American Journal of Respiratory Cell and Molecular Biology

Skeletal muscle dysfunction contributes to poor exercise performance in patients with chronic obstructive pulmonary disease (COPD). Increased oxygen radicals and nitric oxide (NO) have been proposed as mechanisms. In this study, we assessed the levels of protein oxidation (carbonyl formation), lipid peroxidation (4-hydroxy-2-nonenal formation), catalase and Mn-superoxide dismutase (Mn-SOD) expressions, nitric oxide synthases (NOSs), and protein tyrosine nitration in quadriceps muscles of 12 patients with patients with COPD and 6 control subjects. Lipid peroxidation was elevated in muscles of patients with patients with COPD as compared with control subjects, but protein oxidation was not. Muscle Mn-SOD but not catalase protein expression was significantly higher (200%) in patients with patients with COPDas compared with control subjects. Expression of neuronal NOS and endothelial NOS isoforms did not differ between control subjects and patients with COPD, whereas no inducible NOS protein expression was detected in limb muscles of the two groups of subjects. In patients with COPD, neuronal NOS expression correlated negatively with the degree of the airway obstruction (%FEV1 predicted). 3-Nitrotyrosine levels were significantly elevated in muscles of patients with COPDas compared with control subjects, and correlated positively with nNOS protein levels. These results indicate the development of both oxidative and nitrosative stresses in the quadriceps of patients with COPD, suggesting their involvement in muscle dysfunction.

Limb muscle dysfunction has been recognized in the last decade as an important factor in reduced exercise capacity in patients with chronic obstructive pulmonary disease (COPD). Muscle dysfunction is frequently associated with weight loss, which, in turn, contributes to poor quality of life as well as reduced survival (1). Muscle dysfunction is characterized by reduced muscle strength and endurance in addition to muscle wasting. Moreover, limb muscles of patients with COPD develop distinct structural alterations including attenuation of the proportion of type I fibers, reduction in myoglobin levels, and lower number of capillaries per unit surface area compared with control subjects (for review see Ref. 1). These structural changes lead to reduced oxygen delivery within skeletal muscles. In addition, there is evidence that metabolic alterations also occur in limb muscles of patients with COPD and that these alterations are manifested as reduction in the activity of oxidative enzymes, with no significant changes in glycolytic enzymes (2). The etiology of limb muscle dysfunction in patients with COPD is still under investigation; however, many factors have been implicated, including factors related to comorbid conditions such as deconditioning, electrolyte imbalance (alterations in phosphate, calcium, manganese, and chloride metabolism), heart failure, and detraining (1). Furthermore, hypoxia, hypercapnia, poor nutritional status, and a possible increase in inflammatory cytokine levels have also been proposed to contribute to limb muscle dysfunction (3). Finally, oxidative stress has also been suggested to play a role in limb muscle dysfunction in patients with COPD and to contribute to metabolic and contractile dysfunctions of these muscles (4). This proposal is based on the findings that higher levels of lipid peroxidation and oxidized glutathione are present following exercise in arterial blood of patients with COPD as compared with control subjects (5), and that total glutathione is reduced in leg muscles of patients with COPD as compared with control subjects (6). However, no measurements of the effects of reactive oxygen species (ROS) on muscle proteins or lipids have been reported thus far. Therefore, the first objective of this study was to test our hypothesis that oxidative stress develops within peripheral muscles of patients with COPD with normal nutritional status, and that this phenomenon is attributed in part to downregulation of the expression of important antioxidant enzymes such as catalase (responsible for removal of hydrogen peroxide) and Mn-superoxide disumutase (Mn-SOD)(involved in dismutation of superoxide anions). To achieve this objective, we measured protein oxidation (carbonyl formation), lipid peroxidation (4-hyrdroxynonenal protein adduct formation), and protein expression of catalase and Mn-SOD in vastus lateralis muscles of patients with COPD.

