Abstract P1076: Rrad Regulates Cardiomyocyte Cell Cycle Entry By Modulating Calcineurin Activity

Abstract

Background: Cardiomyocytes (CMs) lost during ischemic cardiac injury cannot be replaced due to their limited proliferative capacity, which leads to progressive heart failure. We and others have shown that reduced CM contractility can promote cell cycle induction; however, the underlying mechanisms are still unclear. Calcium (Ca 2+ ), a key regulator of cardiac contractility, is also an important signal transducer that regulates key cellular processes. Ca 2+ enters CMs through the LTCC, which triggers Ca 2+ release from the sarcoplasmic reticulum to initiate CM contractility. The aim of this study is to understand the role of Ca 2+ signaling during CM cell cycle induction. Methods and Results: We performed a drug screening targeting proteins involved in CM calcium cycling in human iPS-derived cardiac organoids. Changes in Ki-67 expression were assessed as an S-G2/M phase marker. The top hit was Nifedipine, an inhibitor of LTCC. To inhibit the LTCC activity, we overexpressed Ras-Related Associated with Diabetes (RRAD), an endogenous regulator of LTCC activity. RRAD overexpression significantly reduced the intracellular calcium amplitude. RRAD overexpression promoted cell cycle induction, increased CM number, and upregulated cell cycle genes in primary neonatal, adult pig, human heart slices, and human iPS-derived cardiac organoids. Moreover, RRAD overexpression significantly augmented the CM response to other cell cycle stimulators such as Cyclin A2, a combination of CDK4 and cyclin D (2F), or a combination of CDK1, CDK4, cyclin D, and cyclin B (4F). Mechanistically, we found that RRAD overexpression reduced the activity of Ca 2+ dependent serine/threonine phosphatase (Calcineurin), as indicated by a significant reduction in calcineurin phosphatase activity in vitro and the expression levels of the calcineurin downstream effectors such as Rcan1 expression as well as translocation of NFAT and Hoxb13 to the CM nucleus. Conclusion: Disruption of Ca 2+ handling by RRAD overexpression promotes cell cycle induction in CMs through modulation of calcineurin activity. Further work in vivo is needed to test whether RRAD overexpression could improve cardiac function after ischemic injury.