1 Department of Pulmonary Diseases and 2 Department of Paediatrics, University Medical Centre Nijmegen, 6500 HB Nijmegen, The Netherlands
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ABSTRACT |
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Recent evidence indicates that hypoxia enhances the generation of oxidants. Little is known about the role of free radicals in contractility of the rat diaphragm during hypoxia. We hypothesized that antioxidants improve contractility of the hypoxic rat diaphragm and that xanthine oxidase (XO) is an important source of free radicals in the hypoxic diaphragm. The effects of N-acetylcysteine (NAC; 18 mM), Tiron (10 mM), and the XO inhibitor allopurinol (250 µM) were studied on isometric and isotonic force generation during hypoxia (PO2 ~7 kPa). NAC and Tiron decreased maximal force generation, slowed the shortening velocity, and decreased the power output. Fatigue rate was decreased in the presence of either NAC or Tiron. Allopurinol did not alter the contractility or fatigability of the diaphragm. During hyperoxia (PO2 ~85 kPa), neither NAC nor allopurinol affected the contractility or fatigability of the diaphragm. Thus free radicals play a significant role in diaphragm contractility during hypoxia. Whether antioxidants exert a beneficial or harmful effect on muscle performance depends on the contraction pattern of the muscle. Free radicals generated by XO do not play a role in diaphragm contractility during either hypoxia or hyperoxia.
isotonic contractions; allopurinol; Tiron; N-acetylcysteine; respiratory muscles
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INTRODUCTION |
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FREE RADICALS ARE IMPORTANT MODULATORS of respiratory muscle function (2, 18, 28). Scavenging free radicals in the unfatigued rat diaphragm impairs in vitro force generation (28), indicating that free radicals are essential for optimal contractile function. However, oxidative stress, an imbalance between oxidants and antioxidants in favor of the former, impairs skeletal muscle contractility (20). Recently, data from our laboratory (13) showed that markers for free radical generation inversely correlate with in vitro force generation of the diaphragm in an animal model of pulmonary emphysema. Fatiguing contractions enhance the generation of free radicals in the rat diaphragm muscle (19), and overproduction of free radicals during contractile activity is associated with the development of respiratory muscle fatigue (30). Scavenging free radicals during strenuous contractions reduces fatigability of the rat diaphragm in vitro (18).
Hypoxia, which is a common feature in several diseases including chronic obstructive pulmonary disease (COPD), has been found to accelerate skeletal muscle fatigue in vitro (31, 38); although in some other studies (10, 31), hypoxia did not affect in vitro fatigability. It has been shown that hypoxia enhances the generation of free radicals in rat cardiac tissue (26). Mohanraj et al. (21) found that free radical scavengers such as N-acetylcysteine (NAC) and Tiron improve in vitro force generation of the rat diaphragm under hypoxic conditions.
To date, no study has evaluated the effect of free radical scavengers on the shortening velocity or power output in striated muscle during hypoxia. This could be important because isotonic contractile properties better reflect diaphragm muscle performance in vivo. In addition, there is no a priori reason to believe why force and velocity should be affected to the same extent because the underlying cellular mechanisms are different. Morrison et al. (24) showed that inhibiting the synthesis of nitric oxide does not affect the maximal force but reduces the maximal shortening velocity (Vmax) and maximal power output of the rat diaphragm in vitro. Based on previous studies (8, 26) showing elevated oxidant production in hypoxic tissues, the first hypothesis of the present study is that free radical scavengers increase the Vmax and maximal power output of the rat diaphragm during severe hypoxia. The effects of scavengers will be more pronounced during fatiguing contractions when the generation of free radicals is further increased. To test this hypothesis, we determined the effects of NAC and Tiron on isometric and isotonic contractile properties of the hypoxic rat diaphragm in vitro.
