Cyclin A2 Induces Human Adult Cardiomyocyte Cytokinesis and Elicits Cardiomyocyte Reprogramming and Dedifferentiation

Abstract

Background: Cyclin A2 (CCNA2), the master regulatory gene of the cell cycle is commonly silenced in postnatal mammalian cardiomyocytes. We have previously demonstrated that it can induce significant cardiac repair in both small and large animals when delivered to the heart via a viral vector. To date, whether CCNA2 gene delivery can induce cytokinesis in isolated cardiomyocytes from adult human hearts has not been investigated. Here we report that CCNA2 delivery can induce cytokinesis in cardiomyocytes isolated from adult human hearts. Methods: We designed a human gene therapy vector featuring a replication-deficient, E1/E3-deleted human adenovirus five encoding human CCNA2 driven by the cardiac Troponin T promoter to enable the expression of CCNA2 in freshly isolated human cardiomyocytes. We utilized time-lapse microscopy with live imaging to study cultured adult human cardiomyocytes isolated from a 21-year-old male, a 41-year-old female, and a 55-year-old male. To elucidate the mechanistic underpinnings of CCNA2-dependent gene regulation in governing cardiomyocyte cytokinesis, we conducted single nucleus transcriptomics (snRNA-seq, 10X Genomics) analysis of hearts isolated from adult transgenic mice that constitutively express CCNA2 in cardiomyocytes (CCNA2-Tg) and non-transgenic mice (nTg). Results: We now report that human adult cardiomyocytes can be induced to undergo complete cytokinesis in response to CCNA2 gene delivery with preservation of sarcomere integrity in the resulting daughter cells and maintaining active calcium mobilization in redifferentiated cardiomyocytes. Remarkably, snRNA-seq analysis revealed a subpopulation of cardiomyocytes enriched with cytokinesis, proliferative and reprogramming genes in hearts obtained from CCNA2-Tg mice as compared to hearts obtained from nTg mice. Additionally, bulk RNA sequencing of human adult and fetal hearts identified key reprogramming genes relevant for understanding the mechanisms of CCNA2-induced effects observed in our experimental models. Conclusion: These results provide a compelling path forward for the clinical development of cardiac regenerative therapy based on strategic manipulation of CCNA2 in cardiomyocytes.