Oxidative Stress and Respiratory Muscle Dysfunction in Severe ...

Oxidative Stress and Respiratory Muscle Dysfunction

in Severe Chronic Obstructive Pulmonary Disease

Esther Barreiro, Beatriz de la Puente, Joan Minguella, Josep M. Corominas, Sergi Serrano,

Sabah N. A. Hussain, and Joaquim Gea



Muscle Research and Respiratory System Unit, Respiratory Medicine, Surgery, and Pathology Departments, IMIM–Hospital del Mar, Universidad

Pompeu Fabra and Universidad Autonoma, Barcelona, Spain; Critical Care and Respiratory Divisions, Royal Victoria Hospital and

`

Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada





Rationale: Oxidative stress is involved in the skeletal muscle dysfunc- than that of healthy subjects with comparable lung volumes. In

tion observed in patients with severe chronic obstructive pulmonary keeping with this finding, several investigators have demonstrated

disease (COPD). We hypothesized that the diaphragms of such that diaphragms from patients with severe COPD undergo adap-

patients might generate greater levels of oxidants than those neu- tive modifications (3–6). In contrast, Levine and colleagues (7)

tralized by antioxidants. Objectives: To assess the levels of both have recently demonstrated that diaphragm fibers from patients

oxidative and nitrosative stress and different antioxidants in the with severe COPD produce intrinsically less force than that gener-

diaphragms of those patients, and to analyze potential relation-

ated by control muscles. So far, the mechanisms whereby the

ships with lung and respiratory muscle dysfunctions. Methods and

diaphragm of patients with severe COPD may be exposed to a

Measurements: We conducted a case-control study in which reactive

remodeling process are still controversial and remain to be fully

carbonyl groups, hydroxynonenal-protein adducts, antioxidant

enzyme levels, nitric oxide synthases, and 3-nitrotyrosine formation

elucidated.

were detected using immunoblotting and immunhistochemistry in Although the etiology of skeletal muscle dysfunction in COPD

diaphragm specimens (thoracotomy) obtained from six patients with is still under investigation, several factors, such as comorbid condi-

severe COPD, six patients with moderate COPD, and seven control tions, hypoxia, hypercapnia, nutritional status, medication, in-

subjects. Main Results: Diaphragms of patients with severe COPD flammation, and oxidative stress (for review, see Reference 8),

showed both higher protein carbonyl groups and hydroxynonenal- have already been implicated. Furthermore, patients with COPD

protein adducts than control subjects. When only considering patients have shown higher levels of oxidative stress in their blood (9) and

with COPD, negative correlations were found between carbonyl peripheral muscles (10–15). We also demonstrated (16) that both

groups and airway obstruction, and between hydroxynonenal-protein oxidative and nitrosative stress develop in the quadriceps mus-

adducts and respiratory muscle strength. Although diaphragmatic cles of patients with COPD. However, there are no reports in

neuronal nitric oxide synthase did not differ among the three the literature exploring whether oxidative stress contributes to

groups and no inducible nitric oxide synthase was detected in any the impairment of the intrinsic contractile properties of the dia-

muscle, muscle endothelial nitric oxide synthase was lower in pa- phragm fibers in severe COPD. On the grounds that production

tients with severe COPD than in control subjects. Muscle nitrotyro- of reactive oxygen species (ROS) and reactive nitrogen species

sine levels were similar in both patients with severe COPD and

within skeletal muscle fibers is regulated in part by strong muscle

control subjects. Conclusions: This study shows that oxidative stress

contractions, and that patients with severe COPD are chronically

rather than nitric oxide is likely to be involved in the respiratory

muscle dysfunction in severe COPD.

exposed to respiratory overloads, we hypothesized that their

diaphragm fibers might generate greater levels of oxidants than

Keywords: chronic obstructive pulmonary disease; diaphragm strength; those normally neutralized by intracellular antioxidant defenses,

oxidative stress thus leading to the development of oxidative stress. Accordingly,

our aims were to evaluate whether oxidative stress and nitric

Muscle dysfunction in patients with severe chronic obstructive oxide (NO)–mediated deleterious effects develop in the dia-

pulmonary disease (COPD) is characterized by reduced muscle phragm muscle of patients with severe COPD, on the one hand,

strength and endurance, probably from the interaction of differ- and whether these two phenomena are associated with the respi-

ent systemic and local factors, and is also highly dependent on ratory muscle dysfunction of such patients on the other. Some

the specific function of the muscle. For instance, lower limb of the results of this study have been previously reported in the

muscles, most likely because of disuse or deconditioning, have form of an abstract (17, 18).

