Canonical Wnt Signaling in Pathogenesis and Rescue of ARVC

描述(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，包括致心律失常的右室心肌病(ARVC)。ARVC的特点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。除了心脏移植外，目前还没有有效的治疗ARVC的方法。ARVC主要由编码桥粒蛋白的基因突变引起。桥粒与黏附连接(AJ)和缝隙连接一起构成间盘(ICD)。虽然传统上被认为是细胞-细胞黏附结构，但ICD已经成为接触介导的细胞信号的主要调节因子。因此，ICD参与了对河马通路的调控，而河马通路在细胞分化和增殖中发挥着重要作用。初步数据显示，ARVC患者心脏ICD发生了广泛的分子重塑。这些变化与河马激酶级联反应的激活有关，河马蛋白激酶级联反应通过磷酸化效应器分子YAP，通过TEAD抑制转录。HIPPO途径的激活与人类心脏中规范的Wnt信号的抑制有关，这与ARVC的发病机制有关。我们推测河马通路的激活在ARVC中起致病作用。在目标1中，在进一步加强初步数据的基础上，我们将利用心肌细胞和小鼠模型来描述河马通路激活的负责机制。初步数据表明，神经纤维素1(NF2)，又名Merlin，是ARVC模型中ICD激活的一种上游河马蛋白激酶。通过获得和丧失功能(GOF和LOF)的方法，河马途径将在心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挽救作用。在目标2中，我们将描述典型的Wnt信号的致病作用及其抑制机制，在激活的河马途径的背景下。标准的Wnt和Hippo分子的GoF和LoF靶向将被用来确定对心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，并确定河马和Wnt通路的致病作用。这些方法是基因命运图谱和敲门记者追踪，后者是为了旁分泌效应(S)。根据初步数据，候选细胞类型为纤维脂肪前体细胞、心外膜细胞、周细胞、 和肌源性血统。将测试突变的肌细胞对上述细胞和常驻前脂肪细胞的旁分泌作用。河马途径在其向纤维脂肪细胞分化中的致病作用将通过LOF和GOF研究来确定。拟议的研究有望为ARVC的分子发病机制提供见解，并有助于确定一种尚无有效治疗方法的致命疾病的新治疗靶点。

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.