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Muscle Research and Respiratory System Unit, Respiratory Medicine, Surgery
Pathology Departments, IMIMeCHospital del Mar, Universidad Pompeu Fabra and Universidad Aute皀oma, Barcelona, Spain
Critical Care and Respiratory Divisions, Royal Victoria Hospital and Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada
ABSTRACT
Rationale: Oxidative stress is involved in the skeletal muscle dysfunction observed in patients with severe chronic obstructive pulmonary disease (COPD). We hypothesized that the diaphragms of such patients might generate greater levels of oxidants than those neutralized by antioxidants. Objectives: To assess the levels of both oxidative and nitrosative stress and different antioxidants in the diaphragms of those patients, and to analyze potential relationships with lung and respiratory muscle dysfunctions. Methods and Measurements: We conducted a case-control study in which reactive carbonyl groups, hydroxynonenal-protein adducts, antioxidant enzyme levels, nitric oxide synthases, and 3-nitrotyrosine formation were detected using immunoblotting and immunhistochemistry in diaphragm specimens (thoracotomy) obtained from six patients with severe COPD, six patients with moderate COPD, and seven control subjects. Main Results: Diaphragms of patients with severe COPD showed both higher protein carbonyl groups and hydroxynonenal-protein adducts than control subjects. When only considering patients with COPD, negative correlations were found between carbonyl groups and airway obstruction, and between hydroxynonenal-protein adducts and respiratory muscle strength. Although diaphragmatic neuronal nitric oxide synthase did not differ among the three groups and no inducible nitric oxide synthase was detected in any muscle, muscle endothelial nitric oxide synthase was lower in patients with severe COPD than in control subjects. Muscle nitrotyrosine levels were similar in both patients with severe COPD and control subjects. Conclusions: This study shows that oxidative stress rather than nitric oxide is likely to be involved in the respiratory muscle dysfunction in severe COPD.
Key Words: chronic obstructive pulmonary disease diaphragm strength oxidative stress
Muscle dysfunction in patients with severe chronic obstructive pulmonary disease (COPD) is characterized by reduced muscle strength and endurance, probably from the interaction of different systemic and local factors, and is also highly dependent on the specific function of the muscle. For instance, lower limb muscles, most likely because of disuse or deconditioning, have been shown to be more adversely affected (1) than inspiratory muscles. In fact, whether intrinsic ventilatory muscle dysfunction develops in patients with severe COPD remains debatable. For instance, Similowski and coworkers (2) showed that the diaphragm of patients with COPD produced even greater force than that of healthy subjects with comparable lung volumes. In keeping with this finding, several investigators have demonstrated that diaphragms from patients with severe COPD undergo adaptive modifications (3eC6). In contrast, Levine and colleagues (7) have recently demonstrated that diaphragm fibers from patients with severe COPD produce intrinsically less force than that generated by control muscles. So far, the mechanisms whereby the diaphragm of patients with severe COPD may be exposed to a remodeling process are still controversial and remain to be fully elucidated.
Although the etiology of skeletal muscle dysfunction in COPD is still under investigation, several factors, such as comorbid conditions, hypoxia, hypercapnia, nutritional status, medication, inflammation, and oxidative stress (for review, see Reference 8), have already been implicated. Furthermore, patients with COPD have shown higher levels of oxidative stress in their blood (9) and peripheral muscles (10eC15). We also demonstrated (16) that both oxidative and nitrosative stress develop in the quadriceps muscles of patients with COPD. However, there are no reports in the literature exploring whether oxidative stress contributes to the impairment of the intrinsic contractile properties of the diaphragm fibers in severe COPD. On the grounds that production of reactive oxygen species (ROS) and reactive nitrogen species within skeletal muscle fibers is regulated in part by strong muscle contractions, and that patients with severe COPD are chronically exposed to respiratory overloads, we hypothesized that their diaphragm fibers might generate greater levels of oxidants than those normally neutralized by intracellular antioxidant defenses, thus leading to the development of oxidative stress. Accordingly, our aims were to evaluate whether oxidative stress and nitric oxide (NO)eCmediated deleterious effects develop in the diaphragm muscle of patients with severe COPD, on the one hand, and whether these two phenomena are associated with the respiratory muscle dysfunction of such patients on the other. Some of the results of this study have been previously reported in the form of an abstract (17, 18).
