The Establishment And Application Of A Skeletal Muscle Specific Overexpression Model Of PGC-1?

Objectives: We proposed to establish a model of PGC-1? in FVB/N mice skeletal muscle with using the technique of injection and electroporation.In this research.Methods: 1.Cell model:C2C12 cell were transfected,and the and overexpressed using by Flag-PGC-1?,after 48 hours,the expression of PGC-1? protein was detected to determine whether the plasmid work.2.Animal model: Male 6-8 months old FVB/N mice were randomly divided into3 groups: injection 5 days group(5D),injection 10 days group(10D),injection 15 days group(15D),10 of mices in each group were tested for PGC-1? protein expression to determine the best detection time after plasmid injected of skeletal muscle.3.Specific PGC-1? overexpression Skeletal muscle model establishment: 6-8months' FVB/N mices,were randomly divided into 3 groups: Negative Control(NC),GFP and Flag-PGC-1? plasmid groups.each group of 10.Flag-PGC-1? plasmid group was injected with Flag-PGC-1? plasmid(2 ?g/?l,40?l)and electroporation to promote transfection.GFP group was injected with GFP plasmid(2 ?g/?l,40?l)in the TA muscle and promoted by electroporation.After transfection,NC group was injected with pUC DNA 3.1 empty plasmid(2 ?g/?l,40?l)and electroporation to promote transfection.Ten days after injection,the experimental animals were sacrificed,and the TA muscle and the myocardium were extracted for detection.4.Identification of skeletal muscle-specific PGC-1? overexpression model: 1)Histological examination(HE staining): The TA muscle of the normal group(Control group),NC group,and Flag-PGC-1? group were respectively performed.HE staining,pathological morphology and analysis of skeletal muscle fibers were performed.2)Western blot analysis was used to detect the expression of related proteins in the TA muscle and myocardium: PGC-1?,GFP,mtTFA,NF-?B,MnSOD,FNDC5.Results:1.Flag-PGC-1? plasmids were transfected into C2C12 myoblasts after48 h.Western blot showed that the overall expression level of PGC-1? protein wassignificantly higher than the control group,indicating that the efficiency of Flag-PGC-1? plasmid transfection was worked.2.Compared the expression of PGC-1? protein in the TA muscles of 5D,10 D and 15 D mice,the expression of PGC-1? protein in 10 D group was the highest,indicating that 10 days after the injection of Flag-PGC-1? plasmid was the best detection time.3.Analysis of GFP protein expression in NC and GFP group showed that the GFP protein was successfully expressed in the GFP group but not in the NC group.4.HE staining results showed that necrosis occurred in the NC group compared with the C group's TA muscle fibers,whereas the muscle fibers in the Flag-PGC-1?injection group tend to heal with respected to the NC group.The protein level of the TA muscle in the experimental mice was detected.The expression level of mtTFA in the Flag-PGC-1? was significantly higher than that of the NC group;the expression level of the FDNC5 was significantly higher in the Flag-PGC-1? group compared with the NC group;There was no significant difference in the expression levels of NF-?B and MnSOD protein among each groups.6.The protein levels of the myocardium of the experimental mice were detected.Compared with the NC group,the expression of FNDC5 and mtTFA in the Flag-PGC-1? was significantly increased.Conclusions:1.The model of PGC-1? overexpressed in skeletal muscle of mice was successfully established.The expression of PGC-1? in skeletal muscle was increased,and the downstream mitochondrial biosynthesis and muscle factor related protein expression were increased.2.Skeletal muscle-specific overexpression of PGC-1? causes increased expression of myocardial mitochondrial biosynthesis-related proteins,suggesting the existence of skeletal muscle-myocardial interactions,and the specific mechanism needs further research.