Center for Genetic Medicine Research, Children's National Research Institute, Children's National Hospital, Washington, D.C., USA.
School of Exercise and Rehabilitation Sciences, The University of Toledo, Toledo, OH, USA.
Skelet Muscle. 2020 Dec 11;10(1):37. doi: 10.1186/s13395-020-00254-1.
Nonsense or loss-of-function mutations in the non-lysosomal cysteine protease calpain-3 result in limb-girdle muscular dystrophy type 2A (LGMD2A). While calpain-3 is implicated in muscle cell differentiation, sarcomere formation, and muscle cytoskeletal remodeling, the physiological basis for LGMD2A has remained elusive.
Cell growth, gene expression profiling, and mitochondrial content and function were analyzed using muscle and muscle cell cultures established from healthy and calpain-3-deficient mice. Calpain-3-deficient mice were also treated with PPAR-delta agonist (GW501516) to assess mitochondrial function and membrane repair. The unpaired t test was used to assess the significance of the differences observed between the two groups or treatments. ANOVAs were used to assess significance over time.
We find that calpain-3 deficiency causes mitochondrial dysfunction in the muscles and myoblasts. Calpain-3-deficient myoblasts showed increased proliferation, and their gene expression profile showed aberrant mitochondrial biogenesis. Myotube gene expression analysis further revealed altered lipid metabolism in calpain-3-deficient muscle. Mitochondrial defects were validated in vitro and in vivo. We used GW501516 to improve mitochondrial biogenesis in vivo in 7-month-old calpain-3-deficient mice. This treatment improved satellite cell activity as indicated by increased MyoD and Pax7 mRNA expression. It also decreased muscle fatigability and reduced serum creatine kinase levels. The decreased mitochondrial function also impaired sarcolemmal repair in the calpain-3-deficient skeletal muscle. Improving mitochondrial activity by acute pyruvate treatment improved sarcolemmal repair.
Our results provide evidence that calpain-3 deficiency in the skeletal muscle is associated with poor mitochondrial biogenesis and function resulting in poor sarcolemmal repair. Addressing this deficit by drugs that improve mitochondrial activity offers new therapeutic avenues for LGMD2A.
无义突变或功能丧失突变导致非溶酶体半胱氨酸蛋白酶钙蛋白酶-3 会引起肢带型肌营养不良 2A 型（LGMD2A）。虽然钙蛋白酶-3 与肌肉细胞分化、肌节形成和肌肉细胞骨架重塑有关，但 LGMD2A 的生理基础仍然难以捉摸。
利用从健康和钙蛋白酶-3 缺陷小鼠中建立的肌肉和肌母细胞培养物分析细胞生长、基因表达谱以及线粒体含量和功能。还使用过氧化物酶体增殖物激活受体-δ 激动剂（GW501516）治疗钙蛋白酶-3 缺陷型小鼠，以评估线粒体功能和膜修复。使用配对 t 检验评估两组或两种处理之间观察到的差异的显著性。使用方差分析评估随时间的显著性。
我们发现钙蛋白酶-3 缺乏会导致肌肉和肌母细胞中线粒体功能障碍。钙蛋白酶-3 缺陷型肌母细胞增殖增加，其基因表达谱显示出异常的线粒体生物发生。肌管基因表达分析进一步揭示了钙蛋白酶-3 缺陷型肌肉中脂质代谢的改变。体外和体内验证了线粒体缺陷。我们使用 GW501516 在 7 个月大的钙蛋白酶-3 缺陷型小鼠体内改善线粒体生物发生。这种治疗方法增加了 MyoD 和 Pax7 mRNA 表达，从而改善了卫星细胞的活性。它还降低了肌肉疲劳性并降低了血清肌酸激酶水平。减少的线粒体功能也损害了钙蛋白酶-3 缺陷型骨骼肌的肌膜修复。通过急性丙酮酸处理改善线粒体活性可改善肌膜修复。
我们的结果提供了证据，证明骨骼肌中的钙蛋白酶-3 缺乏与不良的线粒体生物发生和功能有关，导致肌膜修复不良。通过改善线粒体活性的药物来解决这一缺陷为 LGMD2A 提供了新的治疗途径。
