ERK1/2-Integrin Signaling in Desmosome-Dyad Crosstalk

项目摘要 致心律失常性右室心肌病（ARVC）是一种遗传性疾病， 心脏浸润、危及生命的室性心律失常和心源性猝死，特别是在 交感神经压力ARVC是年轻运动员心脏性猝死的主要原因， 心血管疾病的症状或诊断。桥粒细胞粘附基因突变构成了 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性心室肌细胞是如何表达的。 桥粒功能障碍促进了心动过速和心脏重塑过程。患者管理 因此，仅限于通过减少心脏病症状来改善生活质量， 晚期心力衰竭为了开始揭示ARVC固有的机制，我们进行了无偏质量 来自具有不同桥粒基因突变的患者的心室样品的光谱分析。我们 初步数据表明，整合素β1D在ARVC中显著下调，导致 RyR 2 ryanodine受体Ca 2+通道的稳定，其定位于心脏二分体连接。机械地说， 我们发现，ERK 1/2激活对桥粒丢失的反应导致泛素依赖性降解， 整合素β1D、RyR 2 Ser-2030磷酸化、肌浆网Ca ~（2+）渗漏和平滑肌细胞生成。 重要的是，我们的整合素β1D敲除小鼠的心脏表现出ARVC样疾病，并伴有RyR 2增加 磷酸化，儿茶酚胺诱导的室性心律失常和心脏纤维化。我们假设 桥粒连接和心脏二分体之间的通讯对于维持Ca 2+稳态是必不可少的 并且ERK 1/2激活诱导的整合素β1D的丢失损害了这种“桥粒-二联体串扰”， 促进ARVC中RyR 2依赖性和儿茶酚胺敏感性的血管生成和纤维化浸润。我们 进一步假设针对该通路的干预可能为治疗ARVC提供有希望的方法。 为了验证我们的假设，我们使用CRISPR-Cas9产生了三种同源基因敲入小鼠模型， 含有与我们从人类ARVC患者中鉴定的突变等同的突变。在目标1中，我们将确定 敲入ARVC突变在再现心脏重构、儿茶酚胺诱导的 突变型ARVC小鼠的胚胎发生和桥粒-二分体串扰。在目标2和3中，我们将检验 突变诱导的ARVC表型可通过ERK 1/2（Aim 2）和RyR 2（Aim 3）有效预防 抑制作用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-二分体串扰和减弱Ca 2+处理来治疗 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.