| Skeletal muscle(SKM)mainly originates from the somites evolved from the paraxial mesoderm.The myogenic progenitors within the somites undergo two stages of primary and secondary myogenesis,and eventually develop into fetal muscles.The myogenic progenitors gradually evolve into SKM stem cells.After birth,the mass and size of fetal muscles continue to grow until they develop into adult SKM.Meanwhile,the number of SKM stem cells decreases within 3 weeks after birth,and they finally reside between the plasma membrane and basal lamina,forming adult SKM stem cell pool,also known as SKM satellite cells.Adult SKM is the largest tissue in the body,accounting for 30-40%of human body weight.Such a huge tissue system can adapt to its physiological function and the stress response caused by various injury stimuli,showing strong plasticity and adaptability.Upon exposure to damage factors,the adaptive response of adult SKM is mainly reflected in two aspects.Firstly,the damage in SKM itself,such as acute trauma,muscular dystrophy,etc.In this case,satellite cells are activated to start the myogenic process and regenerate new muscle fibers or repair existing muscle fibers.Secondly,the changes in muscle morphology caused by factors other than SKM itself,such as cachexia,long-term application of glucocorticoid,denervation,etc.In this case,SKM is manifested as a reduction in mass and size,termed SKM atrophy.In this study,the adaptive process of muscle regeneration and the adaptive process of muscle atrophy were respectively discussed to further explore the molecular mechanism of SKM adaptation under different injury stimuli,so as to provide new potential targets and experimental basis for the clinical treatment of SKM-related diseases.Part Ⅰ Role and mechanism of Pim1 kinase on satellite cell-mediated skeletal muscle regenerationBackgroundSkeletal muscle(SKM)has an ability of regeneration after injury due to its intrinsic stem cell reserve.SKM stem cells,also known as muscle satellite cells(SCs),reside between the plasma membrane and basal lamina in a quiescent state.Once the SKM is damaged,the quiescent SCs are immediately activated and begin to proliferate,differentiate,and finally fuse either to form new multinucleated muscle fibers or to repair damaged parts of existing muscle fibers.The regulation of these processes is closely related to the expression cascade of a series of myogenic regulatory factors in SCs.Quiescent SCs only expressed the muscle-specific regulator Pax7,while the activated SCs followed the specification to the myogenic lineage,repressed Pax7 and began to express Myf5,MyoD,myogenin and MRF4,determining the entry of SCs into the myogenetic differentiation program,which eventually fuse with injured myofibers to accomplish regeneration.Among them,a small part of proliferating SCs gradually down-regulated MyoD,maintained Pax7,re-entered the quiescent state,and updated the reserve of SCs pool.Therefore,the proliferation,differentiation,fusion and self-renewal of SCs play an irreplaceable role in the regeneration and repair of SKM after injury.However,severe SKM trauma or functional defects in SCs will make it difficult to initiate SKM regeneration.So,exploring the molecular mechanisms of SCs self-renewal,proliferation,differentiation and fusion has important practical application value for controlling SCs function and restarting SKM regeneration ability.The functions of SCs are regulated by multiple signal pathways,among which,protein kinase,as a class of extremely important signal proteins in mammalian cells,plays a key role in the regulation of SCs function.For example,ERK and JNK can promote SCs self-renewal,and TAK can promote SCs proliferation and repress differentiation,while p38 MAPK,Lkbl and JAK inhibit SCs proliferation and promote differentiation.It can be seen that the effect of protein kinase reported so far on SCs function is just reflected in one state of SCs,or it can promote one state but inhibit another state.At the same time,proliferation,differentiation and fusion of SCs are a staggered sequential process.Therefore,the usage of these reported protein kinase targets should be paid attention to the selection of the time window of each stage of SKM regeneration,which virtually increases the difficulty of clinical