Role of RNA helicase Ddx5 in pathological cardiac remodeling

RNA解旋酶Ddx5在病理性心脏重塑中的作用

基本信息

批准号：
10718560
负责人：
Veli Kemal Topkara
金额：
$ 55.58万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-01 至 2028-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10718560
关键词：
Affect Alternative Splicing American Atherosclerosis Attenuated Binding Biogenesis Biological Biological Assay Calcium Cardiac Cardiac Myocytes Cardiomyopathies Cells Chronic Clinical Investigator Award Co-Immunoprecipitations Complex Data Defect Dependovirus Devices Dilated Cardiomyopathy Disease Down-Regulation Dystrophin Functional disorder Genes Genetic Transcription Goals Heart Heart Diseases Heart failure Heterogeneous-Nuclear Ribonucleoproteins Homeostasis Human Immunoprecipitation In Vitro Injections Injury Knockout Mice Label Laboratories Left Ventricular Remodeling Luciferases Malignant Neoplasms Mass Spectrum Analysis Mediating Medical Messenger RNA Morbidity - disease rate Mus Muscle Development Myocardial Nonsense-Mediated Decay Pathogenesis Pathologic Patients Phenotype Physiological Play Population Post-Transcriptional Regulation Protein Family Proteins Proteomics RNA RNA Helicase RNA Processing RNA Splicing RNA metabolism Recovery Regulation Reporter Research Ribosomes Role RyR2 Sarcomeres Signal Transduction Skeletal Muscle Stress Tail Testing Therapeutic Tissues Transcript Transcriptional Regulation Transgenic Organisms Translations United States National Institutes of Health Untranslated RNA Veins Wild Type Mouse aorta constriction experimental study gene therapy global health heart function helicase hospitalization rates human disease human model in vivo induced pluripotent stem cell derived cardiomyocytes inherited cardiomyopathy insight mRNA Precursor mRNA Stability member micro-dystrophin mini-dystrophin mortality mouse model mutant novel novel therapeutic intervention novel therapeutics overexpression posttranscriptional pressure prevent transcriptional reprogramming transcriptome sequencing transcriptomics vector virtual

项目摘要

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.

抽象的这项新的 R01 提案探索了心力衰竭 (HF) 中 RNA 调节的新机制，心力衰竭是一种主要疾病。发病率和死亡率高的全球健康问题。 PI 是一位高级心力衰竭心脏病专家，他研究心力衰竭和心肌恢复中的转录调控。基于 NIH 支持下生成的数据 K08项目，我们将探索DEAD-box RNA解旋酶5（Ddx5）在心脏稳态和疾病。 Ddx5 几乎调节 RNA 代谢的每一步，包括选择性剪接、mRNA 稳定性、核糖体生物发生和翻译，但 Ddx5（表达最高的 DEAD-box）的心脏功能人类心脏中的 RNA 解旋酶尚不清楚。我们实验室的初步数据显示 Ddx5 在慢性衰竭的人类心脏和肥大的小鼠心脏中下调。我们证明了小鼠与心肌细胞特异性 Ddx5 缺失 (Ddx5-cKO) 相关的进展性致死性心肌病关键肌节基因中的 RNA 剪接异常，肌营养不良蛋白 mRNA 和蛋白质显着下调，以及心肌细胞收缩力显着降低。免疫共沉淀实验确定了人类和小鼠心肌细胞中 Ddx5 和 hnRNP H1 剪接因子之间的相互作用。我们建议测试新的假设，即 Ddx5 信号传导失调通过破坏导致心力衰竭的发病机制 RNA 剪接和 mRNA 网络的下调对心肌细胞功能至关重要。在目标 1 中，我们将确定 Ddx5 调节心肌细胞 RNA 剪接的机制。使用体外剪接报告基因检测，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 Ddx5 结构以确定哪些域是对于体外剪接调节至关重要。 eCLIP 测序和 RNA 免疫沉淀 qPCR 将直接鉴定心肌细胞中 Ddx5 的 RNA 剪接靶标。在目标 2 中，我们将阐明 Ddx5 的机制使用荧光素酶测定、ChIP-qPCR 和邻近标记质量调节心肌细胞收缩力光谱法来表征 Ddx5 在心脏中的转录和转录后调节功能。为了确定 Ddx5-cKO 小鼠的 HF 表型是否是由于肌营养不良蛋白缺乏所致，我们将尝试使用基于 AAV 的微型肌营养不良蛋白基因疗法来挽救体内表型。在目标 3 中，我们将确定是否 Ddx5 过表达通过心脏特异性 Ddx5 保护小鼠免受病理性心脏重塑转基因小鼠和同窝对照小鼠因横向主动脉缩窄（TAC）引起的压力超负荷。作为另一种体内 Ddx5 过表达策略，我们将评估 TAC 对治疗的野生型小鼠的影响通过尾静脉注射使用 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。这些研究将为RNA提供新的见解基线和应激条件下心脏中的新陈代谢和 Ddx5 信号传导，对以下方面具有潜在影响：治疗不断增长的心力衰竭患者群体的新疗法。