Training Depletes Muscle Glutathione in Patients with

Clinical Investigations Respiration 2006;73:757–761 DOI: 10.1159/000094395

Received: September 5, 2005 Accepted after revision: March 18, 2006 Published online: June 30, 2006

Training Depletes Muscle Glutathione in Patients with Chronic Obstructive Pulmonary Disease and Low Body Mass Index Roberto A. Rabinovich a Esther Ardite a Ana Maria Mayer a Maite Figueras Polo b Jordi Vilaró c Josep M. Argilés b Josep Roca a a

Servei de Pneumologia, Hospital Clínic, Facultat de Medicina, b Grup de Recerca en Càncer, Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, i c EUIF Blanquerna, Universitat Ramon Llull, Barcelona, Spain

For editorial comment see p. 737

Abstract Background: A physiological increase in muscle glutathione after training is not seen in patients with chronic obstructive pulmonary disease (COPD), indicating abnormal peripheral muscle adaptations to exercise. Objective: We hypothesized that oxidative stress is primarily associated with low body mass index (BMI). Methods: Eleven patients with preserved BMI (BMIN: 28.2 8 1.2 kg m–2), 9 patients with low BMI (BMIL: 19.7 8 0.60 kg m–2) and 5 age-matched controls (26.5 8 0.9 kg m–2) were studied before and after 8 weeks of highintensity endurance training. Reduced glutathione (GSH) and -glutamyl cysteine synthase heavy-subunit chain mRNA expression (GCS-HS mRNA) were measured in the vastus lateralis. Results: After training, exercise capacity increased (VO2PEAK , 13 8 5.2%; 10 8 5.6% and 15 8 4.3% in BMIL, BMIN and controls, respectively; p ! 0.05 each). GSH levels decreased in BMIL (from 5.2 8 0.7 to 3.7 8 0.8 nmol/ mg protein, GSH –1.5 8 0.7 nmol/mg protein, p ! 0.05); no changes were seen in BMIN (from 5.4 8 0.7 to 6.7 8 0.9 nmol/mg protein, GSH 1.3 8 0.9 nmol/mg protein), whereas GSH markedly increased in controls (from 4.6 8 1 to 8.7 8 0.4 nmol/mg protein, GSH 4.1 8 1 nmol/mg pro-

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tein, p ! 0.01). GSH in BMIL was different from GSH in BMIN and controls (p ! 0.05, each). Consistent changes were observed in GCS-HS mRNA expression. Conclusions: GSH depletion after training in BMIL may suggest that oxidative stress plays a key role in muscle wasting in COPD patients. Copyright © 2006 S. Karger AG, Basel

Introduction

In the last few years, systemic effects of chronic obstructive pulmonary disease (COPD) [1–3] attract increased interest partly because of the impact of muscle wasting on poor disease prognosis [4]. The underlying mechanisms of weight loss are, however, incompletely understood. We have recently reported [5] that COPD patients show an abnormal regulation of skeletal muscle glutathione (GSH). While high-intensity endurance training markedly enhanced antioxidant buffering in healthy subjects by a twofold increase in muscle GSH, COPD patients showed an increase in oxidized glutathione (GSSG) following training without changes in GSH. Impaired regulation of the redox potential in the patients was closely related with the gain in exercise capacity produced by training [5]. These results suggest that antioxidant buffering was not adapted to the higher rate of exercise-induced reactive oxygen species production in COPD

Roberto Rabinovich, MD Servei de Pneumologia, Hospital Clínic Villarroel 170 ES–08036 Barcelona (Spain) Tel. +34 93 227 5540, Fax +34 93 227 5455, E-Mail [email protected]

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Key Words Chronic obstructive pulmonary disease Glutathione Oxidative stress Skeletal muscle wasting

Characteristics

COPD, BMIN

COPD, BMIL

Controls

Age, years Weight, kg BMI, kg m–2 FEV1, liters FEV1, % of predicted FVC, % of predicted TLC, % of predicted FRC, % of predicted RV, % of predicted PaO2, mm Hg KCO, ml min–1 mm Hg–1 l–1 PaCO2, mm Hg VO2peak, l min–1 kg–1

6781.6 80.284.2 28.281.2 1.380.2c 4185.0c 6886.4b 10586.2 139811.9a 164814.0b 7383.3c 3.1980.4b 4182.4 15.781.1c

6682.1 55.482.3c, d 19.780.6c, d 0.9380.1c 3083.8c 5684.7b 11087.8 159817.3a 192828.0b 6984.2c 2.1680.3b 4281.9 14.882.2c

6282.8 74.581.3 26.580.9 3.380.3 10289.0 9987.4 9385.8 9183.5 8289.1 10083.7 4.2480.3 3581.5 23.780.8

a

p < 0.05; b p < 0.01; c p < 0.001 vs. controls; d p < 0.001, BMIN vs. BMIL .

