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

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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.

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Acknowledgments

The authors thank Dr. Kenneth R. Boheler from the Johns Hopkins University for the constructive suggestions and the manuscript editing and also Dr. Ouyang K from Peking University Shenzhen Graduate School for writing the program for Ca2+ transient measurements. We also acknowledge technical support from Xiang Miao, Yu-Jia Zhai, Shu-Yang Yan, and Hua-Jun Bai from Shanghai Institute of Nutrition and Health. We thank WiCell Research Institute for providing the H7 hESCs.

Funding

This work was supported by grants from the National Key R&D Program of China (2017YFA0103700 to H.T.Y. and Q.D.), Strategic Priority Research Program of the CAS (No. XDA16010201 to H.T.Y.), Major Program of Development Fund for Shanghai Zhangjiang National Innovation Demonstration Zone (ZJ2018-ZD-004 to H.T.Y.), and National Natural Science Foundation of China (81520108004, 81470422 to H.T.Y.).

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Authors and Affiliations

  1. CAS Key Laboratory of Tissue Microenvironment and Tumor, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences (CAS), CAS, Shanghai, People’s Republic of China

    Xiao-Ling Luo, Peng Zhang, Sen-Le Rao & Huang-Tian Yang

  2. CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences (CAS), CAS, Shanghai, People’s Republic of China

    Xiangyuan Liu, Shiqian Huang & Qiurong Ding

  3. Translational Medical Center for Stem Cell Therapy & Institute for Heart Failure and Regenerative Medicine, Shanghai East Hospital, Tongji University School of Medicine and Shanghai Institute of Stem Cell Research and Clinical Translation, Shanghai, 200123, People’s Republic of China

    Huang-Tian Yang

  4. Institute for Stem Cell and Regeneration, CAS, Beijing, 100101, People’s Republic of China

    Qiurong Ding & Huang-Tian Yang

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Contributions

X.L.L, P.Z., and H.T.Y. conceived and designed the research; X.L.L., and P.Z. collected the data; X.L, S.H., and P.Z. constructed the hESC reporter cell line; X.L.L., P.Z., and H.T.Y. analyzed the data; S.L.R. participated in the AP analysis; X.L.L, P.Z., and H.T.Y. interpreted the experimental data and wrote the manuscript; Q.D. supervised the construction of hESC reporter cell line and provided financial support; H.T.Y. approved the manuscript and provided the funding support.

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Correspondence to Huang-Tian Yang.

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This  article is submitted to the special issue on Recent Progresses in hiPSCs for Cardiac Repair, Disease Models, and Drug Discovery in Pflügers Archiv-European Journal of Physiology

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Luo, XL., Zhang, P., Liu, X. et al. Myosin light chain 2 marks differentiating ventricular cardiomyocytes derived from human embryonic stem cells. Pflugers Arch - Eur J Physiol 473, 991–1007 (2021). https://doi.org/10.1007/s00424-021-02578-3

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