Cellular sources for free radicals during hypoxia have not yet been identified. During normoxia, the mitochondrial electron transport chain is an important source of free radicals (5). Xanthine oxidase (XO) can generate superoxide in the presence of (hypo)xanthine, a chemical reaction that is largely triggered and controlled by substrate availability (41). Strenuous contractile activity, especially in the presence of hypoxia, may favor ATP degradation in skeletal muscle, resulting in an accumulation of hypoxanthine (12), which, in turn, may enhance the generation of free radicals by XO (41). Recently, Heunks et al. (15) found that allopurinol (a competitive XO inhibitor) prevents exercise-induced oxidative stress in patients with severe COPD. It is conceivable that this was at least partly the result of blocking XO in skeletal muscle. These and other studies (41) suggest that XO may be involved in the generation of free radicals in skeletal muscle during hypoxia, which, in turn, may affect contractility. The second hypothesis of the present study is that free radicals generated by XO play a prominent role in hypoxia-induced impairment in force generation of the diaphragm.
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METHODS |
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Study Design
The role of free radicals in contractile performance of the hypoxic rat diaphragm in vitro was assessed. In addition, the contribution of XO-derived free radicals on diaphragm contractility during hypoxia was investigated. Accordingly, the effects of two chemically distinct antioxidants, NAC and Tiron, and of the XO blocker allopurinol were tested on contractility and fatigability of the hypoxic rat diaphragm in vitro. Three control groups were studied: hyperoxia control, hyperoxia plus NAC, and hyperoxia plus allopurinol. The study was approved by the Animal Ethics Committee, University of Nijmegen (Nijmegen, The Netherlands).General Procedures
Adult male outbred Wistar rats aged 12-16 wk with a mean weight of 306 ± 6 (SE) g were used. The animals were housed in a specific pathogen-free unit and fed ad libitum. The rats were anesthetized with pentobarbital sodium (70 mg/kg body wt ip). Diaphragm bundles were prepared as previously described (36, 37). Briefly, a tracheotomy was performed, and a polyethylene cannula was inserted. The animals were mechanically ventilated with 100% O2. The diaphragm and adherent lower ribs were quickly excised after a combined laparotomy and thoracotomy and were immediately submersed in cooled oxygenated (95% O2-5% CO2) Krebs solution at pH ~7.4. 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, Bornem, Belgium).From the central costal region of the right and the left hemidiaphragm, one rectangular bundle from each was dissected parallel to the long axis of the muscle fibers. Silk sutures were tied firmly to both ends. The strips were mounted vertically in separate tissue baths containing Krebs solution, maintained at 26°C, and perfused with a 95% O2-5% CO2 mixture. In general, the bundle excised from the right hemidiaphragm was used for isotonic contractile properties and the bundle from the left hemidiaphragm was used in the repetitive twitch experiments (see Repetitive Twitch Experiments). In total, 81 rats were used in the present study.
Isotonic Contractile Properties
The insertion of the muscle bundle at the costal margin was attached to a metal clamp mounted in series with a micromanipulator at the base of the tissue bath. The suture attached to the central tendon was connected to the lever arm of a dual-mode length-force servo control system (Cambridge Technologies model 308B) with a steel hook.The Cambridge system was controlled with Poly-5 based software (Inspektor Research Systems, Amsterdam, The Netherlands) running on a Pentium personal computer. Length and force could be controlled independently, allowing the Cambridge system to operate in either the isometric or isotonic mode, respectively. Length and force outputs were digitized with a data acquisition board (DASH 1602, Keithley) at a sampling frequency of 2.0 kHz.
The muscle was stimulated directly with platinum plate electrodes placed on either side of the muscle bundle. Rectangular current pulses (0.5 ms) were generated by a stimulator (ID-electronics, University of Nijmegen) activated by a personal computer. To ensure supramaximal stimulation, the strips were stimulated at 1.25 times the current needed for maximal activation (~200-250 mA). Muscle preload force was adjusted with the micromanipulator until the optimal fiber length (Lo) for maximal twitch force (Pt) was achieved.
