Titin in Skeletal Muscle Health and Disease

肌联蛋白在骨骼肌健康和疾病中的作用

基本信息

批准号：
10468822
负责人：
Henk L. GRANZIER
金额：
$ 40.08万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10468822
关键词：
Address Affect Atomic Force Microscopy Awareness Back Basic Science Binding Proteins Biological Assay Biology Biopsy Cells Centronuclear myopathy Clinical Complex Coupling Data Disease Dominant-Negative Mutation Excision Exons Gene Expression Health In Vitro Inherited Introns Knowledge Measures Mechanics Microfilaments Molecular Mus Muscle Muscle Weakness Muscle function Mutate Mutation Myopathy Neuromuscular Diseases Organ Patients Physiology Pilot Projects Property Proteins Proteomics RNA RNA Splicing Research Respiratory Failure Respiratory Muscles Role Sarcomeres Skeletal Muscle Spectrum Analysis Splice-Site Mutation Structure Techniques Tertiary Protein Structure Testing Therapeutic Transcript Triad Acrylic Resin Work base connectin disease phenotype disease-causing mutation early onset exon skipping experience experimental study insight mouse model muscle stiffness novel protein expression respiratory single molecule therapeutic target transcriptome sequencing translational goal

项目摘要

There is a rapidly increasing awareness of the importance of titin in causing neuromuscular disorders (titinopathies), two of which we focus on in this work, centronuclear myopathy (CNM) and hereditary myopathy with early respiratory failure (HMERF). Titinopathies frequently present early-onset muscle weakness and respiratory difficulty but understanding their underlying mechanisms is held back by our still limited understanding of the functional roles of titin in skeletal muscle. Titin comprises the third myofilament of muscle and spans along the sarcomere, from Z-disk to M-band. Titin’s I-band region functions as a molecular spring that generates passive stiffness with recent studies indicating that passive stiffness affects active force at sub- maximal activation levels. However, it is not known how important titin’s stiffness is to overall skeletal muscle health and if altered stiffness can cause a myopathy. That the importance might be high is suggested by our pilot studies that reveal deranged titin stiffness and reduced active tensions in titinopathy patients with CNM. Aims 1 and 2 address how altering titin-based stiffness affects both passive and active muscle properties and if it can be disease-causing. We will perform mechanical studies on biopsies from titinopathy patients (focus on CNM) as well as study two contrasting mouse models in which the stiffness of titin’s spring region is either increased (PEVK truncation) or reduced (inactivation of Rbm20). The working hypothesis is that altering titin’s spring region alters both passive muscle stiffness and active tension and that this causes myopathy. Aim 2 also studies a novel mouse model that mimics CNM with splice site mutations in titin’s spring region and we will examine the functional consequences at the RNA, protein, structural and functional levels. We also study the A-band segment of titin, specifically the C-zone. This zone has not been studied, it is clinically important as this is where a large number of disease-causing mutations are found. We investigate in Aim 3 a mutation in the C-zone exon 343 which causes HMERF, a myopathy with respiratory muscle involvement that can be fatal. Using a novel HMERF mouse that we made the mechanistic basis of the disease will be studied. Through excision of the mutated titin exon 343, the therapeutic potential of exon skipping for treating HMERF will be tested. With its basic science and translational goals and its in-depth and integrative approach, this application seeks to continue our track record of cutting-edge titin research. Powerful techniques and novel mouse models are in place, pilot data support the guiding hypotheses, and our research team is highly experienced. The proposal will greatly enhance insights in titin biology, titin’s role in muscle disease, and titin’s potential as a therapeutic target.

人们迅速认识到肌联蛋白在引起神经肌肉疾病中的重要性（钛蛋白病），我们在这项工作中重点关注其中两种疾病：中心核肌病 (CNM) 和遗传性肌病早期呼吸衰竭 (HMERF) 常常表现为早发性肌肉无力和呼吸困难，但我们仍然有限，阻碍了对其潜在机制的了解了解肌联蛋白在骨骼肌中的功能作用肌联蛋白构成肌肉的第三肌丝。沿着肌节，从 Z 盘到 M 带区域，Titin 的 I 带区域起到分子弹簧的作用。产生被动刚度，最近的研究表明被动刚度会影响亚的主动力然而，尚不清楚肌动蛋白的硬度对整体骨骼肌有多重要。我们的研究表明，如果僵硬改变可能会导致肌病，那么其重要性可能会很高。初步研究表明，患有 CNM 的钛蛋白病患者的钛蛋白硬度紊乱，主动张力降低。目标 1 和 2 解决了改变基于肌动蛋白的僵硬度如何影响被动和主动肌肉特性，以及如果它可能会引起疾病。我们将对乳腺病患者的活检进行机械研究（重点是） CNM）以及研究两种对比小鼠模型，其中 titin 弹簧区域的刚度为增加（PEVK 截断）或减少（Rbm20 失活）。弹簧区域会改变被动肌肉僵硬和主动张力，这会导致目标 2 肌病。还研究了一种模仿 CNM 的新型小鼠模型，其在 titin 的弹簧区域具有剪接位点突变，我们我们还将研究 RNA、蛋白质、结构和功能水平的功能后果。肌动蛋白的 A 带部分，特别是 C 区该区域尚未被研究，但它在临床上很重要。这是我们在 Aim 3 中发现大量致病突变的地方。 C 区外显子 343 导致 HMERF，这是一种累及呼吸肌的肌病，可通过我们将使用我们制造的新型 HMERF 小鼠来研究该疾病的机制基础。通过切除突变的 titin 外显子 343，外显子跳跃治疗 HMERF 的治疗潜力其基础科学和转化目标及其深入和综合的方法将受到考验。该应用程序旨在继续我们的尖端钛蛋白研究记录。新颖的小鼠模型已经到位，试点数据支持指导假设，我们的研究团队正在该提案将极大地增强对肌动蛋白生物学、肌动蛋白在肌肉疾病中的作用的认识。以及肌联蛋白作为治疗靶点的潜力。