Free Radical Biology & Medicine 41 (2006) 797–809www.elsevier.com/locate/freeradbiomed

Original Contribution

Aging, sex differences, and oxidative stress in human respiratoryand limb muscles

Esther Barreiro ⁎, Carlos Coronell, Barbara Laviña, Alba Ramírez-Sarmiento,Mauricio Orozco-Levi, Joaquim Geaon behalf of the PENAM Project

Muscle and Respiratory System Research Unit and Respiratory Medicine Department, IMIM Hospital del Mar,Experimental Sciences and Health Department, Universitat Pompeu Fabra, C/Dr. Aiguader, 80, Barcelona E-08003, Spain

Received 9 November 2005; revised 7 April 2006; accepted 23 May 2006Available online 3 June 2006

Abstract

Oxidative stress is involved in the sarcopenia of aging muscles. On the grounds that ventilatory muscles are permanently active, and theiractivity may even increase with aging, we hypothesized that the levels of oxidative stress would probably be increased in the external intercostalsof elderly healthy individuals. We conducted a case–control study in which reactive carbonyl groups, malondialdehyde–protein adducts, 3-nitrotyrosine immunoreactivity, Mn-superoxide dismutase (Mn-SOD), and catalase were detected using immunoblotting in external intercostalsand quadriceps (open muscle biopsies) obtained from 12 healthy elderly and 12 young individuals of both sexes. In elderly subjects, reactivecarbonyls, malondialdehyde–protein adducts, 3-nitrotyrosine, Mn-SOD, and catalase were significantly greater in the external intercostals than inthe young controls. A post hoc analysis, in which men and women from both groups were analyzed separately, revealed that the externalintercostals of elderly women, but not those of elderly men, showed significantly increased levels of reactive carbonyls, malondialdehyde–proteinadducts, 3-nitrotyrosine, and Mn-SOD compared to those of control females. This study suggests that differences in muscle activity might explainthe differential pattern of oxidative stress observed in human respiratory and limb muscles with aging as well as the likely existence of a sex-related regulation of this phenomenon in these muscles.© 2006 Elsevier Inc. All rights reserved.

Keywords: Aging; Respiratory and limb muscles; Sex-related differences; Protein oxidation; Protein tyrosine nitration; Antioxidant enzymes; Free radicals

Aging has been defined as a decline in performance andfitness with advancing age. Even in healthy individuals musclesbecome weaker and less powerful as age progresses, impairingtheir ability to perform essential physical activities of daily life,while increasing prevalence for both falls and morbidity.Indeed, the deleterious effects of aging are best observed inpostmitotic tissues such as skeletal muscles and neurons, inwhich damaged or lost cells cannot be replaced by the mitosis ofintact ones. The overall loss of muscle mass, quality, andstrength is generally known as sarcopenia, which includesamong others type II fiber atrophy, decreased mitochondrialvolume and enzyme content, mitochondrial DNA mutations,and reduced muscle respiratory rate [1–4].

⁎ Corresponding author. Fax: +34 93 221 3237.E-mail address: ebarreiro@imim.es (E. Barreiro).

0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.freeradbiomed.2006.05.027

Although the etiology of sarcopenia of aging muscles is stillunder investigation, several factors have already been impli-cated, such as the loss of growth hormone and a reduction inboth estrogen and androgen production [5], impaired glucoseand/or fatty acid metabolism, nitrogen imbalance, decreasedmuscle protein synthesis, reduced physical activity, andoxidative stress [6–8]. In fact, aging has also been attributedto a greater accumulation of deleterious effects on tissues fromreactive oxygen species (ROS) compared to those normallyneutralized by intracellular antioxidant defenses [9,10]. It isnowadays commonly accepted that oxidative stress is primarilyinvolved in the etiology of aging, especially in those tissueswith high levels of oxygen metabolism such as skeletal muscles[6–8]. For instance, the diaphragm of senescent rodents wasshown to increase its oxidative capacity [11], as well as itscontent of antioxidant enzymes such as superoxide dismutases

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(SOD) [12]. The latter was probably acting as a protectivemechanism against the increased superoxide anion productionreported in aging rodent diaphragms [11,12]. Furthermore, anage-dependent loss in sarcoplasmic reticulum Ca2+-ATPaseisoform 2a (SERCA2a) activity was also shown as a result of 3-nitrotyrosine accumulation in aging rat limb muscles [13–15].

On the other hand, other studies have focused their attentionon the assessment of the implications of oxidative stress inhuman muscles. In this regard, increased levels of oxidizedglutathione, lipid peroxidation, both Mn-SOD and catalaseactivity, protein carbonylation, and DNA oxidation have beenshown in the quadriceps muscles of elderly patients undergoingorthopedic surgery [16–19]. Moreover, Mn-SOD activity wasalso shown to be higher in the rectus abdominis of elderlypatients undergoing abdominal surgery [20], whereas catalaseand glutathione transferase activities were shown to be lower insatellite cells from human quadriceps [21].

