Experimental Study Of ARVC Induced By DSG2 Gene Defect

Part I: Silencing of Desmoglein-2 increases gene expression of adipogenic factors in adipocytes differentiated from P19 cellsBackground : The pathological hallmark of arrhythmogenic right ventricular cardiomyopathy(ARVC) is progressive replacement of cardiac myocytes by fibro-adipocytes. Mutations in genes encoding desmosome proteins are responsible for at least 50% of the ARVC cases. Genetic fate mapping identifies second heart field progenitor cells as a source of adipocytes in arrhythmogenic right ventricular cardiomyopathy. A recent study has suggested that overexpressing PG in cardiac progenitor cells could increase nuclear localization of plakoglobin(PG) which results in enhanced adipogenesis and increased levels of adipogenic factors. In our previous study, we found the downregulation of Desmoglein-2(DSG2) in HL-1 cells lead to markely increased m RNA expression of adipogenic related genes(Adiponectin, PPAR-γ, C/EBP-α) as well as procollagen genes(Col1a1, Col1a2, Col3a1) and elevated apoptosis, recapitulating the molecular pathogenesis of human ARVC. However, it remains unclear whether DSG2 is involved in differentiation of cardiac progenitor cells to adipocytes in ARVC.Objective:To investigate the feasibility and effectiveness of the 96-well plate suspension method for differentiation of P19 cells into adipocytes in vitro and determine the effects of DSG2 deficiency on lipogenic gene expression in adipocytes differentiated from P19 cells.Methods:P19 cells were cultivated in aggregates termed embryoid bodies(EBs) in the 96-well plate containing a thin layer of low-gelling temperature agarose for 7 days with the cultivation medium, supplemented with all-trans retinoic acid(at RA) between the 2nd and the 5th day. The EBs were transferred into culture dishes and cultivated for another 20 days in the presence of insulin and triiodothyronine(T3). Oil-Red-O staining was applied to indicate the generation of adipocytes. Additionally, gene expression of adiponectin, PPAR-γ as well as C/EBP-α was detected by quantitative PCR(q PCR) on the 12 th, 17 th, 22 nd, 27 th day. StableSh DSG2 P19 cell lines were established by transfection of recombinant plasmids p GPU6/GFP/Neo-Sh DSG2(Sh DSG2) with geneticin(G418) selection. The anti-DSG2 effect was verified by q PCR. Furthermore, adipogenic genes expression was measured by q PCR in adipocytes derived from stable P19 cell with optimal suppression effect of DSG2 at the end of adipogenesis phase.Results:The 96-well plate suspending culture facilitated the formation of the EBs in similar size. At the end of this period, part of cells present in the EBs outgrowth formed adipocyte colonies, which were characterized by round shape and small lipid droplets scattered throughout cytoplasm. Adipocyte-like cells were stained with Oil-Red-O for fat droplets. Meanwhile, q PCR results showed that the expression levels of PPAR-γ, C/EBP-α and adiponectin were increased to 50.57 times(P < 0.01), 21.65 times(P < 0.01) and 168.89 times(P < 0.01), respectively, compared with undifferentiated cells. After DSG2 si RNA treatment and selection, the Sh DSG2-1373 P19 cell line was established, which DSG2 m RNA expression decreased by 68.06%(P < 0.01), compared with the negative control. After induction of adipogenesis, as compared with the control group, the m RNA levels of PPAR-γ, C/EBP-α and adiponectin in the Sh DSG2-1373 group were strongly increased by 16.24-fold(P < 0.01), 7.67-fold(P < 0.01), 29.05-fold(P < 0.01), respectively.Conclusions:P19 cell-derived EBs formed in 96-well plate suspension could undergo adipocyte differentiation in vitro. Suppression of DSG2 inducing higher expression level of adipogenic factors suggested that DSG2 may be crucial for adipogenesis in ARVC.Part II: The progressive changes of phenotypes in cardiac-specific DSG2-F536 C transgenic miceBackground: Patients with arrhythmogenic right ventricular cardiomyopathy(ARVC) in one extended Chinese family present abnormal 12-lead electrocardiograph(ECG) and prominent changes in echocardiography. Direct sequencing of the five desmosomal genes(JUP, DSP, PKP2, DSG2 and DSC2) was performed in this kindred group and one novel DSG2 missense mutation(c.1592T>G) was identified, which was predicted to replace a cysteine with an alanine at position 531. Then the cardiac-specific DSG2-F536 C overexpression transgenic(Tg) mice was established, and Tg mice > 6-month old showed progressive reduction of ejection fraction as well as fractional shortening and dense fibrosis with collagen bundle formation in the left ventricle(LV), recapitulating the typical features of ARVC. These observations suggested DSG2-F531 C may be a novel ARVC pathogenic mutation. However, the pathogenic potentials of this mutation in the development of ARVC remain unknown.Objective: To compare the changes in histological features and expression of connexin43 at the intercalated discs(IDs) between 15-week-old and 30-week-old Tg mice.Methods: Cardiac-specific DSG2-F536 C transgenic mice(M35) were taken as Model group, provided cardiac-specific DSG2 transgenic mice(W39) as Control group. Lesions of fibro replacement of the myocardium were detected by Masson’s trichrome straining. Distribution and expression at IDs of connexin43 was evaluated by immunohistochemistry.Results: Fibrous type lesions were confined to LV free wall, in association with little fatty tissue, right ventricle and ventricular septum involvement was not obvious. Little collagen fiber in myocardial interstitial with regular arrangement and structured cell morphology was observed in the hearts of 15-week-old W39 mice, while distributed fibrosis was identified in those of 15-week-old M35 mice. With increasing age, the hearts of 30-week-old W39 mice contained few collagen deposits and extensive normal myocardial cells while those of M35 mice showed evidentmyocyte loss with fibrous replacement, surviving myocytes embedded between the collagen bundles, across the full thickness of the LV free wall. In control group, there was no statistical difference in ventricular collagen volume fraction(CVF) between 15-week-old mice and 30-week-old mice. But CVF in model group was significantly higher than age-matched control group and increased significantly in comparison with control group. Immunohistochemical analysis of myocardial samples showed the signal for connexin43 was presented at IDs and absent from the intracellular space as well as lateral membranes, and expression levels were markedly reduced compared with adult B6CBF1 mice, but comparable between model group and control group.Conclusions: The introduction of the DSG2-F536 C mutation into mice resulted in an age-dependent ARVC phenotype, demonstrating that DSG2-F531 C related ARVC was a progressive cardiomyopathy.