Over the past several years, there has been increasing evidence that in addition to ROS, muscle redox status is strongly influenced by nitric oxide (NO). NO is produced inside skeletal muscle fibers by nitric oxide synthases (NOSs). The main source of NO synthesis in normal muscle fibers is the neuronal (nNOS) isoform, which is localized in close proximity to the sarcolemma (7). NO is also produced by the endothelial (eNOS) isoform, which is localized inside skeletal muscle mitochondria and in endothelial cells of blood vessels (8). Although the inducible (iNOS) isoform is not usually present in normal skeletal muscle fibers, this enzyme is induced in severe sepsis and endotoxin shock. There is increasing evidence that NO plays a major role in regulating muscle glucose metabolism, Ca++ release from the sarcoplasmic reticulum, blood flow, and the defense against oxidative stress (for review, see Ref. 9). Excessive NO production inside skeletal muscle fibers, however, exerts deleterious effects on contractile function and sarcolemmal integrity (9). The iNOS isoform has also been shown to be involved in the formation of peroxynitrite, which in turn targets proteins and lipids and leads to inactivation of various enzymes, including those involved in the defense against oxidative stress (10). Despite the importance of NO in the regulation of muscle function, no information is yet available regarding changes in the expression of constitutive NOS isoforms (nNOS and eNOS) in the limb muscles of patients with COPD, nor is there any data regarding the presence of the iNOS isoform and peroxynitrite inside these muscles. The second objective of this study was, therefore, to test our hypothesis that NO production is enhanced and leads to peroxynitrite formation in leg muscles of patients with COPD, and that constitutive and inducible NOS isoforms are responsible for this elevation in muscle NO production.

Patient Characteristics

Twelve patients with COPD and six control individuals with normal pulmonary functions were selected. The COPD diagnosis was established based on a clinical history compatible with chronic bronchitis and/or emphysema, a long history of cigarette smoking, and pulmonary function testing revealing fixed airflow obstruction (FEV1/FVC ratio < 70% and FEV1 < 75% predicted). All subjects underwent thoracotomy for a localized lung neoplasm. Exclusion criteria included chronic respiratory failure, positive bronchodilator test, bronchial asthma, coronary disease, undernourishment (body mass index [BMI] < 20 kg/m2), chronic metabolic diseases, orthopedic diseases, suspected paraneoplastic or myopathic syndromes, previous abdominal or thoracic surgery, and/or treatment with drugs known to alter muscle structure and/ or function.

Study Design

World Medical Association guidelines for research in humans were followed in this study. The Ethics Committee on Human Investigation at Hospital del Mar-IMIM approved all experiments. Informed written consent was obtained from all individuals after full explanation of the purposes and characteristics of the study. Pulmonary and muscle functions, exercise capacity, and nutritional status were assessed at study entry. Two to three days later, samples from the quadriceps muscle were obtained before thoracotomy.

Nutritional Assessment

BMI was calculated in all subjects as the ratio of weight per height2. Blood analysis of serum cholesterol, triglycerides, total protein, albumin, globulins, albumin/globulins index, and prothrombin consumption time were also determined in all individuals.

Physiologic Studies
Pulmonary function tests.

Forced spirometry was performed using a pneumotachograph (Datospir 92; Sibel, Barcelona, Spain) and standard procedures. Static lung volumes and airway resistance were determined using standard body plethysmography (Masterlab; Jaeger, Würzburg, Germany). Carbon monoxide transfer factor (DlCO) was used to assess the diffusion capacity (Masterlab). Data by Roca and coworkers (11, 12) were used as predicted values. Arterial blood gases were determined using a standard gas analyser (ABL 330; Radiometer, Copenhagen, Denmark).

Peripheral muscle function

This was assessed through the classical handgrip (HG) maneuver, performed by the nondominant hand, using a Collins dynamometer (Herrera, Barcelona, Spain). At least three maneuvers were performed in each case, and the highest value was chosen. Predicted values of Mathiowetz and colleagues (13) were used.

General exercise capacity.

This was assessed using a standardized incremental exercise test performed on a cycloergometer (Monark-Crescent 864; Varberg, Sweden). The test consisted of a 3-min warm-up period at 25 watts, followed by 25-watt increments at 2-min intervals while the subject was pedaling at a constant frequency of 60 rpm. Breathing pattern, electrocardiogram, and transcutaneous oxygen saturation were continuously recorded. The exercise was stopped when at least three of the following criteria were reached: (i) a plateau of oxygen uptake (V̇o2 max) in spite of increasing workload; (ii) a heart rate within ±5% of maximal predicted; (iii) a gas exchange respiratory ratio > 1.1; (iv) when the patients were unable to maintain pedaling frequency at 60 rpm. Maximal mechanical power output (Wmax) and V̇o2 max were determined, and data of Jones and associates (14) were used as reference values.