METHODS
Subjects
Twelve male patients with stable COPD (six severe and six moderate; 68 years) and seven male control individuals (66 years) were included. COPD diagnosis was established on the basis of the Global Initiative for Chronic Obstructive Lung Disease guidelines (19). Exclusion criteria included the following: female sex, chronic respiratory failure, bronchial asthma, coronary disease, severe undernourishment (body mass index < 20 kg/m2), chronic metabolic diseases, suspected paraneoplastic or myopathic syndromes, and/or treatment with drugs known to alter muscle structure and/or function. This is a case-control study designed in accordance with the ethical standards on human experimentation in our institution and the World Medical Association guidelines for research on humans. The Ethics Committee on Human Investigation at Hospital del MareCIMIM approved all experiments. Informed, written consent was obtained from all individuals (see the online supplement for additional information).
Nutritional and Functional Assessment
Nutritional evaluation included body mass index and analytic parameters. Pulmonary and respiratory muscle functions and general exercise capacity were evaluated (see the online supplement for additional information).
Biopsies
During thoracotomy because of localized lung lesions, diaphragm biopsy specimens were obtained from the anterior costal diaphragm lateral to the insertion of the phrenic nerve (4). In all subjects, biopsies were obtained 10 to 14 days after the exercise tests (see the online supplement for additional information).
Biological Muscle Studies
Immunoblotting.
The levels of oxidative and nitrosative stress were assessed as described elsewhere (16). Selective antibodies were used to detect the following: carbonyl groups through derivatization (20) to 2,4-dinitrophenylhydrazone (DNP; antieCDNP moiety antibody; Oxyblot kit; Chemicon International, Inc., Temecula, CA); 4-hydroxy-2-nonenal (HNE-)eCprotein adducts (21) and catalase (anti-HNE and anticatalase antibodies; Calbiochem, San Diego, CA); Mn-superoxide dismutase (antieCMn-superoxide dismutase antibody; StressGen, Victoria, BC, Canada); neuronal, endothelial, and inducible NO synthases (nNOS, eNOS, and iNOS, respectively); heme oxygenase-1 (anti-nNOS, anti-eNOS, anti-iNOS, and antieCheme oxygenase-1 antibodies; Transduction Laboratories, Inc., Lexington, KY); and nitrotyrosine formation (antieC3-nitrotyrosine antibody; Cayman Chemical, Inc., Ann Arbor, MI). Corresponding positive controls were used in each case. Blots were scanned with an imaging densitometer, and optical densities of specific proteins were quantified with Diversity Database 2.1.1 (BioRad, Philadelphia, PA; see the online supplement for additional information).
Immunohistochemistry.
On 3-e muscle paraffin-embedded sections, myosin heavy-chain (MHC) I and II isoforms and capillary density were identified using antimyosin heavy-chain I (clone MHC; Biogenesis, Inc., Poole, UK), antimyosin heavy-chain II (clone MY-32; Sigma, St. Louis, MO), and anti-CD34 (Biomeda, Inc., Hayward, CA) primary antibodies, respectively, as well as markers of oxidative stress (16). Capillary density was quantified as the number of capillaries per muscle fiber.
Statistical Analysis
Data are presented as mean ± SD. One-way analysis of variance, Tukey-corrected for multiple comparisons, was used to compare data obtained within the three groups. Pearson's correlation coefficient was used to assess relationships among different variables within patients with COPD. A Bonferroni-type adjustment was performed to take into account the effect of having multiple comparisons and correlations. A p value of 0.05 or less was considered significant.
RESULTS
Characteristics of the Study Subjects
Table 1 indicates the main characteristics of the study subjects. No significant differences in age and nutritional status as assessed by body mass index and analytic parameters were observed between control subjects and patients with either severe or moderate COPD. However, FEV1, FVC, and the ratio of FEV1 to FVC were significantly lower, whereas residual volume (RV), and the ratio of RV to total lung capacity were significantly higher in both groups of patients with COPD. Both exercise capacity and global respiratory muscle strength were moderately reduced in patients with severe COPD.
