Inhibition Mechanism Of Melatonin On Differentiation In Murine Myoblasts

Melatonin,a pleiotropic hormone secreted from the pineal gland,has been shown to exert beneficial effects in muscle regeneration and repair due to its functional diversity,including anti-inflammation,anti-apoptosis,and anti-oxidative activity.However,little is known about the negative role of melatonin in myogenesis.Therefore,this study designed to explore the effects of different concentrations of melatonin on the proliferation,differentiation and apoptosis of C2C12 myoblasts,and further explore the mechanism of melatonin inhibiting muscle development through high-throughput sequencing.The specific contents and results of the study are as follows:1 Effects of different concentrations of melatonin on proliferation,differentiation and apoptosis of C2C12 myoblastsTo investigate the effect of melatonin on skeletal muscle cells proliferation,different concentrations of melatonin were used to treat C2C12 myoblasts.The result shows that melatonin increases cell viability in a dose-and time-dependent manner.Melatonin 1mM significantly promoted C2C12 myoblasts proliferation from 48 to 96 h(48 h,P<0.05;72 h,P<0.01;96 h,P<0.01).Crystal violet staining verified the role of melatonin in promoting cell proliferation related to ImM melatonin dose.After treating C2C12 myoblasts with ImM melatonin for 48 h,we found the intensity of violet-staining cells increased by 20%.The mRNA levels of Ki67 in group after treatment with ImM melatonin for 48 h were significantly higher than control group.Immunofluorescence analysis of Ki67 showed that treatment group contained ImM melatonin significantly enhanced the proportion of Ki67-positive cells compared to control group for 48 h.To investigate the effect of melatonin on muscle differentiation,different concentrations of melatonin were used to treat C2C12 myoblasts.Interestingly,we found melatonin 0-0.5mM showed no effect on cell differentiation(all NS.),but melatonin 1,2mM inhibited skeletal muscle cells differentiation with decreased mRNA levels of myogenic differentiation markers Myogenin(1mM and 2 mM,P<0.001),eMyhc(1mM and 2 mM,P<0.001)and fusion related markers Cahn-15(2 mM,P<0.01),Capn-1(2 mM,P<0.01),Cav3(0.05-0.5 mM,P<0.05;1mM and 2 mM,P<0.001),as well as Des(1 mM,P<0.05 and 2 mM,P<0.01)for 4 days.Melatonin 2mM has no effect on cell proliferation from 0 to 24 h(all NS.),but inhibited cell proliferation and caused death from 48 to 96 h(48 h,P<0.05;72 h,P<0.01;96 h,P<0.001).No significant apoptotic cells were found in 1mM melatonin by flow cytometry tests.These results suggest that melatonin ImM could promote skeletal muscle cells proliferation and inhibits their differentiation.2 Melatonin significantly reduces muscle differentiation both in C2C12 myoblasts and primary myoblastsInterestingly,morphological analysis showed that both C2C12 myoblasts and primary myoblasts treated with ImM melatonin have smaller and thinner myotubes than those not treated with melatonin.Immunofluorescence analysis of MyHC showed that the ability of C2C12 myoblasts fuse to myotube was significantly decreased in the melatonin-treated samples compared with the control group.Statistical analysis showed that C2C12 myoblasts treated with melatonin had fewer myotubes containing four or more nuclei.Moreover,primary myoblasts treated with melatonin had fewer myotubes compared with the control group.We never observed myotubes with more than four nuclei in the melatonin cultures,whereas～90%of myonuclei in control cultures were contained in myotubes with more than four nuclei.Immnofluorescence analysis of MyoG showed that melatonin-treated group had fewer MyoG-positive cells than those not treated with melatonin,and statistical analysis showed that the proportion of MyoG-positive cells treated with melatonin decreased 80%compared with the control group.The expression levels of myogenic and fusion related genes MyoG(P<0.001),eMyhc(P<0.001),Cav3(P<0.001),and Des(P<0.05)were significantly down-regulated,while the expression of MyoD and Myh8 showed no significant changes in C2C12 myoblasts.Consistent with mRNA levels,western blot analysis showed that melatonin significantly down-regulated the expression of MyoG and MyHC in C2C12 myoblasts,while the expression of MyoD showed no significant changes.Statistical analysis showed that the expression levels of MyoG and MyHC treated with melatonin were significantly decreased compared with the control group,while the expression levels of MyoD showed no significant changes.These results indicated melatonin treatment