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

ABSTRACT: To investigate whether apocynin protects the diaphragm from wasting and oxidative stress during mechanical ventilation (MV).Prospective, randomized, controlled study.Research laboratory.Adult female Sprague-Dawley rats.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.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.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.

SUBMITTER: McClung JM

PROVIDER: S-EPMC2909189 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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

McClung Joseph M JM Van Gammeren Darin D Whidden Melissa A MA Falk Darin J DJ Kavazis Andreas N AN Hudson Matt B MB Gayan-Ramirez Ghislaine G Decramer Marc M DeRuisseau Keith C KC Powers Scott K SK

Critical care medicine 20090401 4

<h4>Objective</h4>To investigate whether apocynin protects the diaphragm from wasting and oxidative stress during mechanical ventilation (MV).<h4>Design</h4>Prospective, randomized, controlled study.<h4>Setting</h4>Research laboratory.<h4>Subjects</h4>Adult female Sprague-Dawley rats.<h4>Interventions</h4>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 ...[more]

PMID: 19242334

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Project description:BackgroundIn intensive care unit (ICU) patients, diaphragmatic dysfunction (DD) can occur on admission or during the subsequent stay. The respective incidence of these two phenomena has not been previously studied in humans. The study was designed to describe temporal trends in diaphragm function in mechanically ventilated (MV) patients.MethodsAncillary study of a prospective, 6-month, observational cohort study conducted in two ICUs. MV patients were studied within 24 h following intubation (day-1) and every 48-72 h thereafter. Diaphragm function was assessed by twitch tracheal pressure (Ptr,stim) in response to bilateral anterior magnetic phrenic nerve stimulation. Diaphragm dysfunction was defined as Ptr,stim < 11 cmH2O. Patients who received MV for at least 5 days were retained, and the first and the last measures were analysed.ResultsForty-three patients were included. Overall, 79 % of patients developed DD at some point during their ICU stay: 23 (53 %) patients presented DD on initiation of mechanical ventilation, 14 (33 %) of whom had persistent DD, while diaphragm function improved in 9 (21 %). Among the remaining 20 (47 %) patients who did not present DD on initiation of MV, 11 (26 %) developed DD during the ICU stay, while 9 (21 %) did not. Mortality was higher in patients with DD either on initiation of mechanical ventilation or during the subsequent ICU stay than in those who never developed DD (35 vs. 0 %, p = 0.04). Duration of MV was higher in patients with DD on initiation of MV that subsequently persisted than in patients who never exhibited diaphragm dysfunction (18 vs. 5 days, p = 0.04). Factors associated with a change in Ptr,stim were: age [linear coefficient regression (Coeff.) -0.097, standard error (SD) 0.047, p = 0.046], PaO2/FiO2 ratio (Coeff. 0.014, SD 0.006, p = 0.0211) and the proportion of the time under MV with sedation (per 10 %, Coeff. -5.359, SD 2.451, p = 0.035).ConclusionsDD is observed in a large majority of MV patients ≥5 days at some point of their ICU stay. Various patterns of DD are observed, including DD on initiation of mechanical ventilation and ICU-acquired DD. Trial registration clinicaltrials.gov Identifier # NCT00786526.

Project description:Diaphragmatic weakness, due to both atrophy and contractile dysfunction, is a well-documented response following prolonged mechanical ventilation. Evidence indicates that activation of the proteases calpain and caspase-3 is essential for mechanical ventilation-induced diaphragmatic weakness to occur. We tested the hypothesis that a regulatory cross-talk exists between calpain and caspase-3 in the diaphragm during prolonged mechanical ventilation. To test this prediction, we determined whether selective pharmacological inhibition of calpain would prevent activation of caspase-3 and conversely whether selective inhibition of caspase-3 would abate calpain activation.Animal study.University Research Laboratory.Female Sprague-Dawley rats.Animals were randomly divided into control or one of three 12-hr mechanical ventilation groups that were treated with/without a selective pharmacological protease inhibitor: 1) control, 2) mechanical ventilation, 3) mechanical ventilation with a selective caspase-3 inhibitor, and 4) mechanical ventilation with a selective calpain inhibitor.Compared to control, mechanical ventilation resulted in calpain and caspase-3 activation in the diaphragm accompanied by atrophy of type I, type IIa, and type IIx/IIb fibers. Independent inhibition of either calpain or caspase-3 prevented this mechanical ventilation-induced atrophy. Pharmacological inhibition of calpain prevented mechanical ventilation-induced activation of diaphragmatic caspase-3 and inhibition of caspase-3 prevented activation of diaphragmatic calpain. Furthermore, calpain inhibition also prevented the activation of caspase-9 and caspase-12, along with the cleavage of Bid to tBid, all upstream signals for caspase-3 activation. Lastly, caspase-3 inhibition prevented the mechanical ventilation-induced degradation of the endogenous calpain inhibitor, calpastatin.Collectively, these results indicate that mechanical ventilation-induced diaphragmatic atrophy is dependent on the activation of both calpain and caspase-3. Importantly, these findings provide the first experimental evidence in diaphragm muscle that calpain inhibition prevents the activation of caspase-3 and vice versa and caspase-3 inhibition prevents the activation of calpain. These findings support our hypothesis that a regulatory calpain/caspase-3 cross-talk exists whereby calpain can promote caspase-3 activation and active caspase-3 can enhance calpain activity in diaphragm muscle during prolonged mechanical ventilation.

