Fortilin, CTNNA3, and the Heart

Fortilin、CTNNA3 和心脏

基本信息

批准号：
10553291
负责人：
Ken Fujise
金额：
$ 66.63万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-01 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10553291
关键词：
Adenylate Cyclase Age American Amino Acids Animals Apoptotic Applications Grants Autopsy Binding Biological Assay Birth Blood capillaries Cardiac Cardiac Myocytes Cardiomyopathies Cell Nucleus Cell physiology Cells Complementary DNA Coronary Arteriosclerosis Cytosol Death Rate Dependovirus Development Diagnosis Echocardiography Enterobacteria phage P1 Cre recombinase Fibrosis Functional disorder GATA4 gene Genes Genetic Genetic Complementation Test Genetic Transcription Goals Heart Heart failure Human Hypertension Immunoprecipitation Intercalated disc Interferometry Intraperitoneal Injections Ischemia Knock-out Knockout Mice Left Ligation LoxP-flanked allele Luciferases MCL1 gene Mediating Messenger RNA Methods Microarray Analysis Molecular Molecular Analysis Molecular Target Mus Myocardial Patients Phenotype Progressive Disease Proteins RNA Recombinant adeno-associated virus (rAAV)Resistance Rhesus Role SERCA2a Sampling Sarcomeres Serotyping Stromal Cell-Derived Factor 1 Structure TPT1 gene Testing Therapeutic Transcript Transgenic Mice Transgenic Organisms Transmission Electron Microscopy Ventricular Western Blotting Wild Type Mouse alphaT catenin differential expression ischemic cardiomyopathy multicatalytic endopeptidase complex myocyte-specific enhancer-binding-factor 2C overexpression pressure prevent promoter protein degradation protein expression randomized, clinical trials sarcoplasmic reticulum calcium ATPase small hairpin RNA sound transcription factor

项目摘要

Project Summary The main goal of this grant proposal titled “Fortilin, CTNNA3, and the Heart” is to test the central hypothesis that the inability of the failing heart to maintain fortilin expression leads to decreased CTNNA3 transcription, increased CTNNA3 protein degradation, and lethal heart failure (HF). Nearly 6 million Americans live with HF, with about 670,000 new cases diagnosed each year. HF is a relentlessly progressive disease that is associated with an unacceptably high death rate—about 40% of patients with severe HF die within a year. The attempts to slow HF progression by overexpression of sarcoplasmic reticulum calcium ATPase (SERCA2a), stromal cell-derived factor-1 (SDF-1), or adenylyl cyclase type 6 (AC6) in humans using adeno associated virus (AAV) have been unsuccessful. New molecular targets for development of HF therapeutics must be identified. Although fortilin, a multifunctional 172-amino acid protein, is one of the most abundantly expressed proteins in the hearts, its role in the heart remained unknown when we found that the myocardial samples from HF patients expressed significantly less fortilin than in those from control patients. To test whether decreased fortilin levels in the failing hearts is the cause or effect of HF, we generated heart- specific fortilin knockout (KO) mice. These mice rapidly develop severe HF and die within 9 weeks of age. Non- biased systematic microarray analyses from KO and wild-type mouse hearts showed that the expression of CTNNA3, which encodes a protein also known as α-T-catenin that is a component of the intercalated disc (ID), was severely decreased in the hearts of KO mice. Transmission electron microscopy showed disrupted IDs in the hearts of KO mice. Strikingly, further molecular analyses revealed that fortilin binds CTNNA3 and that fortilin transcriptionally activates the CTNNA3 gene. We have assembled a team with all expertise needed to test the hypothesis that fortilin prevents HF by maintaining CTNNA3 expression and the integrity of ID in the heart. We will first confirm that fortilin positively regulates CTNNA3 at both protein and mRNA levels using cell-based assays. Additionally, using at least 300 de-identified human heart lysates from HF patients and healthy hearts, we will evaluate the correlation between fortilin and CTNNA3 protein expression (Aim 1). We will then attempt to rescue the HF phenotype of the KO mice by replenishing fortilin or CTNNA3 by either AAV-mediated delivery or by crossing the KO mice with CTNNA3 transgenic mice (Aim 2). Finally, we will test if the transgenic overexpression of fortilin in the hearts of mice protects them against ischemia-induced and pressure-overload-induced through CTNNA3 (Aim 3). Upon completion of the project, we will have fully evaluated the role of fortilin in HF, elucidated the mechanism by which fortilin deficiency causes the heart to fail, and established a sound strategy to reverse the progression of HF by replenishing fortilin and/or CTNNA3 in the failing heart.

项目概要这项题为“Fortilin、CTNNA3 和心脏”的拨款提案的主要目标是测试中央假设衰竭的心脏无法维持福替林表达导致 CTNNA3 转录、CTNNA3 蛋白降解增加和致死性心力衰竭 (HF) 接近 6。数百万美国人患有心力衰竭，每年诊断出约 67 万例心力衰竭新病例。进展性疾病与令人无法接受的高死亡率相关——大约 40% 的患者严重心力衰竭患者在一年内死亡。通过肌浆网过度表达来减缓心力衰竭进展的尝试。人类中的钙 ATP 酶 (SERCA2a)、基质细胞衍生因子 1 (SDF-1) 或腺苷酸环化酶 6 型 (AC6) 使用腺相关病毒（AAV）开发心力衰竭的新分子靶点尚未成功。尽管 Fortilin（一种由 172 个氨基酸组成的多功能蛋白）是最有效的治疗方法之一。在心脏中大量表达的蛋白质，当我们发现它在心脏中的作用仍然未知时心力衰竭患者的心肌样本表达的福替林显着低于对照患者的心肌样本。测试衰竭心脏中福替林水平的降低是否是心力衰竭的原因或影响，我们生成了心脏- 特定的 fortilin 基因敲除 (KO) 小鼠会迅速出现严重心力衰竭并在 9 周内死亡。来自 KO 和野生型小鼠心脏的有偏差的系统微阵列分析表明， CTNNA3，编码一种也称为 α-T-连环蛋白的蛋白质，它是闰盘 (ID) 的组成部分，透射电子显微镜显示 KO 小鼠心脏中的 ID 严重减少。令人惊讶的是，进一步的分子分析表明 fortilin 与 CTNNA3 结合，并且 fortilin 通过转录激活 CTNNA3 基因。我们组建了一支拥有所需所有专业知识的团队。检验福替林通过维持 CTNNA3 表达和完整性来预防心力衰竭的假设我们将首先确认 fortilin 在蛋白质和 mRNA 水平上正向调节 CTNNA3。此外，还使用了至少 300 份来自 HF 患者的未鉴定的人类心脏裂解物。和健康心脏，我们将评估 fortilin 和 CTNNA3 蛋白表达之间的相关性（目标 1）。然后，我们将尝试通过补充 fortilin 或 CTNNA3 来挽救 KO 小鼠的 HF 表型 AAV 介导的递送或通过将 KO 小鼠与 CTNNA3 转基因小鼠杂交（目标 2）。如果福替林在小鼠心脏中的转基因过度表达可以保护它们免受缺血引起的和通过CTNNA3（目标3）引起的压力过载。项目完成后，我们将完全拥有。评估了 fortilin 在心力衰竭中的作用，阐明了 fortilin 缺乏导致心力衰竭的机制心力衰竭，并制定了合理的策略，通过补充福替林来逆转心力衰竭的进展和/或衰竭心脏中的CTNNA3。