Studies On Molecular Mechanisms Of Maduramicin’s Inhibition Of Akt-mediated Blockage Of Autophagic Flux Contributing To Apoptosis In Skeletal Muscle Cells

The present study,using cellular and molecular biology techniques and methods including MDC and DAPI staining,RNA interference,fluorescent labeling,Western Blotting,etc.,and employing C2C12 and L6 cells as experimental objects,systematically investigated the correlation of cell apoptosis to autophagic flux blockage in skeletal muscle cells induced by maduramicin,and deeply dissected the role and mechanisms of Akt signaling in maduramicin-induced blockage of autophagic flux contributing to apoptosis in skeletal muscle cells.The detailed results were summarized as follows:1 Maduramicin induces autophagosome accumulation and autophagic flux blockade in skeletal muscle cellsC2C12 and L6 cells,or C2C12 and L6 cells infected with Ad-GFP-LC3,were treated with maduramicin(0,0.05,0.1,0.2,0.5 and 1 μM)for 24 h,followed by analyzing MDC fluorescence intensity by MDC staining,expression of LC3-Ⅱ and p62 proteins by Western blotting,imaging and quantifying GFP-LC3 puncta.Besides,C2C12 cells and L6 cells infected with recombinant adenovirus expression in tandem mCherry-GFP-LC3(Ad-mCherry-GFP-LC3)were evaluated by manifestation of autophagic flux in the cells induced by maduramicin(0,0.5 and 1 μM)for 24 h.The results showed that maduramicin evoked the increase of MDC fluorescence intensity and GFP-LC3 puncta in C2C12 and L6 cells.Western blotting exhibited that maduramicin elevated expression of LC3-Ⅱ and p62 proteins in a concentratin manner in the cells.Moreover,maduramicin elicited more co-localization of mCherry and GFP-LC3 in the cells.The data suggest that maduramicin-induces autophagosome accumulation and autophagic flux blockade in skeletal muscle cells.2 Maduramicin blocks autophagy flux contributing to apoptosis in skeletal muscle cells.CQ,an autophagy inhibitor,and rapamycin,an autophagy inducer,were used to analyze the role of maduramicin-induced autophagosome accumulation and autophagic flux blockade in apoptosis of skeletal muscle cells.The results showed that CQ potentiated maduramicin-increased autophagic flux blockade contributing to apoptosis in the cells,whereas rapamycin improved the event,thereby protecting skeletal muscle cells from apoptosis.The findings indicate that maduramicin blocks autophagy flux contributing to apoptosis in skeletal muscle cells.3 Maduramicin inhibits Akt-mediated blockage of autophagic flux contributing to apoptosis in skeletal muscle cellsChange of Akt and GSK3β phosphorylation was detected using Western blotting in maduramicin-exposed skeletal muscle cells.Using Akt inhibitor or Ad-myr-Akt for inhibiting or upgrading Akt activity,The manifestations of GFP-LC3 puncta,cell viability,autophagic flux and apoptosis in the cells were investigated.The results exhibited that maduramicin inhibited phosphorylation of Akt and GSK3β.Pretreatment with Akt inhibitors strengthened maduramicin-induced increase of GFP-LC3 puncta,cell viability reduction and apoptosis elevation,and the expressions of LC3-Ⅱ,p62 and cleaved-caspase-3 were further enhanced.However,expression of constitutively active Akt attenuated the above events.The data suggest that maduramicin inhibits Akt-mediated blockage of autophagic flux contributing to apoptosis in skeletal muscle cells.