Developmental Changes Affecting Cardiac Titin Function

描述（由申请人提供）：肌联蛋白是一种在心脏和骨骼肌中发现的3000-4000 kD蛋白质，已提出其在控制这些组织中的静息或被动张力中起主要作用。 主要的发展变化已被发现发生在心脏肌联蛋白作为一个结果的选择性剪接途径。 发现了一种独特的大鼠品系，其常染色体显性突变导致延迟的胎儿至成人肌联蛋白转变模式。 这些动物将用于使用超声心动图和压力-容积关系测量来测试肌联蛋白在完整动物中心脏血流动力学中的作用。 对单个心肌细胞的机械实验将测试关于肌联蛋白在静息张力和Frank-Starling关系中的作用的假设。 来自肌联蛋白的可延伸PEVK区域的带正电荷的PPAK肽与带负电荷的聚E肽之间的相互作用将使用色谱、圆二色性、电泳和表面等离子体共振技术进行测试。 将设计研究以确定PPAK和聚E肽的结合是否对序列中的相邻区域具有特异性，或者是否可能发生多种类型的相互作用。 还将检查富含谷氨酸的非肌联蛋白肽的结合。 先前提出的磷酸化位点将在表达的肌联蛋白多肽中鉴定，并且这些位点也将在完整的胎儿和成人肌联蛋白中测定磷酸化状态。 这些实验将检验某些位点必须被磷酸化以进行适当组装和/或功能的假设。 将进行基因定位研究，以定位导致延迟发育肌联蛋白亚型程序的突变位点。 这些研究将为这种巨大蛋白质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。 这项工作也可能提供新的见解机制的选择性剪接，一个领域的日益重要的理解蛋白质组。

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.