Mechanism Of FUNDC1 In Exercise-induced Skeletal Muscle Mitophagy

Objective: An EPS model was established to observe the effects of it on mitochondrial structure,function and autophagy in C2C12 cells.Western blotting and immunofluorescence techniques were used to investigate the mechanical of electric pulse stimulation(EPS)on mitophagy in skeletal muscle C2C12 cells and the role of FUNDC1.Methods: C2C12 mouse skeletal muscle myoblasts were resuscitated and subcultured for 2-3 passages.The cells were divided into control group(C),DMSO group(D),AICAR group(A),and Compound C group(Co).FK506 group(F),electric pulse stimulation group(E),electrical pulse stimulation+Compound C group(E+Co),electrical pulse stimulation+FK506 group(E+F),collected and tested after cell intervention.The content of mitochondrial quantitative enzyme CS,the biochain complex COX-I protein,and the biosynthetic marker PGC-1? protein were detected,analyzing the changes in the number and function of mitochondria.The expressions of AMPK?,p-AMPK?(Thr172),ULK1 and p-ULK1(Ser555)were detected by Western blot,and the changes of energy metabolism index were analyzed.The colocalization of LC3/COXIV,LC3/FUNDC and FUNDC1/COXIV was observed by immunofluorescence;The expression of mitophagy-related proteins such as LC3,p62,FUNDC1 and PGAM5 was detected by Western blot to verify the role of FUNDC1 in mitophagy.The experimental data were analyzed by one-way ANOVA.Results:(1)15V,1Hz,2ms,1h electrical pulse stimulation did not cause an increase in mitochondrial quantitative enzyme CS content in C2C12 cells(p>0.05);It caused a significant increase in the expression of mitochondrial biosynthesis protein PGC-1? and respiratory chain complex COX-I(p<0.05);it caused a significant increase in autophagy-related protein LC3 expression and a significant decrease in p62 protein expression(p<0.05).(2)FK506 at 6uM did not decrease FUNDC1 protein expression at 24h(p>0.05),decreased FUNDC1 protein expression at 48 h and 72h(p<0.01),and had higher cell activity at 48 h.(3)Inhibition of FUNDC1 protein by using FK506 can decrease its phosphatasePGAM5(p<0.01),LC3 protein expression decreased,p62 protein expression increased,and immunofluorescence double staining FUNDC1/COXIV and LC3/FUNDC1 co-localization decreased;The expression of FUNDC1 and LC3 protein in the EPS group was significantly increased,and the expression of p62 protein was significantly decreased(p<0.05).Immunofluorescence double staining LC3/COXIV,FUNDC1/COXIV,LC3/FUNDC1 co-localization significantly increased(p<0.01);FK506 intervention after EPS can significantly increase the expression of FUNDC1 and LC3 protein,and the expression of p62 protein was significantly decreased(p<0.05).Immunofluorescence double staining LC3/COXIV,FUNDC1/COXIV,LC3/FUNDC1 co-localization significantly increased(p<0.01).However,protein levels and immunofluorescence co-localization levels were reduced compared to the EPS group.(4)AMPK activator AICAR can significantly increase the expression of PGC-1? and COX-I protein(p<0.05).AMPK?,p-AMPK?(Thr172),p-ULK1(Ser555),LC3,p62 protein expression increased,immunofluorescence co-localization LC3/COXIV decreased slightly(p>0.05);EPS can significantly increase the expression of PGC-1?,COX-I,AMPK?,p-AMPK?(Thr172),ULK1,p-ULK1(Ser555),FUNDC1,and LC3,and the expression of p62 protein was significantly decreased(p<0.05).The immunofluorescence LC3/COXIV,FUNDC1/COXIV,LC3/FUNDC1 co-localization levels were significantly increased(p<0.01);Compound C intervention after EPS can significantly increase the expression of AMPK?,p-AMPK?(Thr172),ULK1,p-ULK1(Ser555),FUNDC1 and LC3 in the and the expression of COX-I and p62 protein was significantly decreased(p<0.05).The expression of PGC-1? protein was slightly increased,and the co-localization levels of immunofluorescence LC3/COXIV,FUNDC1/COXIV,and LC3/FUNDC1 were significantly increased(p<0.05).Conclusion:(1)EPS can simulate exercise-induced mitophagy in C2C12 cells.(2)FK506 can effectively reduce the expression of FUNDC1 protein.(3)EPS induces the occurrence of mitophagy by activating the AMPK-ULK1 pathway to simulate exercise.(4)FUNDC1 participates in EPS induced mitophagy via the AMPK-ULK1 pathway.