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

肌联蛋白在机械通气期间膈肌无力病理生理学中的作用

• 批准号: 10659578
• 负责人: Coen Ottenheijm
• 金额: $ 55.61万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10659578
• 关键词: Actins; Adopted; Affect; Animal Model; Atrophic; Attention; Attenuated; Binding; Biochemical; Biological Assay; Biopsy; Contractile Proteins; Data; Depressed mood; Disease; Elasticity; Failure; Filament; Functional disorder; Genetic Engineering; Goals; Hand; Health; Heart Diseases; Hour; Individual; Laparotomy; Length; Light; Lung; Mass Spectrum Analysis; Measures; Mechanical ventilation; Mechanics; Mental Depression; Microfilaments; Modeling; Motor; Mus; Muscle; Muscle Proteins; Muscular Atrophy; Myosin ATPase; Oxygen; Patients; Pharmaceutical Preparations; Phosphorylation; Play; Positioning Attribute; Post-Translational Protein Processing; Production; Protein Dephosphorylation; Proteins; Proteomics; Rattus; Relaxation; Research; Respiration; Respiratory Diaphragm; Respiratory Muscles; Role; Running; Sarcomeres; Skeletal Muscle; Testing; Therapeutic; Ventilator Weaning; Vertebral column; Weaning; Work; X ray diffraction analysis; clinically relevant; connectin; experimental study; in vivo; innovation; insight; interdisciplinary approach; mechanical load; mouse model; nanometer resolution; novel; preservation; programs; response; tool; uptake; ventilation

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.

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，并不断受到机械负荷。变化 机械负荷迅速影响隔膜收缩性，例如，在隔膜卸载后数小时内， ICU患者的机械通气（MV），严重的膈肌无力，导致难以 呼吸机脱机。膈肌无力的病理生理机制还不完全清楚。在提案中， 另一方面，我们将研究肌球蛋白和肌动蛋白这两种关键的收缩蛋白之间抑制相互作用的作用。 在肌纤维中的作用，以及肌联蛋白在强迫抑郁中的作用。我们的试验数据表明，卸载隔膜 导致肌球蛋白马达进入所谓的“超松弛状态”（SRX）。一旦进入SRX状态， 马达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作用 蛋白titin从SRX状态释放肌球蛋白，并研究在MV诱导的卸载过程中， 隔膜，这种机制就会受到干扰。 目的1将确定呼吸机ICU患者膈肌肌球蛋白的SRX状态。我们将用我们 ICU患者的膈肌活检，以研究膈肌肌纤维的收缩力，并确定 在激活过程中附着在肌动蛋白上的肌球蛋白马达的数量。我们将把联合收割机力学与X射线衍射结合起来 和生物化学测定以纳米分辨率分辨肌球蛋白马达相对于肌动蛋白的位置 以及肌球蛋白在SRX状态下的比例。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（RLC）的磷酸化 调节肌球蛋白的SRX状态，我们将在膈肌活检中应用质谱法来确定 磷酸化蛋白质组，我们将建立是否有一个因果关系之间的RLC 通过在患者肌纤维中进行RLC交换实验来检测磷酸化和SRX。为了严格检验 本实验通过对健康大鼠进行机械通气，观察RLC的变化是否由膈肌失活引起， 磷酸化目的3将确定，除了RLC磷酸化，是否直接机械 肌联蛋白对肌球蛋白诱导SRX的影响。基于钛蛋白的被动张力使肌球蛋白丝拉紧， SRX国家。我们将研究是否减少钛基被动张力，由于隔膜卸载 在MV期间，减少肌球蛋白丝的应变并增加SRX。我们将使用小鼠模型， 基因工程增加或减少基于肌联蛋白的被动张力，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焦点和指导性假设，独特的膈肌活检 从患者，其新的工具和小鼠模型，该提案的综合方法预计将导致 我们对横膈膜功能和肌联蛋白在其中的作用的理解向前迈进了一大步。

项目成果

COVID-19 is associated with distinct myopathic features in the diaphragm of critically ill patients.

• DOI: 10.1136/bmjresp-2021-001052
• 发表时间: 2021-09
• 期刊: BMJ open respiratory research
• 影响因子: 4.1
• 作者: Shi Z;Bogaards SJP;Conijn S;Onderwater Y;Espinosa P;Bink DI;van den Berg M;van de Locht M;Bugiani M;van der Hoeven H;Boon RA;Heunks L;Ottenheijm CAC
• 通讯作者: Ottenheijm CAC

Replacement Fibrosis in the Diaphragm of Mechanically Ventilated Critically Ill Patients.

机械通气危重患者隔膜的替代性纤维化。

• DOI: 10.1164/rccm.202208-1608le
• 发表时间: 2023
• 期刊: American journal of respiratory and critical care medicine
• 影响因子: 24.7
• 作者: Shi,Zhonghua;vandenBerg,Marloes;Bogaards,Sylvia;Conijn,Stefan;Paul,Marinus;Beishuizen,Albertus;Heunks,Leo;Ottenheijm,CoenAC
• 通讯作者: Ottenheijm,CoenAC

Titin-based mechanosensing modulates muscle hypertrophy.

• DOI: 10.1002/jcsm.12319
• 发表时间: 2018-10
• 期刊: Journal of cachexia, sarcopenia and muscle
• 作者: van der Pijl R;Strom J;Conijn S;Lindqvist J;Labeit S;Granzier H;Ottenheijm C
• 通讯作者: Ottenheijm C

Titin N2A: More than a signaling node?

titin n2a：不仅仅是信号节点？

• DOI: 10.1085/jgp.202112904
• 发表时间: 2021-07-05
• 期刊: The Journal of general physiology
• 作者: van der Pijl RJ;Ottenheijm CAC
• 通讯作者: Ottenheijm CAC

Muscle ankyrin repeat protein 1 (MARP1) locks titin to the sarcomeric thin filament and is a passive force regulator.

• DOI: 10.1085/jgp.202112925
• 发表时间: 2021-07-05
• 期刊: The Journal of general physiology
• 作者: van der Pijl RJ;van den Berg M;van de Locht M;Shen S;Bogaards SJP;Conijn S;Langlais P;Hooijman PE;Labeit S;Heunks LMA;Granzier H;Ottenheijm CAC
• 通讯作者: Ottenheijm CAC