Abstract：
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.
摘要：
人多能干细胞来源的心肌细胞(hPSC-CMs)对于研究人类心脏发育,药物发现,疾病建模,和细胞疗法。然而,混合心肌细胞亚型(心室-，心房-,和结节样心肌细胞）和hPSC-CM的成熟异质性限制了它们在体外和体内的应用。肌球蛋白轻链2（MYL2，编码心室/心肌同工型MLC2v蛋白）被认为是心肌的心室特异性标志物；然而，它在人类心脏发育过程中对心室的限制定位受到质疑。因此,目前尚不清楚MYL2是否明确标记了心室hESC-CM。在这里，通过使用MYL2-VenushESC报告行，我们表征了分化过程中MYL2-Venus阳性（MLC2v-Venus）hESC-CM的时间依赖性增加。我们还比较了分子，细胞，andfunctionalpropertiesbetweentheMLC2v-Venus+andMYL2-Venusnegative(MLC2v-Venus-)hESC-CMs.AtearlydifferentiationstagesofhESC-CMs,我们报道了MLC2v-Venus-和MLC2v-Venus+CM均表现出心室样特征，但MLC2v-Venus+hESC-CM的心室样细胞表现出比MLC2v-Venus-hESC-CM更发达的动作电位（AP）特性。同时，大约一半的MLC2v-Venus-hESC-CM人群表现出心房样AP特性，一半显示出心室样的AP特性，而只有约20％的MLC2v-Venus-hESC-CM表达心房标记核受体亚家族2组F成员2（NR2F2，也称为COUPTFII）。在较晚的时间点，几乎所有的MLC2v-Venus+hESC-CM都表现出心室样AP特性。进一步的分析表明,MLC2v-Venus+hESC-CM在培养期间MLC2v水平增加时具有增强的Ca2+瞬变。同时，与MLC2v-Venus-hESC-CM相比，MLC2v-Venus-hESC-CM显示出更明确的肌节结构和更好的线粒体功能。此外，与MLC2v-Venus-hESC-CM相比，MLC2v-Venus-hESC-CM对缺氧刺激更敏感。这些结果为人类心室肌细胞的发育提供了新的见解，并揭示了MLC2v的表达谱与心室hESC-CM发育之间的直接相关性。我们的发现MLC2v主要是发育未成熟的hESC-CM的心室标志物，对人类发育有影响。药物筛选,和疾病建模，并且该标记应该被证明有助于克服与hESC-CM异质性相关的问题。