application.So our research intends to explore new key protein kinases regulating the multiple links of SCs,and reveal their effects on SCs function and SKM regeneration,providing new ideas and basis for SKM stem cell tissue engineering research and clinical targeted intervention in SKM regeneration.Methods1.Screening and experimental verification of differentially expressed protein kinases during SKM regeneration1.1 The expression profile microarray data of SKM injury model were downloaded from Gene Expression Omnibus(GEO)database.R language and bioinformatics analysis were used to screen differentially expressed protein kinases.1.2 SKM injury model was established by injecting 10μl notexin(NTX,10μg/ml)into the right tibialis anterior(TA)muscle in 10-12w male C57BL/6J mice.Western blot(WB)and qPCR were performed to detect Piml expression in TA muscle at 3d,7d,14d,28d after NTX injection.2.Function of Piml kinase in muscle injury and regeneration model2.1 The wild type(Pim1+/+)and Pim1 knockout(Pim1-/-)mice were used to measure the body weight and lower limb muscle wet weight,calculating the percentage of muscle wet weight in body weight.The muscle morphology of lower limbs was o’bserved by H&E staining.2.2 Piml+/+ and Piml-/-mice were selected to establish a muscle injury and regeneration model.qPCR were used to detect the expression of Piml in TA muscle.At day 14 after muscle injury,H&E staining was used to measure the cross-sectional area(CSA)of fibers with centrally located nuclei.3.Effect of Piml on proliferation,differentiation and fusion of C2C12 myoblasts and muscle SCs3.1 The lentivirus(Lv)vector carrying mouse Piml shRNA(shPiml)was transfected into C2C12 myoblasts to establish Piml low-expression C2C12 cell lines.WB detected Piml expression.3.2 In the normal C2C12 cells and Piml low-expression C2C12 cells,the CCK8 kit was used to detect the viability of the cells,and the EdU kit was used to detect the cell proliferation rate,and the TUNEL assay kit was used to detect the cell apoptosis rate.WB detected the protein expressions of pBad,Bcl-2 and Bcl-xl.3.3 Lv-shPiml was transfected into primary SCs.IF was used to count Pax7 positive cells and MyoD positive cells.3.4 The expression of Piml was detected by qPCR and WB when C2C12 myoblasts were induced to differentiate.The localization of Piml in muscle cells was observed by IF through labelling MyoD and Piml in proliferated SCs and labelling MyHC and Piml in differentiated SCs.At Id,3d and 5d after differentiation,the expression of Piml protein in cytoplasm and nucleus was detected respectively.3.5 The differentiated C2C12 cells were treated with Piml kinase inhibitor TCS PIM-1 I(TCS)at a final concentration of 25pM and 50μM,and the protein of myogenin,MyHC and myomerger was detected by WB,and the mRNA of Myog,Tnni2,Ckm,Mylpf,Actal,Mymk and Mymx was detected by qPCR,and the differentiation index and fusion index were calculated through fluorescence labelling MyHC and nucleus.3.6 Primary SCs were induced to differentiate in vitro,and TCS was applied to inhibit Pim1 kinase activity.MyHC and nucleus were labeled by IF,and the differentiation index and fusion index were calculated.3.7 C2C12 myoblasts were either transfected with Piml(K67M)plasmid,MyoD plasmid,Renilla luciferase reporter plasmid,and firefly luciferase reporter plasmid promoted by MyoD DNA binding core sequence,or treated with TCS.Luciferase activity was detected.Results1.Piml kinase is up-regulated after SKM injury1.1 GSE67032 was downloaded from the GEO database.The wild-type control group and wild-type NTX treatment group were selected and reanalyzed.The R-language limma package was used to screen the differentially expressed gene between the two groups(fold change≥2,p<0.01).A total of 2703 genes were obtained,including 1634 up-regulated and 1069 down-regulated.According to GO enrichment analysis,73 genes with protein kinase activity involved in protein phosphorylation were obtained,and Piml was one of the up-regulated protein kinases.Based on the latest research progress on the key role of Piml in promoting cell survival and growth,we take Piml kinase as the object of this study.1.2 WB and qPCR results showed that Piml protein and mRNA expression were significantly up-regulated in TA muscle at 3d and 7d after