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

7.总结。 这项建议的长期目标是详细了解横隔膜--主要肌肉--如何 呼吸作用--对机械卸货的反应迅速减弱，以及由此产生的 巨型弹性蛋白Titin会影响这种反应。 横隔膜是一种独特的肌肉，因为它不断地受到机械载荷的作用。近期工作 表明横隔膜的强度对机械卸载非常敏感，就像在 ICU中的机械通风。卸载是如何影响横隔膜强度的，目前还知之甚少。渐增 这一理解是至关重要的：在几个小时内，在机械通风过程中，隔膜卸载 导致ICU患者隔膜无力，导致患者撤机困难 支持并导致死亡。寻找横隔膜发育的分子诱因 机械卸货过程中的疲软仍在继续。机械传感器蛋白的潜在作用，这一联系 隔膜卸载对蛋白质周转的作用尚未被探索，但这是一个令人兴奋的新概念，需要 学习。一种候选的机械传感器是肌动蛋白，它是一种巨大的弹性蛋白质，已被认为可以感知 机械应力，并将其与营养信号通路联系起来。肌动蛋白在横隔膜中的作用 营养性和机械通风过程中的隔膜薄弱是这项赠款提案的核心。 目标1将严格测试肌动蛋白是否影响肌肉的营养性。我会用单侧隔膜去神经 (UDD)，这是一种临床上重要的疾病，它本身就是这项工作的一个很好的工具，因为它导致了 周期性被动牵拉导致失神经支配的横隔膜迅速肥大。我将在两部小说中研究UDD Titin KO小鼠模型：通过删除Ig结构域(Ig KO)和 另一种是通过缺失Titin剪接因子RBM20(RBM20 KO)来降低Titin刚性。我 预计UDD后的肥大反应在Ig KO小鼠中被夸大，并在20元人民币中钝化 KO小鼠，并且这种反应是由改变的肌动蛋白信号介导的。目标2将研究低滴度 刚性保护隔膜在机械通风诱导的卸载过程中不会减弱，并将使用 一种低肌钙蛋白硬度的大鼠模型。如果基于Titin的机械传感调节横隔膜对 机械卸载，然后我预计通过将横隔膜预调节到减少，可以获得较低的斜度 机械感应使这种反应变得迟钝。目标3将研究横隔膜无力的力学基础。 首次使用从ICU患者的活检组织中分离出来的横隔膜纤维进行机械通气。 机械通气的ICU患者。其目的是调查动物研究的结果是否 推论到病人身上。 这一方案的创新之处在于研究重点新颖，指导假设创新，其创新性 小鼠模型、机械通气ICU患者独特的横隔膜活检及其新方法 实验工具。该提案的综合方法预计将导致在#年向前迈出重大一步 我们对横隔膜功能和肌动蛋白在其中的作用的理解。

7. Summary. The long-term goal of this proposal is to gain detailed understanding of how the diaphragm - the main muscle of respiration - rapidly weakens in response to mechanical unloading and of the mechanisms whereby the giant elastic protein titin influences this response. The diaphragm is a unique muscle in that it is constantly subjected to mechanical loading. Recent work suggests that diaphragm strength is remarkably sensitive to mechanical unloading, as occurs during mechanical ventilation in the ICU. How unloading affects diaphragm strength is poorly understood. Increasing this understanding is critically important: within hours, diaphragm unloading during mechanical ventilation causes diaphragm weakness in ICU patients, which leads to difficulties in weaning patients from ventilatory support and contributes to mortality. The search for the molecular triggers for the development of diaphragm weakness during mechanical unloading is ongoing. The potential role of mechanosensor proteins, that link diaphragm unloading to protein turnover, is unexplored but is an exciting and novel concept that needs to be studied. A candidate mechanosensor is titin, a giant elastic protein that has been suggested to sense mechanical stress and link this to trophic signalling pathways. The elucidation of titin's role in diaphragm trophicity and in diaphragm weakness during mechanical ventilation is central to this grant proposal. Aim 1 will critically test whether titin affects muscle trophicity. I will use unilateral diaphragm denervation (UDD), a condition that is clinically important and that presents itself as a great tool for this work as it induces rapid hypertrophy of the denervated hemidiaphragm due to cyclic passive stretch. I will study UDD in two novel titin KO mouse models: one in which titin stiffness is increased through deletion of Ig domains (Ig KO) and another in which titin stiffness is decreased through deletion of the titin splice factor rbm20 (Rbm20 KO). I anticipate that the hypertrophic response following UDD is exaggerated in Ig KO mice and blunted in Rmb20 KO mice, and that this response is mediated by altered titin signaling. Aim 2 will study whether low titin stiffness protects the diaphragm from weakening during mechanical ventilation-induced unloading and will use a rat model with low titin stiffness. If titin-based mechanosensing mediates the response of the diaphragm to mechanical unloading, then I anticipate that low titin stiffness, by preconditioning the diaphragm to reduced mechanosensing, blunts this response. Aim 3 will study the mechanistic basis for diaphragm weakness in mechanically ventilated ICU patients using, for the first time, diaphragm fibers isolated from biopsies of mechanically ventilated ICU patients. The goal is to investigate whether the findings of animal studies extrapolate to patients. The innovation of this proposal lies in the novel research foci with innovative guiding hypotheses, its novel mouse models, unique diaphragm biopsies from mechanically ventilated ICU patients, and its novel experimental tools. The proposal's integrative approach is expected to lead to a significant step forward in our understanding of diaphragm function and the role of titin therein.