| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Pulmonary Diseases, University Hospital Nijmegen, 6500 HB Nijmegen; and 2 Departments of Pharmacology and Toxicology, University of Maastricht, 6200 MD Maastricht, The Netherlands
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Loading of skeletal muscles is associated with increased generation of oxidants, which in turn may impair muscle contractility. We investigated whether the load on the hamster diaphragm imposed by pulmonary emphysema induces oxidative stress, as indicated by glutathione oxidation, and whether the degree of glutathione oxidation is correlated with contractility of the diaphragm. In addition, the effect of 12 wk of treadmill exercise training on contractility and glutathione content in the normal (NH) and emphysematous hamster (EH) diaphragm was investigated. Training started 6 mo after elastase instillation. After the training period, glutathione content and in vitro contractility of the diaphragm were determined. Twitch force and maximal tetanic force were significantly reduced (by ~30 and ~15%, respectively) in EH compared with NH. In sedentary hamsters, the GSSG-to-GSH ratio was significantly elevated in the EH compared with the NH diaphragm. A significant inverse correlation was found between GSSG-to-GSH ratio and twitch force in the diaphragm (P < 0.01). Training improved maximal tetanic force and reduced fatigability of the EH diaphragm but did not alter its glutathione content. In conclusion, 1) emphysema induces oxidative stress in the diaphragm, 2) training improves the contractile properties of the EH diaphragm, and 3) this improvement is not accompanied by changes in glutathione redox status.
oxidative stress; respiratory muscles; pulmonary hyperinflation; contractile properties
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
SKELETAL MUSCLES GENERATE reactive oxygen species (ROS) at rest, and generation is increased during contractile activity (17, 27). ROS are required for optimal contractile function (27). However, overproduction of ROS, so-called oxidative stress, is associated with impaired force generation (6, 28). Overproduction of ROS by skeletal muscle during fatiguing contractions may lead to a nonpathological form of oxidative stress (1). Indeed, in the rat diaphragm, an inverse correlation exists between superoxide anion release during fatiguing contractions and the final stress developed (28).
Antioxidant defenses protect skeletal muscles from deleterious effects of ROS. GSH is probably the most important antioxidant in skeletal muscle. Intracellular glutathione metabolism is regulated by complex pathways (21). GSH serves as an antioxidant by reacting directly with free radicals and by providing substrate for glutathione peroxidase (GPX). Both direct and enzymatic oxidation of GSH results in the formation of GSSG, which is reconverted to GSH by glutathione reductase. Thus tissue glutathione status depends on the direct interaction of GSH with oxidants, the activity of the GPX and glutathione reductase redox cycle, and the ability of tissues to synthesize GSH. Elevated glutathione oxidation is considered a marker for oxidative stress (5). For instance, it has been shown that acute loading of the respiratory muscles by inspiratory resistive breathing increases glutathione oxidation in the rat diaphragm (34).
In contrast to acute loading, little is known about the effects of chronic loading on ROS generation in the diaphragm. However, this may be of major relevance to patients with chronic obstructive pulmonary disease (COPD). Respiratory muscle dysfunction frequently occurs in patients with COPD (25). Pulmonary hyperinflation, which is one of the key features of COPD (10), puts the diaphragm on a disadvantageous position of the length-tension curve and thereby chronically increases the load on the diaphragm.
The first hypothesis of the present study was that impaired contractility of the diaphragm due to pulmonary emphysema is accompanied by elevated glutathione oxidation in the diaphragm. We tested this hypothesis in the emphysematous hamster (EH), which is a frequently used animal model to study the effects of pulmonary hyperinflation on diaphragm function and morphology (12, 20, 36).
During general exercise training sessions, the diaphragm faces an elevated load because of increased ventilation. Previous research in healthy rodents revealed that training-induced elevation in oxidative capacity of the diaphragm is accompanied by increased antioxidant enzyme activity (23, 26). This indicates that the antioxidant screen of the diaphragm adapts to the elevated oxygen consumption rate. However, it is unknown whether training affects the glutathione status of the diaphragm. Only one study has been published on the effects of training on contractility of the EH diaphragm (11). In that study, general exercise training did not affect in vitro contractility of the diaphragm. In contrast to our EH model, in the study by Farkas and Roussos (11), pulmonary emphysema did not impair in vitro contractility of the diaphragm. This difference in baseline contractility of the diaphragm may affect the course of training responses. In a separate study, the same authors reported that training did not increase oxidant capacity of the EH diaphragm as expressed by citrate synthase (CS) activity (12). To our knowledge, the effects of training on antioxidant status in the EH diaphragm have not yet been studied.
The second hypothesis of the present study was that training improves contractility of the EH diaphragm, which is accompanied by a reduction in glutathione oxidation at rest. To answer this question, we trained EH on a motor-driven treadmill for 12 wk. Subsequently, the degree of glutathione oxidation and in vitro contractility of the diaphragm were assessed.
