Effect Of Omi/HtrA2 On The Expression Of Mitochondrial Pathway Related Genes In Skeletal Muscle Of Mice

The main manifestations of skeletal muscle atrophy are progressive contraction of muscle fibers,even its disappearance and decrease of muscle mass and volume.There are complex reasons for muscle atrophy.They include maladjustment of protein synthesis and degradation,chronic inflammatory reactions,oxidative stress and other aspects.Muscle cells are rich in mitochondria,which can undertake the energy supply,reactive oxygen species?ROS?homeostasis,cell signaling and other tasks for regulating muscle cells.Studies have found that the number of mitochondria in submandibular?SS?and interstitial myofibrils?IMF?in the muscles of aged rats gradually decreases.Therefore,in the process of skeletal muscle aging,the effect of mitochondrial function has been paid attention to.MuRF1?Trim63?and MAFbx?atrogin-1?,two important skeletal muscle-specific E3 ligases,are considered as markers of skeletal muscle atrophy.When skeletal muscle atrophy appears,MuRF1 and MAFbx are significantly activated,which promotes ubiquitination of substrate proteins and allows more proteins to be transported to proteasome for degradation.When it is inhibited,muscle loss can been reduce.Therefore,it is believed that the expression of the two may be positively correlated with the degree of muscle atrophy.Omi/HtrA2,a serine protease in the mitochondrial membrane gap,is also a molecular partner with heat shock protein properties.It has an important effect on mitochondrial quality control and maintenance of mitochondrial homeostasis.Omi/HtrA2 gene homozygous deletion mice?-/-?(also called HtrA2^mnd2 mice)has been obtained by missense mutation of Omi/HtrA2 gene S276C using gene knockout technology.Compared with wild-type mice,homozygous mice have a shorter survival time with hemiplegia,tremor and other neurodegenerative disease symptoms.In addition,anatomy has found that the skeletal muscle of homozygous mice has obviously shrunk.At present,reports on Omi/HtrA2 effect in skeletal muscle have not been found.The purpose of this experiment is to investigate the effect of Omi/HtrA2on expression of genes related to mitochondrial pathway in skeletal muscle of mice on the basic of the perspectives of mitochondrial biosynthesis,antioxidant stress and autophagy.Methods:According to genotype,mice were divided into three groups:wild type mice?+/+?,Omi/HtrA2 gene heterozygous deletion mice?+/-?and HtrA2^mnd2 mice?-/-?.1.By HE staining,the morphological changes of skeletal muscle in mice were observed.2.Real-time quantitative PCR was used to detect mRNA levels of skeletal muscle atrophy marker genes,cell cycle,mitochondrial biosynthesis,division and fusion,antioxidant stress and autophagy related genes in mice.3.The expression situation of mitochondrial biosynthesis,fission fusion and autophagy related proteins in skeletal muscle tissues of mice in each group were detected by Western blot method.Results:1.Compared with the wild type?+/+?,the life span of HtrA2^mnd2 mice?-/-?was significantly shortened.The results of HE staining showed that the number of skeletal muscle fibers of 45-day-old HtrA2^mnd2 mice?-/-?was significantly reduced.The relative volume of muscle cells decreased significantly with the irregular shape,which suggested that the loss of Omi/HtrA2 might cause skeletal muscle atrophy of mice.2.The results of real-time quantitative PCR showed that compared with the wild type?+/+?,the mRNA levels of MuRF1 and MAFbx,the markers of skeletal muscle atrophy of HtrA2^mnd2 mice?-/-?,were significantly increased,which further suggested that the loss of Omi/HtrA2 might cause skeletal muscle atrophy of mice.3.The results of real-time quantitative PCR showed that compared with the wild type?+/+?,the mRNA levels of cell cycle genes Asns and Trib3 of skeletal muscle tissues of HtrA2^mnd2 mice?-/-?were significantly reduced,which indicated that the loss of Omi/HtrA2 caused arrest of cell cycle in skeletal muscle tissues of mice.4.The results of real-time quantitative PCR showed that compared with the wild type?+/+?,the mRNA level of mitochondrial biosynthesis gene PGC-?1 in skeletal muscle tissue of HtrA2^mnd2 mice?-/-?was decreased,which indicated that the loss of Omi/HtrA2 caused the decrease of the number of mitochondria in skeletal muscle tissue of mice.5.The results of real-time quantitative PCR showed that compared with the wild type?+/+?,the mRNA levels of antioxidant genes HO-1 and GSTA1 in skeletal muscle tissues of HtrA2^mnd2 mice?-/-?were decreased,which indicated that the loss of Omi/HtrA2 caused the reduction of antioxidant stress ability in skeletal muscle tissues of mice.6.The results of real-time quantitative PCR showed that compared with wild type?+/+?,mRNA levels of split genes Drp1,Fis1,fusion genes Mfn1,Mfn2,Opa1 in skeletal muscle tissues of HtrA2^mnd2 mice?-/-?were significantly reduced.Western Blot method was used to detect the corresponding proteins in skeletal muscle tissues of HtrA2^mnd2 mice?-/-?and the expression was decreased,which indicated that the loss of Omi/HtrA2 caused the mitochondrial division and fusion ability in skeletal muscle tissues of mice weakened.7.The results of real-time quantitative PCR showed that compared with wild type?+/+?,the mRNA level of autophagy related gene P62 in skeletal muscle tissue of HtrA2^mnd2 mice?-/-?decreased,while the mRNA levels of LC3b and Lamp2 increased.Western Blot was used to detect P62 protein in skeletal muscle tissues of HtrA2^mnd2mice?-/-?and the expression level was decreased,which suggested that the loss of Omi/HtrA2 caused autophagy activation in skeletal muscle tissues of mice.Conclusion:1.On the basic of morphology and expression level of skeletal muscle atrophy gene,it is diagnosed that HtrA2^mnd2 mice appear skeletal muscle atrophy,which suggests that Omi/HtrA2 gene mutation is connected with skeletal muscle atrophy.2.Omi/HtrA2 gene mutation can cause imbalance of skeletal muscle oxidation and antioxidation,which indicates that oxidative stress is one of the mechanisms of muscle atrophy caused by Omi/HtrA2 gene mutation.3.Mutation of Omi/HtrA2 gene can result in synthesis is reduced and degradation is enhance for skeletal muscle mitochondria.At the same time,autophagy function is disordered,which indicates that skeletal muscle atrophy caused by Omi/HtrA2 gene mutation is connected with reduction of mitochondrial function and disorder of autophagy function.