Despite this progress in humans, it has never been establishedwhether the deleterious effects of accumulating ROS arespecifically confined to the limb muscles of elderly humans orwhether these effects could also be observed in other musclessuch as the respiratory muscles, which are essential to life andmust remain active throughout the life span of the individual. Inaddition, both lung and respiratory muscle function and structurehave been shown to be altered even in healthy elderly humans,leading to a significant loss in chest wall compliance [22].Although this is unlikely to be clinically relevant under healthyconditions, it may clearly have a significant impact on morbidityand mortality under stress conditions.

On this basis, we hypothesized that the levels of oxidativestress would probably be increased in the intercostal muscles ofphysically active, healthy elderly humans. To test thishypothesis, first we aimed at determining the levels of proteincarbonylation and nitration in both the external intercostal andthe quadriceps muscles of a healthy elderly population asopposed to healthy young individuals. Second, we alsoinvestigated whether oxidative stress levels might differbetween elderly males and females in both respiratory andlimb muscles. Third, we explored the levels of severalantioxidant mechanisms in those muscles.

Materials and methods

Subjects

Twelve healthy elderly volunteer individuals (6 male and 6female, 68 years) and 12 healthy young volunteer controlsubjects (identical gender distribution, 25 years) were recruitedfrom the general population on an outpatient basis. This samplesize was calculated on the basis of previous studies from ourgroup [23,24]. The cut-off age for the elderly individuals of 65years and over was chosen specifically according to datapreviously published [16]. All individuals were Caucasian.Exclusion criteria included current or past smoking habit,chronic respiratory failure, bronchial asthma, coronary disease,severe undernourishment (body mass index <20 kg/m2),chronic metabolic diseases, suspected paraneoplastic or myo-

pathic syndrome, and/or treatment with drugs known to altermuscle structure and/or function. This is a case–control studydesigned in accordance with both the ethical standards onhuman experimentation in our institution and the WorldMedical Association guidelines for research on human beings.The Ethics Committee on Human Investigation at Hospital delMar-IMIM approved all experiments. Informed written consentwas obtained from all individuals.

Clinical, nutritional, and functional assessment

Clinical evaluation included medical history, completephysical examination, thorax radiology, and electrocardiogram.Nutritional evaluation included body mass index and analyticalparameters. Forced spirometry was performed and static lungvolumes were determined in all subjects using standardprocedures, and reference values by Roca et al. [25,26] wereused. Inspiratory muscle strength was assessed throughdetermination of maximal inspiratory pressure at the mouth(Sibelmed-163; Sibel, Barcelona, Spain) during an occludedmaneuver from residual volume. Reference values by Wilson etal. [27] were chosen.

Muscle biopsies

Muscle samples were obtained from the external intercostaland quadriceps (vastus lateralis) as the control muscle by openbiopsy after procedures published elsewhere [23,28–30] andwere immediately frozen in liquid nitrogen and stored at −80°Cfor further analysis. All subjects were prevented from doing anypotentially exhausting physical exercise 10 to 14 days beforecoming to the hospital to undergo the surgical procedures.

Biological muscle studies

Total carbonyl groupsTotal levels of those highly reactive carbonyl groups in the

protein side chains were detected by reaction (derivatization)with 2,4-dinitrophenylhydrazine (DNPH), resulting in theformation of 2,4-dinitrophenylhydrazone (DNP) [31]. TheDNP-derivatized proteins were subsequently separated byelectrophoresis and further subjected to immunoblotting withselective antibodies against the DNP moieties.

Aldehyde–protein adductsThe lipid peroxidation product malondialdehyde (MDA) can

cause further cellular damage by binding to and modifyingproteins, which leads to the formation of aldehyde–proteinadducts. The characteristic feature of these adductions is theintroduction of carbonyl groups into the modified proteins.MDA reacts with lysine residues to form Schiff base adductsthat can also be detected in tissues using a selective antibody[32].

ImmunoblottingThe effects of oxidants on muscle proteins and lipids were

evaluated according to methodologies published elsewhere

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[24,30,33]. Frozen muscle samples from both external inter-costals and quadriceps muscles (n = 12/group) were homoge-nized in a buffer containing Hepes 50 mM, NaCl 150 mM, NaF100 mM, Na pyrophosphate 10 mM, EDTA 5 mM, Triton X-100 0.5%, leupeptin 2 μg/ml, PMSF 100 μg/ml, aprotinin 2 μg/ml, and pepstatin A 10 μg/ml. Samples were then centrifuged at1000g for 30 min. The pellet was discarded and the supernatantwas designated as a crude homogenate. Total muscle proteinlevel in each sample was spectrophotometrically determinedwith the Bradford technique using different runs of triplicates ineach case and bovine serum albumin (BSA) as the standard(Bio-Rad protein reagent; Bio-Rad, Inc., Hercules, CA, USA).The final protein concentration in each sample was calculatedfrom at least two Bradford measurements that were almostidentical. Equal amounts of total protein from crude musclehomogenates were always loaded (20 μg per sample/lane) ontothe gels, as well as identical sample volumes/lanes.

