Role of RNA helicase Ddx5 in pathological cardiac remodeling

摘要 这项新的R 01提案探索了心力衰竭（HF）中RNA调控的新机制，这是心力衰竭的主要机制。 高发病率和高死亡率是全球健康问题。PI是一名高级HF心脏病专家， 转录调控和心肌恢复。基于NIH支持下生成的数据 K 08计划，我们将探索死亡盒RNA解旋酶5（Ddx 5）在心脏稳态中的新作用， 疾病Ddx 5几乎调节RNA代谢的每一步，包括选择性剪接，mRNA稳定性， 核糖体的生物合成和翻译，但心脏功能的Ddx 5，最高表达的死亡盒 人类心脏中的RNA解旋酶是未知的。我们实验室的初步数据显示， 在慢性衰竭的人类心脏和肥大的小鼠心脏中下调。我们证明了老鼠 心肌细胞特异性Ddx 5缺失（Ddx 5-cKO）患者发生了进行性致死性心肌病， 在关键肌节基因中存在异常RNA剪接，肌营养不良蛋白mRNA和蛋白质显著下调， 以及心肌细胞收缩力的显著降低。免疫共沉淀实验鉴定了 人和小鼠心肌细胞中Ddx 5和hnRNP H1剪接因子相互作用我们建议 测试Ddx 5信号转导失调通过破坏而导致HF发病机制的新假设 RNA剪接和下调mRNA网络，这对心肌细胞功能至关重要。目标1： 将确定Ddx 5调节心肌细胞RNA剪接的机制。使用体外剪接 报告基因分析，我们将测试Ddx 5的RNA剪接功能是否依赖于靶内含子序列 和/或与hnRNP H1合作。我们将产生突变的Ddx 5构建体以确定哪些结构域是 对体外剪接调控至关重要。eCLIP测序和RNA免疫沉淀-qPCR将直接鉴定 心肌细胞中Ddx 5的RNA剪接靶点。在目标2中，我们将阐明Ddx 5 使用荧光素酶测定、ChIP-qPCR和邻近标记质量调节心肌细胞收缩性 使用荧光光谱法来表征心脏中Ddx 5的转录和转录后调节功能。 为了确定Ddx 5-cKO小鼠的HF表型是否是由于肌营养不良蛋白缺乏引起的，我们将尝试 使用基于AAV的微型肌养蛋白基因疗法在体内挽救表型。在目标3中，我们将确定 Ddx 5过表达通过使心脏特异性Ddx 5 转基因和同窝对照小鼠对由横向主动脉缩窄（TAC）诱导的压力超负荷的耐受性。作为 另一种体内Ddx 5过表达策略，我们将评估TAC在野生型小鼠中的作用， 用AAV 9-Ddx 5与AAV 9-eGFP载体通过尾静脉注射。Ddx 5的RNA和蛋白质靶点将被 在人类心肌细胞和心脏组织中得到证实。这些研究将为RNA提供新的见解 在基线和应激条件下心脏中的Ddx代谢和Ddx 5信号传导， 治疗不断增长的HF患者人群的新疗法。

Abstract This new R01 proposal explores novel mechanisms underlying RNA regulation in heart failure (HF), a major global health concern with high morbidity and mortality. The PI is an advanced HF cardiologist, who studies transcriptional regulation in HF and myocardial recovery. Based on data generated with support from the NIH K08 program, we will explore new roles for DEAD-box RNA helicase 5 (Ddx5) in cardiac homeostasis and disease. Ddx5 regulates virtually every step of RNA metabolism including alternative splicing, mRNA stability, ribosome biogenesis, and translation, but the cardiac functions of Ddx5, the most highly expressed DEAD-box RNA helicase in the human heart are unknown. Preliminary data from our laboratory showed Ddx5 was downregulated in chronically failing human hearts and in hypertrophic mouse hearts. We demonstrated that mice with cardiomyocyte-specific Ddx5 deletion (Ddx5-cKO) developed progressive lethal cardiomyopathy associated with aberrant RNA splicing in key sarcomere genes, marked downregulation of dystrophin mRNA and protein, and a significant reduction in cardiomyocyte contractility. Co-immunoprecipitation experiments identified an interaction between Ddx5 and hnRNP H1 splicing factor in human and mouse cardiomyocytes. We propose to test the novel hypothesis that dysregulation of Ddx5 signaling contributes to the pathogenesis of HF by disruption of RNA splicing and downregulation of mRNA networks that are critical for cardiomyocyte function. In Aim 1, we will determine the mechanisms by which Ddx5 regulates RNA splicing in cardiomyocytes. Using in vitro splicing reporter assays, we will test whether the RNA splicing function of Ddx5 depends on target intronic sequences and/or cooperation with hnRNP H1. We will generate mutant Ddx5 constructs to determine which domains are critical for splicing regulation in vitro. eCLIP-sequencing and RNA immunoprecipitation-qPCR will identify direct RNA splicing targets of Ddx5 in cardiomyocytes. In Aim 2, we will elucidate the mechanisms by which Ddx5 regulates cardiomyocyte contractility using luciferase assays, ChIP-qPCR, and proximity labeling mass spectrometry to characterize the transcriptional and post-transcriptional regulatory functions of Ddx5 in the heart. To determine whether the HF phenotype of Ddx5-cKO mice is due to dystrophin deficiency, we will attempt to rescue the phenotype in vivo using AAV-based mini-dystrophin gene therapy. In Aim 3, we will determine whether Ddx5 overexpression protects mice from pathological cardiac remodeling by subjecting cardiac-specific Ddx5 transgenic and littermate control mice to pressure overload induced by transverse aortic constriction (TAC). As an alternative in vivo Ddx5 overexpression strategy, we will assess the effects of TAC in wild-type mice treated with AAV9-Ddx5 versus AAV9-eGFP vectors via tail vein injection. RNA and protein targets of Ddx5 will be confirmed in human cardiomyocytes and heart tissue. These studies will provide new insights into RNA metabolism and Ddx5 signaling in the heart under baseline and stress conditions, with potential implications for novel therapies to treat the growing population of HF patients.