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Animals, Induction of Emphysema, and Study Design
All experiments were conducted on male Syrian hamsters. Animals were housed in a specific pathogen-free area, with controlled day and night cycle and were fed ad libitum. Hamsters were studied for the following reasons. First, antioxidant enzyme activities in humans are more similar to those in hamsters compared with rats (9). Second, the EH is a frequently used animal model to study the effects of pulmonary emphysema on diaphragm contractility and morphology. The hamster model has several advantages. The pathological lesions in the lung induced by elastase instillation have been studied in detail (31) and are quite similar to those in patients with emphysema. Furthermore, lung function changes in the EH resemble changes in patients with emphysema: increased pulmonary compliance, functional residual capacity, residual volume, and total lung capacity. In the present study, hamsters at 40 wk of age were intratracheally instilled with either porcine pancreas elastase [24 U/100 g body wt (EPC, Owensville, MI) in 0.50 ml 0.9% NaCl/100 g body wt] or an equal volume of 0.9% saline, as was described in detail previously (36). Six months after instillation, 15 EH and 15 normal hamsters (NH) were trained (EH-Tr and NH-Tr, respectively) on a motor-driven treadmill. Sedentary NH and EH were used as controls (n = 17 each group, NH-Sed and EH-Sed, respectively). These studies were approved by the Animal Ethics Committee, University of Nijmegen.Training
The day-and-night cycle was reversed to train the hamsters during their naturally most active period of the day. Training started 6 mo after instillation. In the week preceding the start of training, NH-Tr and EH-Tr were acclimatized to the treadmill by walking at a low speed for 15 min/day (5° inclination). An electrical grid in the rear of each running compartment was used to encourage running. The animals were exercised 5 days/wk for a 12-wk period. In the first week of the training program, running speed was 11.4 m/min for 40 min/day. In the following 5 wk, running speed and duration were gradually increased up to 20 m/min for 60 min/day. An attempt to increase running speed further was abandoned because EH were not able to maintain this speed. Thus, from the sixth until the twelfth week, both NH and EH ran 20 m/min, 60 min/day at 5° inclination for 5 days/wk. Subsequent experiments were performed 24-36 h after the last training session.General Procedures and Tissue Treatment
The hamsters were anesthetized with pentobarbital sodium (Nembutal, 70 mg/kg ip). A tracheotomy was performed, and a polyethylene cannula was inserted. The animals were mechanically ventilated with 100% O2. The diaphragm and adherent ribs were quickly excised after combined laparotomy and thoracotomy, and they were immediately submersed in cooled oxygenated Krebs solution at pH ~7.40. This Krebs solution consisted of 137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM KH2PO4, 24 mM NaHCO3, 7 mM glucose, and 25 µM d-tubocurarine (Sigma Chemical, Bornem, Belgium). The right middle costal hemidiaphragm was used for in vitro contractile measurements. The remainder of the right costal hemidiaphragm was used for CS activity measurement. From the left hemidiaphragm, ribs and connective tissue were removed, and the remainder was quickly frozen in liquid N2 and stored at
80°C for later glutathione
measurements. Meanwhile, the posterior-inferior area of the liver and
the intact soleus muscles were removed from the animal, quickly frozen
in liquid N2, and stored at 80°C for glutathione
measurements. There is no good peripheral counterpart in terms of
fiber-type composition of the diaphragm. The soleus muscle [80%
type I fibers (37)] was chosen arbitrarily as a control muscle
for the diaphragm to investigate whether any changes in glutathione
concentration of the diaphragm were the result of a systemic response.
Measurement of Contractile Properties
From the middle lateral costal region of the right hemidiaphragm, two rectangular strips were dissected parallel to the long axis of the muscle fibers. Silk sutures were tied firmly to both ends of the strip. The strips were suspended in two tissue baths containing Krebs solution, maintained at 37°C, pH 7.35-7.45, and perfused with a mixture of 95% O2 and 5% CO2. The central tendon end of each strip was connected to an isometric force transducer (model 31/1437-10; Sensotec, Columbus, OH) mounted on a micrometer. Two large platinum stimulation electrodes were placed parallel to the muscle strips. Stimuli were applied with a pulse duration of 0.2 ms and a train duration of 250 ms and were delivered by a stimulator (ID-electronics; University of Nijmegen, Nijmegen, The Netherlands) activated by a personal computer. To ensure supramaximal stimulation, the strips were stimulated at ~20% above the voltage at which maximal forces were obtained (~6 V applied through the stimulating electrodes). Data acquisition and storage of the amplified signal were done with a Dash-16 interface (Twist-trigger software, ID-electronics, University of Nijmegen) on a personal computer. Both strips were placed at their optimal length, defined as the length at which peak twitch tension (Pt) was obtained. The following protocols were performed in either both or one of the strips.Twitch characteristics. In each strip, two twitches were recorded to determine maximal Pt, contraction time (CT), and half relaxation time.