Biological Muscle Studies

Muscle samples were obtained from the quadriceps (vastus lateralis) by open muscle biopsy, and were either immediately frozen in liquid nitrogen and stored at –80°C for further analysis or embedded in paraffin and used for immunohistochemistry. In all subjects, biopsies were obtained 10–14 d after the maximal exercise test.

Immunoblotting.

Frozen muscle samples were homogenized in a buffer containing tris-maleate 10 mM, EGTA 3 mM, sucrose 275 mM, DTT 0.1 mM, leupeptin 2 μg/ml, PMSF 100 μg/ml, aprotinin 2 μg/ml, and pepstatin A 1 mg/100 ml (pH 7.2). Samples were then centrifuged at 1,000 × g for 10 min. The pellet was discarded and the supernatant was designated as a crude homogenate. Total muscle protein level in each sample was determined with the Bradford technique (BioRad Inc., Hercules, CA) Crude muscle homogenates (20 μg per sample) were separated by electrophoresis, transferred to polyvinylidene difluoride membranes, blocked with nonfat milk, and incubated overnight with selective monoclonal antibodies to NOS isoforms (Transduction Laboratories Inc., Lexington, KY), monoclonal anti–3-nitrotyrosine antibody (Cayman Chemical Inc., Ann Arbor, MI), polyclonal anti-Mn SOD (StressGen, Victoria, BC, Canada) and polyclonal anti-catalase (Calbiochem Corp., San Diego, CA) antibodies. Lysates obtained from rat cerebellum, endothelial cells, and cytokine-activated macrophages were used as positive controls for nNOS, eNOS, and iNOS protein expression, respectively. Specificity of anti–3-nitrotyrosine antibody was evaluated by preincubation of this antibody with either 10 mM of nitrotyrosine or 10-fold excess of peroxynitrite-tyrosine nitrated bovine serum albumin (generously provided by Dr. Ischiropoulos, University of Pennsylvania). 4-Hydroxy-2-nonenal (HNE) is an α,β-unsaturated aldehyde and is the most cytotoxic product of lipid peroxidation. HNE levels were evaluated by probing the membranes with polyclonal anti-HNE antibody (Calbiochem, San Diego, CA) (15). Specific proteins were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies and a chemiluminescence kit. Negative control experiments were also performed in which primary antibodies were omitted and membranes were probed with secondary antibodies only. Blots were scanned with an imaging densitometer and optical densities (OD) of specific proteins were quantified with ImagePro Plus (Media Cybernetics, Silver Spring, MD). Total 3-nitrotyrosine and total HNE OD values were calculated for each sample by adding OD of individual positive protein bands. To validate equal protein loading among various lanes, polyvinylidene difluoride membranes were stripped and reprobed with a monoclonal anti-sarcomeric α-actinin antibody (Sigma Inc., St. Louis, MO).

Protein oxidation.

We assessed protein oxidation by measuring protein carbonyls (Oxyblot kit; Intergen Inc., Purchase, NY). Protein carbonyls are sensitive indices of oxidative injury (16). Carbonyls were detected in muscle protein samples (15 μg per sample) using the immunoblotting procedure described by Taillé and coworkers (17). To evaluate the selectivity of carbonyl measurements, muscle protein samples also underwent protein carbonyl detection procedure without the derivatization step (negative controls). Total carbonyls in each muscle sample were calculated by adding ODs of individual positive protein bands.

Immunohistochemistry.

Muscle samples were immersed in subsequent baths of different degrees of alcohol, formol, and xylol, to be finally embedded in paraffin. Slides were then fixed in amino propyl-triethoxilane and acetone, and dried by heat (60°C). Three-micrometer muscle paraffin-embedded sections were obtained using a microtome. All sections were deparaffinazed, and incubated with citric acid solution in a pressure cooker (antigen retrieval protocol). Slides were then blocked in 6% H2O2, incubated for 1 h at 37°C in a humid chamber with monoclonal anti-3-nitrotyrosine (1/10 dilution), monoclonal anti-MyHC-I (clone MHC, Biogenesis Inc., England, 1/25 dilution), monoclonal anti–MyHC-II (clone MY-32, 1/150 dilution; Sigma) or polyclonal anti-HNE (1/100 dilution) antibodies. After several washes in phosphate-buffered saline, slides were incubated for 1 h with biotinoylated secondary antibodies followed by HRP-conjugated streptavidin and diaminobenzidine (Dako Corporation, Carpinteria, CA) as a substrate. Negative control slides were exposed only to secondary antibodies. Slides were counterstained with hematoxylin, dehydrated, and mounted for conventional microscopy.

