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

肌动蛋白是骨骼肌的第三种肌丝，它横跨肌节的I带和A带区域。 已有多个titin突变被描述为导致衰弱的肌病，突显了titin的重要性 骨骼肌中的肌动蛋白，以及全面了解肌动蛋白的所有功能的必要性。我们目前对Titin的理解是 主要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-Band片段与Thin 收缩或病变肌肉中的细丝，改变肌蛋白的延伸性，使其与被动肌肉中的延伸性发生变化并产生影响 被动力和粗丝激活。可能的细丝相互作用位点是PEVK元件和 N2a，后者是最近发现的一种新的僵硬调节机制的一部分，该机制 涉及MARP1，一种应激反应蛋白。使用小鼠模型AIMS 1和AIMS 2重点介绍N2A的作用 以及PEVK元件在调节骨骼肌Titin僵硬中的作用，包括上调MARP的效果。 我们还研究了Titin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(一个较新的发现)。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到DRX的转换打开了粗丝，促进了 收缩。已经提出了几种机制来调节骨骼肌厚的开启状态 灯丝，包括一种机械传感机制，涉及粗丝应变。我们之前已经 获得了基于Titin的被动力拉伸骨骼肌粗丝的证据。目标三将测试 假设这会将骨骼肌中的SRX转化为DRX肌球蛋白，并将粗丝从关闭 至On。高分辨率的ATP周转分析表明，尽管SRX状态发生在每一个A- 骨骼肌带区(D区、C区和P区)，C区水平最高。此外 根据Titin的说法，C区含有MyBP-C。目标4将研究每一项在SRX中的重要性。它还将解决 局部扰动Titin株(通过删除单个C带结构域)对骨骼肌中SRX的影响。这部作品 具有很高的新颖性，并解决了具有临床意义的基本问题。所有必需的型号和 工具可用，经验丰富的协作者团队已就位，广泛的试点数据支持该指南 拟议研究的假设。这项建议是朝着我们的长期目标迈出的重要一步，即 详细了解titin在被动和主动骨骼肌中的作用，并为我们的 了解骨骼肌疾病的机制基础。

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.