Maximal tetanic force. Both bundles were stimulated twice at 160 Hz to obtain a plateau in force generation. The maximal force was defined as the maximal tetanic force (Po).
Force-frequency characteristics. One of the bundles was randomly selected for studying force-frequency characteristics. The bundle was stimulated with 2-min intervals at the following frequencies: 15, 25, 50, 80, and 120 Hz. Both the absolute force and the percentage of Po were calculated.
Fatigability. To avoid impairment in force generation at the start of the fatigue protocol because of the measurement of force-frequency characteristics, in vitro fatigability was studied in the strip not used in force-frequency experiments. The strip was stimulated once every 2 s at 25 Hz, 330-ms train duration, for 120 s. After the completion of these measurements, the length of each strip was measured twice by using a micrometer (model 560-128, Mitutuoyo, Veenendaal, The Netherlands), and the strips were weighed. The cross-sectional area (CSA) was calculated by dividing diaphragm strip weight (g) by strip length (cm) times specific density (1.056). Forces were expressed per CSA (N/cm2), and the Pt-to-Po ratio was calculated for each muscle strip.
Verification of Emphysema
After the diaphragm was removed, the trachea was exposed, and an endotracheal cannula was inserted and held in place with silk sutures. Subsequently, the lungs were inflated with 0.9% saline to a pressure of 25 cmH2O. Fluid displacement was assessed as an estimation of lung volume.Biochemical Measurements
Glutathione.
GSSG and total glutathione (T-Glu) were measured enzymatically,
basically as described by Anderson (2). GSH concentration was
calculated by [T-Glu] 2 × [GSSG],
where brackets denote concentration. Because validation experiments
revealed that prolonged storage of tissue samples results in artificial
glutathione oxidation, samples were analyzed within 1 wk after excision
from the animals. In short, the tissues were thawed, minced, and
subsequently homogenized in phosphate-buffered saline. The samples were
diluted ten times in 1.3% 5-sulphosalicylic acid (in 10 mM HCl) to
prevent oxidation. For measurement of T-Glu, 100 µl of a
5,5'-dithiobis-2-nitrobenzoic acid-NADPH solution (final
concentration 0.2 and 0.16 µM, respectively) and 50 µl GSSG
reductase (final concentration 1 U/ml) were added to 50 µl of the
samples. All reagents were dissolved in 145 mM phosphate buffer (pH
7.4) to neutralize the acid. 5-Thio-nitrobenzoic acid was determined at
405 nm in a microplate reader.
CS. CS activity was measured in the remains of the right hemidiaphragm after two muscle strips were excised. Frozen muscles were thawed in ice-cooled buffer containing 250 mM sucrose, 2 mM EDTA, and 10 mM Tris · HCl (pH 7.4). In this buffer, muscle homogenates (5% wt/vol) were prepared. CS activity was measured at 25°C as described by Shepherd and Garland (30) and was expressed in micromoles of coenzyme A formed per minute per gram of tissue.
Data Analysis
Single data (i.e., Pt, Po, GSSG, CS) were compared among groups by using one-way ANOVA. If appropriate, Student Newman Keuls post hoc tests were performed. Repeated-measurements ANOVA was used for comparing force-frequency characteristics and fatigability. The bivariate Pearson correlation coefficient was calculated to test for significant correlations between glutathione oxidation and force generation. All tests were performed by using the SPSS/PC+ package version 8.0 (Chicago, IL) Results were considered significant at P < 0.05. All data are expressed as means ± SE.| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Animal Characteristics
One of the EH did not complete the training program because of serious limb injury. The remaining 29 hamsters successfully completed all training sessions. After several days of treadmill exercise, electrical stimulation was needed only sporadically to encourage running. At the end of each training session, EH were severely fatigued as indicated by tachypnea and lethargy. Some EH were evidently cyanotic, and wheezing was audible after the training session. Instillation with elastase had profound effects on body weight and lung volume of hamsters (Table 1). Elastase treatment resulted in severe pulmonary emphysema, as indicated by an ~35% increase in lung volume. Exercise training further increased lung volume in EH.
|
In Vitro Contractile Properties of the Diaphragm
Pulmonary emphysema significantly reduced Pt, Po, and optimal length of the diaphragm strips compared with NH-Sed (Table 2). Pt and Po were ~30 and ~15% lower in EH-Sed compared with NH-Sed, respectively. In the EH-Sed, normalized force-frequency responses were significantly impaired at frequencies up to 60 Hz compared with that in NH-Sed (Fig. 1). This indicates that, because of elastase treatment, besides a downward shift, a rightward shift of the force-frequency curve also occurred. Furthermore, fatigability of the EH-Sed was significantly higher compared with that of the NH-Sed diaphragm (Fig. 2).
|
|
|
General exercise training did not significantly change Pt in NH or EH but significantly increased CT in EH. Maximal force-generating capacity of the EH diaphragm increased by ~15% due to training (P < 0.05, Table 2). In fact, training abolished the detrimental effects of emphysema on Po. In addition, training did not affect the shape of the normalized force-frequency curve in NH or EH. Training had a small but significant beneficial effect on fatigability of the EH diaphragm.