Capillary density measurement.

Paraffin-embedded sections were deparaffinized, rehydrated, washed, and incubated with a monoclonal anti-CD34 antibody (Biomeda Inc., Hayward, CA). Slides were then blocked with H2O2, probed with HRP-conjugated anti-mouse antibody, and incubated with a mixture of diaminobenzidine and chromogen solution (Dako). Capillary density was quantified as the number of capillaries per muscle fiber.

Statistical Analysis

Data is presented as mean ± SD. ANOVA was used to compare different groups. Relationships between various parameters were studied by calculating the Spearman's correlation coefficient. Bonferroni adjustment was performed by taking into account the effect of multiple comparisons. A P value ⩽ 0.05 was considered significant.

Table 1

TABLE 1 Patient characteristics and functional variables




Control Subjects
 (n = 6)

Patients with COPD
 (n = 12)
Age, yr66 ± 1067 ± 8
Weight, Kg72 ± 1271 ± 12
BMI, Kg / m226 ± 325 ± 3
FEV1, % pred. *86 ± 954 ± 14
FVC, % pred. *85 ± 362 ± 12
FEV1/FVC, % *74 ± 361 ± 7
TLC, % pred.90 ± 597 ± 21
FRC, % pred.99 ± 17110 ± 42
RV, % pred. *94 ± 18141 ± 60
DlCO, % pred81 ± 1179 ± 12
PaO2, mm Hg85 ± 1279 ± 13
PaCO2, mm Hg36 ± 140 ± 3
o2max, % pred.*87 ± 1173 ± 11
Wmax, % pred.*88 ± 1171 ± 14
Handgrip, % pred.**92 ± 1574 ± 16
Type I fibers, %49 ± 940 ± 14
Type I fiber area, μm22208 ± 9122058 ± 482
Type II fiber area, μm22211 ± 8132608 ± 1412
Number of Capillaries/ fiber2.18 ± 0.822.46 ± 1.1
Total serum proteins, g/dl6.70 ± 0.846.50 ± 0.76
Serum albumin, g/dl4.22 ± 0.744.10 ± 0.53
Serum cholesterol, mg/dl208.6 ± 58.8206.4 ± 33.9
Serum triglycerids, mg/dl
129 ± 17.6
118 ± 59.5

*P ⩽ 0.05.

**P ⩽ 0.01.

Definition of abbreviations: BMI, body mass index; pred, predicted; TLC, total lung capacity; FRC, functional residual capacity; RV, residual volume; DlCO, carbon monoxide transfer; PaO2, arterial oxygen pressure; PaCO2, arterial carbon dioxide pressure; V̇o2max, peak exercise oxygen uptake; Wmax, maximal mechanical power output.

indicates characteristics of control subjects and patients with COPD. No differences in age, weight, BMI, nutritional status, and arterial blood gases were observed between control subjects and patients with COPD. FEV1, FVC, and FEV1/FVC were significantly lower in patients with COPD as compared with control subjects. In addition, both peripheral muscle strength and exercise capacity were mildly reduced in patients with COPD. No nutritional abnormalities were detected in patients with COPD or control subjects. No differences were observed in patients with COPD and control subjects with respect to quadriceps fiber type distributions, fiber sizes, or capillary densities.

In control subjects, protein oxidation was limited to two main 40- and 30-kD protein bands (large arrows in Figure 1)

. Weaker protein bands were also detected at 38 and 58 kD. The intensity of these oxidized protein bands varied considerably in muscle samples of patients with COPD, with the highest intensities detected in two patients with COPD (FEV1 of 49% and 43% predicted). Furthermore, an additional oxidized protein band with apparent mass of 9 kD was detectable in muscle samples of several patients with COPD (small arrow in Figure 1). Mean values of total carbonyl contents in patients with COPD were not significantly different from those detected in the muscles of control subjects.

