RyR1-mediated moderate endoplasmic reticulum stress is required for myogenic differentiation of myoblasts

Abstract BackgroundCytosolic Ca2+ plays vital roles in myogenesis and muscle development. Key mutations of ryanodine receptor 1 (RyR1), a major Ca2+ release channel of endoplasmic reticulum (ER), are main causes of severe congenital myopathies. The role of RyR1 in myogenic differentiation has attracted intense research interest, however, it remains unclear. MethodsThis study employed RyR1-knockdown myoblasts and CRISPR/Cas9-based RyR1-knockout myoblasts cells to explore the role of RyR1 in myogenic differentiation, myotube formation as well as the potential mechanism of RyR1-related myopathies.ResultsCytoplasmic Ca2+ concentration was significantly elevated during myogenic differentiation of both primary myogenic cells and myoblasts C2C12 cells, accompanied with a dramatic increase in RyR1 expression and resultant ER stress. Inhibition of RyR1 by siRNA-mediated silence or chemical inhibitor, dantrolene, significantly reduced cytosolic Ca2+, alleviated ER stress, and blocked multinucleated myotube formation. Moderate activation of ER stress effectively relieved myogenic differentiation stagnation induced by RyR1 suppression and demonstrated that RyR1 modulates myogenic differentiation via activation of Ca2+ -induced ER stress signaling. RyR1 knockout-induced Ca2+ leakage led to severe ER stress and excessive unfolded protein response, and drove cell fate from differentiation into apoptosis. ConclusionsTherefore, we concluded that dramatic increase in RyR1 expression is required for myogenic differentiation, and RyR1-mediated Ca2+ release leading to the activation of ER stress signaling serves a double-edged sword role during myogenic differentiation. This study contributes to a novel understanding of the role of RyR1 in muscle development and related congenital myopathies, and provides a potential target for regulation of muscle regeneration and tissue engineering.