Project description:BackgroundMechanical ventilation is a life-saving intervention used to provide adequate pulmonary ventilation in patients suffering from respiratory failure. However, prolonged mechanical ventilation is associated with significant diaphragmatic weakness resulting from both myofiber atrophy and contractile dysfunction. Although several signaling pathways contribute to diaphragm weakness during mechanical ventilation, it is established that oxidative stress is required for diaphragmatic weakness to occur. Therefore, identifying the site(s) of mechanical ventilation- induced reactive oxygen species production in the diaphragm is important.ObjectiveThese experiments tested the hypothesis that elevated mitochondrial reactive oxygen species emission is required for mechanical ventilation-induced oxidative stress, atrophy, and contractile dysfunction in the diaphragm.DesignCause and effect was determined by preventing mechanical ventilation-induced mitochondrial reactive oxygen species emission in the diaphragm of rats using a novel mitochondria-targeted antioxidant (SS-31).InterventionsNone.Measurements and main resultsCompared to mechanically ventilated animals treated with saline, animals treated with SS-31 were protected against mechanical ventilation-induced mitochondrial dysfunction, oxidative stress, and protease activation in the diaphragm. Importantly, treatment of animals with the mitochondrial antioxidant also protected the diaphragm against mechanical ventilation-induced myofiber atrophy and contractile dysfunction.ConclusionsThese results reveal that prevention of mechanical ventilation-induced increases in diaphragmatic mitochondrial reactive oxygen species emission protects the diaphragm from mechanical ventilation-induced diaphragmatic weakness. This important new finding indicates that mitochondria are a primary source of reactive oxygen species production in the diaphragm during prolonged mechanical ventilation. These results could lead to the development of a therapeutic intervention to impede mechanical ventilation-induced diaphragmatic weakness.

Project description:Previous workers have demonstrated that controlled mechanical ventilation results in diaphragm inactivity and elicits a rapid development of diaphragm weakness as a result of both contractile dysfunction and fiber atrophy. Limited data exist regarding the impact of pressure support ventilation, a commonly used mode of mechanical ventilation-that permits partial mechanical activity of the diaphragm-on diaphragm structure and function. We carried out the present study to test the hypothesis that high-level pressure support ventilation decreases the diaphragm pathology associated with CMV.Sprague-Dawley rats were randomly assigned to one of the following five groups:1) control (no mechanical ventilation); 2) 12 hrs of controlled mechanical ventilation (12CMV); 3) 18 hrs of controlled mechanical ventilation (18CMV); 4) 12 hrs of pressure support ventilation (12PSV); or 5) 18 hrs of pressure support ventilation (18PSV).We carried out the following measurements on diaphragm specimens: 4-hydroxynonenal-a marker of oxidative stress, active caspase-3 (casp-3), active calpain-1 (calp-1), fiber type cross-sectional area, and specific force (sp F). Compared with the control, both 12PSV and 18PSV promoted a significant decrement in diaphragmatic specific force production, but to a lesser degree than 12CMV and 18CMV. Furthermore, 12CMV, 18PSV, and 18CMV resulted in significant atrophy in all diaphragm fiber types as well as significant increases in a biomarker of oxidative stress (4-hydroxynonenal) and increased proteolytic activity (20S proteasome, calpain-1, and caspase-3). Furthermore, although no inspiratory effort occurs during controlled mechanical ventilation, it was observed that pressure support ventilation resulted in large decrement, approximately 96%, in inspiratory effort compared with spontaneously breathing animals.High levels of prolonged pressure support ventilation promote diaphragmatic atrophy and contractile dysfunction. Furthermore, similar to controlled mechanical ventilation, pressure support ventilation-induced diaphragmatic atrophy and weakness are associated with both diaphragmatic oxidative stress and protease activation.

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

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

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