Function of Giant Sarcomere Matrix Proteins in Muscle

说明(申请人提供)：Titin是一种多功能的巨型丝状蛋白，对肌肉的结构和功能有许多重要的影响。该项目的长期目标是确定Titin影响被动和主动肌肉生理学的机制，特别是在心血管系统方面。我们对心肌的研究表明，Titin的L带弹力区包含独特的可扩展元素，这些元素既塑造了Titin的短期属性，又通过替代剪接塑造了其长期适应。这些不同的区域是在心肌和骨骼肌中发现的串联Ig和PEVK元件，以及仅在心肌中发现的N2B元件。最近的证据表明，PEVK和N2B元件独特地调节被动心肌僵硬，这种调节对于塑造心脏功能和适应非常重要。我们将重点阐明这些独特的心肌力学机制，包括S100A1在调节基于PEVK的机制中的作用。我们将使用新的敲除(KO)模型，其中PEVK或N2B元件已被切除，以及一个在S100A1(SKO)中缺失的模型。我们还将通过研究PEVK KO和N2B KO小鼠模型(高被动刚性)以及表达巨型Titin亚型的对比大鼠模型(低被动刚性)，对Titin调节主动心肌力发育的假设进行批判性检验。我们将测量钙敏感性的长度依赖关系以及Titin对肌丝结构的影响(使用小角X射线衍射)。我们将使用多方面的方法完成拟议的研究，在单分子、单细胞、整个肌肉和完整心脏的水平上使用实验技术。我们的研究将有助于理解被动僵硬调节，包括它在缺血再灌注损伤中的作用，以及基于Titin的被动僵硬和主动力量发展之间的相互作用。被动心肌硬度是舒张期充盈和Frank-Starling机制应用的重要决定因素，我们的研究将有助于理解Titin在舒张期和收缩期功能障碍中的作用。

DESCRIPTION (provided by applicant): Titin is multi-functional giant filamentous protein with many important effects on structure and function of muscle. The long-term goal of this project is to determine the mechanisms whereby titin influences passive and active muscle physiology, particularly with respect to the cardiovascular system. Our work in cardiac muscle has shown that titin's l-band spring region contains distinct extensible elements that shape both its short-term properties and, through alternative splicing, its long-term adaptation. These distinct regions are the tandem Ig and PEVK elements found in both cardiac and skeletal muscle and the N2B element found only in cardiac muscle. Recent evidence suggests that the PEVK and N2B elements uniquely modulate passive cardiac muscle stiffness, and that this modulation is important for shaping heart function and adaptation. We will focus on elucidating these unique mechanisms in myocardial mechanics, including the role of S100A1 in regulating the PEVK-based mechanism. We will use novel knock-out (KO) models in which the PEVK or N2B element has been excised and a model that is deficient in S100A1 (SKO). We will also critically test the hypothesis that titin regulates active myocardial force development by studying the PEVK KO and N2B KO mouse models (high passive stiffness) as well as a contrasting rat model that expresses a giant titin isoform (low passive stiffness). We will measure the length dependence of calcium sensitivity and the effect of titin on myofilament structure (using low angle X-ray diffraction). We will accomplish the proposed research using a multi-faceted approach, with experimental techniques at levels ranging across the single molecule, single cell, whole muscle, and the intact heart. Our research will contribute to understanding passive stiffness modulation, including its role in ischemia-reperfusion injury, and the interplay between titin-based passive stiffness and active force development. Passive myocardial stiffness is an important determinant of diastolic filling and utilization of the Frank-Starling mechanism, and our research will contribute to understanding the roles of titin in both diastolic and systolic dysfunction.