The Cambridge system was first set for length control (isometric mode). After 15 min of thermoequilibration, both Pt and maximal tetanic force (Po) at 100 Hz were determined twice, with a 2-min interval between subsequent stimulations. Next, the Krebs solution and gas mixture were changed to the appropriate experimental condition (see Effects of Free Radical Scavengers and XO Inhibition on Rat Diaphragm Isotonic Contractile Properties During Hypoxia for experimental groups). After 60 min of drug equilibration, measurements of Pt and Po were repeated. Subsequently, the Cambridge system was set for force control (isotonic mode). The muscle was stimulated at 100 Hz (330-ms train duration) while force was clamped at different levels ranging from 1 to 100% of Po. There was a 2-min interval between each force clamp level. The muscle shortening velocity at each clamp level was calculated as the change in muscle length during a 30-ms period and is expressed as muscle lengths per second (Lo/s).
To determine isotonic fatigue, the load clamp level was set for maximum power output (assumed a priori to be ~33.3% of Po in all groups), and the muscle was stimulated at 100 Hz (330-ms train duration) every 2 s. Stimulation continued until no muscle shortening could be observed, and this was defined as the isotonic endurance time.
Repetitive Twitch Experiments
A previous study (2) demonstrated that free radicals exert time-dependent effects on skeletal muscle contractility. Because this may also be the case for antioxidants, we tested the effects of antioxidants on Pt for a 90-min period. The origin of the muscle was tied to a glass hook fixed to the bottom of the tissue bath. The central tendon end was connected to an isometric force transducer (model 31/1437-10, Sensotec, Columbus, OH) mounted on a micrometer. Two large platinum electrodes were placed parallel to the bundles. Stimuli were applied with a pulse duration of 0.2 ms and duration of 400 ms and were delivered by a stimulator (ID-electronics) activated by a personal computer. Data acquisition and storage of the amplified signal were performed with a DASH 1602 interface on a personal computer (Twist-trigger software, ID-electronics). The strip was placed at Lo, and after a 15-min thermoequilibration period, baseline Po and Pt were determined. After the gas mixture and Krebs solution were changed to the appropriate experimental condition (see Effects of Free Radical Scavengers and XO Inhibition on Rat Diaphragm Isotonic Contractile Properties During Hypoxia), Pt was measured at 2-min intervals for 90 min in three different experimental groups, namely, hypoxia control (n = 9 bundles), hypoxia plus NAC (n = 8 bundles), and hypoxia plus allopurinol (n = 9 bundles). The pH of the Krebs solution was measured at regular intervals and maintained between 7.35 and 7.45.Gas Tension Analysis
In pilot experiments, gas tension and pH of the Krebs solution were measured at regular intervals. After 15 min of thermoequilibration, the gas mixture in the "hypoxic groups" was switched to 5% CO2 and 95% N2. As shown in Fig. 1, this resulted in a rapid decline in O2 tension of the Krebs solution (n = 8 bundles). In the hyperoxic groups, the gas mixture was maintained at 95% O2 and 5% CO2. In these groups, the PO2 of the Krebs solution did not change significantly throughout the experiments. In both groups, pH was maintained between 7.35 and 7.45.
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To verify the experimental conditions in the present studies, pH, PCO2, and PO2 of the Krebs solutions were measured after completion of the contractile experiments (isotonic or repetitive twitch).
