Crosstalk between autophagy and oxidative stress regulates proteolysis in the diaphragm during mechanical ventilation

Highlights

• Mechanical ventilation induces increased autophagic signaling and oxidative stress in the diaphragm.

• Inhibition of autophagy is sufficient to prevent ventilator-induced diaphragm dysfunction.

• Mechanical ventilation-induced autophagy is required for diaphragm mitochondrial dysfunction and oxidative stress.

• Mitochondrial reactive oxygen species emission promotes autophagosome formation in the diaphragm.

Abstract

Mechanical ventilation (MV) results in the rapid development of ventilator-induced diaphragm dysfunction (VIDD). While the mechanisms responsible for VIDD are not fully understood, recent data reveal that prolonged MV activates autophagy in the diaphragm, which may occur as a result of increased cellular reactive oxygen species (ROS) production. Therefore, we tested the hypothesis that (1) accelerated autophagy is a key contributor to VIDD; and that (2) oxidative stress is required to increase the expression of autophagy genes in the diaphragm. Our findings reveal that targeted inhibition of autophagy in the rat diaphragm prevented MV-induced muscle atrophy and contractile dysfunction. Attenuation of VIDD in these animals occurred as a result of increased diaphragm concentration of the antioxidant catalase and reduced mitochondrial ROS emission, which corresponded to reductions in the activity of calpain and caspase-3. To determine if increased ROS production is required for the upregulation of autophagy biomarkers in the diaphragm, rats that were administered the mitochondrial-targeted peptide SS-31 during MV. Results from this study demonstrated that mitochondrial ROS production in the diaphragm during MV is required for the increased expression of key autophagy genes (i.e. LC3, Atg7, Atg12, Beclin1 and p62), as well as for increased activity of cathepsin L. Together, these data reveal that autophagy is required for VIDD, and that autophagy inhibition reduces MV-induced diaphragm ROS production and prevents a positive feedback loop whereby increased autophagy is stimulated by oxidative stress, resulting in further increases in ROS and autophagy.

Introduction

Mechanical ventilation (MV) is used to maintain adequate alveolar ventilation in patients unable to maintain blood gas homeostasis with spontaneous breathing. While MV is a life-saving intervention for critically ill patients, prolonged MV results in the rapid development of diaphragm weakness due to both myofiber atrophy and contractile dysfunction; this condition is termed ventilator-induced diaphragm dysfunction (VIDD) [1], [2], [3]. VIDD is important because diaphragm weakness is predicted to contribute to difficulties in weaning patients from the ventilator [4], [5], [6]. Unfortunately, there is currently no standard therapy to prevent VIDD. Therefore, improving our understanding of the cellular processes that promote VIDD is essential to develop a therapeutic strategy to protect against MV-induced diaphragm weakness.

Although the molecular underpinnings that cause VIDD remain unclear, it is well established that prolonged MV results in accelerated proteolysis in the diaphragm [7]. Specifically, our laboratory has shown that MV-induced activation of calpain and the proteasome system in the diaphragm are associated with the breakdown of myofibrillar proteins [8], [9], [10], and caspase-3 activity is associated with both contractile protein cleavage and apoptosis [8], [11]. In addition to these proteolytic pathways, autophagy is also activated in the diaphragm of both rodents and humans during prolonged MV [12], [13]. However, the role that autophagy plays in the development of VIDD remains unclear. In this regard, evidence suggests that increased autophagy can be a double-edged sword. In theory, accelerated autophagy could produce protective or deleterious effects on skeletal muscle depending on the conditions [14], [15]. For example, MV-induced accelerated autophagy could promote diaphragm atrophy by eliminating healthy organelles and cytosolic proteins from muscle fibers [16]. In contrast, MV-induced autophagy could be protective by removing damaged mitochondria and aggregated proteins within diaphragm myofibers [16], [17].

In addition to the downstream effects of accelerated autophagy on diaphragm muscle function, the upstream trigger promoting accelerated autophagy is unknown. In this regard, it has been demonstrated that MV-induced increases in ROS production in the diaphragm leads to activation of several proteases along with oxidative modification of muscle proteins making them more susceptible to degradation [18], [19], [20], [21]. The degradation of both cytosolic and myofibrillar proteins is a multistep process that requires the cooperation of several proteolytic components including the calpain, caspase-3, ubiquitin-proteasome and autophagy/lysosomal proteolytic systems [18], [19], [20], [21]. We have recently discovered that prevention of MV-induced increases in mitochondrial ROS emission is sufficient to prevent the activation of the calpain, caspase-3 and ubiquitin-proteasome proteolytic systems in the diaphragm during MV [22]. However, the role that mitochondrial ROS emission plays in activating the autophagy/lysosomal system in the diaphragm during prolonged MV remains unknown. Therefore, the goal of these experiments was twofold: (1) to determine the effects of accelerated autophagy on VIDD and; (2) to determine if MV-induced increases in mitochondrial ROS emission is required to increase the expression of autophagy biomarkers in the diaphragm. Specifically, we tested the hypothesis that accelerated autophagy is required for VIDD and that MV-induced increases in mitochondrial ROS emission promote autophagy gene expression and activate autophagy in the diaphragm.