Role of CHF1 in Cardiovascular Development

描述（申请人的逐字描述）：心脏的发展 来自原始中胚层的肌细胞，心房和心室的特化 心肌细胞，心肌细胞的终末分化是 脊椎动物胚胎发育的关键事件。此外， 心肌，如缺血性心脏病和心力衰竭，是 西方社会的主要死因。梗死后，心肌细胞 不能再生，由于它们的有丝分裂后，终末分化 状态对调节心脏的转录程序的理解 肌细胞个体发育可能导致更好的治疗心血管疾病。我们 最近发现了心血管碱性螺旋-环-螺旋蛋白1 （CHF 1），一种新的蛋白质，是远亲的毛茸茸的家庭， 转录抑制因子毛茸茸的家庭成员以玩耍而闻名 在控制神经元细胞命运和神经元细胞凋亡时间中的重要作用 分化CHF 1主要在发育中的心室中表达， 与MLC2v同时，MLC2v是已知最早的心室肌细胞标志物。 这种表达一直持续到出生，并且随着心脏发育而下调。 肌细胞终末分化并退出细胞周期。我们 假设CHF 1在心肌细胞中起重要作用 分化，大概是通过与细胞内蛋白质相互作用。我们 实验目的：目的1：研究CHF 1对心肌表型的影响 体外测定我们假设CHF 1在 心室肌细胞命运测定。我们将获得 小鼠胚胎干细胞的功能和功能丧失研究， 分化成心肌细胞。我们将比较 野生型、杂合型和纯合型CHF 1敲除细胞分化 体外植入心肌细胞。此外，我们将感染野生型ES细胞， 用过表达CHF 1的重组腺病毒，然后测量 体外分化为心肌细胞的时间。目标2：研究 CHF-1对心室肌细胞表型影响我们假设 CHF 1在心血管系统的发育中起着重要作用。 我们将使用Aim 1中描述的杂合敲除ES细胞来产生 基因敲除小鼠缺乏CHF 1。此外，我们还将培育出转基因小鼠 在α-MHC启动子的控制下表达CHF 1，以评估 在成人心肌中持续表达的效果。目标3：确定 细胞内相互作用伙伴的CHF 1介导其影响。我们 假设CHF 1通过相互作用影响心室表型 与其他细胞蛋白质结合。我们将用酵母双杂交筛选， CHF1的全长、bHLH和阻遏物结构域，以确定潜在的 存在于小鼠中的异源二聚化配偶体和转录辅阻遏物 胚胎图书馆

DESCRIPTION (the applicant's description verbatim): The development of cardiac myocytes from primordial mesoderm, the specification of atrial and ventricular cardiac myocytes, and the terminal differentiation of cardiac myocytes are critical events in vertebrate embryonic development. In addition, diseases of the myocardium, such as ischemic heart disease and heart failure, are the leading causes of death in western society. After infarction, cardiac myocytes are unable to regenerate, due to their postmitotic, terminally differentiated state. An understanding of the transcriptional programs that regulate cardiac myocyte ontogeny may lead to better therapies for cardiovascular disease. We have recently identified cardiovascular basic helix-loop-helix protein 1 (CHF1), a novel protein that is distantly related to the hairy family of transcriptional repressors. Members of the hairy family are known to play important roles in control of neuronal cell fate and timing of neuronal differentiation. CHF1 is expressed primarily in the developing ventricle, concurrently with MLC2v, the earliest known marker of ventricular myocytes. This expression persists until birth, and is downregulated as the cardiac myocytes terminally differentiate and withdraw from the cell cycle. We hypothesize that CHF1 plays an important role in cardiac myocyte differentiation, presumably by interacting with intracellular proteins. Our experimental aims are: Aim 1: Study the effect of CHF1 on cardiac phenotype determination in vitro. We hypothesize that CHF1 plays an important role in cardiac ventricular myocyte cell fate determination. We will perform gain of function and loss of function studies in mouse embryonic stem cells that can be made to differentiate into cardiac myocytes. We will compare the ability of wild type, heterozygous and homozygous CHF1 knockout cells to differentiate into cardiac myocytes in vitro. In addition, we will infect wild type ES cells with a recombinant adenovirus that overexpresses CHF1 and then measure the timing of differentiation into cardiac myocytes in vitro. Aim 2: Study the effect of CHF1 on ventricular myocyte phenotype in vivo. We hypothesize that CHF1 plays an important role in the development of the cardiovascular system. We will use the heterozygous knockout ES cells described in Aim 1 to generate knockout mice that lack CHF1. In addition, we will generate transgenic mice that express CHF1 under the control of the alpha-MHC promoter to assess the effect of persistent expression in the adult myocardium. Aim 3: Determine the intracellular interaction partners of CHF1 that mediate its effects. We hypothesize that CHF1 exerts an effect on ventricular phenotype by interaction with other cellular proteins. We will perform a yeast 2-hybrid screen with the full length, bHLH and repressor domains of CHF1 to identify potential heterodimerization partners and transcriptional corepressors present in a mouse embryonic library.