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

描述（由申请人提供）：Titin是一种巨大的蛋白质，横跨半个肌节，作为分子弹簧的作用，是维持收缩肌节结构完整性的被动和恢复力的基础。titin对心肌被动肌肉僵硬的贡献已经得到了很好的研究，但相比之下，titin在骨骼肌中的作用却知之甚少。由于骨骼肌和心肌之间存在许多差异，需要对每种肌肉类型进行单独的研究。骨骼肌被动僵硬严重影响功能活动和生活质量，因此，需要研究被动僵硬产生的分子机制以及titin在这种僵硬中的作用。我们将首先在几个复杂层次（从剥皮纤维到整个肌肉）进行刚度测量，并确定titin对每个层次被动刚度的贡献。我们将研究titin在不同肌肉中对被动僵硬的贡献是如何变化的，这取决于titin的不同剪接和翻译后修饰。为了提高测定不同肌肉类型中表达的titin同种异构体序列的速度和准确性，我们开发了一种titin外显子微阵列，它包含了在一系列物种中发现的所有titin外显子（人类中有363个外显子）。我们预计我们的工作将大大增加对titin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坚实的基础，我们将在下一篇文章中讨论。我们将建立titin在过度负荷诱导的肥大和废用诱导的萎缩中相对于细胞外基质（胶原蛋白和弹性蛋白）变化的刚度。还将在tenascin-X （TNX）缺乏患者（ehers - danlos综合征的一种）和TNX KO小鼠中研究Titin在疾病中的作用；我们的初步数据表明，增加的titin为基础的刚度，作为代偿反应，以抵消tnx缺乏肌肉的胶原蛋白刚度下降。我们的最后一个目标将严格测试titin作为触发肥大的生物力学传感器的建议。主要焦点将放在PEVK外显子219-225 （PEVK KO）缺失的基因工程小鼠模型上。（PEVK是titin弹簧弹性的重要来源）。初步数据显示PEVK KO骨骼肌明显肥大，我们将研究其中的信号通路。我们将使用一种候选方法，包括研究titin和mTOR信号通路之间的相互作用（该通路先前已被证明在被动肌肉拉伸时增加蛋白质合成），以及titin结合蛋白先前与肥大信号通路相关的作用，并在PEVK KO中高度上调。我们将通过将PEVK KO与这些蛋白被删除的模型杂交来剖析它们在肥大信号传导中的作用。了解调节肌肉肥大的机制在临床上是很重要的，因为肌肉萎缩通常是疾病的后果，它使老年人和卧床不起的病人虚弱。总的来说，拟议的工作将是朝着我们的长期目标迈出的重要一步，该目标是详细了解titin在骨骼肌结构和功能，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. PUBLIC HEALTH RELEVANCE: Passive stiffness determines the length range at which skeletal muscle operates and the maximal joint range of motion, both of which greatly influence functional activities and quality of life; we will study the role of the giant elastic protein titin in muscle stiffness of normal and diseased muscle. Our studies include a focus on atrophic muscle and on Ehlers-Danlos syndrome, a disease in which the matrix that 'glues' muscle fibers to each other is weakened and in which a range of muscle abnormalities is present. Our preliminary studies suggest that changes in titin can play a beneficial role towards normalizing muscle function. We also study the role of titin in hypertrophy, which controls the mass of muscle. Understanding the mechanisms that regulate muscle hypertrophy is clinically important because loss of mass is often a consequence of diseases such as cancer and AIDS and it debilitates the elderly and bedridden patients. We hope that ultimately larger muscle masses can be maintained as a result of our studies on the role of titin in hypertrophy signaling.