Prolonged mechanical ventilation (MV) results in diaphragmatic weakness due to fiber atrophy and contractile dysfunction. Recent work reveals that activation of the proteases calpain and caspase-3 is required for MV-induced diaphragmatic atrophy and contractile dysfunction. However, the mechanism(s) responsible for activation of these proteases remains unknown. To address this issue, we tested the hypothesis that oxidative stress is essential for the activation of calpain and caspase-3 in the diaphragm during MV. Cause-and-effect was established by prevention of MV-induced diaphragmatic oxidative stress using the antioxidant Trolox. Treatment of animals with Trolox prevented MV-induced protein oxidation and lipid peroxidation in the diaphragm. Importantly, the Trolox-mediated protection from MV-induced oxidative stress prevented the activation of calpain and caspase-3 in the diaphragm during MV. Furthermore, the avoidance of MV-induced oxidative stress not only averted the activation of these proteases but also rescued the diaphragm from MV-induced diaphragmatic myofiber atrophy and contractile dysfunction. Collectively, these findings support the prediction that oxidative stress is required for MV-induced activation of calpain and caspase-3 in the diaphragm and are consistent with the concept that antioxidant therapy can retard MV-induced diaphragmatic weakness.

Oxidative stress promotes controlled mechanical ventilation (MV)‐induced diaphragmatic atrophy. Nonetheless, the signalling pathways responsible for oxidative stress‐induced muscle atrophy remain unknown. We tested the hypothesis that oxidative stress down‐regulates insulin‐like growth factor‐1–phosphotidylinositol 3‐kinase–protein kinase B serine threonine kinase (IGF‐1–PI3K–Akt) signalling and activates the forkhead box O (FoxO) class of transcription factors in diaphragm fibres during MV‐induced diaphragm inactivity. Sprague–Dawley rats were randomly assigned to one of five experimental groups: (1) control (Con), (2) 6 h of MV, (3) 6 h of MV with infusion of the antioxidant Trolox, (4) 18 h of MV, (5) 18 h of MV with Trolox. Following 6 h and 18 h of MV, diaphragmatic Akt activation decreased in parallel with increased nuclear localization and transcriptional activation of FoxO1 and decreased nuclear localization of FoxO3 and FoxO4, culminating in increased expression of the muscle‐specific ubiquitin ligases, muscle atrophy factor (MAFbx) and muscle ring finger‐1 (MuRF‐1). Interestingly, following 18 h of MV, antioxidant administration was associated with attenuation of MV‐induced atrophy in type I, type IIa and type IIb/IIx myofibres. Collectively, these data reveal that the antioxidant Trolox attenuates MV‐induced diaphragmatic atrophy independent of alterations in Akt regulation of FoxO transcription factors and expression of MAFbx or MuRF‐1. Further, these results also indicate that differential regulation of diaphragmatic IGF‐1–PI3K–Akt signalling exists during the early and late stages of MV.

Objective-To investigate whether apocynin protects the diaphragm from wasting and oxidative stress during mechanical ventilation (MV).Design-Prospective, randomized, controlled study. Setting-Research laboratory. Subjects-Adult female Sprague-Dawley rats.Interventions-Rats were randomly assigned to one of five experimental groups: 1) acutely anesthetized control, 2) spontaneous breathing control, 3) spontaneously breathing control with administration of the nicotinamide adenine dinucleotide phosphate oxidase inhibitor, apocynin, 4) mechanically ventilated, and 5) mechanically ventilated with apocynin. Measurements and MainResults-Apocynin attenuated MV-induced diaphragmatic oxidative stress, contractile dysfunction, and type I, type IIa, and type IIb/IIx myofiber atrophy. The apocynin-induced attenuation of MV-induced diaphragmatic atrophy and contractile dysfunction occurred in conjunction with a reduction in the small increase in nicotinamide adenine dinucleotide phosphate oxidase activity as well as the preservation of total glutathione levels, glutathione peroxidase protein abundance, and a decrease in the activation of the cysteine proteases, calpain-1 and caspase-3. Interestingly, independent of MV, apocynin increased diaphragmatic levels of calpastatin, an endogenous calpain inhibitor. Furthermore, treatment of skeletal muscle cells in culture (C2C12 myotubes) with apocynin resulted in an increase in both calpastatin mRNA levels and protein abundance.Conclusions-Our results suggest that the protective effects of apocynin on the diaphragm during prolonged MV seem to be linked to both its functions as an antioxidant and role in cellular signaling regulating the cysteine protease inhibitor calpastatin.Keywords skeletal muscle; NADPH oxidase; protease; atrophy; oxidative stress; antioxidantThe first two authors contributed equally to this work.The authors have not disclosed any potential conflicts of interest.For information regarding this article, joseph.mcclung@duke.edu NIH Public Access Author ManuscriptCrit Care Med. Author manuscript; available in PMC 2010 July 23. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptMechanical ventilation (MV) is used to maintain adequate alveolar ventilation in patients who are incapable of doing so on their own. Evidence indicates that MV-induced oxidative stress in the diaphragm is a major contributor to diaphragmatic atrophy and contractile dysfunction (1,2) and leads to difficulties in removing patients from the ventilator (i.e., "weaning"). Therefore, circumventing reactive oxygen species (ROS) generation or enhancing the free radical scavenging capabilities of the diaphragm via the administration of antioxidant compounds possesses exciting potential for protecting the diaphragm during MV.Our laboratory and others have suggested that oxidative stress is a critical upstream signaling event leading to the activation of cysteine proteases (i.e., calpains and caspases) involved in myofilament disassembly as well as the deactivation of endo...