Canonical Wnt Signaling in Pathogenesis and Rescue of ARVC Phenotype

描述（由申请人提供）：总体目标是描述心律失常性右室心肌病（ARVC）的分子发病机制，以预防、减弱和逆转其进化表型。ARVC是一种罕见的遗传性疾病，由至少五种桥粒体蛋白突变引起。ARVC患者通常表现为心律失常、心源性猝死（SCD）和晚期整体心力衰竭。在大约25%的病例中，SCD是该病的第一表现。ARVC的表型特征是纤维脂肪细胞浸润心肌，并伴有心肌细胞凋亡。ARVC的分子发病机制和纤维脂肪的细胞起源尚不清楚。ARVC作为一种桥粒蛋白突变的疾病，其遗传基础的阐明为描述其表型的发病机制提供了机会。基于plakhemoglobin (PG)（一种桥胞体蛋白，也是犰狳家族成员）和2-catenin（典型Wnt信号的信号换能器，也是一种犰狳蛋白）在结构和功能上的相似性，我们假设PG（又名3-catenin）和2-catenin之间的竞争性相互作用抑制了典型Wnt信号，导致肌细胞凋亡和脂肪生成增强。为了验证这一假设，我们制造了心脏限制性桥蛋白（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的核易位，典型Wnt信号的抑制，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起源，描述被抑制的典型Wnt信号的分子基础，并通过激活典型Wnt信号来预防表型。目标是：1。通过体内遗传命运定位和共培养研究，确定ARVC中过量脂肪细胞的细胞起源；2. 通过在转基因小鼠心脏中表达可测定水平的突变桥粒蛋白来确定被抑制的典型Wnt信号是否为ARVC发病的共同机制；3. 通过研究蛋白-蛋白相互作用和PG对Wnt核心蛋白复合物有效组装的影响，揭示核PG抑制典型Wnt信号传导的机制；4. 通过条件激活抗降解2-catenin和药理激活激活典型Wnt信号，从而在体内和体内挽救ARVC表型，从而满足Koch的因果关系假设。在转基因小鼠中，转导心肌细胞和肌成纤维细胞的研究旨在描述每种目标的组成部分。研究结果有助于阐明桥粒ARVC的分子发病机制，为人类ARVC的治疗和预防提供新的分子诊断标记和新的药理靶点。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是年轻人猝死的一个重要原因。目前，没有针对ARVC的具体治疗或预防方法。

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.