RNA Interference Of Desmocollin-2 Gene And Its Effects On Cardiac Muscle Differentiation Of P19 Cells

Objective To investigate the effects of down-regulation of DSC2 gene expression on the ultrastructure, apoptosis, adipogenesis and fibrogenesis of cardiac myocyte differentiated from P19 cells. Methods A set of DSC2-specific siRNAs and control siRNA (siNC) were synthesized and cloned into pGPU6/GFP/Neo vector. The recombinant plasmids (ShDSC2 and ShNC) were transfected into P19 cells, and the positive clones were selected with G418. The efficiency of DSC2 knock down on the levels of mRNA and protein expression were determined by RT-PCR and Western blot. P19 cells were induced to differentiate into cardiac myocyte. The changes of cell apoptosis and ultrastructure were evaluated by flow cytometry and transmission electron microscope (TEM), respectively. RT-PCR was used to measure the expression of adipogenesis and fibrogenesis related genes on mRNA level. Results Five recombinant ShDSC2 plasmids were constructed successfully and transfected into P19 cells, and stable cell lines were established by RNA interference. Compared with the control group, the expression levels of DSC2 on mRNA and protein in ShDSC2-613 group were decreased by 69.47%(P<0.01) and 65.62%(P<0.01), respectively. P19 cells were successfully induced to differentiate into cardiomyocytes. In ShDSC2-613 group, TEM showed that fat droplet, cellular vacuoles, swollen mitochondria and disappeared cristae and Flow cytometry further demonstrated that the apoptosis was increased. A marked increase in expression levels of two major regulators of adipogenesis, namely PPAR-γ, and C/EBP-α, and their target gene adiponectin, were also observed. The expression levels of procollagen genes Collal, Colla2 and Col3al were increased remarkably. Conclusions We established DSC2-deficient P19 cell line, which exhibited increased apoptosis, adipogenesis and fibrogenesis, thus recapitulating the phenotype of human arrhythmogenic right ventricular cardiomyopathy. This cellular model could provide the opportunity to explore the pathogenesis and novel therapeutic studies of ARVC in vitro.