Laboratory of Cell Physiology, Institute of Neuroscience, Université Catholique de Louvain, 55/40 av. Hippocrate, 1200 Brussels, Belgium.
J Biol Chem. 2012 Apr 27;287(18):14524-34. doi: 10.1074/jbc.M112.341784. Epub 2012 Mar 6.
We previously showed in vitro that calcium entry through Trpc1 ion channels regulates myoblast migration and differentiation. In the present work, we used primary cell cultures and isolated muscles from Trpc1(-/-) and Trpc1(+/+) murine model to investigate the role of Trpc1 in myoblast differentiation and in muscle regeneration. In these models, we studied regeneration consecutive to cardiotoxin-induced muscle injury and observed a significant hypotrophy and a delayed regeneration in Trpc1(-/-) muscles consisting in smaller fiber size and increased proportion of centrally nucleated fibers. This was accompanied by a decreased expression of myogenic factors such as MyoD, Myf5, and myogenin and of one of their targets, the developmental MHC (MHCd). Consequently, muscle tension was systematically lower in muscles from Trpc1(-/-) mice. Importantly, the PI3K/Akt/mTOR/p70S6K pathway, which plays a crucial role in muscle growth and regeneration, was down-regulated in regenerating Trpc1(-/-) muscles. Indeed, phosphorylation of both Akt and p70S6K proteins was decreased as well as the activation of PI3K, the main upstream regulator of the Akt. This effect was independent of insulin-like growth factor expression. Akt phosphorylation also was reduced in Trpc1(-/-) primary myoblasts and in control myoblasts differentiated in the absence of extracellular Ca(2+) or pretreated with EGTA-AM or wortmannin, suggesting that the entry of Ca(2+) through Trpc1 channels enhanced the activity of PI3K. Our results emphasize the involvement of Trpc1 channels in skeletal muscle development in vitro and in vivo, and identify a Ca(2+)-dependent activation of the PI3K/Akt/mTOR/p70S6K pathway during myoblast differentiation and muscle regeneration.
我们之前在体外实验中证明，通过 Trpc1 离子通道的钙离子内流调节成肌细胞的迁移和分化。在本工作中，我们使用原代细胞培养物和 Trpc1(-/-)和 Trpc1(+/+) 小鼠模型的分离肌肉，研究 Trpc1 在成肌细胞分化和肌肉再生中的作用。在这些模型中，我们研究了心脏毒素诱导的肌肉损伤后的再生，观察到 Trpc1(-/-)肌肉发生显著的萎缩和延迟再生，表现为纤维尺寸减小和中央核纤维比例增加。这伴随着成肌因子如 MyoD、Myf5 和 myogenin 的表达减少，以及它们的一个靶标，即发育性 MHC（MHCd）的表达减少。因此，Trpc1(-/-)小鼠肌肉的肌肉张力普遍较低。重要的是，在再生的 Trpc1(-/-)肌肉中，PI3K/Akt/mTOR/p70S6K 通路的表达下调，该通路在肌肉生长和再生中发挥关键作用。事实上，Akt 和 p70S6K 蛋白的磷酸化以及 PI3K 的激活都减少了，PI3K 是 Akt 的主要上游调节剂。这种效应与胰岛素样生长因子的表达无关。Akt 磷酸化也在 Trpc1(-/-)原代成肌细胞和在没有细胞外 Ca(2+)或用 EGTA-AM 或 wortmannin 预处理的情况下分化的对照成肌细胞中减少，表明 Ca(2+)通过 Trpc1 通道的内流增强了 PI3K 的活性。我们的结果强调了 Trpc1 通道在体外和体内骨骼肌肉发育中的作用，并确定了 Ca(2+)依赖性激活在成肌细胞分化和肌肉再生过程中的 PI3K/Akt/mTOR/p70S6K 通路。
