Ventilator-induced diaphragmatic atrophy: role of autophagy

描述(申请人提供)：每年有数以百万计的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风(MV)来支持呼吸系统。虽然机械通气通常是一种挽救生命的干预措施，但延长机械通气时间会导致患者在脱离呼吸机后出现问题。虽然有几个因素可能会导致脱机困难，但吸气肌无力是一个主要因素。在这一点上，MV导致横隔肌缺乏活动，导致由于横隔肌萎缩和收缩功能障碍(称为呼吸机诱导的横隔膜功能障碍(VIDD))而导致吸气肌无力的快速发展。虽然MV诱导的横隔膜萎缩是由于蛋白质分解增加和蛋白质合成减少所致，但蛋白质分解起主导作用。已经证实，在延长的MV期间，所有四种主要的蛋白水解系统都在隔膜被激活，证据表明泛素-蛋白酶体系统、钙蛋白酶和caspase-3都参与了VIDD的发生。虽然第四个主要的蛋白分解系统，自噬，在延长的MV期间在隔膜中被激活，但目前尚不清楚自噬是促进VIDD还是通过清除受损的蛋白质和细胞器起到保护机制的作用。假设：根据初步结果，我们预测，阻止MV诱导的隔膜自噬增加将保护VIDD，并将抑制导致VIDD的关键蛋白分解系统(即钙蛋白酶和caspase-3)的激活。方法：我们将使用成熟的MV动物模型和创新的分子工具来验证这一假设，以防止MV诱导的横隔膜自噬增加。具体地说，将通过使用腺相关病毒载体进行基因传递来确定因果关系，以表达自噬基因5的显性负突变，该突变将防止在延长的MV期间激活横隔膜中的自噬。为了确定自噬增加对VIDD的影响是否具有时间依赖性，我们将动物暴露在不同持续时间的MV中。具体目的：Aim 1将检验这样一种假设，即防止MV诱导的横隔膜自噬增加对VIDD具有保护作用。目的2将验证一种假设，即防止MV诱导的横隔膜自噬将阻止导致VIDD的其他主要蛋白分解系统(即钙蛋白酶和caspase-3)的激活。意义：VIDD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。我们提出的实验将提供有关自噬在VIDD中所起作用的新的和重要的信息。总的来说，这些实验可以在预防VIDD方面带来新的治疗策略。

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.