Abstract
Cell death and differentiation appear to share similar cellular features. In this study, we aimed to investigate whether differentiation and mitochondrial cell death use a common pathway. We assessed the hallmarks of apoptosis during cardiomyocyte differentiation of human embryonic stem cells and found remarkable changes in P53, reactive oxygen species, apoptotic protease-activating factor 1, poly[ADP-ribose]polymerase 1, cellular adenosine triphosphate, and mitochondrial complex I activity. Furthermore, we observed reversible mitochondrial membrane permeabilization during cardiomyocyte differentiation accompanied by reversible loss of mitochondrial membrane potential, and these changes coincided with the fluctuating patterns of cytosolic cytochrome c accumulation and subsequent caspase-9 and -3/7 activation. Moreover, the use of apoptosis inhibitors (BCL2-associated X protein [BAX] inhibitor and caspase-3/7 inhibitor) during differentiation impaired cardiomyocyte development, resulting in substantial downregulation of T, MESP1, NKX2.5, and α-MHC. Additionally, although the expression of specific differentiation markers (T, MESP1, NKX2.5, MEF2C, GATA4, and SOX17) was enhanced in doxorubicin-induced human embryonic stem cells, the stemness-specific markers (OCT4 and NANOG) showed significant downregulation. With increasing doxorubicin concentration (0.03-0.6 µM; IC50 = 0.5 µM), we observed a marked increase in the expression of mesoderm and endoderm markers. In summary, we suggest that reversible mitochondrial outer membrane permeabilization promotes cardiomyocyte differentiation through an attenuated mitochondria-mediated apoptosis-like pathway.