Developmental Changes Affecting Cardiac Titin Function

描述（由申请人提供）：Titin是一种存在于心脏和骨骼肌中的3000-4000 kD蛋白，被认为在控制这些组织的静息或被动张力中起主要作用。主要的发育变化已被发现发生在心脏titin作为选择性剪接途径的结果。一种独特的大鼠菌株被发现具有常染色体显性突变，导致胎儿到成人的titin过渡模式延迟。这些动物将使用超声心动图和压力-容量关系测量来测试titin在完整动物心脏血流动力学中的作用。单个心肌细胞的机械实验将检验关于titin在静息张力和Frank-Starling关系中的作用的假设。titin可扩展的PEVK区带正电荷的PPAK肽与带负电荷的polyye肽之间的相互作用将通过色谱、圆二色、电泳和表面等离子体共振技术进行测试。研究将被设计来确定PPAK和polyye肽的结合是否对序列中的相邻区域具有特异性，或者是否会发生多种类型的相互作用。富含谷氨酸的Nontitin肽也将被用于结合。先前提出的磷酸化位点将在表达的titin多肽中确定，并且这些位点也将在完整的胎儿和成人titin中检测磷酸化状态。这些实验将检验这样一种假设，即某些分子必须经过磷酸化才能进行适当的组装和/或发挥作用。基因定位研究将用于定位导致发育迟缓的titin异构体程序的突变位点。这些研究将为该巨蛋白的结构和功能及其与人类健康和心血管疾病的关系提供新的信息。这项工作也可能为选择性剪接机制提供新的见解，这是一个对理解蛋白质组学越来越重要的领域。

DESCRIPTION (provided by applicant): Titin is a 3000-4000 kD protein found in heart and skeletal muscle, and it has been proposed to play a major role in controlling the resting or passive tension in these tissues. Major developmental changes have been found to occur in cardiac titin as a result of alternative splicing pathways. A unique rat strain has been discovered with an autosomal dominant mutation that leads to a delayed fetal-to-adult titin transition pattern. These animals will be used to test the role of titin in cardiac hemodynamics in the intact animal using echocardiography and pressure-volume relationship measurements. Mechanical experiments on single cardiomyocytes will test hypotheses regarding titin's role in rest tension and the Frank-Starling relationship. Interactions betweer_ positively charged PPAK peptides from titin's extensible PEVK region with negatively charged polyE peptides will be tested using chromatographic, circular dichroism, electrophoretic, and surface plasmon resonance techniques. Studies will be designed to determine if binding of the PPAK and polyE peptides is specific to adjacent regions in the sequence or if multiple types of interactions can occur. Nontitin peptides rich in glutamic acid will also be examined for binding. Previously proposed phosphorylation sites will be identified in expressed titin polypeptides, and these sites will also be assayed for )hosphorylation state in intact fetal and adult titins. These experiments will test the hypothesis that certain _ites must be phosphorylated for proper assembly and/or function. Gene mapping studies will be conducted to localize the mutation site leading to the delayed developmental titin isoform program. The proposed studies will provide new information regarding the structure and function of this giant protein and its relation to human health and cardiovascular disease. The work may also provide novel insights on mechanisms of alternative splicing, an area of increasing importance in understanding the proteome.