Tissue Glutathione Measurements
Emphysema did not affect glutathione levels in the diaphragm (Table 3). Although GSSG concentration in the EH-Sed was ~34% higher compared with that in NH-Sed, this did not reach statistical significance (P = 0.18, ANOVA). However, the GSSG-to-GSH ratio (GSSG/GSH) was significantly higher in the EH diaphragm compared with that in the NH diaphragm (9.1 ± 0.8 vs. 5.9 ± 0.6%, respectively, Fig. 3A).
|
|
Endurance training did not significantly affect GSH or GSSG levels in the diaphragm of NH or EH. Also, the GSSG/GSH in the diaphragm was not affected by training (7.8 ± 0.7 vs. 10.5 ± 1.1% in NH-Tr and EH-Tr, respectively).
Neither emphysema nor training had significant effects on GSH or GSSG levels or GSSG/GSH in soleus muscle or liver (Table 3, Fig. 3, B and C). T-Glu, GSH, and GSSG levels were significantly higher in the diaphragm compared with that in soleus muscle for all groups. Furthermore, liver glutathione content was significantly higher compared with that in diaphragm and soleus muscle in all groups.
Correlation Between Force Generation and Glutathione Oxidation
To test the first hypothesis of the present study, we made further correlations between glutathione oxidation and in vitro force generation of the diaphragm. If oxidative stress impairs contractility of the diaphragm, an inverse correlation should be present between markers for these variables. Indeed, we found a significant inverse correlation between GSSG/GSH and Pt (Pearson correlation coefficient =0.48, P < 0.01, Fig.
4A). No correlation exits between
GSSG/GSH and Po (Pearson correlation coefficient = 0.26, P > 0.05, Fig. 4B).
|
Oxidative Capacity of the Diaphragm
CS activity was used as a marker of oxidative capacity of the diaphragm. No significant differences in CS activity were observed between NH-Sed and EH-Sed (1.24 ± 0.12 vs. 0.98 ± 0.11, respectively, P > 0.05).General exercise training did not affect CS activity in the NH diaphragm (1.24 ± 0.12 vs. 1.02 ± 0.60 in NH-Sed and NH-Tr, respectively, P > 0.05). However, in the EH, training increased oxidative capacity of the diaphragm (0.98 ± 0.11 vs. 1.35 ± 0.14 in EH-Sed and EH-Tr, respectively, P < 0.05, Student t-test).
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The results of our experiments show that, in hamsters, the load on the respiratory muscles imposed by pulmonary emphysema impairs in vitro contractility of the diaphragm, which is accompanied by increased generation of ROS, as indicated by elevated glutathione oxidation in the diaphragm. In fact, a significant inverse correlation exists between GSSG/GSH and Pt in the diaphragm. Second, general exercise training improves maximal force-generating capacity of the diaphragm in EH. However, contrary to our expectation, exercise training did not affect glutathione redox status of the diaphragm in EH.
Effects of Emphysema on Diaphragm Force Generation and Glutathione Content
Reduction in diaphragm muscle strip length and impairment in in vitro contractility after treatment with elastase have been reported previously by others and by studies from our laboratory (19, 20, 36), although some other groups did not find changes in in vitro contractility (11, 33). Possible explanations for the reduction in contractility due to emphysema have been discussed previously (20), such as alterations in the contractile protein composition, changes in noncontractile proteins (sarcoplasmic, stromal, and mitochondrial), and changes in the intrinsic characteristics of muscle fibers or disturbances in calcium uptake and release. Changes in fiber-type frequency or CSA may also contribute. Data on fiber-type composition in the EH diaphragm are conflicting. It has been shown that the relative contribution of type I fibers is increased in the EH diaphragm (12), whereas other studies reported hypertrophy of type II fibers in the EH diaphragm (20). We did not find any effect of emphysema on diaphragm muscle fiber frequency or CSA (36). Thus, in our animal model of emphysema, it is unlikely that changes in diaphragm morphology fully explain impaired in vitro force generation. It is likely that other factors contributed to the negative effects of emphysema on contractility, such as oxidative stress.GSSG/GSH is frequently used as a marker for oxidative stress. The latter is defined as a disturbance between the prooxidant and antioxidant balance, in favor of the former. GSSG/GSH is a good indicator of oxidative stress (5) and is considered an important regulator of the function of enzymes, receptors, transporters, and transcription factors (14). Accurate measurement of GSH/GSSG is difficult because of GSH autooxidation. Because GSSG is only present in minimal amounts compared with GSH, a small GSH autooxidation during sample processing can give erroneously high GSSG levels. To prevent GSH autooxidation, GSH trapping agents, such as N-ethylmaleimide and 2-vinyl pyridine, have been used. Although HPLC is assumed to be the state-of-the-art technique for measurement of GSH and GSSG, it has been shown that, at least in solid tissue, an enzymatic assay as used in the present study is a valid alternative to the much more time-consuming HPLC technique (13).