In addition to protein oxidation, we evaluated whether lipid peroxidation is elevated in limb muscles of patients with COPD by measuring the level of HNE protein adducts (Figure 2A)

. The anti-HNE antibody detected several positive protein bands with apparent mass of 55, 52, 48, 44, 40, 35, 32, and 30 kD (Figure 2A). In most samples, the 30-kD protein band is the most intense among all protein bands. The intensities of the majority of HNE-positive bands and total HNE OD were significantly greater in muscles of patients with COPD compared with control subjects (Figure 2C, P < 0.05). Positive HNE staining was localized in close proximity to the sarcolemma in muscle fibers of patients with COPD and control subjects (Figure 2D). Few muscle fibers also showed positive cytosolic HNE staining (middle panel of Figure 2D). Removal of the primary anti-HNE antibody completely eliminated positive HNE staining (right panel of Figure 2D).

Figure 3

illustrates the changes in catalase and Mn-SOD protein expressions in vastus lateralis muscles of control subjects and patients with COPD. No differences could be detected in the expression of catalase between control subjects and patients with COPD; however, catalase ODs showed a strong tendency for positive correlation with FEV1 in patients with COPD (Figure 3C). Mn-SOD expression in patients with COPD averaged ∼ 200% of that detected in control subjects (P < 0.05).

NOS protein expressions in vastus lateralis muscle samples are shown in Figure 4

. The anti-nNOS antibody detected a prominent band at 165 kD in muscles of control subjects. Likewise, the same band was also detected in the muscles of patients with COPD. Although no differences in muscle nNOS expression were detected between control subjects and COPD as a group (Figure 4B), patients with COPD with FEV1 < 50% predicted showed a strong tendency to have higher expression of nNOS compared with control subjects. When only patients with COPD were considered, a significant negative correlation between nNOS intensity and relative FEV1 values (%predicted) was detected (Figure 4C). The anti-eNOS antibody detected a positive protein band of 130 kD in vastus lateralis of control subjects (Figure 4A). Mean eNOS OD in muscles of patients with COPD was not different from that of control subjects (Figure 4B). Neither nNOS nor eNOS abundance correlated with muscle capillary density. We were unable to detect iNOS protein expression in vastus lateralis muscles of normal subjects and patients with COPD upon probing muscle lysates with a selective anti-iNOS antibody (Figure 4A).

Figure 5

illustrates representative examples of protein tyrosine nitration in crude muscle homogenates of control subjects and patients with COPD. A selective 3-nitrotyrosine antibody detected five tyrosine-nitrated protein bands with apparent molecular masses of 216, 46, 42, 36, and 30 kD. The intensities of these protein bands, particularly those of 216 and 36 kD, were stronger in the muscles of patients with severe COPD as compared with control subjects. Mean values of total muscle 3-nitrotyrosine OD for all patients with COPD rose to ∼ 200% of those of control subjects (Figure 5B). This increase was particularly evident in muscle samples obtained from patients with severe COPD. Total muscle 3-nitrotyrosine OD in all muscle samples correlated positively and significantly with nNOS OD (r = 0.60, P < 0.05) and negatively with FEV1 (% predicted, r = −0.652, P < 0.05). No such correlation was detected between the muscle tyrosine nitration and eNOS OD. Tyrosine nitration did not correlate with muscle capillary density. Positive tyrosine-nitrated protein bands were undetected when anti–3-nitrotyrosine antibody was preincubated with either pure nitrotyrosine or tyrosine-nitrated bovine serum albumin, confirming the specificity of this antibody. Clear cyotsolic and membrane-associated positive 3-nitrotyrosine staining were evident inside skeletal muscle sections of patients with COPD as well as control subjects (Figure 5C). No clear staining was detectable in negative control sections (Figure 5C).

The main findings of this study are that in the vastus lateralis muscle of patients with COPD as compared with subjects with normal lung function: (i) lipid peroxidation but not protein oxidation was significantly elevated; (ii) the expression of Mn-SOD protein was significantly higher; (iii) the nNOS isoform expression showed a strong tendency to increase in patients with severe COPD; (iv) protein tyrosine nitration averaged ∼ 200% of that of control subjects, and was detectable despite the absence of abundant iNOS protein expression.

