Titin-based stiffness regulation and mechanosensing in activated skeletal muscle.

激活骨骼肌中基于肌联蛋白的刚度调节和机械传感。

• 批准号: 10751746
• 负责人: Henk L. GRANZIER
• 金额: $ 65.34万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-04 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10751746
• 关键词: Actins; Address; Affect; Ankyrin Repeat; Binding; Binding Sites; Biological Assay; Calcium; Contracts; Data; Disease; Elasticity; Elements; Family; Fiber; Generations; Glutamates; Goals; Health; In Vitro; Knockout Mice; Measures; Mechanical Stress; Mechanics; Microfilaments; Modeling; Mus; Muscle; Muscle Contraction; Muscle function; Muscle relaxation phase; Mutate; Mutation; Myopathy; Myosin ATPase; N-terminal; Patients; Pilot Projects; Property; Protein C Deficiency; Proteins; Regulation; Relaxation; Research; Resolution; Roentgen Rays; Role; Sarcomeres; Site; Skeletal Muscle; Stress; Striated Muscles; Testing; Thick Filament; Thin Filament; Viscosity; Work; biological adaptation to stress; cell motility; clinically relevant; connectin; energy efficiency; experience; experimental study; mechanotransduction; mouse model; muscle stiffness; muscle stress; myosin-binding protein C; novel; response; single molecule; therapy development; tool; viscoelasticity

Titin is the third myofilament of skeletal muscle where it spans the I-band and A-band regions of the sarcomere. Multiple titin mutations have been described that result in debilitating myopathies, highlighting titin's importance in skeletal muscle and the need to understand all of titin's functions fully. Our current understanding of titin is largely based on studying passive skeletal muscle and assuming that no established properties change when skeletal muscle is activated. However, recent studies suggest that titin's I-band segment interacts with the thin filament in contracting or diseased muscle, altering titin's extensibility from that in passive muscle and impacting passive force and thick filament activation. Possible thin filament interaction sites are the PEVK element and the N2A of skeletal muscle, the latter is part of a recently discovered novel stiffness regulation mechanism that involves MARP1, a stress response protein. Using mouse models aims 1 and 2 focus on the roles of the N2A and PEVK elements in regulating titin stiffness in skeletal muscle, including the effects of upregulating MARP. We also study the role of titin in activating the thick filament in skeletal muscle. Important work in the myosin field has shown that muscle activation requires thin filament activation (as is well-known) and thick filament activation mechanisms (a more recent discovery). In relaxed skeletal muscle, myosin is either in the super-relaxed (SRX) state or the disordered-relaxed (DRX) state. The conversion of SRX to DRX turns thick filaments ON, promoting contraction. Several mechanisms have been proposed to regulate the ON state of the skeletal muscle thick filament, including a mechano-sensing mechanism that involves thick filament strain. We have previously obtained evidence that titin-based passive force strains the skeletal muscle thick filament. Aim three will test the hypothesis that this converts SRX to DRX myosin in skeletal muscle and switches the thick filament from OFF to ON. High-resolution ATP turnover assays have revealed that although the SRX state occurs in each of the A- band regions of skeletal muscle (the D-zone, C-zone, and P-zone), the C-zone has the highest level. In addition to titin, the C-zone contains MyBP-C. Aim 4 will study the importance of each in SRX. It will also address the effect of locally perturbing titin strain (by deleting single C-zone domains) on SRX in skeletal muscle. This work has high novelty and addresses fundamental questions that have clinical relevance. All required models and tools are available, an experienced team of collaborators is in place, and extensive pilot data support the guiding hypotheses of the proposed research. This proposal is a significant step towards our long-term goal, which is to gain a detailed understanding of the roles of titin in both passive and active skeletal muscle and contribute to our understanding of the mechanistic basis of skeletal muscle disease.

肌联蛋白是骨骼肌的第三条肌丝，跨越肌节的 I 带和 A 带区域。 多种肌联蛋白突变已被描述，可导致衰弱性肌病，凸显肌联蛋白的重要性 骨骼肌的知识以及充分了解肌动蛋白的所有功能的需要。我们目前对titin的理解是 很大程度上基于对被动骨骼肌的研究，并假设当 骨骼肌被激活。然而，最近的研究表明，titin 的 I 带片段与薄层相互作用。 收缩或患病肌肉中的细丝，改变肌动蛋白在被动肌肉中的伸展性并影响 被动力和粗丝激活。可能的细丝相互作用位点是 PEVK 元件和 骨骼肌的 N2A，后者是最近发现的新型刚度调节机制的一部分，该机制 涉及MARP1，一种应激反应蛋白。使用小鼠模型的目标 1 和 2 重点关注 N2A 的作用 和 PEVK 元件调节骨骼肌肌动蛋白硬度，包括上调 MARP 的作用。 我们还研究了肌联蛋白在激活骨骼肌粗丝中的作用。肌球蛋白领域的重要工作 已经表明肌肉激活需要细丝激活（众所周知）和粗丝激活 机制（最近的发现）。在放松的骨骼肌中，肌球蛋白要么处于超放松状态（SRX） 状态或无序放松（DRX）状态。 SRX 到 DRX 的转换会打开粗细丝，促进 收缩。已经提出了几种机制来调节骨骼肌厚度的ON状态 灯丝，包括涉及粗灯丝应变的机械传感机制。我们之前有过 获得证据表明基于肌动蛋白的被动力使骨骼肌粗丝拉紧。目标三将测试 假设这会将骨骼肌中的 SRX 肌球蛋白转化为 DRX 肌球蛋白，并将粗丝从关闭状态切换 至开。高分辨率 ATP 周转分析表明，尽管 SRX 状态发生在每个 A- 骨骼肌的带状区域（D区、C区、P区），其中C区水平最高。此外 对于 titin，C 区包含 MyBP-C。目标 4 将研究 SRX 中每个因素的重要性。它还将解决 局部扰动肌动蛋白应变（通过删除单个 C 区结构域）对骨骼肌 SRX 的影响。这部作品 具有很高的新颖性，解决了具有临床相关性的基本问题。所有必需的型号和 工具已经可用，经验丰富的合作团队已经就位，并且广泛的试点数据支持指导 拟议研究的假设。该提案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被动和主动骨骼肌中的作用，并有助于我们 了解骨骼肌疾病的机制基础。