The Mechanism Of Mitochondrial Derived ROS Modulates Histone Methylation Modification Of Mitochondrial Homeostasis Related Genes In C2C12 Lines

Objective:Mitochondria are the main site where cells produce ATP by oxidative phosphorylation,the health of mitochondria is critical to the survival of the whole cell.Mitochondrial quality control system is an important mechanism to maintain mitochondrial homeostasis,which primarily includes mitochondrial biosynthesis,mitophagy,UPR^mt and mitochondrial fusion and fission.Research shows mitochondria regulate mitochondrial function and maintain mitochondrial homeostasis by releasing ROS.High levels of ROS cause oxidative stress in mitochondria,while low levels of ROS play a role in signaling molecules in cells.ROS also changes the epigenetic modification of related genes by affecting a variety of epigenetic modifying enzymes,thus affecting gene expression.It is speculated that mitochondria may regulate epigenetic modification enzymes through ROS to change the epigenetic modification,thus maintaining mitochondrial homeostasis.In this study,C2C12 cells were incubated with the mitochondrial complex I inhibitor rotenone to establish mitochondrial low-level ROS induction model,while with the mitochondria-targeted antioxidant mito Q to establish mitochondrial ROS clearance model.1)To observe the effects of ROS simulated within"physiological"levels on the expression of AMPK and epigenetic modification enzymes.2)To observe the effects of simulated"physiological"level of ROS on mitochondrial homeostasis by regulating the epigenetic modification of mitochondrial quality control related genes.3)To explore the role of histone methylation modification in the regulation of mitochondrial homeostasis.Methods:C2C12 cells were divided into control group(DMSO),rotenone group(ROT),mito Q group(mito Q)and rotenone+mito Q(R+M).Rotenone concentration was 20n M,mito Q concentration was 60n M,and the intervention time was 48h.The mitochondrial ROS level were stained by DCF.The mitochondrial membrane potential was stained by JC-1.The MDA were stained by colorimetric.The expression of p-AMPK(Thr 172)?AMPK?JMJD3?EZH2?JARID1B?MLL?PGC-1??PINK1?BNIP3?LC3II/??ATF5?DRP1?MFN2 and OPA1 protein were measured with western-blot.The histone methylation modifications,H3K27me3 and H3K4me3,at the promoters of PGC-1?,PINK1,BNIP3,ATF5,DRP1,MFN2 and OPA1 were measured with Chromatin Immunoprecipitation(Ch IP).Result:(1)Rotenone and mito Q regulate ROS,mitochondrial membrane potential and MDA levelsCompared with the DMSO group,ROS and MDA content were significantly increased(p<0.05),and mitochondrial membrane potential level was significantly decreased(p<0.01)in the ROT group.ROS content was significantly decreased in the mito Q group(p<0.05).Compared with the ROT group,ROS and MDA content was significantly decreased(p<0.05)and mitochondrial membrane potential level was significantly increased(p<0.01)in the R+M group.(2)Rotenone and mito Q regulate the expression of AMPK and epigenetic modification enzymesCompared with the DMSO group,the expression of p-AMPK protein was significantly increased(p<0.05)in the ROT group.Compared with the ROT group,the expression of p-AMPK protein was decreased in the R+M group,but there was no significant difference.Compared with the DMSO group,the expression of JMJD3protein was significantly increased(p<0.05),while MLL and JARID1B were significantly decreased(p<0.05)in the ROT group.The expression of JMJD3 and EZH2 protein were significantly increased in the mito Q group(p<0.05).Compared with ROT group,the expression of JMJD3,EZH2 and MLL protein were significantly increased(p<0.05)in R+M group.(3)Rotenone and mito Q regulate the expression of mitochondrial homeostasis related proteinsCompared with the DMSO group,the expression of PGC-1?,BNIP3,PINK1,ATF5,MFN2,OPA1 and DRP1 protein were significantly increased(p<0.05)in the ROT group.The expression of BNIP3 and LC3II/?protein were significantly decreased(p<0.05),the expression of ATF5 protein was significantly increased(p<0.01)in the mito Q group.Compared with the ROT group,the expression of PGC-1?,BNIP3,LC3?/?,MFN2 and OPA1 protein were significantly decreased(p<0.05)in the R+M group.(4)Rotenone and mito Q regulate H3K27me3 modification of mitochondrial homeostasis related genesCompared with the DMSO group,the PINK1 and DRP1 promoter H3K27me3modification were significantly decreased(p<0.05),and the OPA1 promoter H3K27me3 modification was significantly increased(p<0.01)in the ROT group;The PGC-1?,BNIP3,PINK1,ATF5 and DRP1 promoter H3K27me3 modification were significantly decreased(p<0.05)in the mito Q group.Compared with the ROT group,the BNIP3 promoter H3K27me3 modification was significantly increased(p<0.05),and the ATF5 promoter H3K27me3 modification was significantly decreased(p<0.05)in the R+M group.(5)Rotenone and mito Q regulate H3K27me3 modification of mitochondrial homeostasis related genesCompared with the DMSO group,the PGC-1?,MFN2 and OPA1 promoter H3K4me3 modification were significantly increased(p<0.05)in the ROT group;The ATF5 promoter H3K4me3 modification was significantly increased(p<0.01)in the mito Q group.Compared with the ROT group,the ATF5 promoter H3K4me3modification was significantly increased(p<0.05),and the MFN2 promoter H3K4me3modification was significantly decreased(p<0.05)in the R+M group.Conclusion:1.Our study indicate that low-level ROS induced by rotenone can activate AMPK(p-AMPK),and then up-regulate the demethylase JMJD3 protein,which in turn decrease PINK1 and DRP1 promoter H3K27me3(transcriptional repression effect),and than enhance mitophagy and mito-fission.p-AMPK can also increase down-regulate the demethylase JARID1B and MLL proteins,which in turn increase PGC-1??MFN2 and OPA1 promoter H3K4me3(transcriptional activation effect),and than enhance mitochondrial biogenesis and mito-fusion.2.Furtherly,ROS simulated within"physiological"levels can regulate histone methylation epigenetic modifications through activation of AMPK,which in turn affects the expression of mitochondrial homeostasis-related protein.