Mechanical ventilation, oxidative stress, and diaphragmatic atrophy

描述（由申请人提供）：机械通气（MV）在临床上用作挽救生命的干预措施，以维持无法维持充分肺泡通气的患者（例如，呼吸衰竭患者）。将患者从MV中移除被称为“脱机”，并且脱机中的问题是极其常见的。无法使患者脱离MV导致发病率和死亡率的风险增加，沿着的是更高的医疗保健费用。研究表明，由于萎缩和收缩功能障碍，MV诱导的肌肉无力是导致断奶困难的重要因素。我们的研究表明，延长MV与血管氧化应激有关，并且MV诱导的血管萎缩和收缩功能障碍需要两种蛋白酶，钙蛋白酶和半胱天冬酶-3的激活。在延长的MV期间调节隔膜中这些蛋白酶的活性的机制目前是未知的，并且将在本申请中解决。假设：目的1将测试这一假设，即氧化应激是必不可少的激活钙蛋白酶和半胱天冬酶-3在膈肌在延长MV。目的2将测试的假设，即氧化修饰的血管肌丝蛋白增加其敏感性的钙蛋白酶-1/2和/或半胱天冬酶-3的降解。目的3将测试钙蛋白酶和半胱天冬酶-3之间的调节性串扰在确定这些蛋白酶的个体活性中起着至关重要的作用的假设。方法：我们将测试这些假设使用的实验范式，包括MV的动物模型，肽图谱，并在大鼠原代骨骼肌细胞培养的细胞信号转导实验的组合。因果关系将通过预防MV诱导的膈肌氧化应激和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半胱天冬酶-3来确定。意义和长期目标：无法使患者脱离MV是一个重要的临床问题，呼吸肌无力是导致脱离困难的主要原因。因此，我们的长期目标是基于对MV诱导的膈肌萎缩的细胞机制的理解，开发预防MV诱导的膈肌无力的方法。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，例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）用于维持无法维持充分肺泡通气的患者的肺气体交换;从患者体内撤出MV被称为“撤机”，撤机问题极为常见。大量研究表明，由于萎缩和收缩功能障碍，MV诱导的肌肉无力是导致断奶困难的重要因素。我们的长期目标是基于对MV诱导的血管萎缩和收缩功能障碍的细胞机制的理解，开发预防MV诱导的血管无力的方法。

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.