Canonical Wnt Signaling in Pathogenesis and Rescue of ARVC

描述（由申请人提供）：我们的长期目标是描述遗传性心肌病的分子遗传学和发病机制，包括致瘤性右心室心肌病（ARVC）。ARVC的特征是纤维脂肪细胞取代心肌细胞，导致心律失常、心源性猝死和心力衰竭。ARVC目前没有有效的治疗方法，除了心脏移植。ARVC主要由编码桥粒蛋白的基因突变引起。桥粒沿着粘附连接（AJs）和差距连接构成闰盘（ICD）。虽然传统上被认为是细胞-细胞粘附结构，但ICD已经成为接触介导的细胞信号传导的主要调节剂。因此，ICD涉及调节Hippo通路，其在细胞分化和增殖中起重要作用。初步数据显示，ARVC患者心脏中的ICD发生了广泛的分子重塑。这些变化与Hippo激酶级联的激活有关，Hippo激酶级联通过磷酸化效应分子雅普，通过TEAD抑制转录。Hippo通路的激活与人类心脏中经典Wnt信号传导的抑制相关，这与ARVC的发病机制有关。我们证实Hippo通路的激活在ARVC中起致病作用。在目标1中，在进一步加强初步数据后，我们将利用肌细胞和小鼠模型描述Hippo通路激活的负责机制。初步数据表明，神经纤维蛋白1（NF 2），又名Merlin，是一种上游Hippo激酶，在ARVC模型中在ICD处被激活。通过功能获得和功能丧失（GoF和LoF）方法，Hippo通路将在肌细胞和小鼠模型中失活，并将确定对心脏结构和功能、基因表达和纤维脂肪形成的拯救作用。在目标2中，我们将描述在活跃的Hippo通路的背景下，经典Wnt信号传导的致病作用及其抑制机制。经典Wnt和Hippo分子的GoF和LoF靶向将用于确定对肌细胞和小鼠模型的表型效应。在目标3中，我们将确定ARVC中纤维脂肪细胞的细胞来源，并确定Hippo和Wnt通路的致病作用。这些方法是遗传命运作图和敲入报告子追踪，后者用于旁分泌效应。基于初步数据，候选细胞类型是纤维脂肪细胞祖细胞、心外膜细胞、周细胞， 和肌源性谱系。将测试突变肌细胞对上述细胞加上驻留前脂肪细胞的旁分泌作用。将通过LoF和GoF研究确定Hippo途径在其分化为纤维脂肪细胞中的致病作用。这些拟议的研究预计将为ARVC的分子发病机制提供深入了解，并有助于为目前尚无有效治疗方法的致命疾病确定新的治疗靶点。

DESCRIPTION (provided by applicant): Our long-term objective has been to delineate the molecular genetics and pathogenesis of hereditary cardiomyopathies, including arrhythmogenic right ventricular cardiomyopathy (ARVC). ARVC is characterized by fibro-adipocytes replacing cardiac myocytes, which leads to cardiac arrhythmias, sudden cardiac death and heart failure. There is no effective therapy for ARVC, except for heart transplantation. ARVC is primarily caused by mutations in genes encoding desmosome proteins. Desmosomes along with the adherens junctions (AJs) and the gap junctions constitute the intercalated discs (ICDs). While conventionally recognized as cell-cell adhesion structures, ICDs have emerged as major regulators of contact- mediated cell signaling. Accordingly, ICDs are implicated in regulating the Hippo pathway, which plays an important role in cellular differentiation and proliferation. The Preliminary data show extensive molecular remodeling of the ICDs in the human hearts with ARVC. The changes are associated with activation of the Hippo kinase cascade, which by phosphorylating YAP, the effector molecule, suppresses transcription through TEAD. Activation of the Hippo pathway is associated with suppression of the canonical Wnt signaling in the human hearts, which is implicated in the pathogenesis of ARVC. We posit that activation of the Hippo pathway plays a pathogenic role in ARVC. In aim 1, upon further strengthening of the preliminary data, we will delineate the responsible mechanisms for activation of the Hippo pathway utilizing myocyte and mouse models. Preliminary data points to Neurofibromin 1 (NF2), aka Merlin, as an upstream Hippo kinase that is activated at the ICDs in the ARVC models. Through gain- and loss-of-function (GoF and LoF) approaches, the Hippo pathway will be inactivated in myocytes and mouse models and the rescue effects on cardiac structure and function, gene expression and fibro-adipogenesis will be determined. In aim 2, we will delineate the pathogenic role of the canonical Wnt signaling and the mechanisms responsible for its suppression, in the context of active Hippo pathway. GoF and LoF targeting of the canonical Wnt and Hippo molecules will be used to determine the phenotypic effects on myocytes and mouse models. In aim 3, we will identify the cellular origin of fibro-adipocytes in ARVC and determine the pathogenic role of the Hippo and Wnt pathways. The approaches are genetic fate mapping and knock in reporter tracing, the latter for a paracrine effect(s). The candidate cell types, based on the Preliminary data, are fibro-adipocyte progenitors, epicardial cells, pericytes, and myogenic lineage. The paracrine effects of mutant myocytes on the above cells plus resident pre-adipocytes will be tested. The pathogenic role of the Hippo pathway in their differentiation to fibro-adipocytes will be determined through LoF and GoF studies. The proposed studies are expected to provide insights into the molecular pathogenesis of ARVC and facilitate identification of new therapeutic targets for a deadly disease for which there is no effective treatment.