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

描述（由申请人提供）：肌联蛋白是一种跨越半个肌节的巨大蛋白质，作为分子弹簧发挥作用，是维持收缩肌节结构完整性的被动和恢复力的基础。肌联蛋白对被动肌肉僵硬的贡献已经在心肌中得到了很好的研究，但相比之下，肌联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的许多差异，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，被动僵硬产生的分子机制和肌联蛋白在这种僵硬中的作用需要研究。我们将首先在几个复杂程度（从皮肤纤维到整个肌肉）进行刚度测量，并确定肌联蛋白对每个被动刚度的贡献。我们将研究肌联蛋白的贡献如何在不同肌肉的被动刚度不同，这取决于差异剪接和翻译后修饰肌联蛋白。为了促进确定不同肌肉类型中表达的肌联蛋白异构体序列的速度和准确性，我们开发了肌联蛋白外显子微阵列，其包含在一系列物种中发现的所有肌联蛋白的外显子（人类中363个外显子）。我们预计，我们的工作将大大增加对肌联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坚实的基础，我们将在接下来讨论。我们将建立肌联蛋白的刚度在过载引起的肥大和废用引起的萎缩，相对于细胞外基质（胶原和弹性蛋白）的变化。肌联蛋白在疾病中的作用也将在腱生蛋白-X（TNX）缺陷患者（Ehlers-Danlos综合征的类型之一）和TNX KO小鼠中进行研究;我们的初步数据表明基于肌联蛋白的硬度增加，作为抵消TNX缺陷肌肉的胶原硬度降低的补偿反应。我们的最后一个目标将严格测试的建议，肌联蛋白功能作为一个生物力学传感器，触发肥大。主要重点将放在基因工程小鼠模型，这是缺乏PEVK外显子219-225（PEVK KO）。(The PEVK是钛合金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，我们将研究所涉及的信号通路。我们将使用一种候选方法，包括肌联蛋白和mTOR信号通路之间的相互作用的研究（该通路先前已被证明可以增加蛋白质合成，以响应被动肌肉的拉伸）和肌联蛋白结合蛋白的作用先前与肥大信号传导相关，并在PEVK KO中高度上调。我们将通过将PEVK KO与这些蛋白质已被删除的模型交叉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重要的，因为质量损失通常是疾病的结果，它使老年人和卧床不起的患者变得虚弱。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，即详细了解肌联蛋白在骨骼肌结构和功能中的作用，以及健康和疾病。 公共卫生相关性：被动刚度确定骨骼肌操作的长度范围和最大关节活动范围，这两者都极大地影响功能活动和生活质量;我们将研究巨大弹性蛋白肌联蛋白在正常和患病肌肉的肌肉刚度中的作用。我们的研究包括关注萎缩性肌肉和Ehlers-Danlos综合征，这是一种疾病，其中“粘合”肌肉纤维的基质相互减弱，并且存在一系列肌肉异常。我们的初步研究表明，肌联蛋白的变化可以对正常化肌肉功能发挥有益的作用。我们还研究了肌联蛋白在肥大中的作用，它控制着肌肉的质量。了解调节肌肉肥大的机制在临床上很重要，因为质量损失通常是癌症和艾滋病等疾病的后果，它使老年人和卧床不起的患者变得虚弱。我们希望通过对肌联蛋白在肥大信号传导中的作用的研究，最终能够维持更大的肌肉质量。

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.