Effects of Free Radical Scavengers and XO Inhibition on Rat Diaphragm Isotonic Contractile Properties During Hypoxia
The effects of NAC (FLUIMUCIL, Zambon, Amersfoort, The Netherlands) and Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid; Sigma-Aldrich, Zwijndrecht, The Netherlands) were determined on the contractile properties under hypoxic conditions. The final concentration of NAC was 18 mM and of Tiron was 10 mM. These concentrations have been shown to increase in vitro force generation of the rat diaphragm under hypoxic conditions (21). Allopurinol and its metabolite oxypurinol are well-known competitive XO inhibitors. In patients with COPD, Heunks et al. (15) recently showed that plasma concentrations of 13 µM allopurinol and 60 µM oxypurinol effectively inhibit exercise-induced free radical generation. In a pilot study, diaphragm muscle bundles were incubated in Krebs solution containing 13 µM allopurinol and 60 µM oxypurinol for 60 min under hypoxic conditions, and, subsequently, contractile properties of the bundles were determined. No significant differences were observed in the force-velocity characteristics, force-power characteristics, or isotonic fatigability between hypoxia control and allopurinol and/or oxypurinol-treated bundles (n = 6/group; data not shown). Therefore, the allopurinol (Sigma, St. Louis, MO) concentration in the tissue baths was set at 250 µM to ensure complete blocking of XO. The effects of the antioxidants and allopurinol on contractility during hypoxia were compared with contractility in standard Krebs solution under hypoxic conditions. To assess the effects of hypoxia on rat diaphragm contractility, similar measurements were performed under hyperoxic conditions. In addition, the effects of free radical scavenging and XO inhibition were studied under hyperoxic conditions. Accordingly, seven experimental groups were studied: hypoxia control (n = 8 bundles), hypoxia plus NAC (n = 9 bundles), hypoxia plus Tiron (n = 7 bundles), hypoxia plus allopurinol (n = 9 bundles), hyperoxia control (n = 10 bundles), hyperoxia plus NAC (n = 9 bundles), and hyperoxia plus allopurinol (n = 11 bundles). The diaphragm bundles were randomly allocated to the treatment groups. After a thermoequilibration period, baseline contractile properties (Pt and Po) were determined under hyperoxic conditions (95% O2-5% CO2). Subsequently, perfusion of the tissue baths was either maintained with the hyperoxic gas mixture or switched to a gas mixture containing 5% CO2 and 95% N2 (hypoxia). At the same time, the Krebs solution was substituted with the experimental Krebs solution (i.e., control, NAC, Tiron, or allopurinol). After 60 min of gas and drug equilibration, contractile measurements were performed as described above. The repetitive twitch protocol started immediately after the gas mixture was changed and the experimental solutions were introduced.Purine Measurements
To determine the effects of strenuous contractions and XO blocking on ATP degradation products during hypoxia, hypoxanthine and xanthine concentrations were determined in the rat diaphragm. The allopurinol concentration in the diaphragm muscle bundles was also measured. The remaining muscular diaphragm from the rats in the hypoxic groups was used to determine the baseline (hypo)xanthine concentration in the diaphragm. After completion of the experiments, the bundles used for contractile properties were saved for later (hypo)xanthine and allopurinol measurements. The muscle sample was thoroughly rinsed with saline (0.9%), blotted dry, transferred to liquid nitrogen, and stored atData Treatment and Statistics
After completion of the force measurements, muscle bundle length was measured with a micrometer, the bundles were blotted dry, the central tendon and ribs were removed, and muscle weight was determined. Cross-sectional area was calculated by dividing the diaphragm strip weight (in g) by the strip length (in cm) times the specific density (1.056). Force is expressed per cross-sectional area (in N/cm2).The muscle shortening velocity at each clamp level was calculated as
the change in muscle length during a 30-ms period and is expressed as
Lo/s. To eliminate the effect of muscle
compliance, the time window for shortening velocity measurements was
set to begin 10 ms after the first detectable change in length. Because Po was significantly different between experimental groups
after 60 min of drug equilibration, force-velocity characteristics were plotted with respect to P/Po. The data were fitted
to the Hill (16) equation
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Differences in single baseline contractile properties, bundle
dimensions, and gas pressure between the experimental groups were
analyzed with one-way ANOVA and, if appropriate, Student-Newman-Keuls (SNK) post hoc testing. Parameters requiring repeated measures over
time (i.e., force-velocity, force-power, fatigue) were estimated by
using repeated-measures models and, if appropriate, SNK post hoc
testing. Statistical analysis was performed with the SPSS package
version 9.0 (SPSS, Chicago, IL). Data are expressed as means ± SE. Comparisons were considered significant at P 0.05.
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RESULTS |
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Verification of Tissue Bath Hypoxia and Strip Dimensions
Perfusion of the tissue baths with the hypoxic gas mixture significantly reduced PO2 from 84.8 ± 0.9 to ~6.8 ± 0.2 kPa. pH, which was continuously measured during all experiments, was 7.36 ± 0.01 in the hyperoxic groups and 7.37 ± 0.01 in the hypoxic groups. In addition, PCO2 was not significantly different between hyperoxia and hypoxia (4.8 ± 0.2 and 4.8 ± 0.1 kPa, respectively). No significant differences were found in PO2, PCO2, and pH among the hypoxic and hyperoxic groups.Diaphragm muscle bundle dimensions did not significantly differ between experimental groups (P > 0.05 by one-way ANOVA). Average muscle strip weight was 32.8 ± 0.8 mg, and strip length at Lo was 18.44 ± 0.21 mm.
