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

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 MAIN RESULTS: 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.

The IkappaB kinases IKKalpha and IKKbeta are necessary and sufficient for skeletal muscle atrophy.

Nuclear factor-kappaB (NF-kappaB) signaling is necessary for many types of muscle atrophy, yet only some of the required components have been identified. Gene transfer of a dominant negative (d.n.) IKKbeta into rat soleus muscles showed complete inhibition of 7-day disuse-induced activation of a kappaB reporter gene, while overexpression of wild-type (w.t.) IKKbeta did not. Overexpression of a d.n. IKKbeta-EGFP fusion protein showed that atrophy was inhibited by 50%, indicating that IKKbeta is required for the atrophy process. Overexpression of constitutively active (c.a.) IKKbeta-EGFP showed a marked increase in NF-kappaB activity and a decrease in fiber size of weight-bearing soleus muscles, while muscles overexpressing w.t. IKKbeta-HA had no effect. The same results were found for IKKalpha; overexpression of a d.n. form of the protein decreased unloading-induced NF-kappaB activation and inhibited atrophy by 50%, while overexpression of the w.t. protein had no effect. Overexpression of a c.a. IKKalpha-EGFP fusion protein showed that IKKalpha was sufficient to activate NF-kappaB activity and induce fiber atrophy in muscle. Overexpression of d.n. IKKbeta plus d.n. IKKalpha showed an additive effect on the inhibition of disuse atrophy (70%), suggesting that both kinases of the IKK complex are required for muscle atrophy. These data show that both IKKalpha and IKKbeta are necessary and sufficient for physiological muscle atrophy.

Diaphragmatic nitric oxide synthase is not induced during mechanical ventilation.

Mechanical ventilation (MV) is associated with diaphragmatic oxidative stress that contributes to both diaphragmatic atrophy and contractile dysfunction. However, the pathways responsible for oxidant production in the diaphragm during MV remain unknown. To address this issue, we tested the hypothesis that diaphragmatic nitric oxide synthase (NOS) activity is elevated during MV, resulting in nitration of diaphragmatic proteins. Rats were mechanically ventilated for 18 h, and time-matched, anesthetized but spontaneously breathing animals served as controls. Protein levels of endothelial NOS, inducible NOS, and neuronal NOS were measured in diaphragms from all animals. 3-Nitrotyrosine levels were also measured as an index of protein nitration, and S-nitrosothiol levels were measured as a marker of nitric oxide reactions with molecules containing sulfhydryl groups. Levels of nitrates and nitrites were measured as markers of stable end products of nitric oxide metabolism. Finally, as a marker of oxidative stress, diaphragmatic levels of reduced GSH were also analyzed. MV did not promote an increase in diaphragmatic protein levels of endothelial NOS or neuronal NOS. Moreover, inducible NOS was not detected in the diaphragms of either experimental group. Consistent with these findings, MV did not elevate diaphragmatic 3-nitrotyrosine levels in any subcellular fraction of the diaphragm, including the cytosolic, mitochondrial, membrane, and insoluble protein fractions. Moreover, prolonged MV did not elevate diaphragmatic levels of S-nitrosothiols, nitrate, or nitrite. Finally, prolonged MV significantly reduced diaphragmatic levels of GSH, which is consistent with diaphragmatic oxidative stress. Collectively, these data reveal that MV-induced oxidative stress in the diaphragm is not due to increases in nitric oxide production by NOS.

Diaphragm unloading via controlled mechanical ventilation alters the gene expression profile.

RATIONALE: Prolonged controlled mechanical ventilation results in diaphragmatic inactivity and promotes oxidative injury, atrophy, and contractile dysfunction in this important inspiratory muscle. However, the impact of controlled mechanical ventilation on global mRNA alterations in the diaphragm remains unknown. OBJECTIVES: In these experiments, we used an Affymetrix oligonucleotide array to identify the temporal changes in diaphragmatic gene expression during controlled mechanical ventilation in the rat. METHODS: Adult Sprague-Dawley rats were assigned to either control or mechanical ventilation groups (n = 5/group). Mechanically ventilated animals were anesthetized, tracheostomized, and ventilated with room air for 6 or 18 h. Animals in the control group were acutely anesthetized but not exposed to mechanical ventilation. MEASUREMENTS AND MAIN RESULTS: Compared with control diaphragms, microarray analysis identified 354 differentially expressed, unique gene products after 6 and 18 h of mechanical ventilation. In general, genes in the cell growth/cell maintenance, stress response, and nucleic acid metabolism categories showed predominant upregulation, whereas genes in the structural protein and energy metabolism categories were predominantly downregulated. CONCLUSIONS: We conclude that mechanical ventilation results in rapid changes in diaphragmatic gene expression, and subsequently, many of these changes may contribute to atrophy and muscle fiber remodeling associated with unloading this primary inspiratory muscle. Importantly, this study also provides new insights into why the diaphragm, after the onset of contractile inactivity, atrophies more rapidly than locomotor skeletal muscles and also highlights unique differences that exist between these muscles in the mRNA response to inactivity.

