Abstract Body (Do not enter title and authors here): Background: Cyclin A2 (CCNA2) is a hallmark cell cycle regulator typically silenced in postnatal mammalian cardiomyocytes (CMs). We previously demonstrated successful myocardial regeneration in infarcted porcine hearts, showing a 55% increase in new CMs and significantly enhanced cardiac function following CCNA2 gene therapy.
Hypothesis: We hypothesize that CCNA2 gene delivery can induce cytokinesis in isolated adult human CMs.
Methods and Results: 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 (Ad5-cTnT-hCCNA2, vector lacking CCN2 control). We utilized time-lapse microscopic live imaging of cultured adult human CMs isolated from a 55-year-old male, 41-year-old female, and 21-year-old male. We quantified cytokinesis events in human adult CMs compared to controls and observed a significant increase in cytokinesis rate in the aging heart, particularly in the 55-year-old male (17.52 + 1.72 vs. 8.33 + 2.25, p=0.003) and the 41-year-old female (9.27 + 2.22 vs. 2.5 + 1.16, p=0.005). No significant difference was observed in the 21-year-old male (3.63 + 1.77 vs. 3.00 + 1.52, p=0.789). We measured the calcium flux in transfected CMs compared to controls after cell division under pacing conditions (0.5 Hz and 1 Hz current) and detected sarcomere structures using fluorescence microscopy. Approximately 4-5% of Ad5-cTnT-hCCNA2 transfected adult human CMs exhibited active Ca²⁺ mobilization. We now report that CCNA2 can induce complete cytokinesis in in adult human CMs, preservation of sarcomere integrity in the resultant daughter cells.To understand further CCNA2 signaling pathways involved in CM cytokinesis, we conducted single nucleus transcriptomics analysis in hearts isolated from adult transgenic mice that constitutively express CCNA2 in CM (Tg) and non-transgenic mice (nTg). We identified a subpopulation of CMs enriched with cytokinesis (Aurkb, Anln, Sept7, and Rho), proliferative (Mki67 and Kif23), and reprogramming (Pou5f1, Klf4, Gata4, Myc, and Sox2) genes in Tg mice compared to nTg mice. Bulk RNA sequencing of human adult and fetal hearts unveiled key reprogramming genes involved in CCNA2-induced cytokinesis.
Conclusion: Our findings highlight promising mechanisms for the clinical development of cardiac regenerative therapy through strategic manipulation of the CM cell cycle.