Title: Ventilator-induced diaphragm dysfunction: role of calpain signaling

标题：呼吸机引起的膈肌功能障碍：钙蛋白酶信号传导的作用

• 批准号: 9889038
• 负责人: Scott K. Powers
• 金额: $ 16.78万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-07 至 2021-05-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9889038
• 关键词: Actins; Acute respiratory failure; Address; Animal Model; Atrophic; Autophagocytosis; Biological; CASP3 gene; Calpain; Caspase; Cleaved cell; Clinical; Critical Illness; Cytoskeletal Proteins; Data; Development; Event; FRAP1 gene; Failure; Foundations; Functional disorder; Genetic Transcription; Goals; Hour; Intervention; Life; Measurement; Measures; Mechanical ventilation; Mediating; Molecular; Morbidity - disease rate; Muscle; Muscle Proteins; Muscle Weakness; Myosin ATPase; Operative Surgical Procedures; Pathway interactions; Patients; Play; Prevention; Prevention strategy; Protein Biosynthesis; Proteins; Proteolysis; Public Health; Reporter; Respiratory Diaphragm; Respiratory Muscles; Role; Savings; Signal Transduction; Signaling Protein; System; Testing; Therapeutic; Time; Transgenes; Ubiquitin; Ventilator; Weaning; adeno-associated viral vector; calpastatin; experimental study; in vivo; innovation; mTOR protein; mortality; multicatalytic endopeptidase complex; novel; novel therapeutic intervention; overexpression; pre-clinical; prevent; protein degradation; respiratory; skeletal muscle wasting; tool; transcription factor

BACKGROUND: Mechanical ventilation (MV) is used to provide respiratory support to >15 million patients annually during both critical illnesses and surgery. Although MV is often a life-saving intervention, prolonged MV (≥12 hours) promotes problems in “weaning” patients from the ventilator; this failure to wean significantly increases patient morbidity and mortality. While the cause of difficult weaning can be multifactorial, weak inspiratory muscles (i.e., diaphragm) are a major factor. Indeed, an unintended consequence of ventilator support is that prolonged MV results in diaphragmatic inactivity and the rapid development of diaphragm atrophy and contractile dysfunction (collectively known as ventilator-induced diaphragm dysfunction (VIDD)). VIDD occurs due to both decreased protein synthesis and increased protein breakdown in the diaphragm; however, the upstream regulators that control these events remain unclear. Our compelling preliminary data suggest that the cysteine protease calpain plays an essential signaling role in the development of VIDD. Indeed, our pilot experiments reveal that prevention of calpain activation in the diaphragm protects against VIDD, indicating that calpain is required for the development of VIDD. Importantly, these results challenge the current dogma that the role of the classical calpains (i.e., calpain 1 and 2) in skeletal muscle atrophy is limited to cleavage of cytoskeletal proteins. The mechanisms by which active calpain promotes VIDD remain unknown and are the focus of this R21 application. HYPOTHESIS: Guided by our preliminary experiments, we hypothesize that active calpain is an essential upstream regulator of both protein synthesis and proteolysis in diaphragm muscle during prolonged MV. SPECIFIC AIMS: Aim 1 will determine if active calpain plays an essential role in MV-induced decreases in anabolic signaling (i.e., Akt/mTOR) and protein synthesis in the diaphragm during prolonged MV. Aim 2 will establish whether active calpain regulates MV-induced proteolysis in the diaphragm via activation of caspase-3 and forkhead box O (FoxO) transcription factors. APPROACH: We will test our hypothesis by utilizing a well- established animal model of MV along with innovative molecular tools to prevent MV-induced calpain activation in the diaphragm. Cause and effect will be determined using an adeno-associated virus vector (AAV9) to overexpress a calpastatin transgene in the diaphragm that will prevent MV-induced activation of calpain. Primary dependent measures include key steps in anabolic and proteolytic signaling, in vivo protein synthesis, rate of diaphragm proteolysis, and assessment of both caspase-3 activity along with the transcriptional activity of FoxO in the diaphragm. RELEVANCE: VIDD is a serious clinical problem because inspiratory muscle weakness is a major cause of the inability to wean patients from the ventilator. Unfortunately, no standard clinical therapy exists to prevent VIDD. These exciting exploratory experiments will provide novel and important information regarding the role that calpain signaling plays in VIDD. Successful completion of these important experiments will lead to new therapeutic strategies in the prevention of VIDD.