Isotonic Studies
Baseline contractile properties.
hypoxia.
Baseline Pt and Po were not significantly
different between the experimental groups (P > 0.05)
and averaged 9.3 ± 0.4 and 24.6 ± 0.5 N/cm2,
respectively. Sixty minutes of hypoxia impaired Pt (Fig.
2A) and, to a lesser degree,
Po (Fig. 3A). NAC
tended to decrease Pt, whereas Tiron tended to increase
Pt (P = 0.07 and P = 0.06, respectively, by one-way ANOVA; Fig. 2A). However, in muscle
bundles incubated with either NAC or Tiron, Po was
significantly lower compared with that in the hypoxia control (Fig.
3A). Allopurinol (250 µM) did not significantly
affect Pt or Po compared with that in the
hypoxia control (Figs. 2B and 3B).
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Isotonic contractile properties and fatigability.
hypoxia.
NAC and Tiron both significantly slowed the shortening velocity over a
wide range of loads (Fig. 4A).
The curvature of the force-velocity relationship is described by
a/Po. Tiron altered the curvature of the
force-velocity characteristics (a/Po = 0.23 ± 0.03 in hypoxia control vs. 0.37 ± 0.08 in hypoxia
plus Tiron; P < 0.05). Allopurinol did not affect the
shortening velocity (Fig. 4A) or a/Po
(0.22 ± 0.3 and 0.21 ± 0.02 in hypoxia control and hypoxia
plus allopurinol, respectively; P > 0.05). Both NAC and Tiron significantly depressed the power output over a wide range of
loads (Fig. 5A) The maximal
power outputs during hypoxia were 91 ± 8, 63 ± 2, and
47 ± 5 N/cm2 × Lo/s in
control, NAC, and Tiron, respectively (P < 0.000). However, allopurinol did not affect the force-power characteristics under hypoxic conditions (maximal power output 96 ± 6 N/cm2 × Lo/s;
P > 0.05 compared with hypoxia control; Fig.
5A).
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Isotonic fatigue.
hypoxia.
Repetitive isotonic contractions resulted in a rapid decline in
power output of the rat diaphragm under hypoxic conditions (Fig.
6A). Tiron reduced the rate of
decline in power output under hypoxic conditions (P < 0.05; Fig. 6A). NAC tended to decrease the fatigue rate
(P = 0.07; Fig. 6A). Both NAC and Tiron
significantly prolonged isotonic endurance compared with that in the
hypoxia control (P < 0.000; Fig.
7A). Allopurinol did not
significantly affect fatigability under hypoxic conditions (Figs.
6A and 7A).
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Repetitive Twitch Studies
To ascertain that the antioxidants or allopurinol did not exert differential effects in time, the effects of NAC and allopurinol on Pt were studied under hypoxic conditions for a 90-min period, a time window that corresponds with the total duration of the isotonic protocol (incubation time plus time needed for all contractile experiments). As shown in Fig. 8, the "direction of effect" did not change during this period. In accordance with the data shown above (Fig. 2), in NAC-treated muscle bundles (n = 8), Pt was significantly lower compared with hypoxia control (n = 9 bundles), but allopurinol (n = 9 bundles) did not affect Pt.
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Effects of Allopurinol on ATP Degradation Under Hypoxic Conditions
Allopurinol was present in the muscle bundles from the hypoxia plus allopurinol group, indicating adequate uptake from the Krebs solution (Table 1). In hypoxia control, strenuous contractile activity resulted in elevated levels of xanthine (P < 0.05). Both inosine and hypoxanthine were significantly increased and xanthine was decreased after contractile experiments in the hypoxia plus allopurinol group, which is in agreement with blocking the XO activity of the muscle (Table 1).
