Myosin light chain 2 marks differentiating ventricular cardiomyocytes derived from human embryonic stem cells

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great value for studies of human cardiac development, drug discovery, disease modeling, and cell therapy. However, the mixed cardiomyocyte subtypes (ventricular-, atrial-, and nodal-like myocytes) and the maturation heterogeneity of hPSC-CMs restrain their application in vitro and in vivo. Myosin light chain 2 (MYL2, encoding the ventricular/cardiac muscle isoform MLC2v protein) is regarded as a ventricular-specific marker of cardiac myocardium; however, its restricted localization to ventricles during human heart development has been questioned. Consequently, it is currently unclear whether MYL2 definitively marks ventricular hESC-CMs. Here, by using a MYL2-Venus hESC reporter line, we characterized a time-dependent increase of the MYL2-Venus positive (MLC2v-Venus⁺) hESC-CMs during differentiation. We also compared the molecular, cellular, and functional properties between the MLC2v-Venus⁺ and MYL2-Venus negative (MLC2v-Venus^-) hESC-CMs. At early differentiation stages of hESC-CMs, we reported that both MLC2v-Venus^- and MLC2v-Venus⁺ CMs displayed ventricular-like traits but the ventricular-like cells from MLC2v-Venus⁺ hESC-CMs displayed more developed action potential (AP) properties than that from MLC2v-Venus^- hESC-CMs. Meanwhile, about a half MLC2v-Venus^- hESC-CM population displayed atrial-like AP properties, and a half showed ventricular-like AP properties, whereas only ~ 20% of the MLC2v-Venus^- hESC-CMs expressed the atrial marker nuclear receptor subfamily 2 group F member 2 (NR2F2, also named as COUPTFII). At late time points, almost all MLC2v-Venus⁺ hESC-CMs exhibited ventricular-like AP properties. Further analysis demonstrates that the MLC2v-Venus⁺ hESC-CMs had enhanced Ca²⁺ transients upon increase of the MLC2v level during cultivation. Concomitantly, the MLC2v-Venus⁺ hESC-CMs showed more defined sarcomeric structures and better mitochondrial function than those in the MLC2v-Venus^- hESC-CMs. Moreover, the MLC2v-Venus⁺ hESC-CMs were more sensitive to hypoxic stimulus than the MLC2v-Venus^- hESC-CMs. These results provide new insights into the development of human ventricular myocytes and reveal a direct correlation between the expression profile of MLC2v and ventricular hESC-CM development. Our findings that MLC2v is predominantly a ventricular marker in developmentally immature hESC-CMs have implications for human development, drug screening, and disease modeling, and this marker should prove useful in overcoming issues associated with hESC-CM heterogeneity.