Nesprin-1 Is Essential For Mouse Embryonic Stem Cells Differentiation Into Cardiomyocytes

PART ONENesprin-1 is essential for proper C2C12 myoblast differentiation Objective To investigate the role of nesprin-1, a novel spectrin repeat containing scaffolding protein, in skeletal muscle cell differentiation.Methods1.Differentiated the C2C12 myoblasts into myotubes.2.During in vitro differentiation of C2C12 myoblasts into myotubes, changes in nesprin-1 gene expression and sub-cellular localization were detected using Western blotting and immunofluorescence.3.RNA interference was used to reduce nesprin-1 protein levels to further investigate the importance of nesprin-1 in C2C12 myoblast differentiation.Results:1. Western Blotting demonstrated that the nesprin-1 isoform profile was altered in C2C12 myoblast differentiation.2.The immunofluorescence shew that subcelluar fractionation revealed changes in subcellular compartmentalization of nesprin-1.3.Compared with the control group, the expression level of nesprin-1 was reduced in nesprin-1 SiRNA group.4.Compared with the control group, myotube formation was dramatically reduced after nesprin-1 depletion (P<0.001).Conclusion: Nesprin-1 plays important roles in skeletal muscle cell differentiation. Nesprin-1 isoforms may perform specific functions during this process of C2C12 myoblast differentiation as well as have different roles in the cytoplasm and the nucleus of these cells. PART TWO Nesprin-1 is essential for mouse embryonic stem cells differentiation into cardiomyocytesObjective: To investigate the role of nesprin-1in mouse embryonic stem cells differentiation into cardiomyocyte.Methods:1. In vitro ,differentiated the mouse embryonic stem cells into cardiomyocytes by hanging up culture salvia miltorrhiza and 5-azacytidine as inducer.2. Changes in nesprin-1 gene expression was detected using Western blotting and immunofluorescence.3.RNA interference was used to reduce nesprin-1 protein levels to further investigate the importance of nesprin-1 in mouse embryonic stem cells differentiation , groupⅠ(target sequence AAAGCCAAGCACGCAACTA), groupⅡ(target sequence GGGAACCAACAGTGAGATT), groupⅢ(target sequence ACCAGGACATTGCGTACTA), and groupⅣcontrol group.Results:1.The nesprin-1 isoform profile was altered in mouse embryonic stem cells differentiation.2.Compared with the control group, myotube formation was dramatically reduced after nesprin-1 depletion.3.The rates of differentiation of the four groups were (17.78±1.92)%,(36.67±3.34)%,(44.42±5.08)% , (77.78±1.92)%; respectively .The rate of differentiation of groupⅣwas higher than other RNAi groups and the rate of differentiation was significantly increased(p<0.05).Conclution:This is the first report that nesprin-1 plays important roles in mouse embryonic stem cells differentiation into cardiomyocyte. Nesprin-1 isoforms may perform specific functions during this process of mouse embryonic stem cells differentiation ,and some of them may be much more important.