ROS-Drp1-mitophagy feedback loop regulates myogenic differentiation via actin cytoskeleton remodeling-mediated MRTF-A/SRF axis

Mitochondrial division is one of the main characteristics for the initiation of myogenic differentiation. However, the role and mechanism of Dynamin-related protein 1 (Drp1), the most important protein that regulates mitochondrial fission in mammals, in regulating myogenic differentiation are not well understood. Drp1 siRNAs were transfected to C2C12 cells, or AAV9-shDrp1 were injected to C57BL/6J mice to knockdown Drp1 expression. Then, mitochondrial damage, ROS level, myogenic differentiation, mitophagy and actin/MRTF-A/SRF pathway was detected by quantitative real-time PCR, western blotting, immunofluorescence staining and flow cytometry. The results showed that Drp1 was upregulated after C2C12 differentiation; Drp1 knockdown by siRNA transfection impaired myotube formation. ROS are the upstream activators for Drp1 expression, and Drp1 inversely reduces ROS by facilitating mitophagy to form a ROS-Drp1-mitophagy feedback loop during myogenic differentiation. Knockdown of Drp1 disrupted the ROS-Drp1-mitophagy feedback loop-mediated ROS homeostasis, thereby accelerating F-action depolymerization and blocking MRTF-A nuclear translocation by reducing the phosphorylation of cofilin. A decrease in MRTF-A nuclear translocation impaired SRF activity and hindered myogenic differentiation. In summary, this study revealed the functional mechanism of Drp1 and clarified the interactions among ROS, Drp1-mediated mitophagy and actin cytoskeleton remodeling during myogenic differentiation.

线粒体分裂是肌源性分化启动的主要特征之一。然而，动力相关蛋白1（Dynamin-related protein 1, Drp1）——哺乳动物体内调控线粒体分裂的关键蛋白——在肌源性分化中的作用与机制尚未完全阐明。本研究通过向C2C12细胞转染Drp1小干扰RNA（siRNA），或向C57BL/6J小鼠注射腺相关病毒9型（AAV9）介导的Drp1短发夹RNA（shDrp1）以敲低Drp1的表达。随后，采用实时荧光定量PCR（quantitative real-time PCR）、蛋白质印迹（western blotting）、免疫荧光染色及流式细胞术，检测线粒体损伤、活性氧（Reactive Oxygen Species, ROS）水平、肌源性分化、线粒体自噬（mitophagy）及肌动蛋白/MRTF-A/SRF信号通路的相关变化。研究结果显示，C2C12细胞诱导分化后，Drp1的表达水平显著上调；通过siRNA转染敲低Drp1会显著损害肌管形成过程。活性氧是Drp1表达的上游激活因子，而Drp1则通过促进线粒体自噬反向清除活性氧，进而在肌源性分化过程中形成ROS-Drp1-线粒体自噬反馈环路。敲低Drp1会破坏该反馈环路所介导的ROS稳态，继而加速纤维状肌动蛋白（F-actin）解聚，并通过降低丝切蛋白（cofilin）的磷酸化水平阻断MRTF-A的核转位。MRTF-A核转位的减少会削弱血清反应因子（Serum Response Factor, SRF）的活性，进而阻碍肌源性分化进程。综上，本研究揭示了Drp1调控肌源性分化的功能机制，并阐明了肌源性分化过程中活性氧、Drp1介导的线粒体自噬与肌动蛋白细胞骨架重塑之间的相互作用关系。