Function of Giant Sarcomere Matrix Proteins in Muscle

肌肉中巨肌节基质蛋白的功能

• 批准号: 6678371
• 负责人: Henk L. GRANZIER
• 金额: $ 33.03万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6678371
• 关键词: atomic force microscopy; beta adrenergic agent; calcium ion; cardiac myocytes; elasticity; fluorescence spectrometry; genetically modified animals; heart function; immunoelectron microscopy; laboratory mouse; microarray technology; muscle proteins; muscle tension; myocardium; phosphorylation; polymerase chain reaction; posttranslational modifications; protein isoforms; protein kinase A; protein kinase C; protein structure function; recombinant proteins; sarcomeres; striated muscles

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.

描述（由申请人提供）：我们的长期目标是了解肌联蛋白在心脏功能和疾病中的作用。肌联蛋白是横纹肌的第三根肌丝，包含一个可伸展区域，位于肌节的 I 带中，其功能相当于分子弹簧，当伸展时会产生力。这种力占心脏充盈阶段心肌产生的力的很大一部分。人类心肌表达刚性和顺应性肌联蛋白亚型。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 的肌联蛋白突变。 我们建议研究肌联蛋白延伸性的分子基础，包括翻译后修饰对延伸性的影响。我们将使用代表肌动蛋白各种弹簧元素的重组蛋白，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。将检查钙和磷酸化对力学和结构的影响。由于肌联蛋白亚型表达可能调节钙敏感性和基于肌联蛋白弹性的β-肾上腺素能控制，因此我们将开发肌联蛋白外显子微阵列来表征外显子剪接模式。我们还将开发和表征几种小鼠敲除模型，其中弹簧元件已被切除，以便研究以下假设的功能：1）富含谷氨酸的PEVK元件是钙结合位点，可以以钙依赖性方式改变基于肌动蛋白的被动力； 2) 心脏独特的N2B序列可以通过磷酸化改变基于肌动蛋白的被动力。 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的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”，我们的发现对于了解人类心脏功能和疾病非常重要。