been shown to be more adversely affected (1) than inspiratory

muscles. In fact, whether intrinsic ventilatory muscle dysfunction

METHODS

develops in patients with severe COPD remains debatable. For

instance, Similowski and coworkers (2) showed that the dia- Subjects

phragm of patients with COPD produced even greater force

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

(Received in original form July 9, 2004; accepted in final form February 15, 2005) included the following: female sex, chronic respiratory failure, bronchial

Supported by Plan Nacional I D (SAF 2001-0426), RESPIRA (RTIC C03/11; Spain), asthma, coronary disease, severe undernourishment (body mass index

and QLK6-CT-2002-02285 (E.U.). 20 kg/m2), chronic metabolic diseases, suspected paraneoplastic or

Correspondence and requests for reprints should be addressed to Esther Barreiro, myopathic syndromes, and/or treatment with drugs known to alter

M.D., Ph.D., Muscle and Respiratory System Research Unit, IMIM, C/Dr. Aiguader, muscle structure and/or function. This is a case-control study designed

80, E-08003 Barcelona, Spain. E-mail: ebarreiro@imim.es in accordance with the ethical standards on human experimentation in

This article has an online supplement, which is accessible from this issue’s table our institution and the World Medical Association guidelines for re-

of contents at www.atsjournals.org search on humans. The Ethics Committee on Human Investigation at

Am J Respir Crit Care Med Vol 171. pp 1116–1124, 2005

Hospital del Mar–IMIM approved all experiments. Informed, written

Originally Published in Press as DOI: 10.1164/rccm.200407-887OC on February 25, 2005 consent was obtained from all individuals (see the online supplement

Internet address: www.atsjournals.org for additional information).

Barreiro, de la Puente, Minguella, et al.: Diaphragm Oxidative Stress in COPD 1117



Nutritional and Functional Assessment Inc., Poole, UK), antimyosin heavy-chain II (clone MY-32; Sigma, St.

Louis, MO), and anti-CD34 (Biomeda, Inc., Hayward, CA) primary anti-

Nutritional evaluation included body mass index and analytic parameters. bodies, respectively, as well as markers of oxidative stress (16). Capillary

Pulmonary and respiratory muscle functions and general exercise capacity density was quantified as the number of capillaries per muscle fiber.

were evaluated (see the online supplement for additional information).

Statistical Analysis

Biopsies

Data are presented as mean SD. One-way analysis of variance,

During thoracotomy because of localized lung lesions, diaphragm Tukey-corrected for multiple comparisons, was used to compare data

biopsy specimens were obtained from the anterior costal diaphragm obtained within the three groups. Pearson’s correlation coefficient was

lateral to the insertion of the phrenic nerve (4). In all subjects, biopsies used to assess relationships among different variables within patients

were obtained 10 to 14 days after the exercise tests (see the online with COPD. A Bonferroni-type adjustment was performed to take into

supplement for additional information). account the effect of having multiple comparisons and correlations. A

p value of 0.05 or less was considered significant.

Biological Muscle Studies

Immunoblotting. The levels of oxidative and nitrosative stress were RESULTS

assessed as described elsewhere (16). Selective antibodies were used

to detect the following: carbonyl groups through derivatization (20) to Characteristics of the Study Subjects

2,4-dinitrophenylhydrazone (DNP; anti–DNP moiety antibody; Oxy-

Table 1 indicates the main characteristics of the study subjects.

blot kit; Chemicon International, Inc., Temecula, CA); 4-hydroxy-

2-nonenal (HNE-)–protein adducts (21) and catalase (anti-HNE and No significant differences in age and nutritional status as assessed

anticatalase antibodies; Calbiochem, San Diego, CA); Mn-superoxide by body mass index and analytic parameters were observed

dismutase (anti–Mn-superoxide dismutase antibody; StressGen, Victo- between control subjects and patients with either severe or mod-

ria, BC, Canada); neuronal, endothelial, and inducible NO synthases erate COPD. However, FEV1, FVC, and the ratio of FEV1 to

(nNOS, eNOS, and iNOS, respectively); heme oxygenase-1 (anti-nNOS, FVC were significantly lower, whereas residual volume (RV),

anti-eNOS, anti-iNOS, and anti–heme oxygenase-1 antibodies; Trans- and the ratio of RV to total lung capacity were significantly

duction Laboratories, Inc., Lexington, KY); and nitrotyrosine formation higher in both groups of patients with COPD. Both exercise

(anti–3-nitrotyrosine antibody; Cayman Chemical, Inc., Ann Arbor, capacity and global respiratory muscle strength were moderately

MI). Corresponding positive controls were used in each case. Blots reduced in patients with severe COPD.

were scanned with an imaging densitometer, and optical densities of

specific proteins were quantified with Diversity Database 2.1.1 (BioRad, Muscle Structure

Philadelphia, PA; see the online supplement for additional information).

