Role of the Giant Protein Titin in Skeletal Muscle Structure and Function

描述(申请人提供)：肌动蛋白是一种横跨半个肌节的巨大蛋白质，其功能是作为分子弹簧，支持维持收缩肌节结构完整性的被动和恢复性力量。肌动蛋白对被动肌肉僵硬的作用已经在心肌中得到了很好的研究，但相比之下，肌动蛋白在骨骼肌中的作用却知之甚少。由于骨骼肌和心肌之间有许多不同之处，需要对每种肌肉类型进行单独的研究。骨骼肌的被动僵硬对功能活动和生活质量有很大影响，因此，需要研究被动僵直产生的分子机制以及Titin在这种僵硬中的作用。我们将首先进行几个复杂程度(从皮肤纤维到整个肌肉)的僵硬测量，并确定Titin对每个水平的被动僵硬的贡献。我们将研究Titin对被动僵硬的贡献如何在不同的肌肉中变化，这取决于Titin的差异剪接和翻译后修饰。为了便于快速和准确地确定在不同肌肉类型中表达的Titin亚型的序列，我们开发了一种Titin外显子微阵列，它包含了在一系列物种(人类363个外显子)中发现的所有Titin外显子。我们预计，我们的工作将大大提高对肌钙蛋白在被动肌肉僵硬中的作用的理解，这将为理解其在肌肉疾病中的作用提供可靠的基础，我们将在下一步讨论这一问题。我们将在超负荷引起的肥大和废弃引起的萎缩中建立肌动蛋白的僵硬，与细胞外基质(胶原和弹性蛋白)的变化相关。肌动蛋白在疾病中的作用也将在Tenascin-X(TnX)缺乏的患者(Ehler-Danlos综合征的一种类型)和TnX KO小鼠中进行研究；我们的初步数据表明，基于Titin的僵硬增加，作为补偿反应，抵消TnX缺乏的肌肉胶原僵硬的减少。我们的最后一个目标将对Titin作为生物力学传感器触发肥大的提议进行关键测试。主要焦点将放在缺乏PEVK外显子219-225(PEVK KO)的转基因小鼠模型上。(PEVK是Titin弹簧的重要弹性来源)。初步数据显示，PEVK KO的骨骼肌明显肥大，我们将研究其中涉及的信号通路。我们将使用候选方法，包括研究Titin和mTOR信号通路之间的相互作用(这一通路先前被证明可以增加蛋白质合成以响应被动肌肉的拉伸)，以及先前与肥大信号相关的Titin结合蛋白的作用，并且在PEVK KO中高度上调。我们将通过将PEVK KO与这些蛋白已被删除的模型杂交来剖析它们在肥大信号中的作用。了解肌肉肥大的调节机制具有重要的临床意义，因为体重减轻通常是疾病的结果，它会使老年人和卧床不起的患者虚弱。总体而言，拟议的工作将是朝着我们的长期目标迈出的重要一步，我们的长期目标是详细了解Titin在骨骼肌结构和功能、在健康和疾病中的作用。

DESCRIPTION (provided by applicant): Titin, a giant protein that spans the half sarcomere, functions as a molecular spring that underlies the passive and restorative forces that maintain the structural integrity of the contracting sarcomere. The contribution of titin to passive muscle stiffness has been well studied in cardiac muscle but in contrast titin is poorly understood in skeletal muscle. Due to the many differences between skeletal and cardiac muscle, separate studies are needed for each muscle type. Passive stiffness of skeletal muscle greatly influences functional activities and quality of life, and, thus, the molecular mechanisms of passive stiffness generation and the role of titin in this stiffness need to be studied. We will first perform stiffness measurements at several levels of complexity (from skinned fibers to whole muscle) and determine the contribution of titin to passive stiffness of each. We will investigate how titin's contribution to passive stiffness varies in different muscles, depending on differential splicing and posttranslational modifications in titin. To facilitate the speed and accuracy in determining the sequence of titin isoforms expressed in different muscle types, we developed a titin exon microarray which contains all of titin's exons found in a range of species (363 exons in human). We anticipate that our work will greatly increase the understanding of the role of titin in passive muscle stiffness and that this will provide a sound basis for understanding its role in muscle diseases, which we will address next. We will establish titin's stiffness in over-load induced hypertrophy and disuse-induced atrophy, relative to the changes in the extracellular matrix (collagen and elastin). Titin's role in disease will also be studied in tenascin-X (TNX) deficient patients (one of the types of Ehlers-Danlos syndrome) and TNX KO mice; our preliminary data indicate increased titin-based stiffness, as compensatory response to counteract the decreased collagen- stiffness of TNX-deficient muscle. Our last aim will critically test the proposal that titin functions as a biomechanical sensor that triggers hypertrophy. The main focus will be on a genetically engineered mouse model that is deficient in PEVK exons 219-225 (PEVK KO). (The PEVK is an important source of elasticity of the titin spring). Preliminary data show that skeletal muscles of the PEVK KO are significantly hypertrophied, and we will investigate the signaling pathways involved. We will use a candidate approach, including a study of the interaction between titin and the mTOR signaling pathway (this pathway has previously been shown to increase protein synthesis in response to stretch of passive muscle) and of the role of titin-binding proteins previously linked to hypertrophy signaling and highly upregulated in the PEVK KO. We will dissect their roles in hypertrophy signaling by crossing the PEVK KO with models in which these proteins have been deleted. Understanding the mechanisms that regulate muscle hypertrophy is clinically important because loss of mass is often a consequence of diseases and it debilitates the elderly and bedridden patients. Overall, the proposed work will be a major step towards our long-term goal, which is to gain a detailed understanding of the roles of titin in skeletal muscle structure and function, in health and disease.