[5–7], thus leaving skeletal muscles more susceptible to oxidative stress. We hypothesize that oxidative stress might be a central factor in the complex mechanisms of skeletal muscle wasting shown by COPD patients with low body mass index (BMIL) [8]. A better understanding of the underlying processes may have practical implications to refine evidencebased therapeutic strategies such as rehabilitation/training [9]. The current investigation explores the relationship between abnormal training-induced GSH responses and muscle wasting in COPD. Therefore, we expanded the study group reported by Rabinovich et al. [5] to compare COPD patients with preserved BMI (BMIN), BMIL and age-matched healthy sedentary controls. Patients and Methods Study Group Twenty clinically stable COPD patients (all men) [10] free of oral steroids were included in the study. Five healthy sedentary subjects were recruited to serve as controls (BMI 26.5 8 0.9 kg m–2). Eleven out of the 20 COPD patients (BMIN: 28.2 8 1.2 kg m–2) showed preserved BMI, whereas the remaining 9 patients had low BMI (BMIL: 19.7 8 0.60 kg m–2). Up to 17 patients and all 5 controls had also been investigated in a previous study [5]. All participants signed a written, informed consent approved by the Ethics Committee on Investigations Involving Human Subjects at the Hospital Clínic, Universitat de Barcelona. Study Design Selection procedures for inclusion in the study were: (a) clinical assessment; (b) pulmonary function testing at rest (MasterScreen; Jaeger, Wuerzburg, Germany) [11, 12]; (c) chest X-ray film; (d) general blood analysis, and (e) standard incremental exercise testing. Training-induced physiological changes were

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measured by changes in exercise tolerance. In all the subjects (20 COPD patients and 5 controls), a needle muscle biopsy of the ‘vastus lateralis’ was obtained immediately after a 11-min moderate-intensity (40% before training WPEAK) constant-work rate protocol before and after an 8-week endurance training. Training sessions lasted 60 min and were split into small blocks of 2- to 5-min high-intensity continuous cycling (60 cycles/ min; at approximately 90% of WPEAK at the end of the training program) for an effective period of 30 min at least. Recovery time between the high-intensity periods consisted of cycling at the same speed and !60% of WPEAK before training. The 1st week was used as an adaptation period were the high-intensity blocks were initially 60% of WPEAK . Thereafter, the work level has been raised to reach values close to 90% of WPEAK before training and this intensity level was maintained until the end of the training period. Exercise Testing Incremental Exercise. After 3 min of unloaded pedaling (CardiO2 system; Medical Graphics, St. Paul, Minn., USA), the work rate was increased by 5 or 10 W/min. Arterial blood samples (Seldicath, Plastimed, Saint-Leu-La-Foret, France) were taken every 3 min throughout the test to analyze blood gases and lactate (Ciba Corning 800, Medfield, Mass., USA). Muscle Biopsies A muscle sample (150 mg) was obtained from the vastus lateralis using a Bergström needle. Half of the sample was incubated in Krebs buffer (pH 7.40) for immediate processing, and the remainder was frozen in liquid nitrogen and stored at –70 ° C. The two molecular forms of glutathione, GSH and GSSG, were determined in the homogenate by high-performance liquid chromatography [13]. Lipid peroxidation was assessed using cis-parinaric acid, a naturally fluorescent aliphatic acid containing four double bonds [14, 15]. -Glutamyl cysteine synthase heavy-subunit chain mRNA expression (GCS-HS mRNA) was measured using RT-PCR [16]. Values for GCS-HS mRNA were corrected by 18S mRNA and expressed as GCS-HS/18S mRNA.

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Table 1. Characteristics of the study groups (means 8 SEM)

Data Analysis Results are expressed as means 8 SEM. Training effects within groups were analyzed using Student’s paired t test. Comparisons among groups were made using ANOVA. The Student-Newman-Keuls test was used as a post hoc test for contrast analysis. A p value ! 0.05 was taken as statistically significant.

