Role of RNA helicase Ddx5 in pathological cardiac remodeling
NIH
COLUMBIA UNIVERSITY HEALTH SCIENCES
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. Project Narrative  Heart failure is associated with unacceptably high morbidity and mortality worldwide despite currently available medical and device therapies, highlighting the need for deep mechanistic research in this field. Dead box helicase (Ddx) proteins play important roles in RNA splicing and processing. A key member of this protein family, Ddx5 has not been studied in the heart, and in this proposal, we will elucidate its role in cardiac function and disease.
Non-SBIR/STTR