Two different sets of experiments were conducted. For thepurpose of comparison, external intercostal muscle samplesfrom both elderly and young individuals (24) were always runtogether in the same gel, whereas quadriceps muscle sampleswere run in a different set of experiments, but with an identicalapproach being applied. In the different Western blot analyses,the same samples were always run together and kept in the sameorder. Proteins were then separated by electrophoresis,transferred to polyvinylidene difluoride (PVDF) membranes,blocked with nonfat milk, and incubated overnight withselective antibodies. The following antibodies were used todetect the different antigens and phenomena: anti-DNP moietyantibody (rabbit anti-DNP antibody, from the Oxyblot kit;Chemicon International, Inc., Temecula, CA, USA; dilution 1/150), anti-MDA antibody (Academy Bio-Medical Co., Inc.,Houston, TX, USA; dilution 1/4000), anti-3-nitrotyrosineantibody (Cayman Chemical, Inc., Ann Arbor, MI, USA;dilution 1/1000), anti-Mn-SOD antibody (StressGen, Victoria,BC, Canada; dilution 1/5000), and anti-catalase antibody(Calbiochem, San Diego, CA, USA; dilution 1/2000). Tissuehomogenates obtained from rat brain mitochondria and raterythrocytes were used as positive controls for the enzymes Mn-SOD and catalase, respectively. Specific proteins from allsamples were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies and a chemiluminescence kit.For each of the antigens, samples from the different groups werealways detected in the same film under identical exposure times.The specificity of carbonyl groups was confirmed by avoidingthe derivatization process [31] in some of the samples and byomission of the primary antibody and incubation of themembranes only with secondary antibody (goat anti-rabbitIgG, HRP-conjugated, from the Oxyblot kit; ChemiconInternational Inc.; dilution 1/300). Controls for specific bindingof MDA–protein adducts were performed by omitting theprimary antibody and incubating the membranes only withsecondary antibody (peroxidase-conjugated AffiniPure rabbitanti-goat IgG (H+L); Jackson Immunoresearch Laboratories,Inc., West Grove, PA, USA; dilution 1/6000). Also, native BSA(Roche GmbH, Mannheim, Germany) was loaded onto the gelsas a second negative control for MDA specificity. Finally, the

following controls were also performed to confirm thespecificity of 3-nitrotyrosine immunoreactivity: omission ofthe primary antibody and incubation of the membranes onlywith secondary antibody (HRP-conjugated goat anti-mouseimmunoglobulin-specific polyclonal antibody; BD BiosciencesPharmingen, San José, CA, USA; dilution 1/4000), chemicalreduction of nitrotyrosine to aminotyrosine with 100 mMdithionite (Sigma–Aldrich GmbH, Steinheim, Germany), andnative BSA loading onto some gel lanes [34,35]. Blots werescanned with an imaging densitometer, and optical densities(OD) of specific proteins were quantified with DiversityDatabase 2.1.1 (Bio-Rad, Philadelphia, PA, USA). Values oftotal protein carbonylation, total MDA–protein adducts, andtotal protein tyrosine nitration in a given sample were calculatedby addition of OD of individual protein bands in each case.Final optical densities obtained in each specific group ofsubjects corresponded to the mean values of the differentsamples (lanes) of each of the antigens studied. To validateequal protein loading among various lanes, PVDF membraneswere stripped and reprobed with a mouse anti-α-subunit Na,K-ATPase antibody (Johns Hopkins University, Baltimore, MD,USA) in all cases. Optical densities in each histogram wereexpressed as the ratio of the optical densities of the specificantigen to those of α-subunit Na,K-ATPase.

Statistical analysis

Data are presented as means ± SD. Mann–Whitneynonparametric tests were used for unpaired comparisonsbetween elderly and young individuals. In addition, analysisof variance was used to compare optical densities betweenelderly and young individuals of both sexes. Relationshipsbetween various parameters were studied by calculating theSpearman's correlation coefficient. A p value of 0.05 or lesswas considered significant.

Results

Characteristics of the study subjects

Table 1 indicates the main characteristics of both young andelderly individuals. Elderly individuals were significantly olderand showed a significant reduction in the ratio of forcedexpiratory volume in 1 s to forced vital capacity as well as in theratio of residual volume to total lung capacity. No significantcorrelations were found between physiological and biologicalvariables.

