Role of CHF1 in Cardiovascular Development

描述(申请人逐字描述)：心脏疾病的发展 原始中胚层的肌细胞--心房和心室的特性 心肌细胞和心肌细胞的终末分化 脊椎动物胚胎发育中的关键事件。此外，该病还包括 心肌，如缺血性心脏病和心力衰竭，是 西方社会的主要死因。心肌梗塞后，心肌细胞 不能再生，因为它们是有丝分裂后，终末分化的 州政府。对调节心脏功能的转录程序的理解 肌细胞个体发育可能带来更好的心血管疾病治疗方法。我们 最近发现了心血管碱性螺旋-环-螺旋蛋白1 (CHF1)，一种新的蛋白质，与毛发家族有远缘关系 转录抑制因子。众所周知，毛茸茸的家庭成员会玩 在控制神经细胞命运和神经元时机中的重要作用 差异化。CHF1主要在发育中的脑室表达， 与已知的最早的心肌细胞标志物MLC2v同时存在。 这种表达持续到出生，并被下调为心脏 肌细胞最终分化并退出细胞周期。我们 CHF1在心肌细胞中发挥重要作用的假设 分化，可能是通过与细胞内蛋白质相互作用来实现的。我们的 实验目的：目的1：研究CHF1对心脏表型的影响 体外测定。我们假设CHF1在 心肌细胞命运的测定。我们将实现以下目标 小鼠胚胎干细胞的功能和功能丧失的研究 使其分化为心肌细胞。我们将会比较一下 野生型、杂合型和纯合型CHF1基因敲除细胞的分化 转化为体外培养的心肌细胞。此外，我们还将感染野生型ES细胞 使用过表达CHF1的重组腺病毒，然后测量 体外分化为心肌细胞的时机。目标2：研究 CHF1对在体心肌细胞表型的影响。我们假设 CHF1在心血管系统的发育中起着重要作用。 我们将使用目标1中描述的杂合子敲除ES细胞来产生 缺乏CHF1的基因敲除小鼠。此外，我们还将培育转基因小鼠 在α-MHC启动子的控制下表达CHF1以评估 持续表达对成人心肌的影响。目标3：确定 介导其影响的CHF1的细胞内相互作用伙伴。我们 假设CHF1通过相互作用影响心室表型 与其他细胞蛋白质结合。我们将进行酵母菌2杂交筛选 CHF1的全长、bHLH和阻遏结构域的鉴定 小鼠体内存在的异二聚化伙伴和转录共抑制物 胚胎库。

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.