Immunohistochemistry. On 3- m muscle paraffin-embedded sec- Proportions of type I fibers were higher in the diaphragms of

tions, myosin heavy-chain (MHC) I and II isoforms and capillary density the patients with severe COPD compared with control subjects

were identified using antimyosin heavy-chain I (clone MHC; Biogenesis, (Table 1). When considering all patients with COPD as a group,



TABLE 1. MAIN CHARACTERISTICS AND FUNCTIONAL VARIABLES OF THE STUDY SUBJECTS

Control Subjects Patients with Moderate Patients with Severe

(n 7 ) COPD (n 6) COPD (n 6 )



Age, yr 66 7 68 7 68 4

BMI, kg/m2 26.9 1.9 25.1 3.7 26.4 4.2

Total serum proteins, g/dl 6.7 0.6 6.6 0.4 6.4 1

Serum albumin, g/dl 4.1 0.5 4.3 0.5 3.9 0.6

Serum cholesterol, mg/dl 206 50 211 53 195 19

Serum triglycerides, mg/dl 130 64 94 35 124 75

FEV1, % pred***††† 84 7 62 4 42 7

FVC, % pred*** 83 6 72 11 54 9

FEV1/FVC, %***††† 73 4 63 4 56 5

RV, % pred*† 93 20 146 38 177 63

RV/TLC**†† 37 4 54 3 62 13

DLCO, % pred 87 10 83 11 75 6

PaO2, mm Hg 90 9 85 17 77 9

PaCO2, mm Hg 37.8 1.9 40 1.0 40.3 4.1

˙

VO2max, % pred* 105 19 102 31 70 10

WRmax, % pred** 91 11 80 4 68 14

MIP, % pred‡ 91 18 81 4 67 17

Pesmax, cm H2O 93 12 73 33 79 10

Pdimax, cm H2O 120 23 102 33 102 23

Tlim, min 9 6 8 3 6 5

Type I fibers, %* 53 5 57 4 65 11

No. capillaries/fiber 3.63 0.52 3.40 0.10 3.39 0.51



Definition of abbreviations: BMI body mass index; DLCO carbon monoxide transfer; FRC functional residual capacity; MIP

maximal inspiratory pressure; Pdimax maximal transdiaphragmatic pressure; Pesmax maximal esophageal pressure; RV residual

volume; TLC total lung capacity; Tlim endurance time; Wrmax maximal mechanical power output.

Data are presented as mean SD.

Statistical significance of the results is expressed as follows:

between patients with severe COPD and controls

* p 0.05.

** p 0.01.

*** p 0.001.

between patients with moderate COPD and controls

†

p 0.05.

††

p 0.01.

†††

p 0.001.

1118 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 171 2005



the proportion of type I fibers negatively correlated with FEV1 Protein Carbonylation

(r 0.752, p 0.01) and positively correlated with total levels Total carbonyl group formation. As shown in Figure 1A, anti-

of carbonyl groups (r 0.629, p 0.05). Diaphragmatic capillary DNP antibody detected different positive protein bands, with

density did not differ between patients with COPD and control apparent masses ranging from 67 to 29 kD, in the muscles of

subjects, and did not show any relationship with either lung or both patients and control subjects. The diaphragms of patients

respiratory muscle functions or with exercise tolerance. with severe COPD showed higher levels of total carbonyl content









Figure 1. (A ) Representative

examples of protein oxidation

(total carbonyl groups) in dia-

phragms of control subjects and

patients with moderate and se-

vere chronic obstructive pul-

monary disease (COPD). Sev-

eral protein carbonylated bands

of different molecular weights

(MW) were detected. (B )

Mean values SD of total car-

bonyl formation were higher

in the patients with severe

COPD compared with control

muscles (*p 0.05). Total dia-

phragmatic carbonyl forma-

tion did not differ between

patients with moderate COPD

and control subjects (ns

nonsignificant). Among the

overall patients with COPD,

optical densities of total car-

bonyl group formation signifi-

cantly correlated with FEV1 (%

predicted). Note that 14 pa-

tients with COPD are depicted

(two mild, six moderate, and

six severe) in the correlation

graph. (C) Immunohistochemi-

cal localization of carbonyl-

modified proteins in diaphragm

muscle fibers of one control

subject (panel A) and one pa-

tient with severe COPD (panel

B; 200 ). Anti–2,4-dinitrophe-

nylhydrazone (anti-DNP) anti-

body detected positive staining

diffusely localized within the di-

aphragm fibers (panels A and

B). Avoiding the derivatization

process eliminated positive

carbonyl formation staining

(panels C and D). Furthermore,

removal of primary anti-DNP

antibody completely eliminated

positive carbonyl-modified pro-

tein staining (panels E and F).