背景：机械通气（MV）用于为> 1500万患者提供呼吸支持 尽管MV通常是一种挽救生命的干预措施，但长期MV （≥12小时）促进了呼吸机的“断奶”患者的问题；这种失败的断奶 增加患者的发病率和死亡率。虽然艰难断奶的原因可能是多因素的 灵感肌肉（即diaphragm）是一个主要因素。确实，通风支持的意外后果 长时间的MV会导致diaphragmatic的无效性以及diaphragm萎缩的快速发展和 收缩功能障碍（统称为通风诱导的隔膜功能障碍（VIDD））。 Vidd发生 由于蛋白质的合成降低和隔膜中蛋白质分解增加。但是， 控制这些事件的上游调节器仍不清楚。我们引人入胜的初步数据表明 半胱氨酸蛋白钙蛋白酶在Vidd的发展中起着重要的信号传导作用。确实，我们的飞行员 实验表明，预防膜片中钙蛋白酶激活可预防VIDD，表明 VIDD的开发需要钙蛋白酶。重要的是，这些结果挑战了当前的教条 经典钙钙（即钙蛋白酶1和2）在骨骼肌萎缩中的作用仅限于细胞骨架的裂解 蛋白质。主动钙蛋白酶促进vidd的机制仍然未知，是其中的重点 R21应用程序。假设：在我们的初步实验的指导下，我们假设活跃的钙蛋白是 长时间肌肉肌肉中蛋白质合成和蛋白水解的必不可少的上游调节剂 MV。具体目的：AIM 1将确定主动钙蛋白酶在MV引起的下降中是否起着至关重要的作用 长期MV期间，隔膜中的合成代谢信号传导（即Akt/mTOR）和蛋白质合成。 AIM 2意志 确定活性钙蛋白酶是否通过激活caspase-3调节diaphragm中MV诱导的蛋白水解 和叉子盒O（Foxo）转录因子。方法：我们将通过使用良好来检验我们的假设 建立的MV动物模型以及创新的分子工具，以防止MV诱导的钙蛋白酶激活 因果关系将使用腺相关病毒载体（AAV9）确定 过表达diaphragm中的calpastatin转化，可防止MV诱导的钙蛋白酶激活。基本的 依赖度量包括合成代谢和蛋白水解信号传导的关键步骤，体内蛋白质合成，速率 diaphragm蛋白水解以及两种caspase-3活性以及FOXO的转录活性的评估 在膜片中。相关性：Vidd是一个严重的临床问题，因为吸气肌肉无力是 无法从呼吸机中解除患者的主要原因。不幸的是，没有标准的临床疗法 为了防止vidd。这些令人兴奋的探索性实验将提供有关有关的新颖和重要信息 Calpain信号在VIDD中的作用。这些重要实验的成功完成将导致 预防Vidd的新治疗策略。

项目成果

Disturbances in Calcium Homeostasis Promotes Skeletal Muscle Atrophy: Lessons From Ventilator-Induced Diaphragm Wasting.

• DOI: 10.3389/fphys.2020.615351
• 发表时间: 2020
• 期刊: Frontiers in physiology
• 影响因子: 4
• 作者: Hyatt HW;Powers SK
• 通讯作者: Powers SK

Mitochondrial Dysfunction Is a Common Denominator Linking Skeletal Muscle Wasting Due to Disease, Aging, and Prolonged Inactivity.

• DOI: 10.3390/antiox10040588
• 发表时间: 2021-04-11
• 期刊: Antioxidants (Basel, Switzerland)
• 作者: Hyatt HW;Powers SK
• 通讯作者: Powers SK

Calpains play an essential role in mechanical ventilation-induced diaphragmatic weakness and mitochondrial dysfunction.

• DOI: 10.1016/j.redox.2020.101802
• 发表时间: 2021-01
• 期刊: Redox biology
• 影响因子: 11.4
• 作者: Hyatt HW;Ozdemir M;Yoshihara T;Nguyen BL;Deminice R;Powers SK
• 通讯作者: Powers SK

Activation of Calpain Contributes to Mechanical Ventilation-Induced Depression of Protein Synthesis in Diaphragm Muscle.

• DOI: 10.3390/cells11061028
• 发表时间: 2022-03-18
• 期刊: Cells
• 影响因子: 6
• 作者: Hyatt HW;Ozdemir M;Bomkamp MP;Powers SK
• 通讯作者: Powers SK

Angiotensin 1-7 protects against ventilator-induced diaphragm dysfunction.

• DOI: 10.1111/cts.13015
• 发表时间: 2021-07
• 期刊: Clinical and translational science
• 作者: Yoshihara T;Deminice R;Hyatt HW;Ozdemir M;Nguyen BL;Powers SK
• 通讯作者: Powers SK