Muscle Structure
Proportions of type I fibers were higher in the diaphragms of the patients with severe COPD compared with control subjects (Table 1). When considering all patients with COPD as a group, the proportion of type I fibers negatively correlated with FEV1 (r = eC0.752, p < 0.01) and positively correlated with total levels of carbonyl groups (r = 0.629, p < 0.05). Diaphragmatic capillary density did not differ between patients with COPD and control subjects, and did not show any relationship with either lung or respiratory muscle functions or with exercise tolerance.
Protein Carbonylation
Total carbonyl group formation.
As shown in Figure 1A, anti-DNP antibody detected different positive protein bands, with apparent masses ranging from 67 to 29 kD, in the muscles of both patients and control subjects. The diaphragms of patients with severe COPD showed higher levels of total carbonyl content than control muscles (Figure 1B). Within the patients with COPD, total muscle carbonyl formation negatively correlated with FEV1 (Figure 1B), and also with exercise tolerance, as measured by peak exercise oxygen uptake (r = eC0.685, p < 0.05). Immunostaining with anti-DNP antibody revealed the presence of carbonyl groups diffusely localized within the diaphragm muscle fibers in both patients with severe COPD and control individuals (Figure 1C).
HNE-protein adduct formation.
As illustrated in Figure 2A, anti-HNE antibody detected several protein bands, with apparent masses ranging from 92 to 34 kD, in the muscles of both patients and control subjects. The diaphragms of patients with severe COPD showed higher levels of HNE-protein adducts than control muscles (Figure 2B). Among all patients with COPD, the intensity of total diaphragm HNE-protein adducts negatively correlated with respiratory muscle strength as assessed by either maximal inspiratory or maximal esophageal pressures, and also showed a strong tendency to correlate with maximal transdiaphragmatic pressure (Figure 2B). No other relationships were found between HNE-protein adducts and either lung or respiratory muscle functions, or exercise tolerance. Immunohistochemical analysis revealed positive staining of HNE-protein adducts diffusely localized within the muscle fibers in both patients with severe COPD and control subjects (Figure 2C).
Antioxidant Enzymes
As shown in Figure 3A, Mn-superoxide dismutase, catalase, and heme oxygenase-1 were detected in the diaphragms of patients with COPD and control subjects. No significant differences in the intensity of those enzymes were observed among the three groups (Figure 3B). When considering the overall group of patients with COPD, several relationships were found between muscle Mn-superoxide dismutase optical densities and static lung volumes represented by functional residual capacity (r = 0.635, p < 0.05), between catalase optical densities and both FEV1 (r = 0.562, p = 0.09) and peak exercise oxygen uptake (r = 0.777, p = 0.01), and between muscle heme oxygenase-1 content and FEV1 (r = eC0.613, p < 0.05).
NOS Isoform Expression and Protein Tyrosine Nitration
As shown in Figure 4A, the anti-nNOS and anti-eNOS antibodies detected weak expression of 164 and 140 kD proteins, respectively, in the diaphragms of both patients with COPD and control subjects. Although muscle nNOS did not differ among the three groups, eNOS protein levels were significantly lower in the diaphragms of the patients with severe COPD compared with control subjects (Figure 4B). In addition, among all patients with COPD, eNOS protein levels directly and significantly correlated with maximal inspiratory pressure (r = 0.607, p < 0.05, respectively). No other relationships were found between the content of any of those proteins and lung or respiratory muscle functions, or exercise tolerance. No detectable iNOS was found in the diaphragms of the patients with COPD or control subjects (Figure 4A).
Several tyrosine-nitrated protein bands were detected in the diaphragms of both patients with COPD and control subjects, with apparent masses ranging from 63 to 30 kD (Figure 5A). Total muscle 3-nitrotyrosine optical densities did not differ among the three groups (Figure 5B). No significant correlations were found between muscle protein nitration and functional, exercise capacity, or muscle structure variables when considering all the patients with COPD. Immunohistochemical analysis revealed clear, positive 3-nitrotyrosine staining diffusely localized within the diaphragm fibers in both patients with severe COPD and control subjects (Figure 5C).
