Fortilin, CTNNA3, and the Heart

项目摘要 这项名为“Fortilin、CTNNA3和心脏”的拨款提案的主要目标是测试中央 假设衰竭的心脏不能维持Fortilin的表达导致减少 CTNNA3转录，增加CTNNA3蛋白降解，以及致命性心力衰竭(HF)。近6个 数以百万计的美国人患有心力衰竭，每年约有67万新确诊病例。高频是一个无情的 与不可接受的高死亡率相关的进行性疾病-约40%的患者患有 严重的心衰在一年内死亡。肌浆网过表达延缓心衰进展的实验研究 人类的钙ATPase(SERCA2a)、基质细胞衍生因子-1(SDF-1)或腺苷环化酶6型(AC6) 使用腺相关病毒(AAV)一直没有成功。氢氟酸开发的新分子靶点 必须确定治疗方法。尽管福尔替林，一种由172个氨基酸组成的多功能蛋白质，是最多的 在心脏中大量表达的蛋白质，其在心脏中的作用尚不清楚，当我们发现 心力衰竭患者心肌标本中Fortilin的表达明显低于对照组。至 测试衰竭心脏中的Fortilin水平下降是心力衰竭的原因还是结果，我们产生了心脏- 特异性Fortilin基因敲除(KO)小鼠。这些小鼠迅速发展为严重的心衰，并在9周内死亡。非- 来自KO和野生型小鼠心脏的有偏系统微阵列分析表明， CTNNA3，它编码一种蛋白质，也被称为α-T-连环蛋白，是插入盘(ID)的一种成分， 在KO小鼠的心脏中严重减少。透射电子显微镜显示IDs在 KO小鼠的心脏。令人惊讶的是，进一步的分子分析表明，Fortilin与CTNNA3结合，并且 Fortilin转录激活CTNNA3基因。我们已经组建了一支拥有所有所需专业知识的团队 为了验证Fortilin通过维持CTNNA3的表达和CTNNA3的完整性来预防心衰的假设 身份证在心里。我们将首先确认Fortilin在蛋白质和mRNA水平上正向调节CTNNA3 使用基于细胞的分析。此外，使用至少300个来自心力衰竭患者的未识别的人类心脏裂解物 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介导的传递或将KO小鼠与CTNNA3转基因小鼠杂交(目标2)。最后，我们将测试 如果在小鼠心脏中过表达Fortilin转基因可以保护它们免受缺血诱导的和 通过CTNNA3诱导的压力超负荷(目标3)。项目建成后，我们将完全拥有 评价Fortilin在心衰中的作用，阐明Fortilin缺乏导致心力衰竭的机制 心力衰竭，并建立了合理的策略，通过补充Fortilin来逆转心力衰竭的进展 和/或CTNNA3在衰竭的心脏中。

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.