Role of RNA helicase Ddx5 in pathological cardiac remodeling

摘要 这项新的R01提案探索了在心力衰竭(HF)中RNA调节的新机制，心力衰竭是一种主要的 具有高发病率和高死亡率的全球卫生问题。PI是一位高级心力衰竭心脏病专家，他研究 心衰与心肌恢复中的转录调控。基于在NIH的支持下产生的数据 K08计划，我们将探索死盒RNA解旋酶5(Ddx5)在心脏动态平衡和心肌保护中的新作用。 疾病。Ddx5几乎调控RNA代谢的每一步，包括选择性剪接、mRNA稳定性、 核糖体的生物发生和翻译，但心脏功能的Ddx5，表达最高的死盒 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，Ddx5是 在慢性衰竭的人类心脏和肥大的小鼠心脏中表达下调。我们证明了老鼠 与心肌细胞特异性Ddx5缺失(Ddx5-CKO)相关的进行性致死性心肌病 随着关键肌节基因的RNA剪接异常，dystrophin mRNA和蛋白质显著下调， 心肌细胞的收缩能力显著降低。免疫共沉淀实验确定了一种 Ddx5和hnRNP H1剪接因子在人和小鼠心肌细胞中的相互作用。我们建议 验证Ddx5信号失调通过干扰参与心力衰竭发病机制的新假说 对心肌细胞功能至关重要的RNA剪接和mRNA网络下调。在目标1中，我们 将确定Ddx5调节心肌细胞中RNA剪接的机制。使用体外剪接 记者分析，我们将测试Ddx5的RNA剪接功能是否依赖于目标内含子序列 和/或与hnRNP H1合作。我们将生成突变的Ddx5结构来确定哪些结构域 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 Ddx5在心肌细胞中的RNA剪接靶点。在目标2中，我们将阐明Ddx5 利用荧光素酶分析、芯片定量聚合酶链式反应和邻近标记质量来调节心肌细胞收缩能力 Ddx5在心脏的转录和转录后调节功能的光谱特征。 为了确定Ddx5-CKO小鼠的HF表型是否由于dystrophin缺乏，我们将尝试 以AAV为基础的微型肌营养不良蛋白基因治疗在体内挽救表型。在目标3中，我们将确定是否 Ddx5过表达通过使心脏特异性Ddx5保护小鼠免受病理性心脏重构的影响 转基因小鼠和仔鼠对照小鼠对横断性主动脉缩窄(TAC)所致的压力超负荷。AS 作为体内Ddx5过表达的替代策略，我们将评估TAC对野生型小鼠治疗的影响 经尾静脉注射AAV9-Ddx5与AAV9-EGFP载体。Ddx5的RNA和蛋白质靶点将是 在人类心肌细胞和心脏组织中得到证实。这些研究将提供对RNA的新见解 基线和应激条件下心脏中的代谢和Ddx5信号，及其潜在的意义 治疗不断增长的心力衰竭患者的新疗法。

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.