于亮1, 史霄雨1, 刘子铭2, 王祯1, 李琳1, 高久翔1, 刘晓然3, 王瑞元1,*
1北京体育大学运动人体科学学院，北京 100084；2国家体育总局网球运动管理中心，北京 100010；3首都体育学院运动科学与健康学院，北京 100191
本文旨在探讨不同时长、不同强度运动对大鼠骨骼肌线粒体功能和自噬的影响及FUNDC1的作用。选取60只雄性SD大鼠随机分为2周、4周的安静对照组(Con组)、中等强度运动组(M-ex组，跑台运动16 m/min，1 h/d，6 d/week)和大强度运动组(Hi-ex组，跑台运动35 m/min，20 min/d，6 d/week)，每组10只。干预结束后分离双侧比目鱼肌，石蜡切片制备透射电镜样本，用ELISA检测柠檬酸合成酶(citrate synthase, CS)的含量，用冰冻切片免疫荧光染色法观察微管相关蛋白1轻链3 (microtubule-associated protein 1 light chain 3, LC3)/细胞色素c氧化酶IV亚型(cytochrome c oxidase IV, COX-IV)、FUNDC1/COX-IV及LC3/FUNDC1的共定位情况。提取骨骼肌线粒体，用Western blot检测过氧化物酶体增殖物激活受体γ共激活因子-1α (peroxisome proliferator-activated receptor γ co-activator-1α, PGC-1α)、COX-I、腺苷酸活化蛋白激酶α (AMP-activated protein kinase α, AMPKα)、p-AMPKα、Unc-51样激酶1 (Unc-51 like kinase 1, ULK1)、FUNDC1、LC3、自噬选择性底物(p62)等自噬相关蛋白表达。结果显示，运动可上调线粒体功能，即PGC-1α、COX-I蛋白表达和CS含量，2周Hi-ex组和2周M-ex组之间无差异，而4周Hi-ex组显著低于4周M-ex组。在运动强度相同的情况下，4周运动组大鼠的骨骼肌线粒体自噬激活程度高于2周运动组；在运动时长相同的情况下，Hi-ex组的线粒体自噬激活程度高于M-ex组。2和4周运动干预均可提高LC3/COX-IV、COX-IV/FUNDC1、FUNDC1/LC3共定位。运动可提高LC3-II/LC3-I比值，下调p62蛋白表达水平，上调FUNDC1、ULK1蛋白表达水平和AMPKα磷酸化水平，4周Hi-ex组的这些蛋白表达变化幅度显著大于4周M-ex组。以上结果提示，运动可诱导骨骼肌线粒体自噬，自噬的激活程度与运动时间和强度有关，运动的诱导机制可能是通过AMPK-ULK1通路影响FUNDC1的表达。
分类号：G804.2; Q44; R339.4
Effects of exercises with different durations and intensities on mitochondrial autophagy and FUNDC1 expression in rat skeletal muscles
YU Liang1, SHI Xiao-Yu1, LIU Zi-Ming2, WANG Zhen1, LI Lin1, GAO Jiu-Xiang1, LIU Xiao-Ran3, WANG Rui-Yuan1,*
1College of Human Movement Sciences, Beijing Sports University, Beijing 100084, China；2Tennis Sports Management Center, General Administration of Sport, Beijing 100010, China；3School of Kinesiology and Health, Capital University of Physical Education and Sports, Beijing 100191, China
The aim of the present study was to investigate the effects of exercises with different durations and intensities on mitochondrial autophagy and FUNDC1 in rat skeletal muscles. Sixty male Sprague-Dawley rats were randomly divided into 2- and 4-week control groups (Con), moderate-intensity exercise groups (M-ex groups, treadmill exercise, 16 m/min, 1 h/d, 6 d/week), and high-intensity exercise groups (Hi-ex groups, treadmill exercise, 35 m/min, 20 min/d, 6 d/week). The bilateral soleus muscles were separated after the intervention, and paraffin sections were prepared for transmission electron microscopy. ELISA method was used to detect the content of citrate synthase (CS). The co-localizations of microtubule-associated protein 1 light chain 3 (LC3)/cytochrome c oxidase IV (COX-IV), FUNDC1/COX-IV and LC3/FUNDC1 were observed by immunofluorescent staining in frozen sections. The skeletal muscle mitochondria were extracted, and the expression of autophagy-related proteins, including AMPKα, p-AMPKα, Unc-51 like kinase 1 (ULK1), FUNDC1, LC3 and p62, were detected by Western blot. The results showed that exercise increased mitochondrial function, i.e. peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α), COX-I protein expression levels and CS content. There was no difference of mitochondrial function parameters between 2-week M-ex and 2-week Hi-ex groups, while mitochondrial function of 4-weeks Hi-ex group was significantly lower than that of 4-week M-ex group. Under the same exercise intensity, mitochondrial autophagy activation in skeletal muscle of 4-week exercise was higher than that in 2-week exercise group; Under the same duration of exercise, mitochondrial autophagy activation of Hi-ex group was higher than that in M-ex group. Both 2- and 4-week exercise intervention increased LC3/COX-IV, COX-IV/FUNDC1, and FUNDC1/LC3 co-localizations. Exercise increased LC3-II/LC3-I ratio, down-regulated p62 protein expression level, up-regulated FUNDC1, ULK1 protein expression levels and AMPKα phosphorylation, and the changes of these proteins in 4-week Hi-ex group were significantly greater than those in 4-week M-ex group. These results suggest exercise induces mitochondrial autophagy in skeletal muscles, and the activity of autophagy is related to the duration and intensity of exercise. The induction mechanism of exercise may involve the mediation of FUNDC1 expression through AMPK-ULK1 pathway.
通讯作者：王瑞元 E-mail: email@example.com
于亮, 史霄雨, 刘子铭, 王祯, 李琳, 高久翔, 刘晓然, 王瑞元. 运动时长和强度对大鼠骨骼肌线粒体自噬的影响及其机制[J]. 生理学报 2020; 72 (5): 631-642.
YU Liang, SHI Xiao-Yu, LIU Zi-Ming, WANG Zhen, LI Lin, GAO Jiu-Xiang, LIU Xiao-Ran, WANG Rui-Yuan. Effects of exercises with different durations and intensities on mitochondrial autophagy and FUNDC1 expression in rat skeletal muscles. Acta Physiol Sin 2020; 72 (5): 631-642 (in Chinese with English abstract).