Abstract 12469: Ras-Related Associated With Diabetes (RRAD) Regulates Cardiomyocyte Response to Cell Cycle Stimulation

Abstract

Background: Cardiomyocytes (CMs) lost during ischemic cardiac injury can't be replaced due to their limited proliferative capacity. Our laboratory has shown that overexpression of cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1 in combination (4F) promotes cell division in 10-20% of post-mitotic cardiomyocytes (CMs). Recently, we found that 4F overexpression transiently decreased CM contractility during the mitotic phase. Temporal bulk and single-cell RNAseq data from 4F-transduced human iPSC-derived CMs (hiPS-CMs) or P7 neonatal mouse CMs (NMCM) indicated a transient downregulation of contractile genes during the mitotic phase; among the most differentially regulated genes was Ras-Related Associated with Diabetes (RRAD), an endogenous inhibitor of the L-type calcium channel (LTCC). Objective: RRAD inhibition of LTCC primes CMs to respond to cell cycle stimulation through unloading CMs from their commitment to contractile function. Methods and Results: We used viral approaches to overexpress and knockdown (KD) RRAD in hiPS-CMs and NMCM. Functionally, RRAD overexpression abolished intracellular calcium transients’ amplitude while RRAD KD significantly increased calcium transients’ amplitude in response to electric stimulation in CMs. RRAD overexpression in hiPS-CMs or P7 NMCM augments the cell cycle induction following 4F transduction, while RRAD KD resulted in a decline in the cell cycle induction as indicated by cell cycle markers; EDU and PHH3 (PHH3(%): 4F 26.0± 1, 4F+RRAD 41.15± 6.7, 4F+KD 15.92± 2.6, P<0.05; EDU(%): 4F 29.9± 3.9, 4F+RRAD 61.04± 2.6, 4F+KD 16.8± 2.8; n=6, P<0.05). Nitrendipine, an LTCC blocker, and Mavacamten, which directly suppress cardiomyocyte contractility, reversed the inhibitory effect of RRAD KD on cell cycle induction. More interestingly, RRAD overexpression enhanced the cell cycle induction in presence of only 2 factors (CDK4 and CCND (2F)), which are known to induce S phase (PHH3(%): 2F 5.1± 0.4, 2F+RRAD 10.4± 6.7, 2F+KD 0.4± 0.1, EDU (%); 2F 38.6±15, 2F +RRAD 44.7± 18, 2F+KD 5.6± 2; n=6, P<0.05). Conclusions: These data suggest that RRAD modulation of contractile function modulates the response of CMs to cell cycle induction and can reduce the number of factors needed to induce cell cycle in CMs.