Tumor Suppressors RB1 and CDKN2a Cooperatively Regulate Cell-Cycle Progression and Differentiation During Cardiomyocyte Development and Repair: Implications for Stimulating Neomyogenesis with Cell-Based Therapy.

RATIONALE: Although rare cardiomyogenesis is reported in the adult mammalian heart, whether this results from differentiation or proliferation of cardiomyogenic cells remains controversial. The tumor suppressor genes RB1 and CDKN2a are critical cell-cycle regulators, but their roles in human cardiomyogenesis remains unclear.

OBJECTIVE: We hypothesized that developmental activation of RB1 and CDKN2a cooperatively cause permanent cell-cycle withdrawal of human cardiac precursors (CPCs) driving terminal differentiation into mature cardiomyocytes (CM), and that dual inactivation of these tumor suppressor genes promotes myocyte cell-cycle re-entry.

METHODS AND RESULTS: Directed differentiation of human pluripotent stem cells (hPSCs) into CMs revealed that RB1 and CDKN2a are upregulated at the onset of CPC specification, simultaneously with GATA4 and homeobox genes PBX1 and MEIS1, and remain so until terminal CM differentiation. In both GATA4+ hPSC-CPCs and post-mitotic hPSC-CMs, RB1 is hyperphosphorylated and inactivated. Transient, stage-specific, depletion of RB1 during hPSC differentiation enhances cardiomyogenesis at the CPC stage, but not in terminally differentiated hPSC-CMs, by transiently upregulating GATA4 expression through a cell-cycle regulatory pathway involving CDKN2a. Importantly, cytokinesis in post-mitotic hPSC-CMs can be induced with transient, dual RB1 and CDKN2a silencing. The relevance of this pathway in vivo was suggested by findings in a porcine model of cardiac cell therapy post-MI, whereby dual RB1 and CDKN2a inactivation in adult GATA4+ cells correlates with the degree of scar size reduction and endogenous CMs mitosis, particularly in response to combined transendocardial injection of adult human MSCs and cKit+ cells (CCT).

CONCLUSIONS: Together these findings reveal an important and coordinated role for RB1 and CDKN2a in regulating cell-cycle progression and differentiation during human cardiomyogenesis. Moreover, transient, dual inactivation of RB1 and CDKN2a in endogenous adult GATA4+ cells and CMs mediates, at least in part, the beneficial effects of cell-based therapy in a post-MI large mammalian model, a finding with potential clinical implications.