Yu Liang, Shi Xiao-Yu, Liu Zi-Ming, Wang Zhen, Li Lin, Gao Jiu-Xiang, Liu Xiao-Ran, Wang Rui-Yuan
College of Human Movement Sciences, Beijing Sports University, Beijing 100084, China.
Tennis Sports Management Center, General Administration of Sport, Beijing 100010, China.
Sheng Li Xue Bao. 2020 Oct 25;72(5):631-642.
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.
本研究旨在探讨不同持续时间和强度的运动对大鼠骨骼肌线粒体自噬及FUNDC1的影响。将60只雄性Sprague-Dawley大鼠随机分为2周和4周的对照组（Con）、中等强度运动组（M-ex组，跑步机运动，16米/分钟，1小时/天，6天/周）和高强度运动组（Hi-ex组，跑步机运动，35米/分钟，20分钟/天，6天/周）。干预后分离双侧比目鱼肌，制备石蜡切片用于透射电子显微镜检查。采用ELISA法检测柠檬酸合酶（CS）含量。通过冰冻切片免疫荧光染色观察微管相关蛋白1轻链3（LC3）/细胞色素c氧化酶IV（COX-IV）、FUNDC1/COX-IV和LC3/FUNDC1的共定位。提取骨骼肌线粒体，采用蛋白质免疫印迹法检测自噬相关蛋白包括AMPKα、p-AMPKα、Unc-51样激酶1（ULK1）、FUNDC1、LC3和p62的表达。结果显示，运动增加了线粒体功能，即过氧化物酶体增殖物激活受体γ共激活因子-1α（PGC-1α）、COX-I蛋白表达水平和CS含量。2周M-ex组和2周Hi-ex组之间线粒体功能参数无差异，而4周Hi-ex组的线粒体功能显著低于4周M-ex组。在相同运动强度下，4周运动组骨骼肌中的线粒体自噬激活高于2周运动组；在相同运动持续时间下，Hi-ex组的线粒体自噬激活高于M-ex组。2周和4周的运动干预均增加了LC3/COX-IV、COX-IV/FUNDC1和FUNDC1/LC3的共定位。运动增加了LC3-II/LC3-I比值，下调了p62蛋白表达水平，上调了FUNDC1、ULK1蛋白表达水平和AMPKα磷酸化，且4周Hi-ex组中这些蛋白的变化显著大于4周M-ex组。这些结果表明运动可诱导骨骼肌中的线粒体自噬，且自噬活性与运动的持续时间和强度有关。运动的诱导机制可能涉及通过AMPK-ULK1途径介导FUNDC1表达。
