Mechanical ventilation, oxidative stress, and diaphragmatic atrophy

描述(申请人提供)：机械通气(MV)在临床上被用作维持足够肺泡通气量的患者(如呼吸衰竭患者)维持足够的肺气体交换的救命干预手段。将患者从机械通气中移除被称为“脱机”，脱机过程中出现的问题非常常见。无法使患者脱离MV会导致发病率和死亡率的增加，以及更高的医疗费用。研究表明，由于萎缩和收缩功能障碍，MV引起的横隔肌无力是导致脱机困难的重要因素。我们的研究表明，MV的延长与横隔膜的氧化应激有关，并且在MV诱导的横隔膜萎缩和收缩功能障碍中，需要激活两种蛋白水解酶，即钙蛋白酶和caspase-3。在延长的MV期间，调节隔膜中这些蛋白水解酶活性的机制(S)目前尚不清楚，将在本申请中解决。假设：目标1将检验假设，即氧化应激对于延长MV期间隔膜中的钙蛋白酶和caspase-3的激活是必不可少的。目的2将测试假设，氧化修饰的横隔膜肌丝蛋白增加其对降解的敏感性，由钙蛋白-1/2和/或半胱氨酸氨基转移酶-3。目标3将测试这一假说，即钙蛋白酶和caspase-3之间的调节串扰在决定这些酶的个体活性方面起着至关重要的作用。方法：我们将使用一系列实验范例来验证这些假设，包括MV动物模型、肽图谱以及在大鼠原代骨骼肌细胞培养中进行的细胞信号实验。因果关系将通过预防MV诱导的横隔膜氧化应激，以及通过使用创新方法独立抑制初级骨骼肌肌管中的钙蛋白酶和caspase-3来确定。意义和长期目标：未能使患者脱离机械通气是一个重要的临床问题，呼吸肌无力是导致撤机困难的主要原因。因此，我们的长期目标是在了解MV诱导的横隔膜萎缩的细胞机制的基础上，开发预防MV诱导的横隔膜无力的方法。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，例如由于长时间卧床、制动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)用于维持无法维持足够肺泡通气量的患者的肺气体交换；患者停止机械通气被称为“脱机”，撤机的问题非常常见。大量研究表明，由于萎缩和收缩功能障碍，MV引起的横隔肌无力是导致脱机困难的重要因素。我们的长期目标是在了解MV所致的横隔肌萎缩和收缩功能障碍的细胞机制的基础上，开发预防MV所致的横隔肌无力的方法。

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.