Downhill Running Blocks the Mitophagic Flux and Autophagosome-Lysosome Fusion in Rat Soleus Muscle.

Abstract

This study aimed to investigate the effect of downhill running on mitophagic flux and autophagosome-lysosome fusion in rat soleus muscle. Sprague-Dawley rats were trained on a treadmill at a speed of 16 m·min -1 and a decline of -16° for 90 min, and the soleus muscle was sampled at 0 h, 12 h, 24 h, 48 h, and 72 h after exercise. Mitochondrial ultrastructural changes were observed by using a transmission electron microscope. Protein levels of Cathepsin D, Vacuolar H + -ATPase (V-ATPase), mitochondrial respiratory complex Ⅰ (NDUFB8), complex Ⅲ (UQCRC2), and microtubule-associated protein 1 light chain 3 (LC3) were determined by Western blot. Mitochondria co-localizations with LC3 and lysosomal-associated membrane protein 2 (LAMP2), syntaxin 17 (STX17) co-localizations with LC3, LAMP2, SNAP29, and vesicle-associated membrane protein 8 (VAMP8), as well as the SNAP29 co-localizations with VAMP8, were measured by immunofluorescence. To assess mitophagic flux in vivo , colchicine or saline was injected intraperitoneally 3 d before exercise, and the protein expression of mitochondrial LC3-Ⅱ was detected by Western blot. After downhill running, mitochondrial structure appeared to be abnormal and contained autophagosomes and autophagolysosomes. The expression levels of Cathepsin D, Vacuolar H + -ATPase, and mitochondrial LC3, as well as the co-localizations of STX17 with LC3 and SNAP29 were significantly higher, whereas the expression levels of mitochondrial NDUFB8, UQCRC2, and LAMP2, along with the co-localizations of STX17 with LAMP2 and VAMP8 were significantly lower than those in the control group. Specifically, mitochondrial LC3-Ⅱ flux was significantly lower following downhill running. A bout of downhill running may block mitophagic flux by impairing mitophagosome-lysosome fusion, which is accompanied by increased recruitment of STX17 and SNAP29 to autophagosome and reduced co-localizations of STX17 and SNAP29 with lysosomal VAMP8.