MOLECULAR/CELLULAR BIOLOGY OF CARDIOMYOCTE DEVELOPMENT

我们的研究计划的目标是了解 活体胚胎心肌，特别是肌原纤维的组装 在那个组织里。该项目的中心焦点是两个人的角色 肌球蛋白结合蛋白(MyBP)称为MyBP-C和MyBP-H(C和H蛋白， 分别)，我们假设这对于侧向是必要的 新生A带中粗大的肌丝排列。在完成的工作中 在过去的5年里，我们已经：a)克隆、测序和鉴定了 这两种蛋白的肌球蛋白结合域，并精确定位了 MyBP-C是将该蛋白靶向于 一条带；b)表明缺少肌球蛋白结合的截断突变体 结构域是肌原纤维组装的主要负向；c) 证明MyBP-C在肉瘤细胞中显著表达 胚胎心脏和培养的骨骼肌管中的肌球蛋白，以及 显示MyBP-C的出现与MBP-C的出现 这些细胞中的交叉条纹；d)制备复制缺陷， 逆转录病毒载体已被用于追踪人类的血统 心肌细胞，冠状动脉和心脏的前体 传导系统；e)提出合理的模型来解释 心肌、冠脉和外周的形态发生 体内传导；f)构建了这些逆转录病毒的修改版本 基因输送至胚胎心肌或冠状动脉的载体 前体；g)使用这些载体来证明成纤维细胞生长因子信号转导是 是早期心肌细胞增殖所必需的，但不是肌细胞所必需的 分化；h)产生了钙粘附素结构，破坏了贴壁 I)分离了编码MyBP-H的全长小鼠基因组克隆 并制备了胚胎中同源重组所需的载体 干细胞和随后的小鼠基因敲除。基于这些 成就我们现在提出两组体内研究，一组在 胚胎鸡和另一只转基因小鼠，以测试 MyBP-C和-H在胚胎心脏中的作用 1.使用复制缺陷的逆转录病毒载体，我们将针对 重组形式-包括野生型和突变型-到心脏前 中胚层或胚胎特定阶段的形成心肌 小鸡的发育。我们的目标将是测试：a)早熟 野生型MyBP-C的表达；b)野生型反义抑制 MyBP-C；c)或MyBP-C突变形式的表达改变肌原纤维 卵细胞的组装和心脏形态发生。随后的实验将 重点进行MyBP-H的可比性分析。 2.我们将制备骨骼MyBP-H的转基因小鼠敲除， 心脏MyBP-H和心脏MyBP-C。我们的目标将是分析 小鼠心肌和骨骼肌的发育和功能 这些基因为杂合子或为空。

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.