Mechanical ventilation, oxidative stress, and diaphragmatic atrophy

描述（由申请人提供）：机械通气（MV）在临床上作为一种挽救生命的干预措施，用于无法维持足够肺泡通气的患者（如呼吸衰竭患者）维持足够的肺气体交换。将患者从MV中移除被称为“断奶”，而断奶的问题是非常普遍的。无法使患者戒除MV导致发病率和死亡率的风险增加以及医疗保健费用的增加。研究表明，mv引起的膈肌无力，由于萎缩和收缩功能障碍，是导致脱机困难的重要因素。我们的研究表明，MV延长与膈肌氧化应激有关，MV诱导的膈肌萎缩和收缩功能障碍需要两种蛋白酶calpain和caspase-3的激活。在长时间MV期间调节膈肌中这些蛋白酶活性的机制目前尚不清楚，将在本应用中加以解决。假设：目的1将验证氧化应激对长时间MV期间膈肌钙蛋白酶和caspase-3的激活至关重要的假设。目的2将验证横膈膜肌丝蛋白的氧化修饰增加其对calpain1 /2和/或caspase-3降解的易感性的假设。目的3将验证calpain和caspase-3之间的调节性串扰在决定这些蛋白酶的个体活性方面起着至关重要的作用。方法：我们将使用实验范式的组合来检验这些假设，包括动物MV模型、肽图谱和在大鼠原代骨骼肌细胞培养中进行的细胞信号传导实验。因果关系将通过预防mv诱导的膈膜氧化应激和使用创新方法独立抑制原发性骨骼肌肌管中的钙蛋白酶和caspase-3来确定。意义和长期目标：MV患者不能断奶是一个重要的临床问题，而呼吸肌无力是导致断奶困难的主要因素。因此，我们的长期目标是在了解mv引起膈肌萎缩的细胞机制的基础上，开发预防mv引起的膈肌无力的方法。此外，这些实验的结果应该为更广泛的主题提供重要的信息，如由于长时间卧床休息、固定和疾病状态导致的骨骼肌萎缩。公共卫生相关性：机械通气（MV）用于不能维持足够肺泡通气的患者维持肺气体交换；患者戒除MV称为“脱机”，脱机过程中出现的问题极为普遍。大量研究表明，mv引起的膈肌无力，由于萎缩和收缩功能障碍，是导致脱机困难的重要因素。我们的长期目标是在了解mv引起膈肌萎缩和收缩功能障碍的细胞机制的基础上，开发预防mv引起的膈肌无力的方法。

DESCRIPTION (provided by applicant): Mechanical ventilation (MV) is used clinically as a life saving intervention to sustain adequate pulmonary gas exchange in patients that are incapable of maintaining sufficient alveolar ventilation (e.g., patients in respiratory failure). The removal of patients from MV is termed "weaning" and problems in weaning are extremely common. The inability to wean patients from MV results in increased risk of morbidity and mortality along with higher health care costs. Studies indicate that MV-induced diaphragmatic weakness, due to both atrophy and contractile dysfunction, is a significant contributor to weaning difficulties. Our research reveals that prolonged MV is associated with diaphragmatic oxidative stress and that the activation of two proteases, calpains and caspase-3, is required for MV-induced diaphragmatic atrophy and contractile dysfunction. The mechanism(s) that regulate the activity of these proteases in the diaphragm during prolonged MV are currently unknown and will be addressed in this application. HYPOTHESES: Aim 1 will test the hypothesis that oxidative stress is essential for activation of both calpain and caspase-3 in the diaphragm during prolonged MV. Aim 2 will test the postulate that oxidative modification of diaphragmatic myofilament proteins increases their susceptibility to degradation by calpains-1/2 and/or caspase-3. Aim 3 will test the hypothesis that regulatory cross-talk between calpain and caspase-3 plays a vital role in determining the individual activity of these proteases. APPROACH: We will test these hypotheses using a combination of experimental paradigms including an animal model of MV, peptide mapping, and cell-signaling experiments performed in rat primary skeletal muscle cell culture. Cause and effect will be determined by prevention of MV-induced oxidative stress in the diaphragm and by the independent inhibition of calpains and caspase-3 in primary skeletal muscle myotubes using innovative approaches. SIGNIFICANCE AND LONG-TERM GOAL: Failure to wean patients from MV is an important clinical problem and respiratory muscle weakness is a major contributor to weaning difficulties. Hence, our long-term goal is to develop methods for the prevention of MV-induced diaphragmatic weakness based on an understanding of the cellular mechanisms responsible for MV-induced atrophy in the diaphragm. Moreover, the results of these experiments should provide important information for broader topics such as skeletal muscle wasting due to prolonged bed rest, immobilization, and disease states. PUBLIC HEALTH RELEVANCE: Mechanical ventilation (MV) is used to sustain pulmonary gas exchange in patients that are incapable of maintaining adequate alveolar ventilation; the withdrawal of MV from patients is referred to as "weaning" and problems in weaning from MV are extremely common. Numerous studies reveal that MV-induced diaphragmatic weakness, due to both atrophy and contractile dysfunction, is an important contributor to weaning difficulties. Our long-term goal is to develop methods for the prevention of MV-induced diaphragmatic weakness based on an understanding of the cellular mechanisms responsible for MV-induced diaphragmatic atrophy and contractile dysfunction.