Reactive carbonyl groups

Total protein carbonylationAs shown in Fig. 1A, anti-DNP antibody detected several

protein bands with apparent masses ranging from 118 to 25 kDain both the intercostal and the quadriceps muscles (left andright, respectively) of elderly and young subjects. Total proteincarbonylation levels were significantly greater only in theintercostal muscles of the elderly individuals compared to the

Table 1Main characteristics and functional variables of the study subjects

Subjects Young Elderly

All n = 12 n = 12Age, years 25 ± 4 68 ± 5 ⁎⁎⁎

Weight, kg 65 ± 13 70 ± 7.7BMI, kg/m2 23.4 ± 3.4 23.6 ± 2.6FEV1, % pred 97 ± 11 93 ± 12FEV1/FVC 83 ± 8 74 ± 4 ⁎⁎

RV/TLC 24 ± 5 44 ± 5 ⁎⁎⁎

MIP, % pred 106 ± 21 110 ± 27

Males n = 6 n = 6Age, years 25 ± 5 68 ± 5 ⁎⁎

Weight, kg 69 ± 13 74 ± 8BMI, kg/m2 22.9 ± 4.4 22.8 ± 2.3FEV1, % pred 96 ± 10 92 ± 16FEV1/FVC 82 ± 10 72 ± 4 ⁎

RV/TLC 24 ± 4 43 ± 3 ⁎⁎⁎

MIP, % pred 101 ± 23 112 ± 33

Females n = 6 n = 6Age, years 25 ± 3 69 ± 6 ⁎⁎

Weight, kg 61 ± 12 66 ± 5BMI, kg/m2 23.9 ± 2.5 24.4 ± 2.7FEV1, % pred 97 ± 12 94 ± 9FEV1/FVC 83 ± 7 76 ± 4 ⁎

RV/TLC 23 ± 6 46 ± 6 ⁎⁎⁎

MIP, % pred 111 ± 19 108 ± 19

Data are presented as means ± SD. Abbreviations used: BMI, body mass index;FEV1, forced expiratory volume in 1 s; pred, predicted; FVC, forced vitalcapacity; RV, residual volume; TLC, total lung capacity; MIP, maximalinspiratory pressure. Statistical significance of the results is between young andelderly individuals.

⁎ p ≤ 0.05.⁎⁎ p ≤ 0.01.

⁎⁎⁎ p ≤ 0.001.

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young control subjects, remaining unmodified in the quadriceps(Fig. 1B). In a post hoc analysis in which males and femaleswere analyzed separately, the levels of total protein carbonyl-ation were significantly greater only in the external intercostalsof the elderly women compared to those of the young controlfemales (Fig. 1C). No carbonyl groups were detected whenproteins were not derivatized or when the primary antibody wasomitted (data not shown).

MDA–protein adductsFig. 2A illustrates various MDA–protein adducts with

apparent masses ranging from 71 to 26 kDa in both theintercostal and the quadriceps muscles (left and right,respectively) of elderly and young individuals. Total levels ofMDA–protein adducts were significantly higher in both

Fig. 1. (A) Representative examples of carbonylated proteins in the external intercosubjects. Carbonylated proteins of different molecular weights (MW, kDa) were detecto control for equal loading across various lanes. (B) Optical densities in the histogramof α-Na,K-ATPase (mean values ± SD). Total protein carbonylation levels were scompared to those of the young controls (**p < 0.01), whereas protein carbonylatioOptical densities in the histograms are expressed as the ratio of the optical density of panalysis, total carbonyl group formation was significantly increased (**p < 0.01) ocontrol females (left), whereas in the quadriceps no significant variations were observwere greater in the external intercostals of the young men compared to those of the

muscles in the elderly group of subjects compared to those ofthe young control individuals (Fig. 2B). In the post hoc analysis,total MDA–protein adducts were significantly greater in theexternal intercostals of the senescent women compared to thoseof the young control females, whereas in the quadriceps muscle,MDA–protein adducts were significantly greater in the elderlymales compared to the young control men (Fig. 2C). No MDA–protein adducts were detected when the primary antibody wasomitted or when native BSA was loaded (data not shown).

Protein tyrosine nitration

Several tyrosine-nitrated protein bands with apparent massesranging from 69 to 16 kDa were detected in both the externalintercostals and the quadriceps muscles (left and right,respectively) of elderly and young controls (Fig. 3A). Totalmuscle 3-nitrotyrosine levels were significantly greater in boththe external intercostals and the quadriceps of the elderlysubjects compared to those detected in the young control group(Fig. 3B). In the post hoc analysis, only in the elderly womencompared to the young females was total protein tyrosinenitration significantly greater in both muscles (Fig. 3C). Notyrosine-nitrated proteins were detected when the primaryantibody was omitted, or samples were reduced with dithionite,or when BSA was loaded (data not shown).

