The molecular mechanisms underlying arrhythmogenic right ventricular dysplasia/cardiomyopathy

 描述（由申请人提供）：这是我们的补助金R 01 HL 095780 -01的竞争性续期申请。我们最初的资助是通过产生和表征新的桥粒斑蛋白缺陷模型系统，研究已知的中央桥粒组分桥粒斑蛋白在与遗传性心脏病相关的临床和细胞特征中的作用机制。对于该应用，我们在酵母双杂交筛选中发现突触体相关蛋白29（SNAP 29）是成人心脏中的一种新型桥粒斑蛋白相关蛋白，我们通过利用我们的新型ARVC遗传小鼠和人类心脏模型显示其与ARVC相关。虽然SNAP 29在心脏中的作用是未知的，但我们表明SNAP 29共定位于成年小鼠和人类心脏以及人类诱导多能干细胞衍生的心脏细胞（hiPSC）中的心肌桥粒细胞-细胞连接。此外，SNAP 29在桥粒连接处的定位和/或水平在来自我们的ARVC和ARVC hiPSC衍生的心脏细胞的小鼠模型的心脏中丢失，所述心脏细胞表现出显著的桥粒缺陷和致瘤行为。新型SNAP 29缺陷小鼠模型（全局和心肌细胞特异性）的产生揭示了显著的心脏缺陷，包括（i）心肌细胞连接处的自噬缺陷（自噬/溶酶体标志物和结构在细胞连接处的积累），其特异性影响桥粒蛋白水平和（ii）心脏形态缺陷。来自我们的小鼠ARVC模型的数据为这种机制提供了验证，因为我们揭示了它们的心脏在心肌细胞连接处的自噬控制中表现出类似的缺陷。我们假设SNAP 29通过自噬介导的机制控制桥粒更新来调节心肌中的桥粒蛋白水平和功能，并且其损失将触发桥粒蛋白水平和功能的损失，并最终导致ARVC。我们的目标是确定：（i）通过表征SNAP 29功能丧失小鼠模型，SNAP 29在心脏中的功能作用，（ii）通过表达人ARVC突变和使用hiPSC作为工具，SNAP 29-DSP相互作用在人ARVC和心肌细胞中的相关性，和（iii）控制桥粒水平/转换的自噬中的SNAP 29依赖性事件，通过在体外ARVC模型中使用SNAP 29功能丧失模型和SNAP 29的过表达来分析心脏自噬（诱导和通量）和相关桥粒靶标中的缺陷。

 DESCRIPTION (provided by applicant): This is an application for a competitive renewal of our grant R01 HL095780-01. We were originally funded to investigate mechanisms underlying the role of the known central desmosomal component, desmoplakin, in the clinical and cellular features associated with the genetic-based heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC) that causes sudden death in the young, by generating and characterizing novel desmoplakin deficient model systems. For this application, we uncovered synaptosomal associated protein 29 (SNAP29) as a novel desmoplakin associated protein in the adult human heart in a yeast-two hybrid screen that we show has relevance to ARVC, by leveraging our novel genetic mouse and human cardiac models of ARVC. Although the role of SNAP29 is unknown in the heart, we show that SNAP29 co-localizes to cardiac muscle desmosomal cell-cell junctions in the adult mouse and human heart as well as human induced pluripotent stem cell derived cardiac cells (hiPSC). Furthermore, SNAP29 localization and/or levels at desmosomal junctions are lost in hearts from our mouse model of ARVC and ARVC hiPSC-derived cardiac cells that exhibit striking desmosomal defects and arrhythmogenic behavior. Generation of novel SNAP29 deficient mouse models (global and cardiomyocyte-specific) revealed striking cardiac defects including (i) autophagic defects at the cardiac muscle cell junction (accumulation of autophagic/lysosomal markers and structures at the cell junction) that specifically impacted desmosomal protein levels and (ii) cardiac morphology defects. Data from our mouse model of ARVC provides validation to this mechanism as we reveal that their hearts exhibit similar defects in autophagic control at the cardiac muscle cell junction. We hypothesize that SNAP29 regulates desmosomal protein levels and function in cardiac muscle by controlling desmosomal turnover via autophagy-mediated mechanisms and its loss will trigger loss of desmosomal protein levels as well as function and ultimately cause ARVC. We aim to determine: (i) the functional role of SNAP29 in the heart by characterizing SNAP29 loss of function mouse models, (ii) the relevance of SNAP29-DSP interaction in human ARVC and cardiomyocytes by expressing human ARVC mutations and using hiPSCs as a tool, and (iii) the SNAP29-dependent events in autophagy that control desmosomal levels/turnover, by analyzing defects in cardiac autophagy (induction and flux) and relevant desmosome targets using SNAP29 loss of function models and overexpression of SNAP29 in an in vitro ARVC model.