Department of Kinesiology, Brock University, St. Catharines, Ontario, Canada.
Centre for Bone and Muscle Health, Brock University, St. Catharines, Ontario, Canada.
Am J Physiol Cell Physiol. 2019 Nov 1;317(5):C1025-C1033. doi: 10.1152/ajpcell.00345.2018. Epub 2019 Aug 21.
Calcineurin is a Ca/calmodulin (CaM)-dependent phosphatase that plays a critical role in promoting the slow fiber phenotype and myoblast fusion in skeletal muscle, thereby making calcineurin an attractive cellular target for enhancing fatigue resistance, muscle metabolism, and muscle repair. Neurogranin (Ng) is a CaM-binding protein thought to be expressed solely in brain and neurons, where it inhibits calcineurin signaling by sequestering CaM, thus lowering its cellular availability. Here, we demonstrate for the first time the expression of Ng protein and in mammalian skeletal muscle. Both protein and levels are greater in slow-oxidative compared with fast-glycolytic muscles. Coimmunoprecipitation of CaM with Ng in homogenates of C2C12 myotubes, mouse soleus, and human vastus lateralis suggests that these proteins physically interact. To determine whether Ng inhibits calcineurin signaling in muscle, we used Ng siRNA with C2C12 myotubes to reduce Ng protein levels by 60%. As a result of reduced Ng expression, C2C12 myotubes had enhanced CaM-calcineurin binding and calcineurin signaling as indicated by reduced phosphorylation of nuclear factor of activated T cells and increased utrophin . In addition, calcineurin signaling affects the expression of myogenin and stabilin-2, which are involved in myogenic differentiation and myoblast fusion, respectively. Here, we found that both myogenin and stabilin-2 were significantly elevated by Ng siRNA in C2C12 cells, concomitantly with an increased fusion index. Taken together, these results demonstrate the expression of Ng in mammalian skeletal muscle where it appears to be a novel regulator of calcineurin signaling.
钙调神经磷酸酶是一种 Ca/calmodulin（CaM）依赖性磷酸酶，在促进骨骼肌中慢肌纤维表型和成肌细胞融合方面发挥着关键作用，因此使钙调神经磷酸酶成为增强抗疲劳能力、肌肉代谢和肌肉修复的有吸引力的细胞靶标。神经颗粒蛋白（Ng）是一种 CaM 结合蛋白，据认为仅在大脑和神经元中表达，在那里它通过隔离 CaM 来抑制钙调神经磷酸酶信号，从而降低其细胞可用性。在这里，我们首次证明了 Ng 蛋白和 在哺乳动物骨骼肌中的表达。与快速糖酵解肌肉相比，慢氧化肌肉中的蛋白和 水平更高。C2C12 肌管、小鼠比目鱼肌和人股外侧肌匀浆中 CaM 与 Ng 的共免疫沉淀表明这些蛋白质物理相互作用。为了确定 Ng 是否抑制肌肉中的钙调神经磷酸酶信号，我们使用 Ng siRNA 降低 C2C12 肌管中的 Ng 蛋白水平 60%。由于 Ng 表达减少，C2C12 肌管中 CaM-钙调神经磷酸酶结合和钙调神经磷酸酶信号增强，核因子活化 T 细胞的磷酸化减少，utrophin 增加。此外，钙调神经磷酸酶信号会影响肌生成素和稳定素-2 的表达，它们分别参与肌生成分化和成肌细胞融合。在这里，我们发现 Ng siRNA 在 C2C12 细胞中显著上调了肌生成素和稳定素-2 的表达，同时融合指数增加。总之，这些结果表明 Ng 在哺乳动物骨骼肌中表达，它似乎是钙调神经磷酸酶信号的一种新型调节剂。
