FKBP8在运动诱导骨骼肌线粒体自噬中的调控机制研究

Eccentric exercise can cause skeletal muscle dysfunction and delayed muscle soreness, which is practically significant in prevention and treatment of exercise-induced muscle damage. The previous work found that eccentric exercise caused mitochondrial dysfunction and ultrastructure damage within skeletal muscle and activated the PINK1/Parkin pathway to induce mitophagy, which was regulated by the energy switch AMPK directly during the process. AMPK/mTOR, the key pathway of energy metabolism, plays a pivotal role in initiating exercise adaptation changes. AMPK activates mitophagy by inhibiting mTOR. Mitochondrial outer membrane protein FKBP8, is considered as the endogenous inhibitor of mTOR. It is probable that FKBP8, combined LC3A to mediate autophagy, is important for exercise-induced mitophagy in skeletal muscle. This project is designed for exploring the relationship between FKBP8 and eccentric exercise-induced mitochondrial damage and autophagy in skeletal muscle, and clarifing the interaction between FKBP8 and AMPK/mTOR through animal experiments. By using electrical stimulation as simulation of exercise cell experiment, with the help of activator, inhibitor and RNAi technology to reach interference FKBP8 expression, the project reveals the regulatory mechanism of FKBP8 in exercise-induced skeletal muscle mitophagy, and provides theoretical basis for the prevention and treatment of exercise-induced skeletal muscle damage.

离心运动可致骨骼肌功能下降和延迟性肌肉酸痛，有效防治运动性肌肉损伤的发生发展，具有重要现实意义。前期工作发现，离心运动诱发骨骼肌超微结构和线粒体功能发生紊乱，激活PINK1/Parkin通路诱导线粒体自噬，且能量开关AMPK直接参与调控。能量代谢关键通路AMPK/mTOR在启动运动适应性变化中发挥关键作用，其中AMPK可通过抑制mTOR来激活线粒体自噬。线粒体外膜蛋白FKBP8被视为mTOR的内源性抑制剂，能直接与LC3A结合介导自噬，可能在运动诱导的骨骼肌线粒体自噬中发挥重要作用。故本项目拟通过动物实验探明FKBP8与离心运动诱导骨骼肌线粒体损伤及自噬间的关联，厘清运动干预下FKBP8与AMPK/mTOR的互作关系；通过电刺激模拟运动的细胞实验，借助激活剂、抑制剂及RNAi技术干扰FKBP8表达，揭示其在运动诱导骨骼肌线粒体自噬中的调节机制，为运动性骨骼肌损伤的预防和治疗提供理论依据。

本项目采用蛋白质免疫印迹（Western blot）、免疫荧光双染、酶联免疫吸附法（ELISA）、透射电镜和骨骼肌原位注射腺相关病毒（AAV）等手段研究一次离心运动和4周离心训练对大鼠骨骼肌线粒体结构与功能及自噬的影响。结果发现：（1）4周离心运动可提升大鼠运动能力并促进其代谢适应，但仍诱导骨骼肌过劳性损伤，呈现出线粒体结构和功能受损。运动通过消耗能量而诱导AMPK磷酸化表达升高和mTOR磷酸化降低，使线粒体膜蛋白FKBP8表达及FKBP8与LC3共定位增加，对线粒体进行选择性清除，故4周离心运动诱导骨骼肌线粒体自噬可能是通过AMPK-mTOR途径而上调线粒体FKBP8表达及其与LC3互作，这可能是运动诱导骨骼肌线粒体自噬的重要途径；（2）一次离心运动能引起导致比目鱼肌线粒体超微结构改变，包括线粒体肿胀并在肌膜下大量聚集、出现大量自噬体等，此外线粒体数量减少、功能受损，随时间延长又逐渐恢复，一次离心运动可能通过诱导线粒体自噬以清除受损线粒体，其作用机制与运动激活AMPK/mTOR而上调自噬受体FKBP8表达并促进FKBP8与LC3互作有关；（3）比目鱼肌特异性敲低FKBP8会明显下调运动后线粒体自噬体数量与胞浆CytC浓度并上调线粒体含量，从而提示离心运动诱导骨骼肌线粒体自噬主要是通过自噬受体KFBP8而实现。本项目首次从线粒体外膜蛋白FKBP8角度揭示运动诱导骨骼肌线粒体损伤及自噬的分子机制，为深入研究运动性骨骼肌损伤的线粒体调节机制提供了数据支撑。