Canonical Wnt Signaling in Pathogenesis and Rescue of ARVC Phenotype

描述（由申请人提供）：总体目的是描述致瘤性右心室心肌病（ARVC）的分子发病机制，以预防、减弱和逆转其演变的表型。ARVC是一种罕见的遗传性疾病，由至少五种桥粒蛋白突变引起。ARVC患者通常表现为心律失常、心源性猝死（SCD）和晚期全身心力衰竭。SCD是约25%病例的首发症状。ARVC的表型标志是与心肌细胞凋亡相关的心肌纤维脂肪细胞浸润。ARVC的分子发病机制和纤维脂肪病的细胞起源尚不清楚。ARVC作为一种桥粒蛋白突变疾病，其遗传基础的阐明为阐明其表型的发病机制提供了机会。基于桥粒蛋白和犰狳家族成员斑珠蛋白（PG）与经典Wnt信号转导蛋白和犰狳蛋白2-catenin在结构和功能上的相似性，我们证实PG（又称3-catenin）和2-catenin之间的竞争性相互作用抑制经典Wnt信号转导，导致肌细胞凋亡和脂肪生成增强。为了验证这一假设，我们产生了心脏限制性桥粒斑蛋白（DP）缺陷小鼠和siRNA介导的DP缺陷心房肌细胞。我们发现DP缺乏导致PG核转位，经典Wnt信号抑制，心肌细胞凋亡，纤维化和脂肪形成。我们建议确定心脏中过量脂肪细胞的细胞来源，描绘抑制经典Wnt信号传导的分子基础，并通过激活经典Wnt信号传导来防止表型。目标是：1.通过体内基因定位和共培养研究，确定ARVC中过量脂肪细胞的细胞来源;通过在转基因小鼠心脏中表达可滴定水平的突变桥粒蛋白来确定抑制的经典Wnt信号传导是否是ARVC发病的共同机制; 3.通过研究蛋白质-蛋白质相互作用以及PG对Wnt核心蛋白复合物有效组装的影响，阐明核PG抑制经典Wnt信号传导的机制。通过条件性激活抗降解2-连环蛋白和药理学激活激活经典Wnt信号传导，在体内和体内挽救ARVC表型，从而满足Koch因果关系假设。在转基因小鼠、转导的心肌细胞和肌成纤维细胞中的研究被设计为描绘每个目的的组成部分。该结果可以阐明桥粒ARVC的分子发病机制，并为治疗和预防人类ARVC找到新的分子诊断标志物和新的药理学靶点。我们建议研究人类心肌疾病ARVC的分子基础，并在转基因小鼠模型中预防其发展。ARVC是年轻人猝死的重要原因。目前，对ARVC没有特异性的治疗或预防。

DESCRIPTION (provided by applicant): The overall objective is to delineate the molecular pathogenesis of arrhythmogenic right ventricular cardiomyopathy (ARVC) in order to prevent, attenuate and reverse its evolving phenotype. ARVC is an uncommon genetic disease caused by mutations in at least five desmosomal proteins. Patients with ARVC typically present with cardiac arrhythmias, sudden cardiac death (SCD) and in advances stages with global heart failure. SCD is the first manifestation of the disease in approximately 25% of the cases. The phenotypic hallmark of ARVC is fibro-adipocytic infiltration of the myocardium that occurs in conjunction with myocyte apoptosis. The molecular pathogenesis of ARVC and the cellular origin of fibro-adiposis are unknown. Elucidation of the genetic basis of ARVC, as a disease of mutant desmosomal proteins, has provided the opportunity to delineate the pathogenesis of its phenotype. Based on structural and functional similarities between plakoglobin (PG), a desmosomal protein and a member armadillo family, and 2-catenin, the signal transducer of the canonical Wnt signaling and also an armadillo protein, we posit competitive interactions between PG (aka 3-catenin) and 2-catenin suppresses the canonical Wnt signaling, leads to myocyte apoptosis and enhanced adipogenesis. To test the hypothesis, we generated cardiac-restricted desmoplakin (DP) deficient mice and siRNA-mediated DP-deficient atrial myocytes. We showed DP-deficiency led to nuclear translocation of PG, suppression of the canonical Wnt signaling, myocyte apoptosis, fibrosis and adipogenesis. We propose to identify the cellular origin of excess adipocytes in the heart, delineate the molecular basis of suppressed canonical Wnt signaling and prevent the phenotype by activating the canonical Wnt signaling. The aims are: 1. To identify the cellular origin of excess adipocytes in ARVC through in vivo genetic fate-mapping and co-culture studies; 2. To determine whether suppressed canonical Wnt signaling is a common mechanism for the pathogenesis of ARVC by expression of titratable levels of mutant desmosomal proteins in transgenic mice hearts; 3. To delineate the mechanism by which nuclear PG suppresses the canonical Wnt signaling by studying protein-protein interactions and the effects of PG on effective assembly of Wnt core proteins complex; 4. To rescue the ARVC phenotype in vivo and in vivo by activating the canonical Wnt signaling through conditional activation of degradation-resistant 2-catenin and pharmacological activation and hence, to fulfill the Koch's postulates of causality. Studies in genetically modified mice, transduced cardiac myocytes and myofibroblasts are designed to delineate the components of each aim. The results could elucidate the molecular pathogenesis of desmosomal ARVC and lead to identification of new molecular diagnostic markers and novel pharmacological targets for the treatment and prevention of ARVC in humans. Project Narrative we propose to study the molecular basis of human heart muscle disease ARVC and to prevent its development in genetically modified mouse models. ARVC is an important cause of sudden death in the young. Currently, there is no specific treatment or prevention for ARVC.