Reloading the diaphragm following mechanical ventilation does not promote injury.

STUDY OBJECTIVE: Mechanical ventilation (MV) is used clinically to treat patients who are incapable of maintaining adequate alveolar ventilation. Prolonged MV is associated with diaphragmatic atrophy and a decrement in maximal specific force production (P(O)). Collectively, these alterations may predispose the diaphragm to injury on the return to spontaneous breathing (ie, reloading). Therefore, these experiments tested the hypothesis that reloading the diaphragm following MV exacerbates MV-induced diaphragmatic contractile dysfunction, while causing muscle fiber membrane damage and inflammation. METHODS: To test this postulate, Sprague-Dawley rats were randomly assigned to the following groups: (1) control; (2) 24 h of controlled MV; and (3) 24 h of controlled MV followed by 2 h of anesthetized spontaneous breathing. Controls were anesthetized in the short term but were not exposed to MV, whereas MV animals were anesthetized, tracheostomized, and ventilated. Reloaded animals remained under anesthesia, but were removed from MV and returned to spontaneous breathing for 2 h. RESULTS: Compared to the situation with control animals, MV resulted in a 26% decrement in diaphragmatic specific P(O) without muscle fiber membrane damage, as measured by an increase in membrane permeability (using the procion orange technique). Further, there were no increases in neutrophil or macrophage influx. Two hours of reloading did not exacerbate MV-induced diaphragmatic contractile dysfunction or cause fiber membrane damage, but increased neutrophil infiltration, myeloperoxidase activity, and muscle edema. CONCLUSION: We conclude that the return to spontaneous breathing following 24 h of controlled MV does not exacerbate MV-induced diaphragm contractile dysfunction or result in fiber membrane damage, but increases neutrophil infiltration.

Mechanical ventilation induces alterations of the ubiquitin-proteasome pathway in the diaphragm.

Prolonged mechanical ventilation (MV) results in diaphragmatic atrophy due, in part, to an increase in proteolysis. These experiments tested the hypothesis that MV-induced diaphragmatic proteolysis is accompanied by increased expression of key components of the ubiquitin-proteasome pathway (UPP). To test this postulate, we investigated the effect of prolonged MV on UPP components and determined the trypsin-like and peptidylglutamyl peptide hydrolyzing activities of the 20S proteasome. Adult Sprague-Dawley rats were assigned to either control or 12-h MV groups (n=7/group). MV animals were anesthetized, tracheostomized, and ventilated with room air for 12 h. Animals in the control group were acutely anesthetized but not exposed to MV. Compared with controls, MV animals demonstrated increased diaphragmatic mRNA levels of two ubiquitin ligases, muscle atrophy F-box (+8.3-fold) and muscle ring finger 1 (+19.0-fold). However, MV did not alter mRNA levels of 14-kDa ubiquitin-conjugating enzyme, polyubiquitin, proteasome-activating complex PA28, or 20S alpha-subunit 7. Protein levels of 14-kDa ubiquitin-conjugating enzyme and proteasome-activating complex PA28 were not altered following MV, but 20S alpha-subunit 7 levels declined (-17.7%). MV increased diaphragmatic trypsin-like activity (+31%) but did not alter peptidylglutamyl peptide hydrolyzing activity. Finally, compared with controls, MV increased ubiquitin-protein conjugates in both the myofibrillar (+24.9%) and cytosolic (+54.7%) fractions of the diaphragm. These results are consistent with the hypothesis that prolonged MV increases diaphragmatic levels of key components within the UPP and that increases in 20S proteasome activity contribute to MV-induced diaphragmatic proteolysis and atrophy.

Trolox attenuates mechanical ventilation-induced diaphragmatic dysfunction and proteolysis.

