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

总结 我们的长期目标是了解健康和疾病中的膈肌收缩力，以及肌联蛋白在其中的作用。 膈肌驱动呼吸并不断承受机械负荷。变化 机械负荷会迅速影响膈肌收缩力，例如，在隔膜卸载后的数小时内 ICU 患者进行机械通气 (MV) 时，会出现严重的膈肌无力，导致呼吸困难 呼吸机脱机。膈肌无力的病理生理学尚不完全清楚。在提案中 另一方面，我们将研究肌球蛋白和肌动蛋白（两种关键的收缩蛋白）之间抑制相互作用的作用 肌纤维中的肌纤维，以及肌动蛋白在力抑制中的作用。我们的试验数据表明隔膜的卸载 导致肌球蛋白马达进入所谓的“超放松状态”（SRX）。一旦进入 SRX 状态，肌球蛋白就会减少 电机可用于与肌动蛋白结合，并且可以产生较小的力。我们还将研究巨人的角色 蛋白肌联从 SRX 状态释放肌球蛋白，并研究在 MV 诱导的肌球蛋白卸载过程中是否 隔膜，该机制受到干扰。 目标 1 将确定 ICU 通气患者膈肌肌球蛋白的 SRX 状态。我们将使用我们的 对 ICU 患者进行膈肌活检，研究膈肌纤维的收缩力并确定 激活期间附着于肌动蛋白的肌球蛋白马达的数量。我们将力学与 X 射线衍射结合起来 和生化测定以纳米分辨率解析肌球蛋白马达相对于肌动蛋白的位置 激活期间以及肌球蛋白处于 SRX 状态的比例。在目标 2 中，我们将确定以下角色： 肌球蛋白 SRX 的翻译后修饰。调节轻链 (RLC) 的磷酸化 调节肌球蛋白的 SRX 状态，我们将对隔膜活检应用质谱分析以确定 磷酸化蛋白质组，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 交换实验来磷酸化和 SRX。批判性地测试 RLC的变化是否是由膈肌不活动引起的，我们将对健康大鼠进行通气，并研究RLC 磷酸化。目标 3 将确定除了 RLC 磷酸化之外，是否还可以直接机械化 肌动蛋白对肌球蛋白的影响诱导SRX。基于肌联蛋白的被动张力使肌球蛋白丝拉紧，从而调节 SRX 状态。我们将研究隔膜卸载是否会减少基于肌动蛋白的被动张力 在 MV 期间，减少肌球蛋白丝的应变并增加 SRX。我们将使用鼠标模型 基因工程增加或减少基于 titin 的被动张力，并研究 MV 对 肌球蛋白的 SRX 状态。 该提案的创新之处在于新颖的研究焦点和指导假设、独特的膈肌活检 来自患者及其新颖的工具和小鼠模型，该提案的综合方法预计将引领 我们对膈肌功能和肌动蛋白在其中的作用的理解向前迈出了一大步。

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.