NTX injection,which coincided with the peak of SCs proliferation and differentiation.After NTX treatment of 14d and 28d,Piml expression returned to normal.2.Deletion of Piml hinders muscle regeneration2.1 Although the body weight of Piml-/-mice were significantly lower than that of Pim1+/+ at each week of age,there was no significant difference between TA and GAS in relative muscle wet weight.H&E staining showed no myopathy in Piml-/-mice.2.2 After the establishment of Piml+/+ and Piml-/-mouse muscle injury model,qPCR showed that Piml was absent in TA muscle of Piml-/-mice,which proved that Piml gene had been knocked out.Compared with Piml+/+ littermates,Piml-/mice showed an impaired muscle regeneration,as indicated by a smaller CSA of fibres with centrally located nuclei at 14 days post injury.3.Piml kinase promotes myoblasts proliferation,differentiation and fusion3.1 When Lv-shPiml was transfected into C2C12 myoblasts,Piml expression was significantly down-regulated.3.2 In Piml low-expression C2C12 cell lines,CCK8 results showed that the viability of C2C12 cells were significantly decreased.EdU staining showed that the proliferation of C2C12 cells was significantly reduced.TUNEL staining showed that the apoptosis of C2C12 cells increased significantly.WB showed that Bcl-2,Bcl-xl,pBad proteins were significantly down-regulated.3.3 In primary SCs,the proportion of Pax7 positive and MyoD positive cells decreased significantly after Lv-shPiml transfection.3.4 C2C12 myoblasts were induced to differentiate.qPCR and WB showed that the expression of Piml increased gradually with cell differentiation.EF results showed that Piml was mainly localized in the cytoplasm in proliferated SCs,and after induction of differentiation,Piml translocated into the nucleus.The expression of Piml protein in cytoplasm and nucleus were up-regulated from 3rd day of cell differentiation,and the nuclear Piml was phosphorylated3.5 In differentiated C2C12 cells,TCS significantly inhibited both the protein expression of myogenin,MyHC and myomerger,and the mRNA expression of Myog,Tnni2,Ckm,Mylpf,Actal,Mymk and Mymx.The differentiation index and fusion index of C2C12 cells also decreased significantly after TCS treatment.3.6 In differentiated primary SCs,IF results showed that both differentiation index and fusion index were significantly reduced after TCS treatment.3.7 The results of dual luciferase reporter gene showed that Piml(K671M)or TCS significantly inhibited the transcriptional activity of MyoD in differentiated C2C12 cells.Conclusions1.Piml kinase is up-regulated after muscle injury,and the time window of Piml up-regulation is consistent with the peak of SCs proliferation and differentiation.2.In the muscle injury model,Piml knockout significantly inhibits muscle fiber regeneration and repair.3.Piml kinase promotes myoblast proliferation,differentiation and fusion,so as to play a crucial role in promoting SKM regeneration.4.Piml kinase regulates the expression of SKM-specific genes during myogenic differentiation by mediating the transcriptional activity of myogenic regulator MyoD.Part Ⅱ Effect and mechanism of methyltransferase CARM1 on skeletal muscle atrophyBackgroundAs the ultimate executor of various physical movements,skeletal muscle plays an irreplaceable role in one’s life.However,a variety of chronic diseases,cancer,malnutrition,long-term application of glucocorticoid,loss of neural input,and other pathological conditions can lead to muscular atrophy and functional weakness.Therefore,exploring the molecular mechanism of SKM atrophy is of great clinical significance in maintaining the healthy physiological structure and function of SKM tissues under pathological conditions.Coactivator-associated arginine methyltransferase 1(CARM1),is a member of the protein arginine methyltransferase(PRMT)family.CARM1 plays dual functional roles as a methyltransferase and a coactivator.Functionally,CARM1 and its methyltransferase activity are essential for the ontogenesis of mammals,demonstrating the powerful physiological functions of CARM1.In skeletal muscle cells,previous studies have shown a close relationship between CARM1 and myogenesis.CARM1 exerts a significant positive effect on myocyte division and differentiation.Meanwhile,CARM1 also plays a pathological