ERK1/2-Integrin Signaling in Desmosome-Dyad Crosstalk

项目总结 致心律失常的右室心肌病(ARVC)是一种以纤维脂肪为特征的遗传性疾病 心脏渗透、危及生命的室性心律失常和心脏性猝死，特别是在反应中 对同情性的压力。ARVC是年轻运动员心源性猝死的主要原因 心血管疾病的症状或诊断。桥粒细胞黏附基因突变构成 大多数家族性ARVC病例，但在很大程度上仍不清楚儿茶酚胺敏感的脑室 桥粒功能障碍促进了心动过速和心脏重塑过程。病人管理 因此仅限于通过减少心律失常症状或心脏移植来提高生活质量 晚期心力衰竭。为了开始揭示ARVC固有的机制，我们进行了无偏见的质量 不同桥粒基因突变患者脑室标本的光谱分析。我们的 初步数据显示，整合素β1D在ARVC中显著下调，导致 RyR2 ryanodine受体钙通道定位于心脏二联体连接的稳定性。从机械上讲， 我们发现，桥粒丢失后ERK1/2的激活导致泛素依赖性的降解。 整合素β1D、RyR2Ser-2030磷酸化、肌浆网钙泄漏与心律失常 重要的是，我们的整合素β1D基因敲除小鼠的心脏表现出RyR2增加的ARVC样疾病 磷酸化、儿茶酚胺诱发的室性心律失常和心脏纤维化。我们假设 桥粒连接和心脏二联体之间的通讯对维持钙稳态是必不可少的 而ERK1/2激活导致整合素β1D的缺失，从而削弱了这种“桥粒-二联体串扰” 促进RyR2依赖和儿茶酚胺敏感的ARVC心律失常和纤维化的发生。我们 进一步假设，针对这一通路的干预可能为ARVC的治疗提供一种有前途的方法。 为了验证我们的假设，我们使用CRISPR-Cas9建立了三个同源基因敲入小鼠模型 包含的突变与我们从人类ARVC患者中发现的突变相同。在目标1中，我们将确定 儿茶酚胺诱导的重复性心脏重构中敲入ARVC突变的致病性 突变的arvc小鼠的心律失常发生和桥粒-二联体串扰。在目标2和目标3中，我们将测试 突变诱导的ARVC表型可通过ERK1/2(Aim 2)和RyR2(Aim 3)有效预防 抑制力。我们希望我们的研究表明，危及生命的室性心律失常和心力衰竭 ARVC可以通过调节桥粒-二联体串扰和减弱钙离子的处理来进行治疗 功能障碍。

PROJECT SUMMARY Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is an inherited disease characterized by fibro-fatty infiltration of the heart, life-threatening ventricular arrhythmias and sudden cardiac death, particularly in response to sympathetic stress. ARVC is a leading cause of sudden cardiac death among young athletes with no prior symptoms or diagnosis of cardiovascular disease. Desmosome cell-adhesion gene mutations constitute the majority of familial ARVC cases, but it remains largely unknown how catecholamine-sensitive ventricular tachycardia and cardiac remodeling processes are facilitated by desmosome dysfunction. Patient management is therefore limited to improving quality of life by reducing arrhythmic symptoms or cardiac transplantation upon advanced heart failure. To begin to uncover mechanisms inherent to ARVC, we performed unbiased mass spectrometry analysis of ventricular samples from patients with different desmosomal gene mutations. Our preliminary data demonstrate that integrin β1D is significantly downregulated in ARVC resulting in de- stabilization of RyR2 ryanodine receptor Ca2+ channels that localize to cardiac dyad junctions. Mechanistically, we find that ERK1/2 activation in response to desmosome loss results in ubiquitin-dependent degradation of integrin β1D, RyR2 Ser-2030 phosphorylation, sarcoplasmic reticulum Ca2+ leak and arrhythmogenesis. Importantly, hearts of our integrin β1D knockout mice exhibit ARVC-like disease with increased RyR2 phosphorylation, catecholamine-induced ventricular arrhythmias and cardiac fibrosis. We hypothesize that communication between desmosome junctions and cardiac dyads is essential for maintaining Ca2+ homeostasis and that ERK1/2 activation-induced loss of integrin β1D impairs this “desmosome-dyad crosstalk” thereby promoting RyR2-dependent and catecholamine-sensitive arrhythmogenesis and fibrotic infiltration in ARVC. We further hypothesize that interventions targeting this pathway may offer a promising approach for treating ARVC. To test our hypothesis, we have generated three congenic knock-in mouse models using CRISPR-Cas9 that contain mutations equivalent to those we identified from human ARVC patients. In Aim 1, we will determine the pathogenicity of knock-in ARVC mutations in recapitulating cardiac remodeling, catecholamine-induced arrhythmogenesis and desmosome-dyad crosstalk in mutant ARVC mice. In Aims 2 and 3, we will test whether mutation-induced ARVC phenotypes can be effectively prevented through ERK1/2 (Aim 2) and RyR2 (Aim 3) inhibition. We expect our studies to show that life-threatening ventricular arrhythmias and heart failure from ARVC can be therapeutically managed by modulating desmosome-dyad crosstalk and attenuating Ca2+ handling dysfunction.