The Effect Of Bves On P19CL6 Cell Differentiation And Cardiomyocytes Proliferation And Its Mechanisms

Objective : We investigated the effect of BVES on P19CL6 cardiomyocytes differentiation and cardiomyocytes proliferation and its functional signaling pathway, providing a basis to the pathogenesis of congenital heart disease.Methods: P19CL6 cell was considered as a useful in vitro model to study cardiomyocytes differentiation which was induced differentiation into mature cardiomyocytes supplemented with 1%DMSO. Overexpression of BVES effectively upregulated P19CL6 cell endogenous BVES expression. The si RNA of BVES effectively inhibited P19CL6 cell endogenous BVES expression. q RT-PCR was performed to detect cardiac TF(Gata4,Nkx2.5,Tbx5)and cardiac gene(MYH6,MYL7)m RNA level expression, western blotting was performed to detect qualitative protein level expression of mature cardiomyocytes cardiac troponin T(c Tn T)、BVES and Rho A protein expression, Immunofluorescence staining was performed to detect quantitative protein level expression of MF20 and Actinin, CCK-8 was performed to test Cell viability,Ed U was used to detect DNA systhesis, Flow cytometry analysis was performed to detected the cell cycle.Results:P19CL6 was successful induced differentiation into mature cardiomyocytes, immune staining showed that Actinin protein position cardiomyocytes was increased, q RT-PCR showed that cardiac TF(Gata4, Nkx2.5, Tbx5)and cardiac gene(MYL7, MYH6)m RNA expression level was induced during P19Cl6 cardiomyocytes differentiation. BVES protein expression was induced during cardiomyogenesisdifferentiation in P19CL6 model. The cardiomyocytes percentage was shrinked in the condition of BVES-overexpressing and enlarged of BVES deficient during the P19CL6 differentiation which was determined by the fluorescence area of MF20. q RT-PCR showed that the m RNA expression of cardiac TF(Gata4, Nkx2.5, Tbx5)and cardiac gene( MYH6, MYL7) were significantly decreased by up-regulation of BVES in the process of P19CL6 differentiation, BVES deficiency had no effect on the cardiac TF(Gata4, Nkx2.5, Tbx5), however, increased cardiac gene(MYH6, MYL7)m RNA expression. western blotting showed that over-expression of BVES in P19CL6 cells inhibited the expression of c Tn T, one of cardiac markers, induced by 1% DMSO at day 8, however, BVES deficiency promoted c Tn T expression on the differentiation of P19CL6 cells. Over-expressing of BVES inhibited cardiomyocytes proliferation which derived P19CL6 differentiation proved by cell cycle, Ed U incorporation assay and CCK8 assay, however, BVES deficient did not influence cell proliferation. Knockdown of Rho A reversed the effect of BVES on P19CL6 differentiation, but not reversed the effect of BVES on cardiomyocytes proliferation.Conclusions:Our findings suggest that BVES normal expression is critical to cardiomyocytes differentiation and proliferation. Rho A signaling pathway was demonstrated as a functional downstream of BVES in the process of P19CL6 cardiomyocytes differentiation.