Antioxidant enzymes

Mn-SODAs shown in Fig. 4A, Mn-SOD was detected in both the

external intercostals and the quadriceps (left and right,respectively) of all subjects. Muscle protein content of thisenzyme was significantly higher only in the intercostals of theelderly subjects compared to those of the young controls (Fig.4B). Levels of this enzyme showed, however, a tendency to begreater in the quadriceps muscles of the elderly group comparedto the young controls (Fig. 4B). In the post hoc analysis, muscleMn-SOD protein was significantly greater in the externalintercostals of the elderly females, compared to the youngcontrol women (Fig. 4C). In the quadriceps muscles, Mn-SODcontent showed only a strong tendency to be higher, which didnot reach statistical significance, in the elderly femalescompared to the young control women (Fig. 4C).

CatalaseFig. 5A illustrates the presence of the enzyme catalase in

both external intercostals and quadriceps (left and right,respectively) of all individuals. Muscle catalase protein content

stal muscles (left) and vastus lateralis (right) of both elderly and young controlted in both muscles. Monoclonal anti-α-subunit Na,K-ATPase antibody was useds are expressed as the ratio of the optical density of protein carbonylation to thatignificantly higher in the external intercostals (left) of the elderly individualsn remained unchanged in the aging quadriceps (n.s., nonsignificant) (right). (C)rotein carbonylation to that of α-Na,K-ATPase (mean values ± SD). In a post hocnly in the external intercostals of the senescent women compared to the younged among groups (right). It is worth mentioning that protein carbonylation levelsyoung females (*p < 0.05) (left).

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was significantly higher in both muscles in the elderly group ofsubjects compared to the young controls (Fig. 5B). In the posthoc analysis, the levels of catalase showed a only tendency to behigher in both intercostals and quadriceps in the elderly mencompared to the young control males (Fig. 5C).

Discussion

The main findings in this study are that in skeletal muscles ofhealthy elderly individuals compared to muscles of a healthyyoung population: (1) total carbonyl group formation, MDA–

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protein adducts, and protein tyrosine nitration were significantlygreater in the aging external intercostals, whereas only MDA–protein adducts and 3-nitrotyrosine immunoreactivity weresignificantly higher in the senescent quadriceps; (2) whereas

both Mn-SOD and catalase proteins were significantly higher inthe aging external intercostals, only catalase, and not Mn-SOD,was greater in the senescent quadriceps; and (3) a post hocanalysis specifically revealed that elderly women showed

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significantly greater levels of both total protein carbonylationand nitration, as well as Mn-SOD content, in their intercostalmuscles, whereas elderly men showed an increase only inMDA–protein adducts in their quadriceps and a strong tendencytoward higher catalase levels in their respiratory and limbmuscles.

It has been proposed that accumulation of the deleteriouseffects of ROS throughout the entire life of the individualcould be largely responsible for aging [6,8,9]. Indeed, thedetrimental effects of aging are best seen in postmitotic cellssuch as neurons and muscle fibers. ROS-mediated proteindamage was said to be involved in the aging process as a resultof studies showing that exposure of enzymes from younganimals to ROS led to changes in their activity similar to thoseobserved in aging tissues [36,37]. In the light of both thecurrent literature [6–12,16–21] and the results encountered inour study, it is possible to conclude that accumulation of ROSis likely to be involved in the etiology of aging-inducedreduced muscle performance. It is also noteworthy that on thebasis of both the reported decline in mitochondrial functionwith aging and the fact that mitochondria are the main oxygenradical source in muscle fibers, the reactive species superoxideanion is likely to be the major player in this process. Forinstance, it has been well established that in aging,mitochondrial function is characterized by increased electronleakage from the respiratory chain, slowed turnover, andreduced efficiency [38–42]. Also, as the half-life of superoxideanion is very short and it does not cross membranes, itsoxidative effects will probably occur within the mitochondria,thus further impairing mitochondrial function.

The present study is the first to provide evidence of thepresence of ROS-mediated protein modifications in one of themain respiratory muscles, the external intercostal, in activesenescent humans with no comorbidity. It should be emphasizedthat in this study, the three indirect indices employed to analyzemuscle oxidative stress levels, protein carbonylation asmeasured by the DNPH assay, protein tyrosine nitration, andMDA–protein adducts, were clearly increased in this respira-tory muscle in the elderly humans compared to muscles fromthe young controls. It is also worth mentioning that all threeoxidative stress indices as well as both Mn-SOD and catalasecontent were markedly greater in the aging intercostals, whereasthis was not exactly the case for the senescent quadriceps of thesame individuals. The fact that external intercostals must bepermanently active and that certain significant pulmonaryfunction impairment was shown in our healthy elderlyindividuals might help account for these findings. Actually,

Fig. 2. (A) Representative examples of MDA–protein adducts in the external intercosubjects. MDA–protein adducts of different molecular weights (MW, kDa) were deused to control for equal loading across various lanes. (B) Optical densities in the histto that of α-Na,K-ATPase (mean values ± SD). Total MDA–protein adduct levels wquadriceps muscles (*p < 0.05, right) of the elderly individuals compared to those ofratio of the optical density of MDA–protein adducts to that of α-Na,K-ATPase (msignificantly increased (**p < 0.01) in the external intercostals of the senescent womeprotein adducts were significantly greater (*p < 0.05) in the elderly males compared toshowed a tendency to be higher, which did not reach statistical significance (p = 0.08(left).

