Role of titin in the pathophysiology of diaphragm weakness during mechanical ventilation

摘要 我们的长期目标是了解横隔膜的收缩能力在健康和疾病中的作用，以及肌动蛋白在其中的作用。 横隔肌驱动呼吸，不断地承受机械负荷。中的更改 机械加载会迅速影响隔膜的收缩能力，例如在隔膜卸载后的数小时内 机械通气(MV)在ICU患者中，随后会出现严重的横隔膜无力，导致困难 呼吸机脱机。横隔肌无力的病理生理学机制尚不完全清楚。在建议书中， 另一方面，我们将研究肌球蛋白和肌动蛋白这两种关键收缩蛋白之间相互作用减弱的作用。 肌纤维中的肌动蛋白，以及肌动蛋白在力量抑制中的作用。我们的试验数据表明，隔膜的卸载 使肌球蛋白马达进入所谓的“超松弛状态”(SRX)。一旦进入SRX状态，肌球蛋白就会减少 电机可以与肌动蛋白结合，并且可以产生更少的力。我们还将研究巨人的角色 肌球蛋白在SRX状态中的释放，并研究在MV诱导的卸载过程中 隔膜，这个机构是受干扰的。 目的1测定呼吸机ICU患者隔膜肌球蛋白的SRX状态。我们将使用我们的 目的：研究重症监护病房(ICU)患者横隔肌纤维的收缩力，并探讨其与ICU患者的关系。 在激活过程中附着到肌动蛋白的肌球蛋白马达的数量。我们将把力学和X射线衍射结合起来 和生化分析，以纳米分辨率解析肌球蛋白马达相对于肌动蛋白的位置 在激活过程中，肌球蛋白的比例处于SRX状态。在目标2中，我们将确定 肌球蛋白SRX的翻译后修饰。调节轻链的磷酸化(RLC) 调节肌球蛋白的SRX状态，我们将在横隔膜活检上应用质谱仪来确定 我们将确定RLC之间是否存在因果关系 通过对患者的肌纤维进行RLC交换实验来实现磷酸化和SRX。进行批判性测试 RLC的改变是否由横隔膜不活动引起，我们将对健康大鼠进行机械通气，并研究RLC 磷酸化。目标3将确定，除了RLC磷酸化外，直接机械化 肌动蛋白对肌球蛋白诱导SRX的影响。肌球蛋白被动张力对肌球蛋白细丝的调节作用 SRX状态。我们将研究是否由于横隔膜的卸载而减少了TiN基被动张力 在MV期间，降低了肌球蛋白细丝中的应变，增加了SRX。我们将使用鼠标模型 基因工程增加或降低基于肌动蛋白的被动张力，并研究MV对 肌球蛋白的SRX状态。 该方案的创新之处在于新颖的研究重点和指导性假设，独特的横隔膜活检 从患者及其新工具和老鼠模型来看，该提案的综合方法有望引领 使我们对横隔膜功能和肌动蛋白在其中的作用的理解向前迈进了一大步。

Summary Our long-term goal is to understand diaphragm contractility in health and disease, and the role of titin therein. The diaphragm muscle drives respiration and is constantly subjected to mechanical loading. Changes in mechanical loading rapidly affect diaphragm contractility, e.g., within hours of diaphragm unloading during mechanical ventilation (MV) in ICU patients, severe diaphragm weakness ensues, contributing to difficult ventilator weaning. The pathophysiology of diaphragm weakness is incompletely understood. In the proposal at hand, we will study the role of depressed interactions between myosin and actin, the two key contractile proteins in myofibers, and the role of titin in force depression. Our pilot data suggest that unloading of the diaphragm causes the myosin motors to adopt the so-called ‘super-relaxed state’ (SRX). Once in the SRX state, less myosin motors are available for binding to actin, and less force can be generated. We will also study the role of the giant protein titin in releasing myosin from the SRX state and investigate whether during MV-induced unloading of the diaphragm, this mechanism is perturbed. Aim 1 will determine the SRX state of myosin in the diaphragm of ventilated ICU patients. We will use our diaphragm biopsies of ICU patients to study the contractile force of diaphragm myofibers and determine the number of myosin motors that attach to actin during activation. We will combine mechanics with X-ray diffraction and biochemical assays to resolve with nanometer resolution the position of myosin motors relative to actin during activation and the proportion of myosin in the SRX state. In Aim 2 we will determine the role of posttranslational modifications in the SRX of myosin. Phosphorylation of regulatory light chains (RLC) regulates the SRX state of myosin and we will apply mass-spectrometry on the diaphragm biopsies to determine the phospho-proteome, and we will establish whether there is a cause-and-effect relation between RLC phosphorylation and SRX by performing RLC exchange experiments into patients’ myofibers. To critically test whether changes in RLC are caused by diaphragm inactivity, we will ventilated healthy rats, and study RLC phosphorylation. Aim 3 will determine whether, in addition to RLC phosphorylation, direct mechanical effects of titin on myosin induce SRX. Titin-based passive tension strains the myosin filament which regulates the SRX state. We will study whether the reduction in titin-based passive tension, due to diaphragm unloading during MV, reduces the strain in the myosin filament and increases SRX. We will use mouse models with genetically engineered increased or decreased titin-based passive tension and study the effect of MV on the SRX state of myosin. The innovation of this proposal lies in the novel research foci and guiding hypotheses, unique diaphragm biopsies from patients, and its novel tools and mouse models, The proposal’s integrative approach is expected to lead to a major step forward in our understanding of diaphragm function and titin’s role therein.