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.

Caspase-3 regulation of diaphragm myonuclear domain during mechanical ventilation-induced atrophy.

RATIONALE: Unloading the diaphragm via mechanical ventilation (MV) results in rapid diaphragmatic fiber atrophy. It is unknown whether the myonuclear domain (cytoplasmic myofiber volume/myonucleus) of diaphragm myofibers is altered during MV. OBJECTIVE: We tested the hypothesis that MV-induced diaphragmatic atrophy is associated with a loss of myonuclei via a caspase-3-mediated, apoptotic-like mechanism resulting in a constant myonuclear domain. METHODS: To test this postulate, Sprague-Dawley rats were randomly assigned to a control group or to experimental groups exposed to 6 or 12 h of MV with or without administration of a caspase-3 inhibitor. MEASUREMENTS AND MAIN RESULTS: After 12 h of MV, type I and type IIa diaphragm myofiber areas were decreased by 17 and 23%, respectively, and caspase-3 inhibition attenuated this decrease. Diaphragmatic myonuclear content decreased after 12 h of MV and resulted in the maintenance of a constant myonuclear domain in all fiber types. Both 6 and 12 h of MV resulted in caspase-3-dependent increases in apoptotic markers in the diaphragm (e.g., number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive nuclei and DNA fragmentation). Caspase-3-dependent increases in apoptotic markers occurred after 6 h of MV, before the onset of myofiber atrophy. CONCLUSIONS: Collectively, these data support the hypothesis that the myonuclear domain of diaphragm myofibers is maintained during prolonged MV and that caspase-3-mediated myonuclear apoptosis contributes to this process.

Moderate caloric restriction increases diaphragmatic antioxidant enzyme mRNA, but not when combined with lifelong exercise.

Diaphragmatic antioxidant enzymes are upregulated following acute and long-term treadmill exercise, but the effect of lifelong voluntary exercise (E) on diaphragmatic antioxidants is unknown. Therefore, 10-week old Fisher 344 rats were assigned to either: (a) sedentary ad libitum (AL) fed (24AL; n = 6); (b) E + 8% caloric restriction (24ECR; n = 9); or (c) sedentary + 8% caloric restriction (24CR; n = 9) groups. Diaphragms were harvested from animals at 24 months of age. Heme oxygenase-1 (HO-1) mRNA in addition to catalase (CAT), glutathione peroxidase (GPX), copper-zinc superoxide dismutase (Cu-ZnSOD) and manganese superoxide dismutase (MnSOD) mRNA and protein levels were measured. Reduced glutathione (GSH) and citrate synthase (CS) activity were measured to assess antioxidant status and oxidative capacity, respectively. The 24CR group demonstrated increased GPX, HO-1, MnSOD, and CAT mRNA compared to 24AL and 24ECR. Interestingly, the increased mRNA in 24CR animals did not result in elevated protein levels. No group differences in Cu-ZnSOD mRNA, CS activity, or GSH were observed, although GSH was 30% greater in 24CR animals (p = 0.085). In summary, although CR elevated the mRNA of key antioxidant enzymes in the diaphragm, lifelong CR alone or in combination with voluntary exercise did not alter diaphragm CS activity, antioxidant protein quantity, or GSH levels.

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.

Localization and early time course of TGF-beta 1 mRNA expression in dystrophic muscle.

Fibrosis is a common pathological feature observed in muscle from patients with Duchenne muscular dystrophy (DMD). In the dystrophic (mdx) mouse model of DMD, the diaphragm is more severely affected than other skeletal muscles. The level of transforming growth factor-beta1 (TGF-beta1), an inflammatory cytokine, is significantly elevated in mdx diaphragm. However, little is known about the onset of TGF-beta1 messenger ribonucleic acid (mRNA) expression, or which cells express the mRNA. In this study, we characterized the location and time course of expression of TGF-beta1 mRNA in diaphragm from mdx mice. TGF-beta1 mRNA was significantly elevated in mdx diaphragm at 6 and 9 but not 12 weeks of age, and these changes corresponded with changes in type I collagen mRNA and hydroxyproline concentration. Mononucleated cells localized to areas of fiber necrosis highly expressed the TGF-beta1 transcript in mdx diaphragm. Neutralization of TGF-beta1 by decorin administration resulted in a 40% reduction in the level of diaphragm muscle type I collagen mRNA. These findings support a role for TGF-beta1 during the early stages of fibrogenesis in dystrophic diaphragm muscle. Therapeutic interventions aimed at neutralizing this cytokine may be beneficial in slowing the development of fibrosis in DMD.