MOLECULAR/CELLULAR BIOLOGY OF CARDIOMYOCTE DEVELOPMENT

我们研究计划的目标是了解形态发生 体内胚胎心肌，特别是肌原纤维的组装 在那个组织里。 该项目的核心焦点是两个角色的作用 肌球蛋白结合蛋白 (MyBP) 称为 MyBP-C 和 MyBP-H（C 和 H 蛋白， 分别），我们假设这对于横向是必要的 新生 A 带中粗肌丝的排列。 在完成的工作中 在过去的 5 年里，我们：a) 克隆、测序并鉴定了 两种蛋白质的肌球蛋白结合域并精确定位了 MyBP-C 对于将该蛋白靶向至 一支乐队； b)表明缺乏肌球蛋白结合的截短突变体 域作为肌原纤维组装的显性失活； c) 证明 MyBP-C 在肌节后显着表达 胚胎心脏和培养的骨骼肌管中的肌球蛋白，以及 结果表明 MyBP-C 的出现与 这些细胞中的交叉条纹； d) 准备好的复制缺陷， 逆转录病毒载体已被用于追踪谱系 心肌细胞、冠状血管前体和心脏 传导系统； e) 提出合理的模型来解释 心肌、冠状血管和外周血管的形态发生 体内传导； f) 构建这些逆转录病毒的修饰版本 用于将基因递送至胚胎心肌或冠状动脉的载体 前体； g) 使用这些载体证明 FGF 信号传导是 早期肌细胞增殖所需，但肌细胞不需要 差异化； h) 创建了破坏粘附的钙粘蛋白结构 路口； i) 分离出编码 MyBP-H 的全长小鼠基因组克隆 并制备了胚胎同源重组所需的载体 干细胞（ES）和随后的小鼠基因敲除。 基于这些 取得的成就我们现在提出两组体内研究，一组在 胚胎鸡和另一种转基因小鼠，以测试 MyBP-C 和-H 在胚胎心脏中的功能。 1. 使用复制缺陷型逆转录病毒载体，我们将靶向 重组形式 - 野生型和突变型 - 针对心前病变 中胚层或胚胎选定阶段形成的心肌 雏鸡发育。 我们的目标是测试是否：a）早熟 野生型MyBP-C的表达； b) 野生型的反义抑制 MyBP-C； c) 或MyBP-C突变形式的表达改变肌原纤维 卵内的组装和心脏形态发生。 后续实验将 重点关注 MyBP-H 的可比分析。 2. 我们将准备骨骼MyBP-H基因敲除的转基因小鼠， 心脏 MyBP-H 和心脏 MyBP-C。 我们的目标是分析 小鼠心肌和骨骼肌的发育和功能 这些基因是杂合的或无效的。

The goal of our research program is to understand the morphogenesis of embryonic cardiac muscle in vivo, particularly the assembly of myofibrils in that tissue. The central focus of the project is the role of two myosin-binding proteins(MyBPs) termed MyBP-C and MyBP-H (C and H protein, respectively), which we hypothesize to be necessary for the lateral alignment of thick myofilaments in nascent A bands. In work accomplished over the past 5 years, we have: a) cloned, sequenced and identified the myosin binding domains of both proteins and pinpointed the region of MyBP-C both necessary and sufficient for targeting of this protein to the A band; b) shown that a truncation mutant lacking the myosin binding domain acts as a dominant negative for myofibril assembly; c) demonstrated that MyBP-C is expressed significantly after sarcomeric myosin in the embryonic heart and in cultured skeletal myotubes, and shown that the appearance of MyBP-C correlates with the emergence of cross-striations in those cells; d) prepared replication-defective, retroviral vectors which have been used to trace the lineage of cardiomyocytes, precursors of the coronary vessels, and the cardiac conduction system; e) proposed plausible models to explain the morphogenesis of the myocardium, coronary vessels and the peripheral conduction in vivo; f) constructed modified versions of these retroviral vectors for gene delivery to the embryonic myocardium or to coronary precursors; g) used those vectors to prove that FGF signaling is required for early myocyte proliferation but not for myocyte differentiation; h) created cadherin constructs which disrupt adherent junctions; i) isolated a full-length mouse genomic clone encoding MyBP-H and prepared the vector needed for homologous recombination in embryonic stem (ES) cells and subsequent mouse gene knockout. Based on these accomplishments we now propose two sets of in vivo studies, one in embryonic chickens and the other in transgenic mice, to test the functions of MyBP-C and -H in the embryonic heart. 1. Using replication-deficient, retroviral vectors, we will target recombinant forms - both wild-type and mutant - to the precardiac mesoderm or to the forming myocardium at selected stages of embryonic chick development. Our goal will be to test whether: a) precocious expression of wild-type MyBP-C; b) anti-sense suppression of wild-type MyBP-C; c) or the expression of mutant forms of MyBP-C alter myofibril assembly and cardiac morphogenesis in ovo. Subsequent experiments will focus on comparable analyses of MyBP-H. 2. We will prepare transgenic mouse knockouts for skeletal MyBP-H, cardiac MyBP-H and cardiac MyBP-C. Our goal will be to analyze the development and function of cardiac and skeletal muscle in mice either heterozygous or null for these genes.