Cardiomyocyte differentiation from iPS cells is delayed following knockout of Bcl-2

Abstract Anti-apoptotic B-cell lymphoma 2 (Bcl-2) regulates a wide array of cellular functions involved in cell death, cell survival decisions and autophagy. Bcl-2 acts by both direct interaction with different components of the pathways involved and by intervening in intracellular Ca 2+ signalling. The function of Bcl-2 is in turn regulated by post-translational modifications including phosphorylation at different sites by various protein kinases. Besides functions in cell death and apoptosis, Bcl-2 regulates cell differentiation processes, including of cardiomyocytes, although its mechanism of action in this process is not fully elucidated. To further address the role of Bcl-2 in cardiomyocyte differentiation, we investigated the effect of its genetic knockout by CRISPR/Cas9 on the differentiation and functional maturation trajectory of human induced pluripotent stem cells (hiPSC) to cardiomyocytes. Our results indicate that differentiation of hiPSC to cardiomyocytes is delayed by Bcl-2 KO. This effect was associated with smaller areas of spontaneously beating cells. At the cell population level this exhibited in reduced expression and activity of the cardiomyocyte Ca 2+ toolkit. However, when restricting the comparison to the active areas exclusively Bcl-2 KO did not show to significantly alter the functionality of the differentiated cardiomyocytes. Finally, Bcl-2 KO reduced c-Myc expression early in the cardiac differentiation process which may account at least in part for the here observed delay in cardiac differentiation resulting reduced efficiency of the differentiation process. These data are supportive of a pivotal role for Bcl-2 in cardiomyocyte differentiation and maturation.