In the present study, GSSG/GSH in the diaphragm was significantly higher in EH-Sed compared with NH-Sed. This indicates that, even at rest, the EH diaphragm is in a more oxidized state compared with the NH diaphragm. In vitro studies using rat diaphragm showed that oxidants accumulate intracellularly and contribute directly to the loss of contractile function, which occurs in muscular fatigue (27). Other studies have confirmed the association between increased generation of ROS and muscular fatigue (17), but the present study is the first to investigate the oxidant status of the EH diaphragm. In other models for loading of the respiratory muscles, such as inspiratory resistive breathing, in vitro force generation is also impaired, and glutathione oxidation increased (4, 34). In these latter studies, GSSG/GSH in the rat diaphragm increased up to ~28%, whereas in the present study this ratio was ~9% in the EH. These differences in the degree of glutathione oxidation are probably the result of differences in the duration and severity of loading. In inspiratory resistive breathing, a strenuous load is acutely applied, resulting in ventilatory failure and apnea within ~30 min, whereas in the EH a less severe load is applied continuously for a prolonged period of time. Indeed, when inspiratory resistive breathing was discontinued before the development of apnea (7) or when the loading was applied by tracheal banding for 4-12 days (32), changes in diaphragm glutathione content were less severe (7). The association between oxidant status and force generation is also demonstrated by the significant correlation between GSSG/GSH and Pt in the diaphragm in the present study (Fig. 4A), although no such correlation existed for GSSG/GSH and Po (Fig. 4B). It has been recognized previously that submaximal force is more sensitive to oxidative stress than maximal force. For instance, prolonged exposure of mouse skeletal muscle fibers to H2O2 reduced maximal force generation by 23%, whereas submaximal force was reduced by 79% (3). Reduction in (sub)maximal force generation by oxidants could arise from modification of different steps in the excitation-contraction coupling process, including oxidation of hyperreactive thiols on the myosin head (24), modification of intracellular sulphydryl groups on Ca2+ release channels (8), and reduced Ca2+ sensitivity (3). The present study was not designed to identify the target for elevated generation of oxidants. However, because in addition to a downward shift a rightward shift of the force-frequency curve also occurred (Fig. 1), it is likely that oxidants have effects on noncontractile proteins as well.
The liver plays a prominent role in GSH homeostasis, and hepatic export of GSH is the major source for plasma glutathione (22). Thus, theoretically, it is possible that emphysema altered GSH status of the liver. However, this did not occur in the present study, indicating that the liver could fulfill the glutathione demands of the other tissues.
Soleus muscle glutathione content and GSSG/GSH were not affected by emphysema. This indicates that the alterations in GSSG/GSH in the diaphragm were not the result of a systemic inflammatory response following elastase instillation. Although EH had significantly lower body weight compared with NH, it is unlikely that a catabolic response attributed to changes in glutathione status in the diaphragm, because no such changes were observed in the limb skeletal muscles. Thus changes in diaphragm GSSG/GSH in EH are most likely the direct result of the increased loading.
CS activity in the diaphragm tended to be lower in the EH-Sed compared with NH-Sed. This is in apparent contrast to other studies (12, 20) in which CS activity was increased. However, our findings are in line with the increased fatigue rate of the EH compared with NH diaphragm found in the present study. The degree of hyperinflation due to elastase treatment was less severe in the present study compared with previously published studies (i.e., Ref. 20). This may have contributed to the lack of response of CS activity in the diaphragm due to hyperinflation.