Prolonged mechanical ventilation results in diaphragmatic oxidative injury, elevated proteolysis, fiber atrophy, and reduced force-generating capacity. We tested the hypothesis that antioxidant infusion during mechanical ventilation would function as an antioxidant to maintain redox balance within diaphragm muscle fibers and therefore prevent oxidative stress and subsequent proteolysis and contractile dysfunction. Sprague-Dawley rats were anesthetized, tracheostomized, and mechanically ventilated with 21% O(2) for 12 hours. The antioxidant Trolox was intravenously infused in a subset of ventilated animals. Compared with acutely anesthetized, nonventilated control animals, mechanical ventilation resulted in a significant reduction (-17%) in diaphragmatic maximal tetanic force. Importantly, Trolox completely attenuated this mechanical ventilation-induced diaphragmatic contractile deficit. Total diaphragmatic proteolysis was increased 105% in mechanical ventilation animals compared with controls. In contrast, diaphragmatic proteolysis did not differ between controls and mechanical ventilation-Trolox animals. Moreover, 20S proteasome activity in the diaphragm was elevated in the mechanical ventilation animals (+76%); Trolox treatment attenuated this mechanical ventilation-induced rise in protease activity. These results are consistent with the hypothesis that mechanical ventilation-induced oxidative stress is an important factor regulating mechanical ventilation-induced diaphragmatic proteolysis and contractile dysfunction. Our findings suggest that antioxidant therapy could be beneficial during prolonged mechanical ventilation.

Mechanical ventilation depresses protein synthesis in the rat diaphragm.

Prolonged mechanical ventilation results in diaphragmatic atrophy and contractile dysfunction in animals. We hypothesized that mechanical ventilation-induced diaphragmatic atrophy is associated with decreased synthesis of both mixed muscle protein and myosin heavy chain protein in the diaphragm. To test this postulate, adult rats were mechanically ventilated for 6, 12, or 18 hours and diaphragmatic protein synthesis was measured in vivo. Six hours of mechanical ventilation resulted in a 30% decrease (p < 0.05) in the rate of mixed muscle protein synthesis and a 65% decrease (p < 0.05) in the rate of myosin heavy chain protein synthesis; this depression in diaphragmatic protein synthesis persisted throughout 18 hours of mechanical ventilation. Real-time polymerase chain reaction analyses revealed that mechanical ventilation, in comparison with time-matched controls, did not alter diaphragmatic levels of Type I and IIx myosin heavy chain messenger ribonucleic acid levels in the diaphragm. These data support the hypothesis that mechanical ventilation results in a decrease in both mixed muscle protein and myosin heavy chain protein synthesis in the diaphragm. Further, the decline in myosin heavy chain protein synthesis does not appear to be associated with a decrease in myosin heavy chain messenger ribonucleic acid.

Cumulative effects of aging and mechanical ventilation on in vitro diaphragm function.

STUDY OBJECTIVE: Unloading the diaphragm, via mechanical ventilation (MV), results in significant diaphragmatic atrophy, contractile dysfunction, and oxidative stress in young adult animals. Since aging increases skeletal muscle susceptibility to atrophy and injury, we tested the hypothesis that MV-induced diaphragmatic contractile dysfunction would be exacerbated in aging rats. METHODS: Fisher 344/Brown Norway hybrid rats (4 months old [young] and 30 months old [old]) were assigned to either control or MV groups. MV rats were anesthetized, tracheostomized, and ventilated with 21% O(2) for 12 h. Arterial BP, pH, and blood gas homeostasis were maintained in the MV animals throughout the experimental period. Animals in the control group were acutely anesthetized, and the diaphragms were immediately removed. Muscle strips from the mid-costal diaphragm were removed from each experimental animal, and contractile properties were studied in vitro. RESULTS: Compared to young control animals, aging (old control animals) was associated with a 13% decrease in maximal isometric tension (24.5 N/cm(2) vs 21.3 N/cm(2)). Although, MV induced similar relative losses (24%) in diaphragmatic isometric tension in both young and old animals receiving MV, the combined effects of aging and MV resulted in a 34% decrement in diaphragmatic isometric tension compared to young control animals (24.5 N/cm(2) vs 16.1 N/cm(2)). CONCLUSIONS: These data do not support the hypothesis that aging exacerbates the relative MV-induced impairment in diaphragmatic isometric tension. Nonetheless, the additive effects of aging and MV have dramatic effects on diaphragmatic force reserve. This could exacerbate weaning difficulties in older individuals receiving MV.

