Effect Of Kirrel1 Protein On Myoblast Differentiation And Fusion

Background Cell-cell fusion is essential for the development of multicellular organisms.Skeletal muscle is a unique tissue composed of many muscle fibers,each of which is the product of the fusion of hundreds or thousands of myoblasts.Myoblast fusion is critical not only for skeletal muscle formation,but also for muscle regeneration mediated by adult muscle satellite cells.In order for myoblast fusion to occur,the two fusion partners must recognize each other,adhere to their plasma membranes,open fusion pores to allow the exchange of substances within the cell,and eventually merge into one cell.In Drosophila embryos,there are two different types of myoblasts,founder cells(FCs)and fusion competent myoblasts(FCMs),which fuse together toform fusion muscles in a segmented repeat pattern.In FCs,two paralogs,Dumbfounded(Duf)also known as Kin-of-Irre(Kirre)and Roughest(Rst,also known as Irre C).In FCMs,Sticks and stones(Sns)acts as the main cell adhesion molecule(CAM),its function is partially compensated by its collateral homologous substance Hibris(Hbs).Similar to muscle-developing embryos,multinuclear Drosophila adult muscles are fused by FCMs expressing Sns-Hbs around the FCs expressing Duf-Rst.The mammalian homologous of Rst and Kirre is the Kirrel gene family,including Kirrel1,Kirrel2 and Kirrel3..Kirrel1 has two subtypes,Kirrel A and Kirrel B.The mammalian homologue of Sns is Nephrin gene,and our previous work has demonstrated that Nephrin is important for myoblast fusion in zebrafish.The process of fusion of skeletal muscle in Drosophila and zebrafish is well known,but little is known about mammalian myoblast fusion.Therefore,we propose examine the role of a Kirrel family member,Kirrel1 in skeletal myoblast differentiation and fusionObjective 1.To investigate the m RNA expression level of Kirrel A and Kirrel B during the differentiation and fusion of mouse skeletal muscle myoblast C2C12 and in vivo during the repair and reconstruction of injured muscle in mice;2.To investigate the effects of knocking down Kirrel1,Kirrel A and Kirrel B on the differentiation and fusion of mouse skeletal muscle myoblasts C2C12.Methods RT-PCR and RT-q PCR were used to detect the m RNA expression level of Kirrel A and Kirrel B during the differentiation and fusion of myoblast C2C12 and during muscle repair after CTX mouse injury.C2C12 cells were transfected with Control-,Kirrel A-,Kirrel B-and Kirrel1-sh RNA plasmids,and stable cell lines were established by drug selection,establishment of stable cell line was verified by RT-PCR,RT-q PCR and Western Blot.Immunofluorescence assay was used to detect the expression of myosin in Control-,Kirrel A-,Kirrel B-,Kirrel1-sh RNA cell lines on the Day2(D2)and Day 5(D5),and to calculate the fusion index;Western Blot was used to detect the expression of myosin and myogenin proteins in the above sh RNA cell lines during differentiation and fusion.(D0~D5)(myosin is mainly expressed in myotubes,reflecting the fusion competency of myoblasts;myogenin is a differentiation-promoting factor that initiates cell differentiation;Fusion index refers to the ratio of the number of nuclei in the myotubes to the total number of nuclei in the same microscopic field of view.)Results 1.RT-PCR and RT-q PCR results showed that-Kirrel A and Kirrel B m RNA expression levels were significantly increased during C2C12 cell differentiation and fusion.And during muscle reconstruction after cardiotoxin in mice.(p < 0.05,n = 3).2.Control-,Kirrel A-,Kirrel B-and Kirrel1-sh RNA expressing stable cell lines established and knocking down effects were verified.Myosin immunofluorescence results showed that the fusion index of Kirrel A-,Kirrel B-,and Kirrel1-sh RNA was lower than that of the control group in D2 and D5 of C2C12 cells,and it was statistically significant(p < 0.05,n = 6).3.Control-,Kirrel A-,Kirrel B-,Kirrel1-sh RNA expression plasmids were transfected into the C2C12 cell line.Myosin Western Blot results showed that the expression levels of D0~D5 Kirrel A-,Kirrel B-,Kirrel1-sh RNA myosin protein in C2C12 cells were lower than those in the control group(p < 0.05,n = 3);myogenin protein expression level was different from the control group but it was not statistically significant.Conclusions 1.Kirrel A and Kirrel B are involved in the process of myoblast fusion and muscle damage reconstruction.2.Knockdown of Kirrel1,Kirrel A,and Kirrel B inhibits C2C12 cell fusion.