Effects of Training on Diaphragm Contractility and Glutathione Status
The beneficial effects of exercise training on contractility of the EH diaphragm are in apparent contrast to previous studies by Farkas and Roussos (11). These differences in outcome are probably the result of differences in experimental setup. As mentioned earlier, in the study by Farkas and Roussos, elastase instillation did not affect contractility of the diaphragm at optimal fiber length, indicating the absence of respiratory muscle dysfunction in their hamster model. Possibly the load on the respiratory muscles of the EH was higher in our study. It has been postulated that the threshold for obtaining a training effect is much higher in the diaphragm compared with limb skeletal muscle (16). Because of the absence of respiratory muscle dysfunction, it is possible that this threshold was not exceeded in the study by Farkas and Roussos (11), and thus no effect of training on contractility was achieved. Second, in the present study, training started 6 mo after instillation with elastase, whereas Farkas and Roussos started training 3 wk after instillation. It has been demonstrated that changes in lung volume occur up to 26 wk after instillation with elastase (31). Thus part of the differences in outcome between their and our data may be explained by differences in the experimental setup.Training reduced fatigability of the EH diaphragm, which is in accordance with training-induced elevation in CS activity of the EH diaphragm. In contrast, contractility of the NH diaphragm was not affected by endurance exercise training. This is probably due to the fact that the training stimulus was not strenuous enough for the NH diaphragm. This is in line with our expectations, as the training program was designed for EH. Indeed, increasing running speed beyond 20 m/min was abandoned because the EH could not sustain this speed. In contrast to the NH, the EH appeared severely fatigued at the end of each training session.
Training-induced elevation in CS activity in the diaphragm of EH increases oxygen flux through the mitochondria, which may favor generation of ROS. Despite the effects of training on contractility and oxidative capacity of the EH diaphragm, training did not affect glutathione status of the diaphragm at rest. No other studies have been published regarding the effects of exercise training on glutathione status in the (EH) diaphragm. In addition, scarce literature is available concerning the effects of exercise training on glutathione status of limb skeletal muscles. In line with our observations, training did not affect the T-Glu level of highly oxidative rat limb skeletal muscles such as the red gastrocnemius (29) and the soleus muscle (18). However, the absence of a response on muscle glutathione status after training does not necessarily imply that antioxidant capacity was unaffected. Sen et al. (29) found that GPX activity increased in response to training, whereas glutathione content was unaltered. Upregulation of antioxidant enzyme activity was reported in other studies as well. Superoxide dismutase activity of the diaphragm increased after 10 wk of endurance training in rats (26) and mice (23). Also, in rats GPX activity of the diaphragm increased after training (23, 26). Moreover, a significant correlation existed between GPX and CS activity (26). Thus it is conceivable that endurance training increases enzymatic antioxidant capacity of skeletal muscles, including the diaphragm, without affecting glutathione status at rest. This suggests that, after training, the diaphragm is better equipped to sustain increased generation of oxidants, for instance, as induced by an acute bout of exercise.
Clinical Relevance
Pulmonary hyperinflation, which is a key feature of COPD, has detrimental effects on diaphragm function (10). This is of importance because a significant correlation exists between respiratory muscle strength and exercise tolerance in COPD (15). Thus strategies to improve respiratory muscle function in COPD are of major clinical importance. The present study shows that pulmonary hyperinflation is associated with oxidative stress in the diaphragm and that a significant correlation exists between in vitro contractility and oxidant status of the diaphragm. Based on these findings, we speculate that, in patients with COPD, the diaphragm is continuously exposed to oxidant stress. This implies that treatment with antioxidants might be able to improve respiratory muscle function. Indeed, it has been demonstrated that administration of the antioxidant N-acetylcysteine, a precursor of glutathione, to healthy subjects before inspiratory resistive breathing attenuates the fall in transdiaphragmatic pressure and increases task endurance (35). However, no such studies have been performed in patients with COPD. Furthermore, we showed that endurance training reversed the detrimental effects of hyperinflation on contractility of the diaphragm, indicating that, in patients with COPD, exercise training may improve diaphragm function and thus exercise tolerance.In conclusion, elastase-induced pulmonary emphysema impairs in vitro contractility of the hamster diaphragm, which is accompanied by alterations in the redox status of the diaphragm. The significant inverse correlation between submaximal force generation and GSSG/GSH suggests an important role of oxidative stress in impaired force generation in the diaphragm of EH. Endurance training did not affect the glutathione status of the diaphragm but reversed the detrimental effects of emphysema on maximal force-generating capacity of the diaphragm.
| |
ACKNOWLEDGEMENTS |
|---|
We thank D. Smits, H. van Moerkerk, and I. Dekker for expert biotechnical and biochemical assistance, and Prof. J. Veerkamp, Dept. of Biochemistry, University of Nijmegen, for the advice on biochemical measurements.
| |
FOOTNOTES |
|---|
This study was financially supported by Dutch Asthma Foundation Grant 97.34.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. N. R. Dekhuijzen, Dept. of Pulmonary Diseases, Univ. Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands (E-mail: R.Dekhuijzen{at}long.azn.nl).
Received 15 September 1999; accepted in final form 9 February 2000.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
American Thoracic Society and European Respiratory Society.
Skeletal muscle dysfunction in chronic obstructive pulmonary disease.
