Cytosolic Ca2+ plays vital roles in myogenesis and muscle development. As a major Ca2+ release channel of endoplasmic reticulum (ER), ryanodine receptor 1 (RyR1) key mutations are main causes of severe congenital myopathies. The role of RyR1 in myogenic differentiation has attracted intense research interest but remains unclear. In the present study, both RyR1-knockdown myoblasts and CRISPR/Cas9-based RyR1-knockout myoblasts were employed to explore the role of RyR1 in myogenic differentiation, myotube formation as well as the potential mechanism of RyR1-related myopathies. We observed that RyR1 expression was dramatically increased during the late stage of myogenic differentiation, accompanied by significantly elevated cytoplasmic Ca2+ concentration. Inhibition of RyR1 by siRNA-mediated knockdown or chemical inhibitor, dantrolene, significantly reduced cytosolic Ca2+ and blocked multinucleated myotube formation. The elevation of cytoplasmic Ca2+ concentration can effectively relieve myogenic differentiation stagnation by RyR1 inhibition, demonstrating that RyR1 modulates myogenic differentiation via regulation of Ca2+ release channel. However, RyR1-knockout-induced Ca2+ leakage led to the severe ER stress and excessive unfolded protein response, and drove myoblasts into apoptosis. Therefore, we concluded that Ca2+ release mediated by dramatic increase in RyR1 expression is required for the late stage of myogenic differentiation and fusion. This study contributes to a novel understanding of the role of RyR1 in myogenic differentiation and related congenital myopathies, and provides a potential target for regulation of muscle characteristics and meat quality.

中文翻译：

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Ryanodine 受体 RyR1 介导的 Ca2+ 浓度升高是肌原性分化和融合晚期所必需的

细胞溶质 Ca2+ 在肌生成和肌肉发育中起着至关重要的作用。作为内质网 (ER) 的主要 Ca2+ 释放通道，兰尼碱受体 1 (RyR1) 关键突变是严重先天性肌病的主要原因。RyR1 在肌原性分化中的作用引起了强烈的研究兴趣，但仍不清楚。在本研究中，RyR1 敲低成肌细胞和基于 CRISPR/Cas9 的 RyR1 敲除成肌细胞均被用于探索 RyR1 在肌原性分化、肌管形成中的作用以及 RyR1 相关肌病的潜在机制。我们观察到 RyR1 表达在成肌分化后期显着增加，伴随着细胞质 Ca2+ 浓度的显着升高。通过 siRNA 介导的敲低或化学抑制剂丹曲林抑制 RyR1，显着降低细胞溶质 Ca2+ 并阻断多核肌管形成。细胞质 Ca2+ 浓度的升高可有效缓解 RyR1 抑制导致的成肌分化停滞，表明 RyR1 通过调节 Ca2+ 释放通道来调节成肌分化。然而，RyR1 敲除诱导的 Ca2+ 渗漏导致严重的 ER 应激和过度的未折叠蛋白反应，并驱使成肌细胞凋亡。因此，我们得出结论，由 RyR1 表达显着增加介导的 Ca2+ 释放是成肌分化和融合晚期所必需的。本研究有助于对 RyR1 在肌源性分化和相关先天性肌病中的作用有新的认识，并为调节肌肉特征和肉质提供潜在的靶点。