could inhibit skeletal muscle cells differentiation.3 Melatonin down-regulated the essential fusion pore components Myomaker and Myomixer in myoblastsTo investigate this phenotype,we next sought to understand how melatonin alters gene expression,through high-throughput sequencing of differentiated C2C12 cells treated with melatonin and those not treated with melatonin for 4 days.After comparing two groups based on the requirement of ’variable importance in the project p<0.001’,a total of 364 up-regulated genes and 774 down-regulated genes were identified.The gene ontology(GO)enrichment was developed to comprehensively depict characteristics of various genes and their products.More than 40%of the differentially expressed proteins belonged to the biological process,and the other two main categories(cellular component and molecular function)of these proteins consisted of the intracellular(79%),cytoplasm(64%)and protein binding(59%)proteins.Genes of myosin heavy chain family were significantly down-regulated in melatonin-treated C2C12 cells,and genes related to fusion were also significantly down-regulated in melatonin-treated C2C12 cells.Intriguingly,among the novel melatonin-induced targets revealed by RNA-seq were two genes essential for myoblast fusion pore formation:Myomaker(also called Tmem8c)and Myomixer(also called GM7325,Myomerger,and Minion).In addition,RNA-seq showed that other factors related to Wnt signaling pathway were induced at least twofold by melatonin in C2C12 myoblasts.These genes were frizzled class receptor 1(Fzdl),secreted frizzled-related protein 2(Sfrp2),WNT1 inducible signaling pathway protein 2(Wisp 2),protein kinase C alpha(Prkca),calciumlcalmodulin-dependent protein kinase Ⅱ alpha(Camk2a),porcupine homolog(Drosophila)(Porcn).The down-regulation of fusogenic genes Myomaker and Myomixer were also confirmed by quantitative real-time PCR(qRT-PCR)and western blot analysis.These results suggested that melatonin might function in modulating fusion pore assembly through Wnt signaling pathway.4 Melatonin phosphorylates GSK-3β at Ser9 to regulate myogenic differentiation,myomaker,and myomixerInterestingly,C2C12 myoblasts treated with melatonin had significantly reduced levels of pGSK-3β(Ser9),suggesting that melatonin treatment had elevated activation of GSK-3β.Immunofluorescence analysis clearly showed that cells treated with melatonin resulted in markedly reduced β-catenin,as compared with control.Next,we investigated if inhibition of GSK-3β with LiCl restores the differentiation of C2C12 myoblasts treated with melatonin.Inhibition of GSK-3β by LiCl promoted myotube formation in C2C12 myoblasts and rescued myotube formation in melatonin-treated C2C12 myoblasts.Quantification of the percentage of myonuclei in mono-versus multinucleated cells showed that Licl partially rescued the differentiation efficiency of melatonin-treated C2C12 myoblasts.LiCl treatment significantly increased the proportions of MyoG-positive cells both in control and melatonin-treated C2C12 myoblasts.The mRNA levels of differentiation and fusion related genes(MyoG,Myh8,eMyhc,Cav3,Des)were up-regulated by LiCl in C2C12 cells and were improved in melatonin-treated C2C12 cells.Interestingly,inhibition of GSK-3β by LiCl boosted both Myomaker and Myomixer mRNA levels in C2C12 Myoblasts,but only Myomaker was saved in melatonin-treated C2C12 myoblasts.The mRNA levels of Myomixer showed no significant changes after LiCl administration in melatonin-treated C2C12 myoblasts.Moreover,fluorescence images showed that inhibition of GSK-3 β by LiCl restored the accumulation of β-catenin in C2C12 myoblasts.Western blot analysis showed that inhibition of GSK-3β not only improved MyHC,MyoG,Myomaker,and Myomixer protein levels in C2C12 myoblasts,but also rescued those proteins other than Myomixer in melatonin-treated C2C12 myoblasts.Immunofluorescence analysis,western blot and real-time PCR results confirmed that inhibition of GSK-3β completely rescued the expression of differentiation marker genes(especially Myomaker)in melatonin treatment group,while the expression of Myomixer cannot be rescued by LiCl.These data demonstrated that melatonin regulates myogenic differentiation,Myomaker and Myomixer through GSK-3β.In our study,we demonstrated that melatonin inhibits muscle differentiation via reducing expression of myogenic transcription markers MyHC,MyoG,and fusogenic micropepptide Myomaker and Myomixer-Myomerger-Minion.Furthermore,we demonstrated that melatonin acts through Wnt/b-catenin pathway to regulate the expression of Myomaker and Myomixer.