Barreiro, de la Puente, Minguella, et al.: Diaphragm Oxidative Stress in COPD 1119









Figure 2. (A) Representative ex-

amples of 4-hydroxy-2-nonenal

(HNE-)–protein adducts and tu-

bulin in diaphragms of control

subjects and patients with mod-

erate and severe COPD. Several

HNE-protein adducts were de-

tected. Monoclonal anti– -

tubulin antibody was used to

control equal loading among

various lanes. (B ) Mean

values SD of total HNE-

protein adducts were higher

in the patients with severe

COPD compared with control

muscles (*p 0.05). Dia-

phragmatic levels of total

HNE-protein adducts did not

differ between patients with

moderate COPD and control

subjects (upper left panel).

Among the overall group of

patients with COPD, optical

densities of total HNE-protein

adduct formation significantly

correlated with respiratory mus-

cle force as measured by maxi-

mal inspiratory pressure (MIP;

five patients with severe and

six patients with moderate

COPD are depicted) (upper right

panel) and maximal esopha-

geal pressure (lower left panel)

and showed a tendency to

correlate with maximal trans-

diaphragmatic pressure (lower

right panel). Note that only six

patients with COPD (four se-

vere and two moderate) ac-

cepted to undergo balloon

catheter placement, and this is

why only six patients with

COPD are depicted in both

graphs. (C ) Immunohisto-

chemical localization of HNE-

protein adducts in diaphragm

muscle fibers of one control

individual (panel A) and one

patients with severe COPD

(panel B; 200 ). Anti-HNE an-

tibody detected positive stain-

ing diffusely localized within

the muscle fibers (panels A and

B ). Removal of anti-HNE anti-

body completely eliminated

positive HNE staining (panels

C and D ).

1120 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 171 2005



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 0.685, p

0.05). Immunostaining with anti-DNP antibody revealed the

presence of carbonyl groups diffusely localized within the dia-

phragm 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 appar-

ent 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 transdia-

phragmatic pressure (Figure 2B). No other relationships were

found between HNE-protein adducts and either lung or respira-

tory muscle functions, or exercise tolerance. Immunohistochemi-

cal 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 pa-

tients 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 0.613, 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, respec-

tively, 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 dia-

phragms 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 maxi-

mal 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 Figure 3. (A ) Representative examples of protein expression of Mn-

superoxide dismutase (Mn-SOD), catalase, heme oxygenase-1 (HO-1),

diaphragms of both patients with COPD and control subjects,

and tubulin in the diaphragms of control subjects and patients with

with apparent masses ranging from 63 to 30 kD (Figure 5A).

moderate and severe COPD. Corresponding positive controls are indi-

Total muscle 3-nitrotyrosine optical densities did not differ cated accordingly ( ve). Monoclonal anti– -tubulin antibody was used

among the three groups (Figure 5B). No significant correlations to control equal loading among various lanes. (B ) Mean SD values

were found between muscle protein nitration and functional, of Mn-SOD, catalase, and HO-1 in diaphragm muscles of control sub-

exercise capacity, or muscle structure variables when considering jects and patients with moderate and severe COPD. No significant

all the patients with COPD. Immunohistochemical analysis re- differences were found among these three groups.

vealed clear, positive 3-nitrotyrosine staining diffusely localized

within the diaphragm fibers in both patients with severe COPD

and control subjects (Figure 5C).

Barreiro, de la Puente, Minguella, et al.: Diaphragm Oxidative Stress in COPD 1121



DISCUSSION Muscle Structure and Function

The major findings of this study are that in the diaphragms of The human diaphragm can develop fatigue (22), and in severe

patients with severe COPD as compared with those from control COPD several underlying molecular and structural adaptive

subjects (1) reactive carbonyl group levels were increased and mechanisms (3–6) have been suggested to produce a more fa-

correlated with the severity of their disease, (2) HNE-protein tigue-resistant diaphragm phenotype. In this regard, as was pre-

viously shown by Levine and coworkers (3), we also report

adduct formation was also elevated and correlated with their respi-

herein increased proportion of type I fibers in the diaphragms

ratory muscle function, (3) constitutive eNOS protein levels were

of our patients with severe COPD, which was associated with

reduced, and (4) protein tyrosine nitration remained unchanged. both the severity of their disease and muscle protein oxidation