Results

Effects of Physical Training Physiologic training effects at peak exercise (and VO2PEAK) were observed in all three groups (p ! 0.05). After training VO2PEAK levels were: 130 8 10 ml min–1 (10%), p ! 0.05, in BMIN; 120 8 10 ml min–1 (13%), p ! 0.05, in BMIL , and, 260 8 70 ml min–1 (15%), p ! 0.05, in healthy sedentary subjects. Changes in the Muscle Redox Status. After training, muscle GSH levels increased in healthy subjects (from 4.6 8 1 to 8.70 8 0.4 nmol/mg protein, p ! 0.01). In contrast, GSH decreased in BMIL (from 5.2 8 0.7 to 3.7 8 0.8 nmol/mg protein, p ! 0.05), whereas no differences were seen in the BMIN (from 5.36 8 0.7 to 6.7 8 0.9 nmol/mg protein). Following training, changes in GSH (GSH; fig. 1a) differed significantly between BMIL (–1.5 8 0.7 nmol/mg protein) and the other two groups (1.3 8 0.9 and 4.1 8 1 nmol/mg protein in BMIN and Muscle Wasting and Glutathione Regulation

Fig. 1. a Individual (symbols) and mean (horizontal lines) changes in muscle reduced glutathione (GSH) before and after training in BMIL (n = 9, o); BMIN (n = 11, d), and healthy sedentary subjects (n = 5, +). After training, GSH was twofold increased in controls; in BMIN GSH did not change, and a significant fall was noted in BMIL (ANOVA, p ! 0.005). GSH in BMIL showed significant differences compared to BMIN and controls (* p ! 0.05 each). b Effects of training on GCS-HS/18S mRNA expression(before vs. after) in BMIL (n = 7); BMIN (n = 8) and controls (n = 5). GCS-HS/18S mRNA expression showed a trend to increase in BMIL , no changes were seen in BMIN, whereas GCS-HS mRNA downregulation was observed in controls. Statistically significant differences were seen between BMIL and healthy sedentary subjects (* p ! 0.05, ANOVA with contrast analysis).

controls, respectively; p ! 0.05 each). None of the three groups showed a training-induced increase in lipid peroxidation (BMIL , from 461 8 154 to 356 8 104 AU; BMIN, from 339 8 82 to 348 8 60 AU, and controls from 358 8 75 to 439 8 72 AU). In the BMIL group, after training, the fall in GSH was accompanied by a strong tendency to an increase in GCS-HS mRNA expression (fig. 1b) in 6 of the 7 patients Respiration 2006;73:757–761

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Assessment before Training Anthropometric characteristics and lung function in the three groups (BMIN, BMIL and controls) are listed in table 1. On average, patients showed a severe obstructive ventilatory defect (FEV1: BMIN 40.5 8 5.0% of predicted; BMIL 30.1 8 3.8% of predicted and controls 102 8 9.0% of predicted) and moderate arterial hypoxemia. Peak exercise tolerance (peak VO2/kg) was severely reduced in COPD patients (BMIN 15.67 8 1.14 and BMIL 14.82 8 2.2 ml min–1 kg–1) compared to controls (23.7 8 0.85 ml min–1 kg–1). No differences among the three groups were seen in glutathione levels before training (GSH: 5.2 8 0.7; 5.36 8 0.7, and 4.6 8 1 nmol/mg protein in BMIL , BMIN and controls, respectively). Oxidized GSH (GSSG) levels and lipid peroxidation index (data not reported) were also similar among groups. As described previously [5], 11 min of moderate-intensity, constant-work-rate exercise did not generate statistically significant changes (before vs. after exercise) in (1) total glutathione levels and (2) lipid peroxidation index. Likewise, no differences in these variables were seen from rest to moderate exercise after training.

