TROPONIN I IN CARDIAC DEVELOPMENT

肌钙蛋白 I 在心脏发育中的作用

• 批准号: 3474162
• 负责人: ANNE Z MURPHY
• 金额: $ 11.1万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 1996-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3474162
• 关键词: cell differentiation; developmental genetics; embryo /fetus tissue /cell culture; embryogenesis; gene expression; genetic library; genetic recombination; genetic regulatory element; genetic transcription; growth /development; heart; heart cell; hypertrophy; immature animal; laboratory rabbit; laboratory rat; mature animal; monoclonal antibody; muscle contraction; mutant; myofibrils; nucleic acid sequence; protein sequence; protein structure function; reporter genes; striated muscles; structural genes; thyroid hormones; tissue /cell culture; transfection; troponin; western blottings

Troponin I, the inhibitory subunit of troponin, is an important regulatory protein in striated muscle. Separate genes encode three tissue-specific isoforms of this protein. In mature fast- and slow-twitch skeletal and cardiac muscle these isoforms are specifically expressed, however, in fetal and neonatal rat and human heart the cardiac and slow skeletal troponin I mRNAs and isoforms are co-expressed. Alterations in the response of the contractile apparatus to acidosis and beta-adrenergic stimulation may result from the presence of slow skeletal troponin I in the immature heart. The long term objectives of-this work are to understand the functional consequences of troponin I isoform variation in the heart, and to understand the cis- and trans-acting factors which allow expression of the slow skeletal and cardiac troponin I mRNAs in immature heart, and those which direct specific expression of cardiac troponin I in the mature heart. The specific aims of this proposal are: 1. To characterize the regulatory regions of the rat cardiac troponin I gene by the use of transient expression assays of reporter gene constructs transfected into primary neonatal rat cardiocytes. The regions to be characterized include elements which regulate expression during cardiac maturation and elements which direct cardiac-specific expression. 2. To obtain genomic clones of rat slow skeletal troponin I by screening rat genomic libraries in lambda vectors. To characterize regulatory regions of this gene by the use of transient expression assays of reporter gene constructs in skeletal muscle cell lines, and in primary cardiocytes. The elements to be characterized include those which confer muscle-specificity, those which inhibit expression during cardiac maturation, and elements responsive to thyroid hormone. 3. To determine if fast skeletal troponin I or an "embryonic" cardiac troponin I isoform is expressed in embryonic or fetal rat heart, and the time course of its expression. If an embryonic gene is found, to determine whether its mRNA is re-expressed in a model for pressure overload hypertrophy created by banding the aorta of adult rats. 4. To purify recombinantly expressed wild type and mutant rat cardiac troponin I in order to assess regions of the cardiac troponin I molecule which result in the greater sensitivity of the contractile apparatus to acidosis when the cardiac isoform in present in the troponin complex.

肌钙蛋白 I 是肌钙蛋白的抑制亚基，是一种重要的肌钙蛋白 横纹肌中的调节蛋白。 不同的基因编码三个 该蛋白质的组织特异性亚型。 在成熟的快速和 慢肌骨骼肌和心肌这些异构体是专门的 然而，在胎儿和新生大鼠以及人类心脏中表达 慢速骨骼肌钙蛋白 I mRNA 和异构体共表达。 收缩器官对酸中毒的反应的改变 β-肾上腺素能刺激可能是由于慢速骨骼肌的存在引起的 肌钙蛋白 I 在未成熟的心脏中。 这项工作的长期目标 是为了了解肌钙蛋白 I 亚型的功能后果 心脏的变异，并了解顺式和反式作用 允许慢骨骼肌肌钙蛋白和心肌肌钙蛋白表达的因素 I 未成熟心脏中的 mRNA，以及那些指导特定表达的 mRNA 成熟心脏中的心肌肌钙蛋白 I。 本次活动的具体目标 建议是： 1. 描述大鼠的调节区域 心肌肌钙蛋白 I 基因的瞬时表达分析 报告基因构建体转染原代新生大鼠 心肌细胞。 要表征的区域包括以下元素 调节心脏成熟过程中的表达以及指导的元件 心脏特异性表达。 2. 获得大鼠慢基因组克隆 通过筛选 lambda 载体中的大鼠基因组文库来检测骨骼肌钙蛋白 I。 通过使用瞬态来表征该基因的调控区域 骨骼肌细胞中报告基因构建体的表达测定 线和原代心肌细胞中。 待表征的元素 包括赋予肌肉特异性的那些、抑制肌肉特异性的那些 心脏成熟过程中的表达以及对甲状腺有反应的元素 激素。 3. 确定快骨骼肌钙蛋白 I 是否为“胚胎” 心肌肌钙蛋白 I 同工型在胚胎或胎鼠心脏中表达， 及其表达的时间过程。 如果发现胚胎基因， 确定其 mRNA 是否在压力模型中重新表达 通过捆扎成年大鼠的主动脉而造成的超负荷肥大。 4. 至 纯化重组表达的野生型和突变型大鼠心肌肌钙蛋白 I 为了评估心肌肌钙蛋白 I 分子的区域 导致收缩装置对 当心肌同种型存在于肌钙蛋白复合物中时，会发生酸中毒。