role in different disease states.For example,it is involved in the development and metabolism of tumors and promotes hyperalgesia induced by peripheral nerve injury.However,the mechanism by which CARM 1 influences skeletal muscle wasting in adults remain unknown.Therefore,our research will explore the role and mechanism of CARM1 in the development of SKM atrophy from the following aspects:1 To detect the expression pattern of CARM1 in SKM atrophy model and explore the relationship between CARM1 and the progression of muscular atrophy.2 To clarify the role of CARM1 in the development of SKM atrophy.3 To study the relationship between CARM1 and Fox03 and reveal the molecular mechanism of CARM1 in promoting muscular atrophy.4 To investigate the role of CARM1 in the autophagy process of muscle fibers.Methods1.Expression of CARM1 in denervation-induced muscle atrophy and its relationship with muscle wet weight1.1 Denervation(Den)-induced muscle atrophy model was established by right sciatic nerve transection in 8-10w male C57BL/6J mice,and the expression of CARM1 in tibialis anterior(TA)and gastrocnemius(GAS)was detected by western blot(WB)at Den 3d,7d,2w and 4w.1.2 The muscle wet weight of TA and GAS muscle were measured.The percentage of the muscle wet weight to the contralateral was calculated to analyze the difference between TA and GAS,and establish the relationship between the change of CARM1 expression and the change of the muscle wet weight.2.Function of CARM1 in denervation-induced muscle atrophy model2.1 Adeno-associated virus(AAV)vector carrying mouse CARM1 shRNA(shCARM1)was transfected into TA muscle by intramuscular injection,and CARM1 expression was detected by WB and qPCR.2.2 After 4w of AAV-shCARM1 injection,Den-induced muscle atrophy model was established.At Den 2w and 4w,the TA muscle wet weight and the muscle fiber cross-sectional area(CSA)was measured.The protein of myosin heavy chain(MyHC)was detected by WB and the mRNA of Atroginl,MuRFl and MUSA1 were detected by qPCR.3.Function of CARM1 in dexamethasone-induced muscle atrophy model3.1 After 4w of AAV-shCARM1 injection,dexamethasone(Dex)-induced muscle atrophy model was established by intraperitoneal injection(10mg/kg/d)for 4w.TA muscle wet weight and myofiber CSA was measured3.2 CARM1 knockdown in C2C12 myotubes was performed by CARM1 targeting siRNA(siCARM1)transfection.Subsequently,a Dex(100RM)-induced C2C12 myotube atrophy model was established.WB and qPCR were used to detect the expression of CARM1,and the diameter of C2C12 myotubes was measured by immunofluorescence(IF).The Atroginl and MuRF1 mRNA was detected by qPCR.4.Modulating mechanism of CARM1 on transcription factor Fox034.1 WB was used to detect Fox03 and pFox03(S253)protein levels in Dex-induced C2C12 myotube atrophy model in vitro or Den-induced SKM atrophy model in vivo4.2 In Dex-induced myotube atrophy model,IF was used to detect the localization of CARM1 and Fox03 to analyze whether there was co-localization.4.3 In Dex-induced C2C12 myotube atrophy model and Den-induced SKM atrophy model,the interaction between CARM1 and Fox03 was determined by co-immunoprecipitation(Co-IP),and the asymmetric di-methylation(me2a)level of Fox03 was detected by immunoprecipitation(IP)5.Effect of CARM1 methyltransferase activity on Fox03-dependent transcriptional activation and myotube atrophy5.1 In Dex-induced C2C12 myotube atrophy model,the mRNA expression of Fox03 target genes Atroginl and MuRF1 were detected by qPCR after CARM1 methyltransferase inhibitor(CARM1i)application.The diameter of C2C12 myotubes was measured by IF.6.Effect of CARM1 on muscle fiber autophagy6.1 After siCARM1 or shCARM1 transfection in muscle atrophy model,WB was used to detect the protein levels of LC3-I and LC3-II,and IF was used to detect the spot aggregation of LC3-Ⅱ in myotubes.The mRNA levels of autophagy-related genes LC3b,Becn1,Bnip3,Cts1,Atg5,Atgl2,Atg13 and Atgl4 were detected by qPCR,and the protein levels of Atg13 and Atg14 were detected by WBResults1.The increase of CARM1 protein is accompanied by the loss of muscle mass upon denervation1.1 At Den 2w and 4w,CARM1 protein in TA muscle was not different from that in the innervation(Inner)group,but CARM1 protein in GAS muscle was significantly increased compared with that