Discussion

Abnormal regulation of the skeletal muscle redox system leading to higher vulnerability to oxidative stress has been shown in COPD patients after training [5] and during high-intensity exercise [6]. The impact of this phenomenon on cell function has not been established yet [17, 18]. The analysis of the individual results reported in 2001 [5] prompted the hypothesis that the abnormal adaptation of the GSH system to training might be primarily associated to the COPD phenotype characterized by weight loss. Cell GSH seems to be an important regulator of myogenic differentiation in murine skeletal muscle C2C12 cells [19]. Maintenance of optimal GSH levels might be a useful strategy to prevent muscle damage in COPD. While adequate cytosol GSH guarantees a successful myogenic program, the maintenance of mitochondrial GSH levels may ensure cell survival as inferred from previous observations [20, 21] in which the selective depletion of mitochondrial GSH sensitizes cells against chemical or cytokine-mediated cell death. The main finding of the current study was that the BMIL group showed a distinctive behavior in terms of the GSH response of the muscle to training compared to BMIN and healthy sedentary subjects. As displayed in figure 1a, GSH clearly decreased in BMIL after training (in 7 of the 9 patients). In contrast, GSH levels almost doubled in healthy sedentary subjects (after training GSH increased in all 5 controls), whereas no changes in GSH were observed in the BMIN patients, on average. Of note, in 6 of the 11 BMIN patients a physiological increase in GSH was observed, whereas GSH fell after training in the remaining 5 patients. The variability observed in BMIN may partly reflect the complex interactions that determine muscle dysfunction in COPD. Muscle wasting is a serious complication in COPD, as well as in other chronic disorders [22], and hence identification of responsible mechanisms may be of utmost importance as muscle wasting is as an independent predic760


tor of mortality. Weight loss in COPD patients may likely reflect the genetic susceptibility as well as the interplay of several factors at pulmonary and peripheral level. An abnormal cytokine response, oxidative stress, tissue hypoxia, physical deconditioning and disease severity have been extensively studied [2, 23, 24] as potential triggers of peripheral muscle dysfunction in COPD. Redox-dependent and redox-independent pathways modulate key transcription factors such as NF-B, which plays a prominent role in regulating myogenesis via the control of cell cycle and muscle-specific transcription factors (i.e. Myo D) [25, 26]. NF-B is also known to regulate cell survival [27–29]. The analysis of these complexities is, however, clearly beyond the scope of this study. Increased GCS-HS mRNA expression after training in BMIL (fig. 1b) was interpreted as an adaptive mechanism to counteract exercise-induced oxidative stress in these patients. -GCS-HS is known to be upregulated in response to divergent stressful conditions including oxidative stress or GSH depletion [16, 30, 31]. GCS-HS mRNA levels showed a trend to a decrease in controls that was analyzed as a negative feedback regulation due to the increase in GSH levels. The negative correlation between GSH and the fall in GCS-HS mRNA levels (r = –0.95) after training supports this hypothesis. In summary, the current results indicate an association between muscle wasting and the abnormal response of the redox system in COPD. The impact of muscle wasting on the prognosis of the disease prompts the need for further studies clarifying the mechanisms involved in weight loss. In this respect, a proper characterization of the genomic determinants underlying the abnormal response of the GSH system may facilitate the identification of pivotal factors modulating the progress of the disease. Moreover, it may also help to guide the debate on the rationale for interventions increasing the redox potential of muscle cells in the subset of COPD patients with low BMI. Whether or not new pharmacological antioxidant agents now under development might be useful to enhance physiological adaptations to endurance training remains speculative.

Acknowledgments The authors would like to thank Felip Burgos, Conxi Gistau and Jose Luis Valera and all the technical staff of the Lung Function Laboratory for their skillful support during the study. Anna Capitán, Cristina Gonzalez and Eduard Vilar from EUIF Blanquerna are acknowledged for their outstanding work supervising the training sessions. We thank Carme Hernandez, coordinator nurse of the Home Care Unit, for her support in the logistics of

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(p = 0.06). In contrast, after training, the increase in GSH observed in healthy subjects was accompanied by a simultaneous tendency to a fall in GCS-HS mRNA expression in 4 of the 5 subjects (p = 0.09). No changes in GCS-HS mRNA expression were observed in BMIN (n = 8). The contrast analysis among groups (ANOVA, p = 0.05) showed statistically significant differences in GCS-HS mRNA regulation between each of the COPD groups (BMIL and BMIN) and healthy sedentary subjects.

the study. Finally, the authors acknowledge the material support received from Erich Jaeger to conduct the study. The study was supported by grants FIS 00/0281 from the Fondo de Investigaciones Sanitarias; E-Remedy (IST-2000-25146) from the European Union; Comissionat per a Universitats i Recerca de la Generalitat

de Catalunya (1999 SGR 00228) and Red Respira C03/011. R.A.R. was a Research Fellow supported by the European Respiratory Society, 2000, and A.M.M. was a Research Fellow supported by the Fundación CAPES, Brasil, 2001-02.

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