respiratory muscle function may be increased in elderlyindividuals as a result of modifications in respiratory mechanics[43–45]. In line with this, it has been shown that the mechanicsof the respiratory system undergo structural and functionalchanges with aging, which somehow mimic those occurring inpulmonary emphysema, but at a much lower intensity [43–46].In fact, as lung compliance increases because of reduced lungelasticity, chest wall compliance steadily declines withadvancing age [22,45]. Hence, it is plausible that themaintenance of an adequate ventilation in humans is achievedat the expense of an increased activity of the respiratorymuscles, even in healthy aging.

In aging quadriceps, however, catalase and both MDA–protein adducts and protein tyrosine nitration, but not carbonylgroups from the DNPH assay or Mn-SOD content, weresignificantly increased in the elderly subjects compared toyoung controls. Given the fact that the widely used DNPH assayindiscriminately detects total protein carbonyl groups formed bydiverse species such as hydroxyl and alkylperoxyl radicals,MDA, and hydroxynonenal [47], and that lipid peroxidation is acommon phenomenon of oxidative stress [48,49], we decidedspecifically to detect whether MDA–protein adducts wereincreased in aging muscles. Indeed, MDA–protein adductswere found to be greater in both the intercostals and thequadriceps of the elderly human subjects compared with theyoung controls, suggesting that peroxidation of polyunsaturatedmembrane lipids is likely to be a continuing event during theaging process, at least in muscles. In line with this, other studies[16–19] have already shown that lipid peroxidation, oxidizedglutathione, protein oxidation as measured by the DNPH assay,and DNA oxidation were increased in the vastus lateralis ofhospitalized patients undergoing orthopedic surgery (hipreplacement, bone lesions) at ages above 65 years [16–20].As to the activity of Mn-SOD enzyme, this was reported to beincreased in senescent quadriceps in some studies [16,17],whereas no significant variations in its activity were reported ina most recent study [20]. Mn-SOD activity was actuallyincreased in the rectus abdominis of patients of the study inquestion, who underwent abdominal surgery for various reasons[20]. In all of these studies, catalase activity remainedunchanged in the senescent muscles analyzed [16,17,20]. Inour study, however, catalase was significantly increased in bothmuscles of the elderly individuals, whereas Mn-SOD contentshowed only a tendency to be greater in the quadriceps and wassignificantly increased only in the external intercostals of thesame subjects. The facts that hospitalized patients undergoing asurgical procedure (with certain previous immobilization) were

stal muscles (left) and vastus lateralis (right) of both elderly and young controltected in both muscles. Monoclonal anti-α-subunit Na,K-ATPase antibody wasograms are expressed as the ratio of the optical density of MDA–protein adductsere significantly higher (**p < 0.01) in the external intercostals (left) and in thethe young controls. (C) Optical densities in the histograms are expressed as theean values ± SD). In a post hoc analysis, total MDA–protein adducts were

n compared to the young control females (left), whereas in the quadriceps MDA–the young control men (right). It is worth mentioning that MDA–protein adducts), in the external intercostals of the young males compared to the young females

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Fig. 4. (A) Representative examples of protein expression of Mn-SOD in both the external intercostal muscles (left) and the vastus lateralis (right) of both elderly andyoung control subjects. Monoclonal anti-α-subunit Na,K-ATPase antibody was used to control for equal loading across various lanes. Corresponding positive control(+C, rat brain mitochondria) is indicated. (B) Optical densities in the histograms are expressed as the ratio of the optical density of Mn-SOD to that of α-Na,K-ATPase(mean values ± SD). Mn-SOD protein content was significantly higher (***p < 0.001) in the external intercostals (left) of the elderly subjects compared to those of theyoung controls. In the quadriceps, however, Mn-SOD levels showed only a tendency to be greater, which did not reach statistical significance (p = 0.1), in the elderlycompared to the young controls (right). (C) Optical densities in the histograms are expressed as the ratio of the optical density of Mn-SOD to that of α-Na,K-ATPase(mean values ± SD). In a post hoc analysis, Mn-SOD levels were significantly increased (**p < 0.01) in the external intercostals (left) of the elderly women comparedto those in the young control females. In the vastus lateralis (right), Mn-SOD levels showed only a tendency to be greater, which did not reach statistical significance(p = 0.1), in the elderly women compared to the young control females.

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assessed in the former studies, whereas physically activehealthy elderly subjects from the general population wereused in ours, and that protein content, and not enzyme activity,was explored in our study might help account for thesediscrepancies.

