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.

Titin是一种在心脏和骨骼肌中发现的3000 kD蛋白，据信它在很大程度上负责这些组织的细胞静息张力。 将进行研究以确定来自不同物种的心肌细胞之间静息张力的差异是由于钛同工型的表达改变或来自单个Titin基因的盒式盒式表达改变。 这些研究将采用与对应于Titin中央I波段部分相对应的探针的插槽印迹。 将确定大鼠和狗心肌的主要同工型的PEVK区域的cDNA序列。 TITIN N2A和N2B类型消息的频率将通过使用迄今为止观察到的所有心脏滴定素中常见的N2B序列进行隔离和测序来自大鼠和狗cDNA库中的多个克隆的频率。 这些物种的组织切片的特定抗体和定量免疫荧光将用于估计N2A和N2B同工型类的同工型的蛋白质比例。 类似的方法将用于检查不同大鼠菌株（包括自发性高血压-SHR）和慢性起搏后肥大的狗心脏中的钛同工型表达。 包含PEVK区域与肌动蛋白的表达的滴定片段的结合将由共同测量测定和表面等离子体共振确定。 此类碎片还将测试对皮肤单一心肌细胞的影响。 通过测量几种激酶（蛋白激酶A，蛋白激酶C，酪蛋白激酶II）对皮肤大鼠肌细胞静息张力的影响，将通过测量几种激酶（蛋白激酶A，蛋白激酶C，酪蛋白激酶II）的影响来确定静息张力的调节。 不同的纯化激酶（蛋白激酶A，蛋白激酶C，酪蛋白激酶II）将通过使用标记为ATP的伽马32P磷酸化的心脏细胞中磷酸化钛的能力进行测试，这种磷酸化与细胞中的任何机械变化有关。磷酸化的肽（S）将被分离和测序，并将其位于肌节映射。 由2D肽图确定的磷酸化模式也将与用β-激动剂或磷酸酶抑制剂治疗完整的心肌细胞后获得的磷酸化模式。 肥大与正常大鼠和狗心脏的磷酸化状态将使用相似的方法进行评估。 通过改变同工型表达或通过改变Titin的磷酸化状态来调节静止张力可能是改变体内心脏功能的重要机制。 TITIN的变化可能与高血压通常引起的肥厚反应有关，因此是人类健康中常见的问题。