DISCUSSION
The major findings of this study are that in the diaphragms of patients with severe COPD as compared with those from control subjects (1) reactive carbonyl group levels were increased and correlated with the severity of their disease, (2) HNE-protein adduct formation was also elevated and correlated with their respiratory muscle function, (3) constitutive eNOS protein levels were reduced, and (4) protein tyrosine nitration remained unchanged.
Muscle Structure and Function
The human diaphragm can develop fatigue (22), and in severe COPD several underlying molecular and structural adaptive mechanisms (3eC6) have been suggested to produce a more fatigue-resistant diaphragm phenotype. In this regard, as was previously shown by Levine and coworkers (3), we also report herein increased proportion of type I fibers in the diaphragms of our patients with severe COPD, which was associated with both the severity of their disease and muscle protein oxidation levels. One could argue that those diaphragms with higher content of oxidative fibers (type I) are those likely to suffer most from protein oxidation. However, it remains debatable whether this and other adaptive mechanisms (3eC6) are sufficient to make the diaphragms of patients with severe COPD more efficient. For instance, and in line with other investigators (2, 23, 24), we have found that the respiratory muscle function was impaired in our patients with severe COPD. What remains a matter of speculation is whether this respiratory muscle dysfunction is solely from abnormalities in the geometric configuration of the thoracic cage in severe COPD, or whether it also includes impaired intrinsic contractile properties of the diaphragm of these patients (7). The causes of this intrinsic impairment, however, are still unknown.
Oxidative Stress and Its Relationships with Function
The present study is the first to provide evidence of the effects of ROS on muscle proteins and lipids of the human diaphragm both in patients with COPD and subjects with normal lung function. Oxidative stress has been implicated in the pathogenesis of a wide range of conditions and in chronic degenerative diseases (25eC28). Furthermore, over the last decade, a growing body of evidence has shown that oxidative stress is one of the mechanisms clearly involved in the skeletal muscle dysfunction of patients with COPD (8eC16). Carbonyl formation (ketones and aldehydes) is an important detectable marker of protein oxidation. Carbonyl groups can be formed by direct reaction of proteins with ROS, leading to the formation of protein derivatives containing highly reactive carbonyl groups (29eC34), on the one hand. On the other, carbonyl groups may also be introduced into proteins by Michael-addition reactions of lysine, cysteine, or histidine residues with unsaturated aldehydes (hydroxynonenal and malondialdehyde) formed during the peroxidation of polyunsaturated fatty acids (29eC34). Our study is the first to demonstrate that the diaphragms of patients with severe COPD have greater levels of oxidative stress than those detected in control muscles. Furthermore, this finding might partly account for the etiology of the impaired intrinsic contractile properties of those muscle fibers, recently shown by Levine and colleagues (7). The association found between the diaphragmatic levels of protein oxidation and respiratory muscle function strongly supports this conclusion. Finally, diaphragm proteins targeted by ROS led to increased levels of both total carbonyls and HNE-protein adducts, whereas in the quadriceps of a more heterogeneous population of patients with COPD, HNE-protein adducts were the sole index of protein oxidation having increased. It could be argued that increased workloads imposed on the diaphragm in severe COPD might account for those differences by enhancing oxidant production, and might also explain the different protein patterns targeted by oxidants in each muscle.
The content of the mitochondrial enzyme Mn-superoxide dismutase did not differ between muscles of patients with severe COPD and control subjects. However, it showed a significant relationship with air trapping. On the basis of this finding, we could hypothesize that Mn-superoxide dismutase might act as a cellular defense mechanism against ROS-mediated deleterious effects on those muscles only in the patients with more severe COPD. Furthermore, we already showed (16) that the content of Mn-superoxide dismutase was increased in the quadriceps muscles of patients with COPD. It is very likely that, in COPD, local mechanisms developing in each muscle might account for the intermuscle differences.