Am J Respir Crit Care Med
159:
S1-S40,
1999
2.
Anderson, ME.
Determination of glutathione and glutathione disulfide in biological samples.
Methods Enzymol
113:
548-555,
1985[ISI][Medline].
3.
Andrade, FH,
Reid MB,
Allen DG,
and
Westerblad H.
Effect of hydrogen peroxide and dithiothreitol on contractile function of single skeletal muscle fibres from the mouse.
J Physiol (Lond)
509:
565-575,
1998
4.
Anzueto, A,
Andrade FH,
Maxwell LC,
Levine SM,
Lawrence RA,
Gibbons WJ,
and
Jenkinson SG.
Resistive breathing activates the glutathione redox cycle and impairs performance of rat diaphragm.
J Appl Physiol
72:
529-534,
1992
5.
Asensi, M,
Sastre J,
Pallardo FV,
Lloret A,
Lehner M,
Garcia DD-A,
and
Viña J.
Ratio of reduced to oxidized glutathione as indicator of oxidative stress status and DNA damage.
Methods Enzymol
299:
267-276,
1999[ISI][Medline].
6.
Barclay, JK,
and
Hansel M.
Free radicals may contribute to oxidative skeletal muscle fatigue.
Can J Physiol Pharmacol
69:
279-284,
1991[ISI][Medline].
7.
Borzone, G,
Julian MW,
Merola AJ,
and
Clanton TL.
Loss of diaphragm glutathione is associated with respiratory failure induced by resistive breathing.
J Appl Physiol
76:
2825-2831,
1994
8.
Brotto, MAP,
and
Nosek TMRA
Hydrogen peroxide disrupts Ca2+ release from the sarcoplasmic reticulum of rat skeletal muscle fibers.
J Appl Physiol
81:
731-737,
1996
9.
Bryan, CL,
and
Jenkinson SG.
Species variation in lung antioxidant enzyme activities.
J Appl Physiol
63:
597-602,
1987
10.
Decramer, M.
Effects of hyperinflation on the respiratory muscles.
Eur Respir J
2:
299-302,
1989[ISI][Medline].
11.
Farkas, GA,
and
Roussos C.
Adaptability of the hamster diaphragm to exercise and/or emphysema.
J Appl Physiol
53:
1263-1272,
1982
12.
Farkas, GA,
and
Roussos C.
Histochemical and biochemical correlates of ventilatory muscle fatigue in emphysematous hamsters.
J Clin Invest
74:
1214-1220,
1984.
13.
Floreani, M,
Petrone M,
Debetto P,
and
Palatini P.
A comparison between different methods for the determination of reduced and oxidized glutathione in mammalian tissues.
Free Radic Res
26:
449-455,
1997[ISI][Medline].
14.
Gilbert, HF.
Thiol/disulfide exchange equilibria and disulfide bond stability.
Methods Enzymol
251:
8-28,
1995[ISI][Medline].
15.
Gosselink, R,
Troosters T,
and
Decramer M.
Peripheral muscle weakness contributes to exercise limitation in COPD.
Am J Respir Crit Care Med
153:
976-980,
1996[Abstract].
16.
Green, HJ,
Plyley MJ,
Smith DM,
and
Kile JG.
Extreme endurance training and fiber type adaptation in rat diaphragm.
J Appl Physiol
66:
1914-1920,
1989
17.
Kolbeck, RC,
She ZW,
Callahan LA,
and
Nosek TM.
Increased superoxide production during fatigue in the perfused rat diaphragm.
Am J Respir Crit Care Med
156:
140-145,
1997
18.
Leeuwenburgh, C,
Hollander J,
Leichtenweis S,
Griffiths N,
Gore M,
and
Ji LLRA
Adaptations of glutathione antioxidant system to endurance training are tissue and muscle fiber specific.
Am J Physiol Regulatory Integrative Comp Physiol
272:
R363-R369,
1997
19.
Lewis, M,
Monn S,
Zhan W,
and
Sieck G.
Interactive effects of emphysema and malnutrition on diaphragm structure and function.
J Appl Physiol
77:
947-955,
1994
20.
Lewis, MI,
Zhan WZ,
and
Sieck GC.
Adaptations of the diaphragm in emphysema.
J Appl Physiol
72:
934-943,
1992
21.
Meister, A.
Glutathione metabolism.
Methods Enzymol
251:
3-7,
1995[ISI][Medline].
22.
Meister, A,
and
Anderson ME.
Glutathione.
Annu Rev Biochem
52:
711-760,
1983[ISI][Medline].
23.
Ohishi, S,
Kizaki T,
Ookawara T,
Sakurai T,
Izawa T,
Nagata N,
and
Ohno HRA
Endurance training improves the resistance of rat diaphragm to exercise induced oxidative stress.
