TITINS ROLE IN MODULATING CARDIAC PASSIVE TENSION

Titins 在调节心脏被动张力中的作用

• 批准号: 6537569
• 负责人: MARION Lewis GREASER
• 金额: $ 20.25万
• 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 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6537569
• 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.

肌联蛋白是在心脏和骨骼肌中发现的3000 kD蛋白质，并且据信其主要负责来自这些组织的细胞的静息张力。 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否是由于肌联蛋白同种型的表达改变或来自单个肌联蛋白基因的表达盒改变。 这些研究将采用狭缝印迹法，探针对应于肌联蛋白的中央I带部分。 将测定大鼠和犬心肌中主要亚型PEVK区域及其侧翼区域的cDNA序列。 肌联蛋白N2 A和N2 B型信息的频率将通过使用迄今为止观察到的所有心脏肌联蛋白中常见的N2 B序列从大鼠和犬cDNA文库中分离和测序多个克隆来估计。 将使用这些种属组织切片的特异性抗体和定量免疫荧光来估计N2 A和N2 B亚型的蛋白质比例。 类似的方法将用于检查肌联蛋白同种型在不同品系的大鼠（包括具有自发性高血压- SHR的那些）中和在慢性起搏后在肥大的狗心脏中的表达。 表达的含有PEVK区域的肌联蛋白片段与肌动蛋白的结合将通过共沉降测定和表面等离子体共振测定。 还将测试这些片段对去皮的单个心肌细胞的影响。 通过测量几种激酶（蛋白激酶A、蛋白激酶C、酪蛋白激酶II）对去皮大鼠肌细胞静息张力的影响，确定磷酸化状态变化对静息张力的可能调节。 将使用γ 32 P标记的ATP测试不同纯化的激酶（蛋白激酶A、蛋白激酶C、酪蛋白激酶II）在皮肤化的心脏细胞中磷酸化肌联蛋白的能力，并且这种磷酸化与细胞中的任何机械变化相关。磷酸化肽将被分离和测序，并绘制其在肌节中的位置。 还将通过2D肽图谱确定的磷酸化模式与用β-激动剂或磷酸酶抑制剂处理完整心肌细胞后获得的磷酸化模式进行比较。 将使用类似方法评估肥大与正常大鼠和犬心脏的磷酸化状态。 通过改变肌联蛋白亚型表达或改变肌联蛋白磷酸化状态来调节静息张力可能是改变体内心脏功能的重要机制。 肌联蛋白的变化可能与通常由高血压引起的肥大反应有关，因此是人类健康中常见的问题。