Fortilin, CTNNA3, and the Heart

项目摘要 这项名为“Fortilin，CTNNA 3和心脏”的拨款提案的主要目标是测试中央 假设衰竭的心脏不能维持福替林表达， CTNNA 3转录、CTNNA 3蛋白降解增加和致死性心力衰竭（HF）。近6 在美国，有1000万人患有HF，每年诊断出约670，000例新病例。HF是一个无情的 进展性疾病与不可接受的高死亡率相关-约40%的患者 严重的HF在一年内死亡。通过过度表达肌浆网来减缓心力衰竭进展的尝试 钙ATP酶（SERCA 2a）、基质细胞衍生因子-1（SDF-1）或腺苷酸环化酶6型（AC 6） 使用腺相关病毒（AAV）的方法是不成功的。HF发展的新分子靶点 必须确定治疗方法。虽然fortilin是一种多功能的172个氨基酸的蛋白质， 虽然在心脏中大量表达的蛋白质，但当我们发现心脏中的蛋白质在心脏中的作用仍然未知时， HF患者的心肌样品中fortilin的表达显著低于对照患者。到 为了测试衰竭心脏中福替林水平下降是否是HF的原因或影响，我们产生了心脏- 特异性Fortilin敲除（KO）小鼠。这些小鼠迅速发展为严重的HF，并在9周龄内死亡。非 来自KO和野生型小鼠心脏的偏倚系统微阵列分析显示， CTNNA 3，它编码一种蛋白质，也称为α-T-连环蛋白，是闰盘（ID）的组成部分， 在KO小鼠的心脏中严重降低。透射电子显微镜显示， KO小鼠的心脏令人惊讶的是，进一步的分子分析显示，fortilin结合CTNNA 3， Fortilin转录激活CTNNA 3基因。我们已经组建了一个团队， 为了验证Fortilin通过维持CTNNA 3的表达和CTNNA 3的完整性来预防HF的假设， 心脏里的身份证。我们将首先证实Fortilin在蛋白和mRNA水平上正调控CTNNA 3 使用基于细胞的分析。此外，使用来自HF患者的至少300个去识别的人心脏裂解物， 和健康心脏，我们将评估Fortilin和CTNNA 3蛋白表达之间的相关性（目的1）。 然后，我们将尝试通过补充fortilin或CTNNA 3来挽救KO小鼠的HF表型， AAV介导的递送或通过使KO小鼠与CTNNA 3转基因小鼠杂交（Aim 2）。最后，我们将测试 如果小鼠心脏中fortilin的转基因过表达保护它们免受缺血诱导的， 通过CTNNA 3诱导的压力超负荷（目的3）。项目完成后，我们将全面 评价了福替林在心力衰竭中的作用，阐明了福替林缺乏引起心力衰竭的机制， 心脏衰竭，并建立了一个健全的战略，以扭转HF的进展，补充福替林 和/或CTNNA 3的基因

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.