Function of Giant Sarcomere Matrix Proteins in Muscle

描述（由申请人提供）：我们的长期目标是了解titin在心功能和疾病中的作用。肌粘连蛋白是横纹肌的第三条肌丝，位于肌节的i带，具有分子弹簧的功能，当拉伸时产生力量。这种力是心肌在心脏充盈期产生的很大一部分力的基础。人心肌同时表达僵硬型和柔顺型titin亚型。在扩张型心肌病（DCM）和冠状动脉疾病患者中发现了异构体表达谱的改变。这些都伴随着被动刚度的改变。此外，最近的研究发现了几种导致DCM的titin突变。

DESCRIPTION (provided by applicant): Our long-term objective is to understand the roles of titin in cardiac function and disease. Titin is the third myofilament of striated muscle and contains an extensible region, located in the I-band of the sarcomere, which functions as a molecular spring that, when extended, develops force. This force underlies a large fraction of the force developed by myocardium during the filling phase of the heart. Human myocardium expresses both stiff and compliant titin isoforms. Altered isoform expression profiles have been found in patients with dilated cardiomyopathy (DCM) and coronary artery disease. These are accompanied by altered passive stiffness. Furthermore, recent studies have discovered several titin mutations that lead to DCM. We propose to study the molecular basis of titin's extensibility, including the effects of posttranslational modifications on extensibility. We will use recombinant proteins representing titin's various spring elements and study their mechanical and structural properties, using atomic force microscopy and spectroscopic techniques. The effects of calcium and phosphorylation on mechanics and structure will be examined. Because titin isoform expression may regulate calcium sensitivity and beta-adrenergic control of titin-based elasticity, we will develop a titin exon microarray to characterize exon-splicing patterns. We will also develop and characterize several mouse knockout models in which spring elements have been excised in order to study the following hypothesized functions: 1) glutamate rich PEVK elements are calcium binding sites that can alter titin-based passive force in a calcium-dependent manner; 2) the cardiac unique N2B sequence can alter titin-based passive force via phosphorylation. This research will provide important and novel insights into the molecular basis of titin's elasticity and its adjustments via calcium and phosphorylation. Considering the multitude of 'titinopathies' that are rapidly being discovered, our findings will be important for understanding human cardiac function and disease.