Oxidative Stress

The development of oxidative stress in skeletal muscles of patients with COPD is still under investigation. A significant rise in blood malondialdehyde (index of lipid peroxidation) and elevated oxidized glutathione/reduced glutathione ratios have been observed in patients with COPD in response to exhaustive exercise (5). Couillard and coworkers have reported an increase in muscle oxidation as measured by lipid peroxidation and protein oxidation following local exercise (18). In line with this, Allaire and colleagues (19) reported that lipofuscin inclusion (as a marker of oxidative damage) was increased in vastus lateralis muscles of severe patients with COPD. However, Rabinovich and associates (20) did not confirm the presence of oxidative stress in the peripheral muscles of severe patients with COPD. Our study strongly suggests the presence of ROS-mediated cellular damage and enhanced lipid peroxidation in leg muscles of patients with COPD with a wide range of disease severity. We also report that the levels of muscle carbonyls were not different between muscles of control subjects and patients with COPD, suggesting that ROS in muscles of patients with COPD are preferentially targeting membrane lipids rather than proteins. This selective targeting is likely to be influenced by the molecular sources and sites of ROS production inside muscle fibers. ROS formation inside skeletal muscle fibers is traditionally attributed to electron carriers on the inner mitochondrial membrane, membrane-bound oxidoreductases, the cyclooxygenase pathways, and xanthine oxidase. More recently, a membrane-bound nonphagocytic NADPH oxidase complex has also been identified inside skeletal muscle fibers (21). Although the exact contribution of each of these pathways to lipid peroxidation in skeletal muscles of patients with COPD remains unknown, we speculate, on the basis of localization of NADPH oxidase in close proximity to the sarcolemma, that this enzyme complex, rather than xanthine oxidase and mitochondrial oxidoreductases, is responsible for increased lipid peroxidation in leg muscles of patients with COPD. However, this speculation, along with possible mechanisms responsible for activation of the NAPDH oxidase enzyme complex inside muscle fibers of patients with COPD, needs to be confirmed.

Little is known about changes in antioxidant defenses inside skeletal muscles of patients with COPD. Only recently has it been shown that local glutathione peroxidase activity failed to increase in patients with COPD following exercise (18). Our study provides first evidence that the expression of an important mitochondrial enzyme responsible for the dismutation of superoxide anions, Mn-SOD, is significantly elevated in leg muscles of patients with COPD. This enhanced expression may represent a response to increased ROS production inside the mitochondria of these muscles. Although increased mitochondrial density can also lead to increased muscle Mn-SOD protein level in total muscle lysates, this is not a likely explanation for our finding because limb muscles of patients with COPD have been reported to have lower percentage of mitochondrial rich (type I) fibers compared with control subjects (1).

The NO Pathway

One of the major findings of our study is that leg muscles of patients with COPD did not express significant iNOS protein, suggesting that the level of inflammatory processes and/or the numbers of inflammatory cells inside these muscles are not sufficient to evoke iNOS expression. We did not observe significant differences between control subjects and patients with COPD with respect to constitutive NOS enzyme expression in their quadriceps muscles. However, we found that the severity of the airway obstruction correlates significantly and positively with vastus lateralis nNOS protein levels. Although it is difficult to directly assess the functional relevance of this correlation, we propose two hypotheses. First, an increase in constitutive NOS expression, such as nNOS in the quadriceps of patients with severe COPD, may be a compensatory mechanism designed to improve muscle function. Indeed, constitutive NO production in skeletal muscles promotes glucose uptake, enhances fuel oxidation, improves muscle perfusion, inhibits proteases, and promotes muscle repair and sarcomere addition (9, 2224). Activity of nNOS also improves muscle contractility by protecting Cyst3635 of ryanodine-sensitive sarcoplasmic reticulum channels from oxidation by reactive oxygen species (9). Second, an alternative interpretation is that an increase in muscle nNOS expression in patients with severe COPD is a pathologic process whose combination with excessive superoxide anion production leads to the formation of the highly reactive oxidant, peroxynitrite, and the development of nitrosative stress. This interpretation is supported by the presence of elevated 3-nitrotyrosine levels (footprint of peroxynitrite) and a positive correlation between 3-nitrotyrosine intensity and nNOS expression in leg muscles of patients with COPD. This correlation along with the absence of abundant iNOS protein expression suggests that nNOS rather than iNOS is primarily responsible for 3-nitrotyosine formation in leg muscles of patients with COPD. We should emphasize that our study does not exclude the possibility that an additional mechanism other than peroxynitrite might have been involved in the formation of 3-nitrotyrosine, namely, myeloperoxidase derived from infiltrating phagocytes. This enzyme is capable of utilizing both NO2 and H2O2 to produce nitration of tyrosine residues (25).