Fig. 3. (A) Representative examples of protein tyrosine nitration (3-nitrotyrosine immof both elderly and young control subjects. Tyrosine-nitrated proteins of different mosubunit Na,K-ATPase antibody was used to control equal loading across various lanedensity of protein tyrosine nitration to that of α-Na,K-ATPase (mean values ± SD). Texternal intercostals (left) and in the quadriceps muscles (right) of the elderly indihistograms are expressed as the ratio of the optical density of protein tyrosine nitratprotein tyrosine nitration levels were significantly increased (**p < 0.01) in both thecompared to the young control females. It is worth mentioning that total protein tyrsignificance (p = 0.01), in the quadriceps of the young males compared to the youn

Clearly, the content of the mitochondrial isoform Mn-SODis increased in senescent human muscles probably tocounteract the deleterious effects (compensatory mechanism)produced by enhanced levels of superoxide anion in thosemuscles. Also, in our study this is more evident in the

unoreactivity) in the external intercostal muscles (left) and vastus lateralis (right)lecular weights (MW, kDa) were detected in both muscles. Monoclonal anti-α-s. (B) Optical densities in the histograms are expressed as the ratio of the opticalotal protein tyrosine nitration levels were significantly higher (**p < 0.01) in theviduals compared to those of the young controls. (C) Optical densities in theion to that of α-Na,K-ATPase (mean values ± SD). In a post hoc analysis, totalexternal intercostals (left) and the vastus lateralis (right) of the senescent womenosine nitration showed a tendency to be higher, which did not reach statisticalg females (right).

Fig. 5. (A) Representative examples of protein expression of catalase in both the external intercostal muscles (left) and the vastus lateralis (right) of both elderly andyoung control subjects. Monoclonal anti-α-subunit Na,K-ATPase antibody was used to control for equal loading across various lanes. Corresponding positive control(+C, erythrocytes) is indicated. (B) Optical densities in the histograms are expressed as the ratio of the optical density of catalase to that of α-Na,K-ATPase (meanvalues ± SD). Catalase protein content was significantly higher (*p < 0.05) in both the external intercostals (left) and the quadriceps (right) of the elderly subjectscompared to those of the young controls. (C) Optical densities in the histograms are expressed as the ratio of the optical density of catalase to that of α-Na,K-ATPase(mean values ± SD). In a post hoc analysis, catalase levels showed a tendency to be greater, which did not reach statistical significance (p = 0.09), in both the externalintercostals (left) and the quadriceps (right) of the elderly males compared to those in the young control men.

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external intercostal muscle, where its increased activity due tomodifications of the respiratory mechanics with age [22,43–46] led to greater levels of oxidative stress than thoseobserved in the quadriceps muscles of the same individuals.Furthermore, this finding also reinforces the concept thatsuperoxide anion is probably the main oxygen speciesinvolved in aging-induced reduced muscle performance.Finally, catalase content was increased in both the respiratoryand the limb muscles of the elderly humans in the presentstudy, indicating that the hydrogen peroxide formed by theaction of Mn-SOD will be scavenged by catalase, aubiquitous enzyme that catalyzes the dismutation of hydrogenperoxide into water and molecular oxygen.

Our study is also the first to provide evidence of increasedlevels of tyrosine protein nitration in both respiratory and limbmuscles of healthy elderly humans as opposed to muscles fromyoung control individuals. Viner et al. [13,15] alreadydemonstrated that, in senescent rat muscles, 3-nitrotyrosineselectively accumulated on the SERCA2a, probably throughperoxynitrite formation, even in the presence of an excess ofSERCA1 isoform [14]. Collectively, it can be concluded that invivo nitration of senescent muscles might be mediated byinduced activity of nitric oxide synthases (NOS) such as theconstitutive neuronal NOS, whose levels were shown to beincreased in aging muscles [50]. It has been clearly establishedthat nitric oxide reacts extremely fast with superoxide anion (the

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reaction is six times faster than the scavenging of superoxide bysuperoxide dismutases) to form peroxynitrite [51], furtherleading to protein tyrosine nitration in tissues. Future studies toexplore the nature and function of the different tyrosine-modifiedproteins in human senescent muscles are clearly required.

Another remarkable finding in our study is that total carbonylgroups as measured by the DNPH assay, MDA–proteinadducts, protein tyrosine nitration, and Mn-SOD levels of theexternal intercostals on the one hand, and 3-nitrotyrosineimmunoreactivity of the quadriceps on the other, weresignificantly modified only in the elderly postmenopausalwomen and not in the elderly men. This might imply thepresence of a sex-related mechanism in the redox regulation ofthese aging muscles. Indeed, there is evidence that estrogenshave strong protective antioxidant properties against oxidativestress in muscles and other tissues [52–55]. This suggests thatmuscles from young females, as opposed to males of a similarage, are strongly protected against ROS-mediated deleteriouseffects. Actually, the fact that in the external intercostals of theyoung males as opposed to the young females, proteincarbonylation was significantly greater and that MDA–proteinadducts showed a tendency to be higher, strongly supports thishypothesis. In fact, although levels of these three indices ofoxidative stress were indeed increased in the intercostals of theelderly males, they did not reach statistical significancecompared to those of the young males, because levels in themuscles of the latter were already relatively higher as opposedto those of the young females. Furthermore, probably as aprotective compensatory mechanism, the content of Mn-SODwas also increased in the intercostal muscles and showed atendency to be higher in the quadriceps of the postmenopausalfemales compared to that observed in the young controls.Clearly, future studies are required to fully elucidate the natureof the sex-related differences regarding oxidative stressdevelopment in human senescent muscles.

