TROPONIN I IN CARDIAC DEVELOPMENT

肌钙蛋白I，肌钙蛋白的抑制亚单位，是一种重要的 横纹肌中的调节蛋白。单独的基因编码三个 这种蛋白质的组织特有的异构体。在成熟的快速-和 慢抽动骨骼肌和心肌这些异构体是特定的 然而，在胎儿和新生大鼠以及人的心脏中表达的心脏 慢速骨骼肌钙蛋白ImRNAs和异构体共表达。 收缩装置对酸中毒反应的改变和 β-肾上腺素能刺激可能是缓慢骨骼的存在所致 幼稚心脏中的肌钙蛋白I。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，并理解顺式和反式作用 允许慢骨骼肌钙蛋白和心肌肌钙蛋白表达的因素 ImRNAs在未成熟心脏中的表达，以及那些直接特异性表达 成熟心脏中的心肌肌钙蛋白I。这样做的具体目的是 建议如下：1.确定大鼠的调控区域 心肌肌钙蛋白I基因的瞬时表达分析 报告基因载体导入原代新生大鼠的研究 心肌细胞。要表征的区域包括以下元素 心脏成熟过程中基因表达的调控及其调控因素 心脏特异表达。2.大鼠慢速基因组克隆的获得 通过在lambda载体中筛选大鼠基因组文库获得骨骼肌钙蛋白I。 通过使用瞬变信号来表征该基因的调节区 报告基因在骨骼肌细胞中的表达分析 在原代培养的心肌细胞中。要刻画的要素 包括那些赋予肌肉特异性、抑制 心脏成熟过程中的表达及其对甲状腺的反应 荷尔蒙。3.确定快速骨骼肌钙蛋白I或“胚胎” 心肌肌钙蛋白I亚型在胚胎或胎鼠心脏中表达， 以及它的表达的时间进程。如果发现了胚胎基因， 确定其mRNA是否在压力模型中重新表达 成年大鼠主动脉环扎造成的超负荷肥大。4.至 重组表达的野生型和突变型大鼠心肌肌钙蛋白的纯化 为了评估心肌肌钙蛋白I分子的哪些区域 从而使收缩装置对 肌钙蛋白复合体中存在心脏亚型时的酸中毒。

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.