Selenoprotein K Mediating Myogenic Differentiation Of Satellite Cells Regulates Skeletal Muscle Postjured Repair In Mice

Selenium（Selenium,Se）is one of the essential trace elements for life,and has biological functions such as regulation of immunity,metabolism,growth and development,and resistance to harmful stimuli.Selenium mainly exerts physiological functions in the form of selenoproteins in the body.Selenoprotein K（SelK）is a small transmembrane selenoprotein located in the endoplasmic reticulum,which has been proved to have antioxidant,alleviation of endoplasmic reticulum stress and anti-apoptosis.Skeletal muscle is the largest tissue in adult mammals and is mainly composed of muscle fibers formed by muscle satellite cells through myogenic differentiation and fusion.Studies have shown that selenium deficiency can cause skeletal muscle damage,including white muscle disease.SelK is abundantly expressed in skeletal muscle,but the role of SelK in the skeletal muscle postinjured repair has not been reported.Based on this,in the experiment,10-week-old wild-type and SelK knockout C57B/L6 mice were used as the test objects.The skeletal muscle injury model was replicated by injecting 1.2%Ba Cl₂solution into the right tibialis anterior muscle,and the in vitro model of myogenic differentiation was established by stimulated C2C12 myoblasts with culture medium containing 2%horse serum.H&E staining,immunofluorescence staining,immunohistochemical staining,antioxidant index detection,TUNEL staining,CCK-8 detection,flow cytometry,real-time quantitative PCR and western blot analysis were used to determine the changes of SelK expression after skeletal muscle injury,to observe the morphological changes of skeletal muscle tissue regeneration after SelK deletion,and to detect the effect of SelK silencing on myoblast proliferation and myogenic differentiation,levels of oxidative stress,endoplasmic reticulum stress,apoptosis and autophagy and related genes expression.This experiment aimed to reveal that SelK can participate in the skeletal muscle postinjured repair in mice,and to clarify its biological function and mechanism in skeletal muscle regeneration.The main results were as follows:（1）The Results of localization analysis and expression detection of SelK showed that under normal physiological conditions,the expression of SelK in quiescent satellite cells was not significant.After skeletal muscle injury,satellite cells were activated and began to differentiate,and the expression of SelK in myogenic cells was significantly upregulated.At the cellular level,the expression of SelK in C2C12 cells increased with the progression of myogenic differentiation.The results suggested that SelK may be involved in satellite cell-mediated skeletal muscle postinjury repair.（2）The result of morphological observation and analysis showed that the myofibers size and integrity were similar in skeletal muscle of uninjured wild-type and SelK-knockout mice.After induced injury,the myofibers of wild-type mice first fragmented and disintegrated accompanied with inflammatory cell infiltration,and then regenerated multinucleated muscle fibers fully filled the damaged area for repair.The SelK knockout mice also showed a phenotype of muscle fiber regeneration,but the proportion and size of multinucleated myofibers were significantly reduced.The results showed that SelK is essential for ensuring normal regeneration of skeletal muscle in mice after injury.（3）The detection results of satellite cell proliferation and differentiation condition showed that the proportions of myogenic differentiated cells（Pax7^-/Myo D⁺,Myo D⁺/Myo G⁺and Myo D^-/Myo G⁺）in the injured skeletal muscle of SelK knockout mice were significantly reduced,and the myogenic proliferating cells（Ki67⁺/Pax7⁺and Pax7⁺/Myo D⁺）ratios were not significantly changed.At the cellular level,SelK knockdown significantly inhibited the percentages of Myo G⁺and My HC⁺cells and the myotube formation of C2C12 cells cultured in differentiation medium,but did not alter cell viability and cell cycle of C2C12 cells in growth medium.Meanwhile,quantitative analysis showed that SelK knockdown decreased the expression levels of myogenesis-related genes（Myo D,Myo G,and My HC）in muscle tissue and C2C12 cells.The results indicated that SelK knockout could inhibit myogenic differentiation.（4）The results of TUNEL staining showed that the degree of apoptosis in the injured skeletal muscle of SelK knockout mice was significantly increased compared with that of wild-type mice,and the expression of anti-apoptotic gene Bcl2 protein was decreased,and the protein expressions of pro-apoptotic genes Bax and cle-Cas3 were increased.At the cellular level,SelK knockout also increased the AO/EB positive ratio and the expression changes of apoptosis-related genes in C2C12 cells under myogenic differentiation.The results indicated that SelK knockout aggravated apoptosis.（5）Compared with wild-type mice,the expression of autophagy marker LC3-II protein expression in skeletal muscle of SelK knockout mice was increased,and the protein expression of degradation substrate P62 was decreased.Meanwhile,SelK knockout increased the expression changes of autophagy-related genes under myogenic differentiation of C2C12 cells.The results indicated that SelK knockout aggravated autophagy.（6）Compared with wild-type mice,the activities of antioxidant enzymes SOD,CAT and T-AOC in the injured skeletal muscle of SelK knockout mice were significantly down-regulated,and the contents of peroxide H₂O₂and MDA were increased.At the cellular level,SelK knockout promoted SOD1,SOD2,and CAT m RNA expression and increased ROS content under myogenic differentiation of C2C12 cells,while treatment with ROS inhibitors NAC and SF could significantly alleviate the changes of antioxidant gene expression and ROS content in SelK knockout cells,extenuate apoptosis and autophagy occurrence,and partially restore the percentage of inhibited Myo G⁺and My HC⁺cells and myotube formation.The results indicated that SelK knockout resulted in the inhibition of myoblast differentiation by aggravating oxidative stress.（7）Compared with wild-type mice,the protein levels of endoplasmic reticulum stress marker genes CHOP,ATF6 and GRP78 and the phosphorylation levels of e IF2α,PERK and IRE1 in the injured skeletal muscle of SelK knockout mice,were significantly increased.During myogenic differentiation of C2C12 cells,SelK knockout also promoted the expression of ER stress-related genes,and ER stress inhibitors 4-PBA and TUDCA could significantly alleviate the expression changes of endoplasmic reticulum stress marker genes in SelK knockout cells,extenuate apoptosis and autophagy occurrence,and partially restored the percentage of inhibited Myo G⁺and My HC⁺cells and myotube formation.The results indicated that SelK knockout resulted in the inhibition of myoblast differentiation by aggravating endoplasmic reticulum stress.（8）During myogenic differentiation of SelK knockout C2C12 cells,NAC and SF treatment had no effect on the protein levels of CHOP,ATF6 and GRP78 and the phosphorylation levels of e IF2α,PERK and IRE1 in the cells,while 4-PBA and TUDCA treatment significantly alleviated the elevated SOD1,SOD2 and CAT expression and the elevated ROS content caused by SelK knockout.The results indicated that SelK knockout could regulate oxidative stress by promoting endoplasmic reticulum stress.In conclusion,the SelK expression is activated during the repair of skeletal muscle injury in mice,and SelK knockout can inhibit the regeneration of skeletal muscle after injury.Further experimental results confirmed the role of SelK in satellite cell-regulated myogenic differentiation,that is,satellite cells regulate the activation level of endoplasmic reticulum stress by upregulating SelK expression to limit ROS production,and prevent excessive endoplasmic reticulum stress,oxidative stress,apoptosis and autophagy occurrence,thereby ensuring myogenic differentiation regulated by muscle satellite cells and maintaining skeletal muscle regeneration and repair.The results of this study provide new therapeutic targets for the treatment of diseases involving muscle damage,and provide a reference for further research on the function of SelK.