GSNO induced mitochondrial Cx43 nitrosylation in cardiomyocyte differentiation from mouse ES cells in vitro

S-nitrosoglutathione (GSNO), considered vital to S-nitrosylation of proteins, has been found fundamentally important to the cardiomyocytes (CMs) maturation. Our previous studies demonstrated that GSNO treatment significantly enhanced the S-nitrosylation of 104 proteins during the differentiation of mouse embryonic stem cells (ESCs) into CMs. Mitochondrial Cx43 (mtCx43), a membrane protein implicated in the intercellular communication, also plays a pivotal role in CMs regeneration from stem cells. However, the involvement of mtCx43 S-nitrosylation in GSNO-induced myocardial differentiation has not been fully elucidated. In this study, we employed an ESCs-derived CMs differentiation model to elucidate the mechanisms underlying GSNO-induced cardiogenesis. Our findings revealed that GSNO treatment significantly up-regulated mitochondrial transmembrane potential, ATP production, reactive oxygen species (ROS) levels, respiratory chain complex Ι activity and mtCx43 hemichannel permeability in embryoid bodies (EBs). Furthermore, S-nitrosylation of mtCx43 was markedly enhanced in differentiating EBs after GSNO treatment. Overexpression of mtCx43 further amplified the pro-mitochondrial maturation effects of GSNO, whereas overexpression of a mutant form, mtCx43^C271A attenuated this effect. To investigate the functional role of mtCx43 hemichannels, we pretreated EBs with Gap19, a specific mtCx43 hemichannel blocker, followed by GSNO administration. Gap19 significantly reduced in mitofusin 2 (Mfn2) expression, thereby impairing mitochondrial maturation and function. In addition, Gap19 treatment abrogated the pro-cardiogenic effects of mtCx43 S-nitrosylation. Furthermore, we demonstrated that mtCx43 S-nitrosylation-induced cardiac differentiation was dependent on mitochondrial Ca²⁺ uptake. In conclusion, GSNO-induced S-nitrosylation of mtCx43 enhances mitochondrial function in EBs by promoting the opening of mtCx43 hemichannels, thus facilitating the targeted differentiation of ESCs into CMs. These findings provide novel insights into the role of mtCx43 S-nitrosylation in mitochondrial regulation and cardiac lineage commitment.