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种肌丝，它跨越肌节的I带和A带区域。 多个肌联蛋白突变已被描述，导致衰弱性肌病，突出肌联蛋白的重要性 以及完全了解肌联蛋白所有功能的必要性。我们目前对肌联蛋白的理解是 这主要是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，肌联蛋白的I带片段与薄的 收缩或患病肌肉中的肌丝，改变肌联蛋白的可伸展性， 被动力和粗丝激活。可能的细丝相互作用位点是PEVK元件和 N2 A，后者是最近发现的一种新的刚度调节机制的一部分， MARP 1是一种应激反应蛋白。使用小鼠模型，目的1和2关注N2 A的作用 和PEVK元件在调节肌联蛋白刚度在骨骼肌，包括上调MARP的影响。 我们还研究了肌联蛋白在激活骨骼肌粗肌丝中的作用。肌球蛋白领域的重要工作 已经表明肌肉激活需要细细丝激活（如众所周知的）和粗细丝激活 机制（最近的发现）。在松弛的骨骼肌中，肌球蛋白要么处于超松弛状态（SRX）， 状态或无序松弛（DRX）状态。SRX向DRX的转化使粗丝接通，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。 细丝，包括涉及粗细丝应变的机械感测机制。我们先前已经 获得的证据表明，肌联蛋白为基础的被动力量应变骨骼肌粗丝。目标三将测试 这一假设将骨骼肌中的SRX转化为DRX肌球蛋白，并将粗丝从关闭状态转换为关闭状态， 高分辨率ATP周转试验表明，虽然SRX状态发生在每个A- 在骨骼肌的三个带区（D区、C区和P区）中，C区的水平最高。此外 C区含有MyBP-C。目标4将研究每一个在SRX中的重要性。它还将解决 局部扰动肌联蛋白应变（通过删除单个C区结构域）对骨骼肌中SRX的影响。这项工作 具有高度的新奇，并解决了具有临床相关性的基本问题。所有必需的模型和 工具可用，经验丰富的合作者团队到位，广泛的试点数据支持指导 提出的研究假设。这一建议是朝着我们的长期目标迈出的重要一步， 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。