Effect Of Downhill Running On Mitophagic Flux In Rat Skeletal Muscle

PURPOSE: To probe the effect of downhill treadmill running on mitophagic flux and lysosome function in rat skeletal muscle. METHODS: Male Sprague-Dawley rats were subjected to an exercise protocol of a 90-min downhill run with a slope of -16° and a speed of 16 m/min, and then the soleus muscle was sampled at 0, 12, 24, 48, and 72 h after exercise. Mitochondrial ultrastructural changes in skeletal muscle were observed by using a transmission electron microscope. Protein expression levels of skeletal muscle Cathepsin D (CTSD) and Vacuolar H+-ATPase (V-ATPase) were determined by Western blot. Mitochondria co-localizations with sequestosome 1 (p62), microtubule-associated protein 1 light chain 3 (LC3) and lysosome-associated membrane protein-2 (LAMP2) were measured by the immunofluorescence technique. To assess mitophagic flux in vivo, colchicines or saline was injected intraperitoneally 3d before the exercise experiment, and the protein level of mitochondrial LC3-II was measured by Western blot. One-way and two-way ANOVAs were used for data analysis. RESULTS: After downhill treadmill running, skeletal muscle mitochondrial structure appeared to be abnormal and contained a large number of mitophagosomes and a few mitophagolysosomes; the protein expression levels of CTSD (E24 = 1.90 ± 0.56) and V-ATPase (E12 = 2.06 ± 0.54) and the co-localizations of mitochondria with p62 (E12 = 2.27 ± 0.28) and LC3 (E12 = 2.97 ± 0.41) were significantly higher, whereas the co-localization of mitochondria with LAMP2 (E0 = 0.97 ± 0.01) was significantly lower at 0 h and changed to be significantly higher at 48 h (E48 = 1.02 ± 0.01) than those in the control group (p < 0.05 to p < 0.01). Importantly, mitochondrial LC3-II flux (E12 = 0.11 ± 0.41) was significantly lower after downhill running compared with that in the control group (p < 0.05). CONCLUSIONS: A bout of downhill treadmill running inhibited mitophagic flux without impairing lysosome function, which may be related to the blocking fusion between mitophagosomes and lysosomes. Natural Science Foundation of China grant 31900842