The Molecular Mechanism And New Diagnosis Marker Of Arrhythmogenic Right Ventricular Cardiomyopathy

Background:Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC) is a genetic disease over half patients caused by mutations in desmosomal proteins. The phenotypic hallmark of ARVC is fibroadipocytic replacement of cardiac myocytes, which is a unique phenotype selected involved in right ventricle with a yet-to-be-defined molecular mechanism. Canonical Wnt signaling not only regulates cell proliferation and right ventricle formation but also is a major transcriptional switch regulator of myogenesis versus adipogenesis. Animal models of ARVC showed suppressed canonical Wnt signaling by nuclear plakoglobin (PG) is associated With ARVC. Furthermore, the molecular mechanism of ARVC remain not fully understood and the diagnosis of ARVC be challenging due to lack distinguish makers and optional biopsy sites.Objectives:To develop the new molecular mechanism and diagnosis makers of ARVCMethods:Whole exome sequencing selected ARVC patients with desmosome gene mutations and ARVC patients with extra-desmosome gene mutations to involved in this study. Electron Microscopy detect the ultrastructure of the cardiomyocytes. Expression of PG in nuclear and cytosol detected by immunoblotting and immunofluorescence. Co-immunoprecipitation coupled with mass spectrometry (IP/MS) screened the protein interacted with TCF7L2. Immunoblotting and Immunofluorescence detected β-catenin, phosphorylation β-catenin and GSK-3β, IP/MS detect the proteins bingding with phosphorylation P-catenin. RT-PCR detected the target genes in Wnt signaling. Immunofluorescence and Flow cytometry detected transitional from myogenic to an adipogenic state fate CPCs in five different biopsy locations(right ventricular outflow tract, right ventricular antero-apical, inferior-sub-tricuspid, right-sided ventricular septum (D) and the left ventricular free wall). Comparative three diagnosis methods(Ⅰ, Assessment of residue myocardium; Ⅱ, immunohistochemical analysis of PG; Ⅲ Islet1+/C/EBP-α+cells) in the heart tissue bank including21ARVC,53other cardiornyopathy and8nornal hearts to test the diagnosis ability.Results:ARVC patients with desmosome gene mutations (DSG2,DSG2,PKP1, PKP3/DSG2,n=4) and ARVC patients with extra-desmosome gene mutations (TGFβ3,RYR2,TGFβ3,TMEM43,n=4). Electron Microscopy showed that desmosome destroyed in patients with desmosome gene mutations. PG translocated into nuclus interacted with TCF7L2and suppresses canonical Wnt/β-catenin signaling in ARVC patients with desmosome gene mutation carriers. The key molecule of wnt/β-catenin signaling pathway have obviously phosphorylation also caused the downstream of this pathway inhibited in extra-desmosome gene mutation carriers.The "final pathway" of ARVC was suppresses canonical Wnt/β-catenin signaling pathway, which caused he second heart field cardiac progenitor cells (CPCs) inducd a transcriptional switch to adipogenesis in ARVC. AUC (area under curve) of ROC (receiver operating characteristic curve), sensitivity, specificity, positive predictive values and negative predictive values of detecting Islet1+CEBP/α+cells, assessment of residue myocardium and immunohistochemical PG analysis in most accurate sites for ARVC diagnosis (right ventricular outflow tract) were0.912,0.86,0.97,0.90,0.95;0.766,0.76,0.77,0.53,0.90;and0.731,0.91,0.58,0.41,0.94; respectively. Highest AUC was obtained using tissues from right ventricular outflow tract regardless the diagnostic method and Islet l+/C/EBP-α+cells,which was a highly sensitive and specific diagnostic marker for ARVC.Conclusions:Wnt/β-catenin signaling pathway repressed caused the second heart field cardiac progenitor cells (CPCs) inducd a transcriptional switch to adipogenesis in ARVC, which lead to the right ventricle were involved by adipocytes.The transitional from myogenic to an adipogenic state fate CPCs----Islet1+/C/EBP-α+cells,which was a highly sensitive and specific diagnostic marker for ARVC. Right ventricular outflow tract is the optimal