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.

Dual human iPSC-derived cardiac lineage cell-seeding extracellular matrix patches promote regeneration and long-term repair of infarcted hearts.

Human pluripotent stem cell-derived cardiovascular progenitor cells (hCVPCs) and cardiomyocytes (hCMs) possess therapeutic potential for infarcted hearts; however, their efficacy needs to be enhanced. Here we tested the hypotheses that the combination of decellularized porcine small intestinal submucosal extracellular matrix (SIS-ECM) with hCVPCs, hCMs, or dual of them (Mix, 1:1) could provide better therapeutic effects than the SIS alone, and dual hCVPCs with hCMs would exert synergic effects in cardiac repair. The data showed that the SIS patch well supported the growth of hCVPCs and hCMs. Epicardially implanted SIS-hCVPC, SIS-hCM, or SIS-Mix patches at 7-day post-myocardial infarction significantly ameliorated functional worsening, ventricular dilation and scar formation at 28- and 90-day post-implantation in C57/B6 mice, whereas the SIS only mildly improved function at 90-day post-implantation. Moreover, the SIS and SIS-cell patches improved vascularization and suppressed MI-induced cardiomyocyte hypertrophy and expression of Col1 and Col3, but only the SIS-hCM and the SIS-Mix patches increased the ratio of collagen III/I fibers in the infarcted hearts. Further, the SIS-cell patches stimulated cardiomyocyte proliferation via paracrine action. Notably, the SIS-Mix had better improvements in cardiac function and structure, engraftments, and cardiomyocyte proliferation. Proteomic analysis showed distinct biological functions of exclusive proteins secreted from hCVPCs and hCMs, and more exclusive proteins secreted from co-cultivated hCVPCs and hCMs than mono-cells involving in various functional processes essential for infarct repair. These findings are the first to demonstrate the efficacy and mechanisms of mono- and dual-hCVPC- and hCM-seeding SIS-ECM for repair of infarcted hearts based on the side-by-side comparison.

Strontium ions protect hearts against myocardial ischemia/reperfusion injury.

Timely restoration of blood supply following myocardial infarction is critical to save the infarcted myocardium, while reperfusion would cause additional damage. Strontium ions have been shown to promote angiogenesis, but it is unknown whether they can save the damaged myocardium. We report that myocardial ischemia/reperfusion (I/R)-induced functional deterioration and scar formation were notably attenuated by injection of strontium ion-containing composite hydrogels into murine infarcted myocardium at 20 minutes of reperfusion following 60 minutes of ischemia. These beneficial effects were accompanied by reduced cardiomyocyte apoptosis and increased angiogenesis. The effects of strontium ions were further confirmed by the enhanced viability of cardiomyocytes and stimulated angiogenesis in vitro. These findings are the first to reveal the cardioprotective effects of strontium ions against I/R injury, which may provide a new therapeutic approach to ischemic heart disease at a lower cost, with higher stability, and with potentially greater safety.