Identifying Cardiomyopathy Genes in Mice and Drosophila

说明(申请人提供)：钙调神经磷酸酶是一种钙激活的蛋白磷酸酶，对于心脏肥厚表型的发展是必要和充分的。在哺乳动物中，钙调神经磷酸酶依赖的活化T细胞核因子(NFAT)转录因子的去磷酸化导致肥大基因的诱导。相反，果蝇不包含钙调神经磷酸酶调节的NFAT亚型，这表明存在NFAT不依赖机制的想法。事实上，另一种转录因子，心肌细胞增强因子2(MEF2)，也与钙调神经磷酸酶介导的心肌肥大有关。在这一竞争更新中，我们建议在果蝇中进行缺失和EMS突变筛选，以确定钙调神经磷酸酶信号通路中新的NFAT非依赖性增强和抑制基因。为了实现这一目标，我们将使用Sullivan和Rubin以及Gajeski和Schulz进行的筛查中已经获得的信息，他们已经确定了果蝇基因组的大区域，这些区域在由结构性活性钙调神经磷酸酶(CanAAct)敏化的果蝇筛查中扮演着抑制和增强基因座的角色。因此，我们假设钙调神经磷酸酶介导的心肌肥厚可以通过NFAT非依赖的途径发展，并且在果蝇中的遗传筛选将识别诱导心肌肥厚的新的分子机制。我们提出了以下的具体目标：目的1.在果蝇中寻找钙调神经磷酸酶诱导的肥大信号的新的抑制基因。NFAT非依赖的钙调神经磷酸酶信号基因将通过使用表达心脏特异性CanAAct的致敏苍蝇对先前映射的抑制基因进行缺失筛选来识别。目的2.鉴定钙调神经磷酸酶诱导果蝇肥大信号的新的增强子基因。NFAT非依赖性钙调神经磷酸酶信号基因将通过使用表达心脏特异性CanAAct的致敏苍蝇，通过对先前定位的增强子基因的缺失筛选来鉴定。目的3.寻找EMS诱变诱导果蝇钙调神经磷酸酶诱导的肥大信号的新的遗传修饰物。EMS化学突变筛选在表达肌动蛋白-CanAAct的致敏果蝇中诱导整个果蝇基因组的突变。我们预计，拟议的研究将确定导致心肌肥大的新基因和途径，并将更好地确定钙调神经磷酸酶在肥厚过程中的作用。此外，拟议的研究将导致治疗人类心肌肥大和心力衰竭的新靶点。 公共卫生相关性：拟议的实验将扩大我们对心肌肥厚分子机制的了解，我们相信这将为心力衰竭患者的识别和治疗带来新的靶点。钙调神经磷酸酶是心肌肥厚的结节信号通路。更简单的模型系统，如果蝇，提供了独特的能力来进行遗传筛选，以确定钙调神经磷酸酶途径的遗传修饰物，否则在哺乳动物系统中将很难做到这一点。

DESCRIPTION (provided by applicant): Calcineurin is a calcium-activated protein phosphatase that is both necessary and sufficient for the development of a cardiac hypertrophic phenotype. In mammals, calcineurin-dependent dephosphorylation of nuclear factor of activated T cells (NFAT) transcription factors lead to the induction of hypertrophic genes. In contrast, Drosophila do not contain calcineurin-regulated isoforms of NFAT suggesting the idea that NFAT-independent mechanisms exist. Indeed, another transcription factor, myocyte enhancer factor 2 (MEF2), has also been implicated in calcineurin-mediated cardiac hypertrophy. In this competing renewal we propose to carry out a deficiency and EMS mutagenesis screen in Drosophila to identify novel NFAT-independent enhancer and suppressor genes of the calcineurin signaling pathway. To accomplish this goal we will use information already available from screens performed by Sullivan and Rubin and Gajewski and Schulz who have identified large regions of fly genome that act as suppressor and enhancer loci in a Drosophila screen sensitized by constitutive active calcineurin (CanAAct). Accordingly, we hypothesize that calcineurin-mediated cardiac hypertrophy can develop through an NFAT-independent pathway, and that a genetic screen in the fly will identify new molecular mechanisms that induce cardiac hypertrophy. We propose the following specific aims: Aim 1. Identify novel Suppressor genes of Calcineurin-induced hypertrophic signaling in Drosophila. NFAT-independent calcineurin signaling genes will be identified by a deficiency screen of previously mapped suppressor loci using a sensitized fly expressing cardiac-specific CanAAct. Aim 2. Identify novel Enhancer genes of Calcineurin-induced hypertrophic signaling in Drosophila. NFAT-independent calcineurin signaling genes will be identified with by a deficiency screen of previously mapped enhancer loci using a sensitized fly expressing cardiac-specific CanAAct. Aim 3. Identify novel genetic modifiers of Calcineurin-induced hypertrophic signaling induced by EMS mutagenesis in Drosophila. EMS chemical mutagenesis screen to induce mutations across the entire Drosophila genome in a sensitized fly expressing actin- CanAAct. We anticipate that the proposed investigation will identify novel genes and pathways that lead to cardiac hypertrophy and will better define the role of calcineurin in the hypertrophic process. Furthermore, the proposed studies will lead to new targets to treat cardiac hypertrophy and heart failure in humans. PUBLIC HEALTH RELEVANCE: The proposed experiments will expand our knowledge of the molecular mechanisms of cardiac hypertrophy, which we believe will lead to new targets for the identification and treatment of patients with heart failure. Calcineurin is a nodal signaling pathway for cardiac hypertrophy. Simpler model systems, such as Drosophila, provide the unique ability to perform genetic screens to identify genetic modifiers of the Calcineurin pathway that otherwise would be very difficult to do in mammalian systems.