Mechanical ventilation-induced oxidative stress in the diaphragm.

Prolonged mechanical ventilation (MV) results in oxidative damage in the diaphragm; however, it is unclear whether this MV-induced oxidative injury occurs rapidly or develops slowly over time. Furthermore, it is unknown whether both soluble (cytosolic) and insoluble (myofibrillar) proteins are equally susceptible to oxidation during MV. These experiments tested two hypotheses: 1). MV-induced oxidative injury in the diaphragm occurs within the first 6 h after the initiation of MV; and 2). MV is associated with oxidative modification of both soluble and insoluble proteins. Adult Sprague-Dawley rats were randomly divided into one of seven experimental groups: 1) control (n = 8); 2) 3-h MV (n = 8); 3). 6-h MV (n = 6); 4). 18-h MV (n = 8); 5). 3-h anesthesia-spontaneous breathing (n = 8); 6). 6-h anesthesia-spontaneous breathing (n = 6); and 7). 18-h anesthesia-spontaneous breathing (n = 8). Markers of oxidative injury in the diaphragm included the measurement of reactive (protein) carbonyl derivatives (RCD) and total lipid hydroperoxides. Three hours of MV did not result in oxidative injury in the diaphragm. In contrast, both 6 and 18 h of MV promoted oxidative injury in the diaphragm, as indicated by increases in both protein RCD and lipid hydroperoxides. Electrophoretic separation of soluble and insoluble proteins indicated that the MV-induced accumulation of RCD was limited to insoluble proteins with molecular masses of approximately 200, 120, 80, and 40 kDa. We conclude that MV results in a rapid onset of oxidative injury in the diaphragm and that insoluble proteins are primary targets of MV-induced protein oxidation.

Effects of norandrostenedione and norandrostenediol in resistance-trained men.

The purpose of this study was to determine the effects of norsteroid supplementation (224 mg of 19-nor-4-androstene-3,17-dione and 120 mg of 19-nor-4-androstene-3,17-diol, total daily dose = 344 mg) on body composition and strength in resistance-trained men. In a placebo-controlled, double-blind, randomized fashion, 10 subjects received the norsteroid (11 capsules containing a combination of both norsteroids) or a placebo for 8 wk (five subjects per group). Each subject participated in resistance training an average of 4 d/wk for the duration of the study. Body composition was determined via dual-energy x-ray absorptiometry. Strength was determined using a one-repetition maximum bench press and a one-repetition maximum biceps curl. With regard to all measures in both groups, there were no significant changes between before and after the study.Therefore, in this small sample of resistance-trained men, 344 mg/d of norsteroid supplementation had no effect on strength or body composition.

Mechanical ventilation results in progressive contractile dysfunction in the diaphragm.

These experiments tested the hypothesis that a relatively short duration of controlled mechanical ventilation (MV) will impair diaphragmatic maximal specific force generation (specific P(o)) and that this force deficit will be exacerbated with increased time on the ventilator. To test this postulate, adult Sprague-Dawley rats were randomly divided into one of six experimental groups: 1) control (n = 12); 2) 12 h of MV (n = 4); 3) 18 h of MV (n = 4); 4) 18 h of anesthesia and spontaneous breathing (n = 4); 5) 24 h of MV (n = 7); and 6) 24 h of anesthesia and spontaneous breathing (n = 4). MV animals were anesthetized, tracheostomized, and ventilated with room air. Animals in the control group were acutely anesthetized but were not exposed to MV. Animals in two spontaneous breathing groups were anesthetized and breathed spontaneously for either 18 or 24 h. No differences (P > 0.05) existed in diaphragmatic specific P(o) between control and the two spontaneous breathing groups. In contrast, compared with control, all durations of MV resulted in a reduction (P < 0.05) in diaphragmatic specific tension at stimulation frequencies ranging from 15 to 160 Hz. Furthermore, the MV-induced decrease in diaphragmatic specific P(o) was time dependent, with specific P(o) being approximately 18 and approximately 46% lower (P < 0.05) in animals mechanically ventilated for 12 and 24 h, respectively. These data support the hypothesis that relatively short-term MV impairs diaphragmatic contractile function and that the magnitude of MV-induced force deficit increases with time on the ventilator.