Core Research Facilities, The Jikei University School of Medicine, Tokyo, Japan.
Department of Molecular Physiology, The Jikei University School of Medicine, Tokyo, Japan.
J Gen Physiol. 2022 Nov 7;154(11). doi: 10.1085/jgp.202213136. Epub 2022 Oct 6.
Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum (SR) of the skeletal muscle and plays a critical role in excitation-contraction coupling. Mutations in RYR1 cause severe muscle diseases, such as malignant hyperthermia, a disorder of Ca2+-induced Ca2+ release (CICR) through RYR1 from the SR. We recently reported that volatile anesthetics induce malignant hyperthermia (MH)-like episodes through enhanced CICR in heterozygous R2509C-RYR1 mice. However, the characterization of Ca2+ dynamics has yet to be investigated in skeletal muscle cells from homozygous mice because these animals die in utero. In the present study, we generated primary cultured skeletal myocytes from R2509C-RYR1 mice. No differences in cellular morphology were detected between wild type (WT) and mutant myocytes. Spontaneous Ca2+ transients and cellular contractions occurred in WT and heterozygous myocytes, but not in homozygous myocytes. Electron microscopic observation revealed that the sarcomere length was shortened to ∼1.7 µm in homozygous myocytes, as compared to ∼2.2 and ∼2.3 µm in WT and heterozygous myocytes, respectively. Consistently, the resting intracellular Ca2+ concentration was higher in homozygous myocytes than in WT or heterozygous myocytes, which may be coupled with a reduced Ca2+ concentration in the SR. Finally, using infrared laser-based microheating, we found that heterozygous myocytes showed larger heat-induced Ca2+ transients than WT myocytes. Our findings suggest that the R2509C mutation in RYR1 causes dysfunctional Ca2+ dynamics in a mutant-gene dose-dependent manner in the skeletal muscles, in turn provoking MH-like episodes and embryonic lethality in heterozygous and homozygous mice, respectively.
类型 1 兰尼碱受体 (RYR1) 是骨骼肌肌浆网 (SR) 中的 Ca2+释放通道，在兴奋-收缩偶联中起着关键作用。RYR1 突变会导致严重的肌肉疾病，如恶性高热，这是一种由 SR 中的 RYR1 引起的 Ca2+诱导的 Ca2+释放 (CICR) 紊乱。我们最近报道挥发性麻醉剂通过增强杂合 R2509C-RYR1 小鼠的 CICR 引起恶性高热 (MH) 样发作。然而，由于这些动物在子宫内死亡，尚未对同源小鼠的骨骼肌细胞中的 Ca2+动力学特征进行研究。在本研究中，我们从 R2509C-RYR1 小鼠中生成了原代培养的骨骼肌细胞。野生型 (WT) 和突变型肌细胞的细胞形态无差异。WT 和杂合型肌细胞中发生自发 Ca2+瞬变和细胞收缩，但在纯合型肌细胞中未发生。电子显微镜观察显示，与 WT 和杂合型肌细胞分别为 ∼2.2 和 ∼2.3 µm 相比，纯合型肌细胞的肌节长度缩短至 ∼1.7 µm。一致地，纯合型肌细胞的细胞内静息 Ca2+浓度高于 WT 或杂合型肌细胞，这可能与 SR 中的 Ca2+浓度降低有关。最后，使用基于红外激光的微加热，我们发现杂合型肌细胞比 WT 肌细胞表现出更大的热诱导 Ca2+瞬变。我们的研究结果表明，RYR1 中的 R2509C 突变以突变等位基因剂量依赖性方式导致骨骼肌中 Ca2+动力学功能障碍，进而分别在杂合子和纯合子小鼠中引发 MH 样发作和胚胎致死。