site for endomyocardial biopsy. Background:Isolated Arrhythmogenic Left Ventricular Cardiomyopathy (IALVC) is characterized pathlogically by fibroadipose of left ventricle (LV), while sparing the right side of the heart. The pathological features and molecular mechanism of IALVC remain not fully understood.Objectives:To depict the distinctive pathological features and molecular mechanism of IALVC.Methods:4hearts pathological diagnosis with IALVC underwent heart transplantation due to the end-stage heart failure and4normal hearts,4ARVC hearts with LV inlolvement as control were involved in this study. Masson staining detected the pathological features and coimmunoprecipitation showed PG (plakoglobin) and β-catenin compete for nuclear localization; RT-PCR detected the target genes of Wnt signaling; Immunofluorescence and Flow cytometry detect cells coexpressed adipogenic transcription factors and the epicardial progenitors markers.Results:Masson staining found IALVC have instinctive pathogenesis that fibroadipose developing from the outer epicardial myocardium to the inner endocardium. myocardium was replaced by mature fibrofatty tissues completely. However, the remaining myocardial tissue became island-like and its structure was disordered The LV of ARVC with LV involved showed that remnant cardiac muscle was located sporadically in fibrofatty tissues. Immunofluorescence and Immunoblotting can detect the Wtl express in IALVC tissues but rare in LV of ARVC. Furthermore we served the desmosomal proteins with signal functions PG (plakoglobin) reduced. Subcellular protein fractionation and immunofluorescence showed PG nuclear localization of in IALVC. Coimmunoprecipitation showed PG and β-catenin compete for nuclear localization, binding to Tcf/Lef transcription factors, which caused the suppression of the canonical Wnt signaling. This promotes adipogenesis but inhibit epithelial/endothelial to mesenchymal transition (EMT) and myogenesis in left ventricular myocardium of IALVC. Immunofluorescence and Flow cytometry found that cells coexpressed adipogenic transcription factors and the epicardial progenitors markers wtl (cells in transition from a myogenic to an adipogenic fate) in human myocardium from patients with IALVC.Conclusions:Adipocytes in IALVC at least in part, originate from the wt1+epicardial progenitors, which switch to an adipogenic fate because of suppressed canonical Wnt signaling by nuclear plakoglobin. Background:Conventional Coronary angiography (CCA) has been considered as a "gold standard" for the diagnosis of coronary artery diseases (CAD), however its diagnostic accuracy is still unknown.Objectives:To test the diagnostic accuracy of CCA in the real world.Methods:From July2004and December2011,97patients underwent CCA before heart transplantation in Fuwai hospital. A "head-to-head comparison" study was conducted to examine the diagnostic accuracy of CCA as compared with that of pathological coronary artery anatomy (PCA) in patient,vessel and segment based level.Results:As confirmed by PCA,44(45.4%) patients had CAD. The patient-based diagnostic accuracy evaluation showed that the AUC of CCA for detecting≥50%stenosis was0.91, with a sensitivity of91%and a specificity of93%. A per-vessel analysis of291vessels yielded an AUC of0.79. The AUC was0.88for proximal and middle segments and was0.62for distal segments. The patient-and vessel-based evaluations showed similar diagnostic accuracy of CCA in detecting≥75%stenosis. Per-segment evaluation found CCA was more accurate for detecting≥50%and≥75%stenosis in proximal and middle segments than distal segments and the diagnosis ability decreased in more serve stenosis segments and more complex lesions. Disease complex was confirmed by "anatomic" SYNTAX Score found that CCA have a poor performance in score>33group (P=0.97).Conclusions:The accuracy of CCA is quite high in detecting coronary artery stenosis in patients-and vessels-based level. However, the diagnosis ability decreased in more serve and complex lesions, especially for distal segments.