The functional significance of protein tyrosine nitration in regulating skeletal muscle function remains unclear. Many studies have documented tyrosine nitration of proteins involved in energy production, apoptosis, fatty acid metabolism, oxidative stress, and structural integrity (26). In the majority of these proteins, tyrosine nitration causes loss of protein function primarily as a result of nitration of critical tyrosine residues involved in the catalytic activity of these proteins (as for example in Mn-SOD) or as a result of increased proteolytic degradation of these proteins (27). Further studies are needed to identify tyrosine-nitrated proteins inside human skeletal muscles and to explore the functional significance of protein tyrosine nitration to various muscle functions including contractile performance and the redox state.

The exact mechanisms responsible for the elevation of constitutive NOS expression in leg muscles of severe patients with COPD are unknown. We propose that hypoxia, which induces significant upregulation of both eNOS and nNOS expression in skeletal muscles, might be involved (28). The presence of normal PaO2 at rest in our patients does not exclude the possibility that muscle hypoxia may develop during exercise and may contribute to increased gene expression of both nNOS and eNOS, though we have not found an increase in the latter. Another possible mechanism is proinflammatory cytokines such as tumor necrosis factors and interleukins, which have been proposed to mediate limb muscle dysfunction in patients with COPD (1). In pathologic conditions where these cytokines are elevated, such as in severe sepsis, an increase in muscle eNOS and nNOS expression has been reported (29). Disuse or lack of physical activity are not likely to have contributed to the rise in constitutive NOS expression in the leg muscle of patients with COPD because of the strong association between increased muscle activity and muscle nNOS expression (30).

Limitations of the Study

One limitation of our study is related to a relatively small population (6 control subjects and 12 patients with COPD). This is because we employed very restrictive inclusion and exclusion criteria. In addition, we chose to obtain muscle samples with an open biopsy procedure and not the commonly used needle biopsy technique. This preference was based on the fact that muscle sample size and quality provided by open biopsies are superior to that of needle biopsies. The fact that all of our patients with COPD and control subjects share a common morbidity, i.e., the presence of small and very localized neoplasm, should be acknowledged. However, we believe that this morbidity did not cause the observed differences in limb muscle oxidative stress and NO production among control subjects and patients with COPD because we excluded all subjects with nutritional abnormalities and paraneoplastic syndromes. Therefore, we propose that all the findings reported herein can be exclusively attributed to the effects of COPD limb muscles.

Another limitation of this study is related to methodologies. Direct confirmation of ROS involvement in muscle dysfunction of patients with COPD is technically difficult, and previous studies focused mainly on measuring antioxidant levels, primarily those of glutathione (reduced and oxidized). Recently, investigators have introduced a new and reliable experimental approach to evaluate protein oxidation as a marker of ROS production within cells that is based on measuring protein carbonyl moieties by either HPLC or an immunoblotting technique (31). Similarly, thus far several methods have been employed to evaluate lipid peroxidation such as malondialdehyde and 8-isoprostane formations; however, these methods require relatively large muscle samples. Indeed, we opted to measure 4-hydroxy-2-nonenal formation in this study because this aldehyde is considered to be the most toxic product of lipid peroxidation and a major mediator of free radical cell damage and to use Western blot analysis to detect HNE protein adducts, because it requires a relatively small amount of muscle sample. HNE crosslinks with various proteins such as Na+-K+-ATPase and glucose-6-phosphate dehydrogenase, resulting in inactivation of these proteins in addition to changes in membrane fluidity and increased nonspecific permeability to ions such as Ca++.

In summary, our study indicates that lipid peroxidation rather than protein oxidation is elevated in vastus lateralis muscles of patients with COPD with different degrees of airway obstruction. These muscles express enhanced protein tyrosine nitration, probably at the expense of nNOS enzyme activity, compared with muscles obtained from control subjects. These results are strongly suggestive of the development of reactive oxygen and nitrogen species-derived limb muscle dysfunction.

S.N.A.H. is a Chercheur Nationaux of the F.R.S.Q. The authors are thankful to Ms. Anna Llorens and to Mr. L. Franchi for their technical assistance. E.B. was supported by ASTRA, SOCAP (Spain), Red Respira (RTIC C03/11, FIS, Instituto de Salud Carlos III), and BIOMED BMH4-CT98–3406 (EU). This study was supported by a grant from the Canadian Institute of Health Research.

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Address correspondence to: Dr. S. Hussain, Room L3.05, 687 Pine Ave. West, Montreal, PQ, H3A 1A1 Canada. E-mail:

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