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

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 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 导致危重病人的横隔膜无力，导致脱机失败。关于寻找 导致横隔膜虚弱的分子诱因仍在继续。的潜在作用 机械传感器蛋白质将卸货与蛋白质周转联系在一起，这一概念尚未得到充分研究，但却是一个令人兴奋的概念。 这一点需要研究。一种候选的机械传感器是Titin，一种已被提出的巨大的弹性蛋白质 来感知机械压力，并将其与营养信号通路联系起来。这项提议的目的是理解 横隔膜中的机械传感在健康和疾病中的作用以及肌动蛋白在其中的作用。 目标1将关键地测试肌联蛋白如何影响肌肉的营养性。我们将采用单侧隔膜神经切断。 (UDD)。在UDD期间可以观察到的一个特征是初始肥厚反应，我们已经证明 这种失神经支配的横隔肌肥大是由横隔膜纤维的周期性被动伸展引起的。 Titin的弹性性质决定了反应的大小。在这个目标中，我们将确定基于Titin的 涉及的信号通路。目的2确定肌动蛋白弹性在呼气末正压通气诱导中的作用 纵隔膜萎缩。这项工作建立在我们最近发现的机械通风和 PEEP会缩短横隔膜的长度，导致纤维纵向萎缩。试点数据 这表明，基于Titin的机械感觉调节了这种反应。为了批判性地测试Titin的作用，我们将 PEEP对两种Titin KO小鼠模型纵向萎缩的影响 一个是僵硬的，另一个是降低的。在目标3中，我们将使用危重患者独特的横隔膜活检。 患者验证目标1和目标2的结果，并研究基于Titin的机械传感是否有助于 横隔膜无力。将确定上调/下调的Titin结合蛋白，其意义是 通过基因缺失在小鼠模型中进行测试。 这一方案的创新之处在于创新的研究重点和创新的指导假设，其创新性 小鼠模型，机械通气危重患者独特的横隔膜活检组织，及其新的 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 critically ill patients, contributing to weaning failure. The search for the molecular triggers for the development of diaphragm weakness is ongoing. The potential role of mechanosensor proteins, that link unloading to protein turnover, is under-explored but an exciting 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. This proposal’s aims to understand mechanosensing in the diaphragm in health and disease, and the role of titin therein. Aim 1 will critically test how titin affects muscle trophicity. We will use unilateral diaphragm denervation (UDD). A property that can be observed during UDD is an initial hypertrophy response and we have shown that this hypertrophy of the denervated hemidiaphragm is caused by cyclic passive stretch of diaphragm fibers, and that titin’s elastic properties dictate the magnitude of the response. In this Aim we will identify the titin-based signalling pathways involved. Aim 2 determines the role of titin’s elasticity in PEEP ventilation-induced longitudinal diaphragm atrophy. This work builds on our recent finding that mechanical ventilation with PEEP, which unloads the diaphragm at a shortened length, causes longitudinal atrophy of fibers. Pilot data suggest that titin-based mechanosensing modulates this response. To critically test the role of titin, we will study the effect of PEEP ventilation on longitudinal atrophy in two titin KO mouse models: one with increased titin stiffness and one with lowered. In Aim 3 we will use unique diaphragm biopsies of critically ill patients to validate the findings from aim 1&2 and study whether titin-based mechanosensing contributes to diaphragm weakness. Up/downregulated titin binding proteins will be determined, the significance of which is tested in mouse models through genetic deletion. The innovation of this proposal lies in the novel research foci with innovative guiding hypotheses, its innovative mouse models, unique diaphragm biopsies from mechanically ventilated critically ill 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.