Apocynin attenuates diaphragm oxidative stress and protease activation during prolonged mechanical ventilation

Abstract

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...

Ventilator-induced diaphragm dysfunction: cause and effect

“…However, because the rat and human diaphragm are anatomically alike and contain a similar fiber type composition (76,81), the rat has become the most commonly used animal model to study MV-induced changes in diaphragm fiber size and function. Several studies reveal that as few as 12 h of controlled MV results in a 10 -15% reduction in the cross-sectional area of all rat diaphragm fiber types (i.e., type I, type IIa, and type IIx/b) (67,69,82,103). This MV-induced rat diaphragmatic fiber atrophy increases as a function of time and approaches a 30% reduction in fiber cross-sectional area following 18 -24 h of prolonged MV (33,100,117).…”

“…For example, caspase-3 is active in skeletal muscle during periods of unloading and active caspase-3 can degrade actin/myosin complexes (24,67). Abundant evidence demonstrates that full support MV activates caspase-3 in both the rodent and human diaphragm (58,67,69,75,106). This is important because active caspase-3 is required for myonuclear apoptosis in the diaphragm (67).…”

“…MECHANICAL VENTILATION AND DIAPHRAGM chondrion (30,69,82,111,116). For example, as few as 12 h of MV results in a large increase in ROS emission from rat diaphragm mitochondria during both state 3 and state 4 respiration (50,82).…”

Mechanistic Links Between Oxidative Stress and Disuse Muscle Atrophy

“…The mechanisms responsible for this inactivity-induced increase in mitochondria ROS production remain unknown. It is also possible that both xanthine oxidase and NADPH oxidase make small contributions to inactivity-induced ROS production in muscle (44,71) (Fig. 2).Hence, it appears that inactivity-induced ROS production in skeletal muscle is derived from several different sites and additional work is required to complete our understanding of the control of ROS producing pathways in skeletal muscle during prolonged periods of inactivity.…”

Mechanical Ventilation-Induced Oxidative Stress in the Diaphragm

“…13 Additionally, oxidative stress can activate several key proteases (eg, calpain and caspase-3), and activation of these proteases is an important contributor to the MV-induced diaphragmatic atrophy and contractile dysfunction. [19][20][21][22] Therefore, understanding the interplay between oxidant production and antioxidant action in the diaphragm during prolonged MV is important. In this context, the current experiment focused on the role of heme oxygenase (HO)-1 as a regulator of redox balance in the diaphragm during MV.…”