Am J Respir Crit Care Med
156:
1579-1585,
1997
24.
Perkins, WJ,
Han YS,
and
Sieck GC.
Skeletal muscle force and actomyosin ATPase activity reduced by nitric oxide donor.
J Appl Physiol
83:
1326-1332,
1997
25.
Polkey, MI,
Kyroussis D,
Hamnegard CH,
Mills GH,
Green M,
and
Moxham J.
Diaphragm strength in chronic obstructive pulmonary disease.
Am J Respir Crit Care Med
154:
1310-1317,
1996[Abstract].
26.
Powers, SK,
Criswell D,
Lawler J,
Martin D,
Ji LL,
Herb RA,
and
Dudley G.
Regional training-induced alterations in diaphragmatic oxidative and antioxidant enzymes.
Respir Physiol
95:
227-237,
1994[ISI][Medline].
27.
Reid, MB,
Haack KE,
Franchek KM,
Valberg PA,
Kobzik L,
and
West MS.
Reactive oxygen in skeletal muscle. I. Intracellular oxidant kinetics and fatigue in vitro.
J Appl Physiol
73:
1797-1804,
1992
28.
Reid, MB,
Shoji T,
Moody MR,
and
Entman ML.
Reactive oxygen in skeletal muscle. II. Extracellular release of free radicals.
J Appl Physiol
73:
1805-1809,
1992
29.
Sen, CK,
Marin E,
Kretzschmar M,
and
Hanninen O.
Skeletal muscle and liver glutathione homeostasis in response to training, exercise, and immobilization.
J Appl Physiol
73:
1265-1272,
1992
30.
Shepherd, D,
and
Garland PD.
Citrate synthase from rat liver.
Methods Enzymol
13:
11-16,
1969.
31.
Snider, GL,
and
Sherter CB.
A one-year study of the evolution of elastase-induced emphysema in hamsters.
J Appl Physiol
43:
721-729,
1977
32.
Supinski, G,
Nethery D,
Stofan D,
Hirschfield W,
and
DiMarco A.
Diaphragmatic lipid peroxidation in chronically loaded rats.
J Appl Physiol
86:
651-658,
1999
33.
Supinski, GS,
and
Kelsen SG.
Effect of elastase-induced emphysema on the force-generating ability of the diaphragm.
J Clin Invest
70:
978-988,
1982.
34.
Supinski, GS,
Stofan D,
Ciufo R,
and
DiMarco A.
N-acetylcysteine administration and loaded breathing.
J Appl Physiol
79:
340-347,
1995
35.
Travaline, JM,
Sudarshan S,
Roy BG,
Cordova F,
Leyenson V,
and
Criner J.
Effect of N-acetylcysteine on human diaphragm strength and fatigability.
Am J Respir Crit Care Med
156:
1567-1571,
1997
36.
Van der Heijden, HF,
Dekhuijzen PN,
Folgering H,
Ginsel LA,
and
van Herwaarden CL.
Long-term effects of clenbuterol on diaphragm morphology and contractile properties in emphysematous hamsters.
J Appl Physiol
85:
215-222,
1998
37.
Wilcox, PG,
Hards JM,
Bockhold K,
Bressler B,
and
Pardy RL.
Pathologic changes and contractile properties of the diaphragm in corticosteroid myopathy in hamsters: comparison to peripheral muscle.
Am J Respir Cell Mol Biol
1:
191-199,
1989.
This article has been cited by other articles:
|
|
 |
|
  M. Kristensen and T. Hansen Statistical analyses of repeated measures in physiological research: a tutorial Advan Physiol Educ, March 1, 2004; 28(1): 2 - 14. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.W. Boots, G.R.M.M. Haenen, and A. Bast Oxidant metabolism in chronic obstructive pulmonary disease Eur. Respir. J., November 2, 2003; 22(46_suppl): 14s - 27s. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M. A. Heunks, H. A. Machiels, R. de Abreu, X. Ping Zhu, H. F. M. van der Heijden, and P. N. R. Dekhuijzen Free radicals in hypoxic rat diaphragm contractility: no role for xanthine oxidase Am J Physiol Lung Cell Mol Physiol, December 1, 2001; 281(6): L1402 - L1412. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. C. POOLE, C. A. KINDIG, and B. J. BEHNKE Effects of Emphysema on Diaphragm Microvascular Oxygen Pressure Am. J. Respir. Crit. Care Med., April 1, 2001; 163(5): 1081 - 1086. [Abstract] [Full Text]
|
|
|
|
 |
|
 L. M A Heunks and P N R. Dekhuijzen Respiratory muscle function and free radicals: from cell to COPD Thorax, August 1, 2000; 55(8): 704 - 716. [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
significant difference between
EH-Sed and all other groups (P < 0.05, repeated-measurements
ANOVA, SNK post hoc testing).