levels. One could argue that those diaphragms with higher con-

tent of oxidative fibers (type I) are those likely to suffer most

from protein oxidation. However, it remains debatable whether

this and other adaptive mechanisms (3–6) 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 im-

paired 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 func-

tion. Oxidative stress has been implicated in the pathogenesis of

a wide range of conditions and in chronic degenerative diseases

(25–28). Furthermore, over the last decade, a growing body of

evidence has shown that oxidative stress is one of the mecha-

nisms clearly involved in the skeletal muscle dysfunction of pa-

tients with COPD (8–16). Carbonyl formation (ketones and

aldehydes) is an important detectable marker of protein oxida-

tion. Carbonyl groups can be formed by direct reaction of pro-

teins with ROS, leading to the formation of protein derivatives

containing highly reactive carbonyl groups (29–34), 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 poly-

unsaturated fatty acids (29–34). Our study is the first to demon-

strate 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









Figure 4. (A ) Representative examples of neuronal nitric oxide syn-

thases (nNOS), endothelial NOS (eNOS), inducible NOS (iNOS), and

tubulin proteins in diaphragms of control subjects and patients with

moderate and severe COPD. Corresponding positive controls are indi-

cated accordingly ( ve). Note that eNOS protein expression was weak

in all groups. Note that no iNOS protein was detected in the diaphragms

of patients with severe or moderate COPD or control subjects. Mono-

clonal anti– -tubulin antibody was used to control equal loading among

various lanes. (B ) Mean SD values of nNOS and eNOS in diaphragms

of control subjects and patients with moderate and severe COPD. Dia-

phragmatic levels of nNOS did not differ among these three groups.

Intensity of muscle eNOS was lower in the patients with severe COPD

compared with control subjects (*p 0.05).

1122 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 171 2005



association found between the diaphragmatic levels of protein ducts, whereas in the quadriceps of a more heterogeneous popu-

oxidation and respiratory muscle function strongly supports this lation of patients with COPD, HNE-protein adducts were the

conclusion. Finally, diaphragm proteins targeted by ROS led to sole index of protein oxidation having increased. It could be

increased levels of both total carbonyls and HNE-protein ad- 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 skele-

tal 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 ventila-

tory 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









Figure 5. (A ) Representative examples of protein tyrosine nitration (to-

tal 3-nitrotyrosine immunoreactivity) and tubulin in diaphragms of

control subjects and patients with moderate and severe COPD. Several

tyrosine-nitrated proteins were detected. Monoclonal anti– -tubulin

antibody was used to control equal loading among various lanes. (B )

Mean values SD of total 3-nitrotyrosine optical densities in control

subjects and patients with moderate and severe COPD. Diaphragmatic

levels of total 3-nitrotyrosine formation did not differ among these three

groups. (C ) Immunohistochemical localization of 3-nitrotyrosine in dia-

phragm muscles of one control subject (panel A) and one patients with

severe COPD (panel B; 200 ). Anti–3-nitrotyrosine antibody detected

positive staining diffusely localized within the muscle fibers (panels A

and B ). Removal of primary anti–3-nitrotyrosine antibody completely

eliminated the staining (panels C and D).

Barreiro, de la Puente, Minguella, et al.: Diaphragm Oxidative Stress in COPD 1123



related to their respiratory muscle dysfunction. One possible does not have a financial relationship with a commercial entity that has an interest

in the subject of this manuscript; J.G. does not have a financial relationship with

mechanism responsible for decreased muscle eNOS expression a commercial entity that has an interest in the subject of this manuscript.

in the patients with severe COPD might be the existence of a

functional defect in the capillary permeability of their dia- Acknowledgment : The authors thank Mr. Daniel Sanchez and Mrs. Anna Llorens for

´

their technical assistance in the laboratory, Mr. Pablo Peretti for his technical support

phragms, as occurs in other conditions (38), because a proportion in the preparation of the figures, and Mr. Roger Marshall for his editing aid.

of muscle eNOS protein is likely to be derived from endothelial

cells. So far, little is known about transcriptional regulation of

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Conflict of Interest Statement : E.B. does not have a financial relationship with a Am J Respir Crit Care Med 2003;167:A28.

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Hussain SNA, Gea J. Nitric oxide synthases (NOS) and protein tyro-

in the subject of this manuscript; J.M. does not have a financial relationship with

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does not have a financial relationship with a commercial entity that has an interest J Respir Crit Care Med 2004;169:A245.

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a commercial entity that has an interest in the subject of this manuscript; S.N.A.H. prevention of COPD: 2003 update. Eur Respir J 2003;22:1–2.

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