in the Inner group.1.2 The decrease of muscle wet weight in GAS muscle was more obvious than that in TA muscle at Den 2w and 4w,indicating that the more up-regulation of CARM1 protein after Den,the more severe the muscle atrophy.2.CARM1 knockdown can relieve Den-induced SKM atrophy2.1 WB and qPCR showed that AAV-shCARMl transfection could significantly reduce the CARM1 expression in both Inner and Den TA muscles.2.2 After Den 4w,compared with shCtrl group,the ratio of TA muscle wet weight to contralateral in shCARM1 group increased by 8.8%(shCtrl 48.54±0.32%,shCARMl 57.34±1.01%).The percentage of TA muscle average CSA(relative to contralateral)increased by 14.83%(shCtrl 50.49±4.00%,shCARM1 65.32±2.63%).CARM1 knockdown significantly increased the MyHC protein level,and reduced the atrophy-related genes Atroginl and MuRF1 mRNA level.3.CARM1 knockdown confers resistance to Dex-induced muscle atrophy3.1 After Dex-induced muscle atrophy for 4w,AAV-shCARMl transfection could effectively reduce the loss of muscle wet weight.H&E showed that AAV-shCARM transfection significantly increased the mean muscle fiber CSA of TA muscle(shCtrl 1695.3±61.1μm2,shCARM1 1896.6±41.8μm2).3.2 In Dex-induced C2C12 myotube atrophy model,WB and qPCR confirmed that siCARMl transfection induced CARM1 down-regulation.IF showed that siCARM1 transfection significantly prevented the reduction of myotube diameter(siCtrl 12.04±0.51μm,siCARM1 25.46±1.37μm).qPCR showed that Atroginl and MuRF1 mRNA were also significantly down-regulated after siCARM1 transfection.4.CARM1 interacts with and asymmetrically dimethylates the transcription factor Fox034.1 A significant up-regulation of the Fox03 protein was observed after muscle atrophy both in vitro and in vivo,while CARM1 knockdown did not affect the level of protein expression or the level of phosphorylation at Ser253 of Fox03,suggesting that the alleviation of myotube atrophy by CARM1 knockdown does not occur by affecting the protein level and intracellular localization of Fox03.4.2 IF showed that CARM1 is distributed mainly in the myotube nuclei.Double staining revealed the colocalization of CARM1 and Fox03 in the nucleus after Dex-induced myotube atrophy.4.3 Co-IP further revealed the interaction between endogenous CARM1 and Fox03 in C2C12 myotubes or in the GAS muscle.IP showed that Fox03 was dimethylated in C2C12 myotubes,although the degree of methylation was similar in conditions of vehicle and Dex treatment.However,in vivo,the level of Fox03 me2a increased by 2.5-fold in a Den-induced GAS muscle atrophy model.This result may be attributable to the upregulation of the CARM1 protein in denervated GAS muscles5.Methylation by CARM1 is required for FoxO3 transcriptional activity and myotube atrophy5.1 In Dex-induced C2C12 myotube atrophy model,the suppression of Fox03 me2a by CARM1i treatment induced a significant transcriptional reduction of the Fox03 target genes Atroginl and MuRF1.IF showed that CARMli treatment substantially inhibited the diameter reduction in Dex-treated C2C12 myotubes(Ctrl 14.15±1.22μm,CARMli 24.14±1.49μm)6.CARM1 is involved in skeletal muscle fiber autophagy6.1 In C2C12 myotubes,Dex treatment facilitated the conversion of non-lipidated LC3-I to lipidated LC3-Ⅱ,but siCARM1 transfection diminished LC3-Ⅱ formation,as indicated by a decreased LC3-Ⅱ/LC3-Ⅰ ratio.IF also revealed a remarkable decrease in the number of autophagosomes,as indicated by punctate LC3-Ⅱ aggregation upon siCARM1 transfection.In a Den-induced mouse muscle atrophy model,CARM1 knockdown inhibited the conversion of LC3-Ⅰ to LC3-Ⅱ.qPCR and WB showed that the autophagy-related genes Atg13 and Atg14 were significantly down-regulated after shCARM1 transfection.Conclusions1.CARM1 protein is significantly up-regulated in Den-induced muscle atrophy model,and the increase of CARM1 protein is accompanied by the loss of muscle mass.2.Under the atrophy-inducing condition,CARM1 knockdown by RNA interference can effectively alleviate the degree of SKM atrophy.3.CARM1 interacts with and asymmetrically dimethylates transcriptional factor Fox03.4.Methylation by CARM1 is required for Fox03 activity and myotube atrophy.5.CARM1 is involved in skeletal muscle fiber autophagy in vitro and in vivo. |
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