The diaphragmatic content of catalase was associated with exercise capacity as measured by peak exercise oxygen uptake, suggesting that exercise tolerance, which was also reduced in severe COPD, might well be related to catalase content, at least in the diaphragm. As has also been formerly reported in the quadriceps of patients with COPD (16), an almost significant correlation was found between catalase and the degree of the airway obstruction. Systemic effects of COPD on different skeletal muscles might account for these findings.
The present study provides first evidence of the presence of heme oxygenase-1 in the diaphragms of both patients with severe COPD and control subjects. So far, there is little documentary evidence regarding the involvement of heme oxygenase-1 in COPD, and all existing reports have mainly focused on the study of the role of this enzyme as an antioxidant in their lungs (35). Whether this enzyme exerts antioxidant effects on the ventilatory muscles in severe COPD, as shown in other conditions (27), remains an open question. However, our finding of a negative relationship between diaphragmatic heme oxygenase-1 protein content and pulmonary function as measured by FEV1 might support this hypothesis.
The Role of NO
No iNOS protein content could be detected in the diaphragms of our individuals. Accordingly, it is possible that inflammatory events occurring in the diaphragm of patients with COPD are not sufficient to induce iNOS expression as occurs in other conditions (36, 37). Furthermore, we found that, in the diaphragms of the patients with severe COPD, eNOS protein was reduced, and when considering all patients with COPD, those levels were related to their respiratory muscle dysfunction. One possible mechanism responsible for decreased muscle eNOS expression in the patients with severe COPD might be the existence of a functional defect in the capillary permeability of their diaphragms, as occurs in other conditions (38), because a proportion of muscle eNOS protein is likely to be derived from endothelial cells. So far, little is known about transcriptional regulation of muscle eNOS expression. There is also documentary evidence that NO derived from mitochondrial eNOS exerts tonic inhibitory effects on mitochondrial respiration, possibly through reversible inhibition of cytochrome oxidase (39), known to be elevated in COPD (40).
In view of the absence of increased levels of 3-nitrotyrosine formation in the diaphragms of our patients with severe COPD, we can speculate that ventilatory muscle dysfunction in severe COPD is not associated with nitrosative stress, a finding that contrasts with our recent observation (16). We concluded in that study that tyrosine nitration in limb muscles of patients with COPD may be derived from the nNOS protein rather than the iNOS isoform. We should emphasize that such a conclusion also holds in this study because the absence of any rise in diaphragm protein tyrosine nitration was associated with no significant elevation in muscle nNOS protein in patients with severe COPD. In the current study, the different proteins that were tyrosine-nitrated in diaphragms from both control subjects and patients with COPD differ from those detected in the quadriceps. In COPD, the preference of reactive nitrogen species for targeting limb muscle proteins rather than those of the diaphragm might be related to the specific function of each muscle.
Limitations of the Study
Diaphragm muscle biopsies from our study subjects were obtained during thoracotomy because of localized lung lesions, the gold-standard technique to obtain those tissues from different populations. Although lung volume reduction surgery also allows the obtaining of diaphragm specimens, only patients with very severe COPD undergo that type of surgery. Therefore, diagnostic/therapeutic thoracotomy is the only approach available for studying subjects with moderate and mild COPD and subjects with normal lung function. Accordingly, the population of our study shares a common morbidity: the presence of a localized lung neoplasm. Nevertheless, we do not believe that this condition has made any significant contribution to the development of oxidative stress in our diaphragms, because extremely restrictive criteria were used to select our population. Another limitation encountered in our study is directly related to methodologies. In our case, the use of indirect indices of oxidative stress was chosen because direct confirmation of ROS presence is either technically rather complex or unavailable (see the online supplement for additional information).
Conclusions
Our results provide first evidence that, in severe COPD, the diaphragm muscle exhibits increased levels of oxidative stress, and such levels are associated with the degree of impairment of both pulmonary and respiratory muscle functions. These data suggest that oxidative stress might be involved in the intrinsic ventilatory muscle dysfunction recently reported in severe COPD.
Acknowledgments
The authors thank Mr. Daniel Sechez and Mrs. Anna Llorens for their technical assistance in the laboratory, Mr. Pablo Peretti for his technical support in the preparation of the figures, and Mr. Roger Marshall for his editing aid.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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