Study On The Mechanism Of Coupling Of Mitochondria-Myosin Heavy Chain In Myogenic Differentiation Regulated By JMJD3

Objective: Skeletal muscle is a highly plasticity heterogeneous tissue.According to its contractile and metabolic characteristics,skeletal muscle can be divided into slow muscle fibers with mitochondrial aerobic oxidation and fast muscle fibers with glycolytic metabolism,and two types of muscle fibers can conversion.In addition,the muscle fiber type and mitochondrial function of skeletal muscle are coupled with each other,and the increased expression of genes related to slow muscle fibers can simultaneously promote mitochondrial biosynthesis and improve the oxidative phosphorylation ability of mitochondria,so that the oxidative phosphorylation metabolism of mitochondria is more compatible with slow muscle fibers.Epigenetic modification of H3K27me3 is involved in the regulation of muscle fiber types.The epigenetic enzyme JMJD3 is the specific demethylase of H3K27me3,but its mechanism in the regulation of the transformation of muscle fiber types remains unclear.In this study,C2C12 cells were used to investigate the mitochondrial biosynthesis,mitochondrial unfolded protein response(UPRmt)and epigenetic modifications of two types of muscle fiber related genes and the expression of related proteins through siRNA interference and overexpression of JMJD3 by plasmid transfection.To investigate the mitochondrial mechanism of the epigenase JMJD3 in regulating the transformation of muscle fiber type.Methods: In this study,C2C12 cells were selected as experimental subjects,which were divided into control group(MOCK),JMJD3 siRNA group(JMJD3-siRNA),empty vector group(pc DNA3.1(+)-3x FLAG)and JMJD3 overexpression group(pc DNA3.1(+)-Kdm6b-3x FLAG).The transfection efficiency of siRNA was determined by experiment.The transfection intervention lasted for 2 days and the differentiation lasted for 3 days.The mitochondrial membrane potential change ΔΨwas determined by JC-1 probe,the ROS formation level of cells was determined by DCFH-DA probe,and the mitochondrial biosynthesis related proteins PGC-1α and COX Ⅳ were detected by Western-blotting assay.Uprmt-related proteins ATF5,Hsp60,Clp P;The expression levels of MYH4 and MYH7 proteins related to muscle fiber type.The enrichment of transcriptional inhibitory histone H3K27me3 at the promoter of ATF5,PGC-1α,MYH4,and MYH7 genes was detected by chromatin immunoprecipitation(CHIP).Results:(1)Effects of JMJD3 on mitochondrial biosynthesis and UPRMt-related protein expression in C2C12 cellsCompared with Mock group,the expr essions of mitochondrial biosynthesis-related proteins PGC-1α and COX Ⅳ in siRNA-JMJD3 group were significantly decreased(p<0.05).The expressions of UPRMt-related proteins ATF5 and Hsp 60 in the siRNA-JMJD3 groups were significantly decreased(p<0.05),and the expression of Clp P was decreased,but there was no significant difference.Compared with empty plasmid group,the expressions of mitochondrial biosynthesis-related proteins PGC-1α and COX Ⅳ in JMJD3 overexpression group were significantly increased(p<0.05).The expressions of UPRMT-related proteins ATF5 and Hsp60 in JMJD3 overexpression group were significantly increased(p<0.05),while Clp P showed an increasing trend but no significant difference.(2)Effect of JMJD3 on mitochondrial function of C2C12 cellsCompared with Mock group,mitochondrial membrane potential in siRNA-JMJD3 group was significantly decreased(p<0.01).Compared with Mock group,ROS in siRNA-JMJD3 group was significantly increased(p<0.05).Compared with empty plasmid group,mitochondrial membrane potential of JMJD3 overexpression group had no significant change.Compared with empty plasmid group,ROS in JMJD3 overexpression group was significantly increased(p<0.05).(3)Effect of JMJD3 on expression of muscle fiber type related proteins in C2C12cellsCompared with the Mock group,the expression of MYH7 protein(slow muscle fiber)was significantly decreased in siRNA-JMJD3 group(p<0.05),while the expression of MYH4 protein(fast muscle fiber)was significantly increased in Sir NA-JMJD3 group(p<0.05).Compared with empty plasmid group,the expression of MYH4 protein in JMJD3 overexpression group was significantly decreased(p<0.05),and the expression of MYH7 protein in JMJD3 overexpression group was significantly increased compared with empty plasmid group(p<0.05).(4)Effects of JMJD3 on mitochondrial biosynthesis in C2C12 cells,UPRmt and UPRMT modification of the fast and slow muscle related gene H3K27me3Compared with the Mock group,there was no significant change in the H3K27me3 enrichment rate of UPRmt activation key transcription factor ATF5 gene promoter after siRNA-JMJD3 interfered with C2C12 cells.The concentration of H3K27me3 in the promoter of PGC-1α gene,a key transcription factor for mitochondrial biosynthesis,increased after siRNA-JMJD3 interfered with C2C12 cells,but there was no significant change.There was no significant change in the concentration rate of H3K27me3 at the promoter of the slow muscle fiber related gene MYH7 after siRNA-JMJD3 interfered with C2C12 cells.After siRNA-JMJD3 interfered with C2C12 cells,the enrichment rate of H3K27me3 at the promoter of MYH4 gene of fast muscle fiber related gene was significantly decreased(p<0.05),and the transcriptional activity of MYH4 gene was activated.Compared with empty plasmid group,H3K27me3 enrichment rate of UPRmt activated transcription factor ATF5 gene promoter was significantly decreased after transfection with JMJD3 overexpression plasmid(p<0.05),and ATF5 gene transcriptional activity was activated.After transfection of JMJD3 overexpression plasmid into C2C12 cells,the concentration of H3K27me3 at the promoter of PGC-1αgene,a key activating transcription factor for mitochondrial biosynthesis,was significantly decreased(p<0.05),and the transcriptional activity of PGC-1α gene was activated.After transfection of JMJD3 overexpression plasmid into C2C12 cells,the concentration rate of H3K27me3 at the promoter of MYH7 gene associated with slow muscle fiber was significantly decreased(p<0.05),and the transcriptional activity of MYH7 gene was activated.After transfection of JMJD3 overexpression plasmid into C2C12 cells,the enrichment rate of H3K27me3 at the promoter of MYH4 gene related to fast muscle fiber was significantly increased(p<0.05),and the transcriptional activity of MYH4 gene was inhibited.Research conclusion: 1.On the one hand,the epigenase JMJD3 promoted the expression of mitochondrial homeostasis related genes and enhanced mitochondrial homeostasis by reducing the concentration rate of transcriptional inhibitory histone H3K27me3 on mitochondrial homeostasis related genes.On the other hand,the epigenase JMJD3 promoted the expression of slow myosin heavy chain subtype by reducing the transcriptional inhibitory histone H3K27me3 enrichment rate on genes related to slow myosin heavy chain subtype.JMJD3 may conjugate myosin heavy chain isoforms and mitochondrial homeostasis by regulating epigenetic modification of related genes H3K27me3.