TITINS ROLE IN MODULATING CARDIAC PASSIVE TENSION

Titins 在调节心脏被动张力中的作用

• 批准号: 6390322
• 负责人: MARION Lewis GREASER
• 金额: $ 19.73万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6390322
• 关键词: animal tissue; beta adrenergic agent; cardiac myocytes; casein kinase; chickens; heart enlargement; immunologic assay /test; laboratory rabbit; laboratory rat; muscle cells; muscle proteins; muscle tension; nucleic acid sequence; phosphatase inhibitor; phosphorylation; protein binding; protein isoforms; protein kinase A; protein kinase C; protein localization; protein structure function; sarcomeres; species difference; spontaneous hypertensive rat

Titin is a 3000 kD protein found in heart and skeletal muscle, and it is believed to be largely responsible for the resting tension of cells from these tissues. Studies will be conducted to determine if differences in resting tension between cardiomyocytes from different species are due to altered expression of titin isoforms or altered cassette expression from the single titin gene. These studies will employ slot blots with probes corresponding to the central I-band portion of titin. The cDNA sequence for the region including and flanking the PEVK region of the major isoforms in rat and dog cardiac muscle will be determined. The frequency of the titin N2A and N2B type messages will be estimated by isolation and sequencing multiple clones from rat and dog cDNA libraries using the N2B sequence common in all cardiac titins observed to date. Specific antibodies and quantitative immuno-fluorescence of tissue sections from these species will be used to estimate the protein proportion of isoforms of the N2A and N2B isoform classes. A similar approach will be used to examine titin isoform expression in different strains of rats (including those with spontaneous hypertension- SHR) and in hypertrophied dog heart after chronic pacing. The binding of expressed titin fragments containing the PEVK region to actin will be determined by co-sedimentation assays and by surface plasmon resonance. Such fragments will also be tested for effects on skinned single cardiomyocytes. The possible modulation of rest tension by changes in phosphorylation state will be determined by measuring the effects of several kinases (protein kinase A, protein kinase C, casein kinase II) on resting tension of skinned rat myocytes. Different purified kinases (protein kinase A, protein kinase C, casein kinase II) will be tested for their ability to phosphorylate titin in skinned cardiac cells using gamma 32P labeled ATP, and this phosphorylation related to any mechanical changes in the cells. The phosphorylated peptide(s) will be isolated and sequenced and their location in the sarcomere mapped. The phosphorylation patterns as determined by 2D peptide maps will also be compared to those obtained after treatment of intact cardiomyocytes with beta-agonist or phosphatase inhibitors. Phosphorylation status of hypertrophied versus normal rat and dog heart will be assessed using similar methods. The possible modulation of rest tension either by altering isoform expression or by changing titin's phosphorylation state may be important mechanisms for changing cardiac function in vivo. Titin changes may be related to the hypertrophic response that commonly is brought about by high blood pressure and thus is a frequent problem in human health.

肌动蛋白是一种3000kD的蛋白质，存在于心脏和骨骼肌中，被认为是这些组织细胞静息张力的主要原因。将进行研究，以确定不同物种的心肌细胞之间静息张力的差异是由于titin亚型表达的变化还是由于单个titin基因盒表达的变化。这些研究将使用缝隙印迹法，探针对应于Titin的中心I带部分。将确定包括大鼠和狗心肌主要异构体PEVK区及其两侧的区域的cDNAs序列。将通过从大鼠和狗的cDNA文库中分离和测序多个克隆来估计titin N2a和N2b型消息的频率，该克隆使用迄今观察到的所有心脏titin中常见的N2B序列。来自这些物种的特定抗体和组织切片的定量免疫荧光将被用来估计N2A和N2B亚型的亚型的蛋白质比例。类似的方法将被用来检测不同品系的大鼠(包括自发性高血压-自发性高血压大鼠)和长期起搏后肥厚的狗心脏中肌动蛋白亚型的表达。含有PEVK区的表达的Titin片段与肌动蛋白的结合将通过共沉淀法和表面等离子体共振来确定。这些碎片也将被测试对带皮的单个心肌细胞的影响。通过测量几种蛋白激酶(蛋白激酶A、蛋白激酶C、酪蛋白激酶II)对大鼠皮肤细胞静息张力的影响，可以确定磷酸化状态变化对静息张力的可能调节。不同纯化的激酶(蛋白激酶A、蛋白激酶C、酪蛋白激酶II)将使用伽马32P标记的ATP来测试它们对皮肤心肌细胞中肌动蛋白的磷酸化能力，这种磷酸化与细胞中的任何机械变化有关。磷酸化肽(S)将被分离和测序，并绘制它们在肌节中的位置。二维肽图谱确定的磷酸化模式也将与用β-激动剂或磷酸酶抑制剂处理完整的心肌细胞后所获得的模式进行比较。肥厚的大鼠和狗心脏的磷酸化状态将使用类似的方法进行评估。通过改变肌动蛋白亚型的表达或改变肌动蛋白的磷酸化状态来调节静息张力可能是体内改变心功能的重要机制。肌动蛋白的变化可能与肥大反应有关，而肥大反应通常是由高血压引起的，因此是人类健康的常见问题。