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肌动蛋白在心脏功能和疾病中的作用。肌动蛋白是横纹肌的第三种肌丝，它包含一个可伸展的区域，位于肌节的I-带，其作用是当伸展时产生力量的分子弹簧。这种作用力是心脏充盈阶段心肌产生的很大一部分作用力的基础。人心肌表达刚性和顺应性肌动蛋白亚型。在扩张型心肌病(DCM)和冠状动脉疾病患者中发现了不同亚型的表达谱。伴随着这些变化的是被动僵硬。此外，最近的研究发现了几个导致扩张型心肌梗死的肌动蛋白突变。 我们建议研究Titin延展性的分子基础，包括翻译后修饰对延展性的影响。我们将使用代表Titin的各种弹簧元件的重组蛋白质，并使用原子力显微镜和光谱技术研究它们的机械和结构特性。钙和磷酸化对力学和结构的影响将被检测。由于肌动蛋白异构体的表达可能调节钙敏感性和β-肾上腺素能调控肌动蛋白的弹性，我们将开发一种肌动蛋白外显子微阵列来表征外显子剪接模式。我们还将建立和鉴定几种剔除弹簧元件的小鼠基因敲除模型，以研究以下假设功能：1)富含谷氨酸的PEVK元件是钙结合部位，可以以钙依赖的方式改变基于titin的被动力；2)心脏独特的N2B序列可以通过磷酸化改变基于titin的被动力。 这项研究将为肌动蛋白弹性的分子基础及其通过钙和磷酸化进行调节提供重要而新颖的见解。考虑到正在迅速发现的大量‘钛病’，我们的发现将对了解人类的心脏功能和疾病非常重要。

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.