Genetic Studies of Sarcomere-based Cardiac Diseases

描述（由申请人提供）：肌节是横纹肌的基本收缩单位。虽然肉瘤蛋白的突变与各种人类心血管疾病有关，但尚不清楚为什么不同肉瘤基因的突变甚至同一基因的不同突变会导致不同的疾病。本研究的目的是利用titin作为一个范例来研究肌节组装和肌节性疾病的分子机制。我们已经从突变筛选中鉴定出斑马鱼中的6个titin突变体，并证明其中一个突变等位基因的表型类似于人类扩张型心肌病。我们最近确定了斑马鱼titin （tinl）的全长基因组序列，并克隆了斑马鱼的另一个titin同源物（tin2）。我们的初步数据支持该提案的中心假设，该假设预测Titin的不同结构域参与肌原纤维发生破坏的不同步骤，导致不同的肉瘤疾病。在我们的具体目标1中，我们将研究tinl和tin2的功能分化。通过利用morpholino技术，我们将检验不同的titin亚型在肌原纤维形成中具有不同功能的假设。在我们的特异性目标2中，我们将检验不同结构域在肌原纤维形成中具有不同功能的假设。我们将在体内产生一系列tinl和tin2的截断，以揭示c端结构域的功能，包括激酶结构域。我们还将通过敲除Titin相互作用蛋白Tcap来研究Titin n端结构域的功能。在我们的特异性目的中，我们将确定斑马鱼中不同的titin突变的病理后果。我们将首先确定所有六种titin突变体的突变位置，然后表征胚胎和成年鱼的病理表型。通过产生和表征一系列的titin突变，这些实验将加深我们对为什么不同的titin外显子突变导致肌节不同的结构变化和肌瘤疾病（包括扩张性心肌病、肥厚性心肌病和肌肉萎缩症）不同表型的理解。

DESCRIPTION (provided by applicant): The sarcomere is the basic contractile unit in striated muscle. While mutations in sarcomeric proteins have been linked to various human cardiovascular diseases, it is unclear why mutations in different sarcomeric genes or even different mutations in the same gene result in distinct diseases. The goal of this proposal is to use titin as a paradigm to investigate molecular mechanisms of sarcomere assembly and sarcomeric diseases. We have identified six titin mutants in zebrafish from mutagenesis screens and demonstrated that phenotypes from one mutant allele resemble human dilated cardiomyopathy. We recently identified the full-length genomic sequence for zebrafish titin (tinl) and cloned the other titin homologue (tin2) in zebrafish. Our preliminary data support the central hypothesis of this proposal, which predicts that different domains of Titin participate in distinct steps of myofibrillogenesis disruption of which results in different sarcomeric diseases. In our Specific Aim 1, we will investigate the functional divergence of tinl and tin2. By leveraging morpholino technology, we will test the hypothesis that different isoforms of titin have different functions in myofibrillogenesis. In our Specific Aim 2, we will test the hypothesis that different domains of titin have different functions in myofibrillogenesis. We will generate a series of truncations of both tinl and tin2 in vivo to reveal the functions of the C-terminal domains, including the kinase domain. We will also investigate the function of a Titin N-terminal domain by knocking out a titin interacting protein, Tcap. In our Specific Aim3, we will determine the pathological consequences of different titin mutations in zebrafish. We will first identify the location of mutations in all six titin mutants and then characterize the pathological phenotypes in both embryonic and adult fish. By generating and characterizing a series of titin mutations, these experiments will deepen our understanding of why mutations in different titin exons result in distinct structural changes of the sarcomere and different phenotypes of sarcomeric diseases, including dilated cardiomyopathy, hypertrophic cardiomyopathy, and muscular dystrophy.