Eventually, sarcopenia of old age results in muscle weaknessthat may predispose elderly individuals to a higher risk of fallsand loss of autonomy as well as to increased morbidity. Indeed,Powers et al. [56] already demonstrated that diaphragm forcegeneration was reduced with aging in rats. In another study,maximum transdiaphragmatic pressure was considerably lowerin elderly individuals compared to that in the young controls[57], possibly contributing to further respiratory muscle failure ofsenescent patients with severe lung disease. In line with this, ithas recently been demonstrated that aging has additive effects onprotein oxidation and diaphragm force reserve impairment ofsenescent animals mechanically ventilated [58]. This is of vitalimportance because the difficulty of weaning elderly patientsfrom mechanical ventilation represents a major clinical defeat inintensive care units [59,60]. Moreover, aging was shown to be anindependent predictor of weaning complications of patients aftercardiac surgery [61]. More recently, aging was also shown to bean important incidence predictor of the development of thehighly prevalent chronic obstructive pulmonary disease (COPD)[62], the prevalence of which has also been shown toprogressively increase in the female sex. In conclusion,alterations of the respiratory mechanics described in aging are

unlikely to be clinically relevant under healthy conditions;however, they may clearly have a significant impact on morbidityand mortality under stress conditions. This is important, becauseelderly humans have also been shown to be more prone to sufferfrom respiratory illnesses or to take medication that might alsoinfluence ROS production in muscles [45].

Study critique

The main limitation of our study has to do with the relativelysmall sample size used in contrast to previous reports [16–20], inwhich no clear inclusion criteria were provided to recruit thesubjects. Actually, from an ethical point of view, the utilization ofa population size larger than that required for the obtaining ofsignificant results is not usually recommended. Despite therelatively small number of subjects studied, we found consistentsignificant results in terms of protein expression of the differentindices analyzed. Furthermore, in our study, extremely restrictiveinclusion and exclusion criteria were employed in order tocarefully select healthy elderly and young subjects from thegeneral population. In addition, the volunteer subjects used in thestudy were physically active and had not been hospitalized forany reason in the months previous to study entry, as opposed toformer studies, in which subjects underwent orthopedic surgeryfor different bone lesions, implying a certain degree ofimmobilization both before and during surgery [16–20]. Thisis important, because immobilization might have influencedoxidative stress levels in those muscles, as has been shown inother studies [63–65], despite the fact that biopsies from theyoung controls were also obtained under identical conditions.

In the current study, another remarkable difference fromprevious reports has been the assessment of the aging effects onhuman respiratory muscles and not only on the limb muscles.Because this has involved the use of relatively “invasive”procedures, especially that of the external intercostals, we werecompelled to use a relatively reduced number of subjects in thisstudy, who were sampled on an outpatient basis in order toensure their maximum comfort. Open thoracotomy is the goldstandard technique to obtain biopsies from the diaphragm, themain inspiratory muscle. However, this surgical practice can beapplied to subjects only in the presence of serious illnesses suchas lung neoplasms. Therefore, normal healthy individualscannot be exposed to such an invasive procedure. Furthermore,under certain conditions, the external intercostals or other ribcage muscles have been shown to be progressively recruited[66–68], making them an excellent target for study. Finally,clear accessibility of the external intercostals by means of anopen biopsy on an outpatient basis as well as the fact that thismodel enabled us to carefully select the subjects, excluding anycomorbid condition, clearly justified the design of the currentstudy.

Conclusions

Our study is the first to provide evidence of increased ROS-mediated effects on muscle proteins in the external intercostalsof healthy elderly humans, which may have additional effects

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on other conditions such as mechanical ventilation and COPD.Furthermore, it also suggests the likely existence of a sex-related regulation of the oxidative stress levels in limb musclesand especially in the respiratory muscles. These findings mighteventually offer a target for therapeutic intervention in the agingprocess.

Acknowledgments

This study was supported by the Pan-European Network forAgeing Muscle (PENAM) (QLRT-2000-00417) (EU) andRESPIRA (RTIC C03/11) (Spain). We gratefully acknowledgeFrancesc Sánchez, Sandra Mas, and Esthela Garcia for theirtechnical assistance in the laboratory and Roger Marshall for hisediting aid.

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