA sequencing of reptin mutant and control cardiomyocytes at 3 dpf revealed the unanticipated result that the hyperproliferative reptin mutant cardiomyocytes upregulate both pro-proliferative (e.g. fosl1a & junba) and anti-proliferative (e.g. tp53 & cbx7a) transcripts. Analysis at 4 and 5 dpf revealed that the reptin mutant cardiomyocytes lose their hyperproliferative phenotype by 4 dpf and have significantly reduced proliferation rates compared to controls at 5 dpf. I hypothesize: 1) that reptin mutant cardiomyocytes require AP-1 activity for their initial proliferative burst where DNA damage accumulates and triggers tp53 upregulation to halt cell division, and 2) that compensatory upregulation of the PRC1 complex component cbx7a drives an anti-proliferative chromatin landscape in reptin mutant cardiomyocytes. In Aim 1, I will assess the sufficiency of AP-1 family members to drive cardiomyocyte proliferation in embryonic zebrafish, the accumulation of DNA damage in hyperproliferative reptin mutant cardiomyocytes, and the role of tp53 upregulation in DNA damage signaling and cell cycle arrest in reptin mutant hearts. In Aim 2, I will identify changes in the chromatin landscape that are associated with reptin loss of function and correlate those alterations to changes in gene expression. I will also investigate the ability of cbx7a to dampen cardiomyocyte proliferation in wildtype and reptin mutant backgrounds. The proposed studies will give insight into the regulation of cardiomyocyte proliferation and identify druggable pathways/targets for novel regenerative therapies.