Ventilator-induced diaphragmatic atrophy: role of autophagy

描述（由申请人提供）：每年有数百万患者在手术期间和需要呼吸机支持的危重患者中使用机械通气（MV）来支持呼吸系统。虽然中压通气通常是一种挽救生命的干预措施，但延长中压通气会增加患者“脱离”呼吸机的问题。虽然有几个因素会导致断奶困难，但吸气肌无力是主要因素。在这方面，MV导致膈肌不活动，由于膈肌萎缩和收缩功能障碍（称为呼吸机诱发的膈肌功能障碍（VIDD）），导致吸气肌无力迅速发展。虽然mv引起的膈肌萎缩是由于蛋白质分解增加和蛋白质合成减少，但蛋白质水解起主导作用。研究证实，在长时间MV期间，膈膜中所有四种主要的蛋白水解系统都被激活，证据表明泛素-蛋白酶体系统、calpain和caspase-3都参与了VIDD。虽然第四个主要的蛋白水解系统，自噬，在长时间的MV过程中在隔膜中被激活，但尚不清楚自噬是促进VIDD还是通过去除受损的蛋白质和细胞器作为一种保护机制。假设：在初步结果的指导下，我们预测，预防mv诱导的隔膜自噬增加将防止VIDD，并将抑制导致VIDD的关键蛋白水解系统（即calpain和caspase-3）的激活。方法：我们将使用一个完善的MV动物模型和创新的分子工具来验证这一假设，以防止MV诱导的隔膜自噬增加。具体而言，因果关系将通过使用腺相关病毒载体进行基因传递来确定，以表达自噬基因5的显性负突变，该突变将阻止长时间MV期间隔膜自噬的激活。为了确定增加的自噬对VIDD的影响是否与时间有关，我们将使动物暴露于不同的MV持续时间。具体目的：目的1将检验假设，即预防mv诱导的膈膜自噬增加可以预防VIDD。目的2将验证这样一个假设，即预防mv诱导的隔膜自噬将阻止其他主要蛋白水解系统（即calpain和caspase-3）的激活，这些系统会导致VIDD。意义：肺心病是一个重要的临床问题，因为吸气肌无力是患者无法脱离呼吸机的主要原因。我们提出的实验将为自噬在VIDD中的作用提供新的重要信息。总的来说，这些实验可以导致新的治疗策略，以预防VIDD。

DESCRIPTION (provided by applicant): Mechanical ventilation (MV) is used in millions of patients each year to support the respiratory system during surgery and in critically ill patients requiring ventilator support. Although MV is often a life-saving intervention, prolonged MV promotes problems in "weaning" patients from the ventilator. While several factors can contribute to difficult weaning, weak inspiratory muscles are a major factor. In this regard, MV results in diaphragmatic inactivity resulting in the rapid development of inspiratory muscle weakness due to diaphragmatic atrophy and contractile dysfunction (known as ventilator-induced diaphragm dysfunction (VIDD)). Although MV-induced diaphragmatic atrophy occurs due to both increased protein breakdown and decreased protein synthesis, proteolysis plays a dominant role. It is established that all four major proteolytic systems are activated in the diaphragm during prolonged MV and evidence indicates that the ubiquitin- proteasome system, calpains, and caspase-3 all contribute to VIDD. While the fourth major proteolytic system, autophagy, is activated in the diaphragm during prolonged MV, it remains unknown if autophagy promotes VIDD or serves as a protective mechanism by removing damaged proteins and organelles. HYPOTHESIS: Guided by preliminary results, we predict that prevention of MV-induced increases in autophagy in the diaphragm will protect against VIDD and will suppress the activation of key proteolytic systems (i.e., calpain and caspase-3) that contribute to VIDD. APPROACH: We will test this hypothesis using a well-established animal model of MV and innovative molecular tools to prevent MV-induced increases in autophagy in the diaphragm. Specifically, cause and effect will be determined by using an adeno-associated virus vector for gene delivery to express a dominant negative mutation of autophagy gene 5 that will prevent activation of autophagy in the diaphragm during prolonged MV. To determine if the impact of increased autophagy on VIDD is time dependent, we will expose animals to varying durations of MV. SPECIFIC AIMS: Aim 1 will test the hypothesis that prevention of MV-induced increases in autophagy in the diaphragm is protective against VIDD. Aim 2 will test the hypothesis that prevention of MV-induced autophagy in the diaphragm will prevent the activation of other major proteolytic systems (i.e., calpain and caspase-3) that contribute to VIDD. SIGNIFICANCE: VIDD is an important clinical problem because inspiratory muscle weakness is a major cause of the inability to wean patients from the ventilator. Our proposed experiments will provide new and important information regarding the role that autophagy plays in VIDD. Collectively, these experiments can lead to new therapeutic strategies in the prevention of VIDD.