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DISCUSSION |
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The present study demonstrated that severe hypoxia impairs force generation of the rat diaphragm muscle but does not affect the velocity of shortening. The antioxidants NAC and Tiron impaired force generation, slowed the velocity of shortening, and reduced the fatigability of the diaphragm during hypoxia, indicating that free radicals play a role in cross-bridge recruitment and cross-bridge cycling kinetics under hypoxic conditions. Antioxidants had opposite effects in the unfatigued and fatigued muscles, indicating that the contraction pattern determines whether the effects of antioxidants during hypoxia are beneficial or harmful. The fact that allopurinol did not affect contractile function or fatigability of the diaphragm during hypoxia indicates that free radicals generated by XO are not directly involved in excitation-contraction coupling under hypoxic conditions. Under hyperoxic conditions, NAC and allopurinol did not affect isometric or isotonic contractility of the diaphragm, indicating that in our model, free radicals do not affect muscle performance during hyperoxia.
Allopurinol, NAC, and Tiron
Allopurinol was used to block XO activity in the rat diaphragm. The major pharmacological actions of allopurinol are mediated by its major metabolite oxypurinol. Both are structural analogs of the purine bases hypoxanthine and xanthine and competitively bind to XO. Therefore, they inhibit the conversion of hypoxanthine to xanthine and of xanthine to uric acid and thus the generation of superoxide. Moorhouse et al. (23) reported that allopurinol in concentrations >250 µM may have direct hydroxyl-scavenging properties. Therefore, in the present study, the allopurinol concentration in the Krebs solution was 250 µM. This concentration was sufficient to block XO activity as indicated by the increase in hypoxanthine concentration and the reduction in xanthine concentration in allopurinol-treated bundles during hypoxia.NAC is an antioxidant drug commonly used in clinical practice. Two possible antioxidant mechanisms have been proposed for this thiol-containing agent. First, NAC may have direct free radical-scavenging properties. Free radicals may react with NAC, resulting in the formation of NAC disulfide (3, 22). Although this mechanism of action is of limited relevance in vivo due to the high first-pass effect of NAC (25), it might be important in in vitro studies. Second, NAC may also exert its antioxidant effects indirectly by facilitating the biosynthesis of GSH. The concentration of NAC in the present study was 18 mM and was similar to that in a previous study (21) with this agent in the rat diaphragm. To ensure that the effects of NAC on muscle contractility were the result of its antioxidant properties, similar experiments were conducted with Tiron, a chemically distinct free radical scavenger. Tiron is an intracellular superoxide scavenger and metal chelator and has been shown to effectively scavenge free radicals in the contracting rat diaphragm (34).
Hypoxia and Muscle Function
Van der Heijden et al. (37) have discussed the severity of hypoxia as used in the present study in a recent paper. Briefly, a reduction in PO2 in the Krebs solution from ~85 to 7 kPa should be considered as severe hypoxia because oxygenation of diaphragm muscle bundles is dependent solely on diffusion of O2 into the core region of the bundles. Hypoxia impairs force generation and increases fatigability of the diaphragm in vitro (10, 31, 32, 37, 38). The present studies confirm these effects of hypoxia on isometric contractility. After 60 min, both Pt and Po were significantly lower in hypoxia compared with hyperoxia, indicating that hypoxia impaired cross-bridge recruitment or reduced force per cross bridge. This study also shows that hypoxia does not significantly affect the force-velocity relationship, indicating that hypoxia does not have major effects on cross-bridge cycling rate of the diaphragm. It has been previously shown (9, 17) that variation in maximum force has little effect on the normalized force-velocity relationship. This is not surprising because there is no a priori reason why force and velocity should be reduced to the same extent because the underlying cellular mechanisms are different. The effects of hypoxia on the velocity of shortening and power output in the rat diaphragm have not been previously investigated. However, a study on fatigued and hypoxic muscle (11) demonstrated that the changes in the intracellular milieu under these conditions are remarkably similar. It has been shown (39) that in intact muscle fibers in the early stages of fatigue, isometric force declines significantly, with little effect on the Vmax. This is in line with the results of the present study. It has been proposed that the changes in intracellular inorganic phosphate and Ca2+ concentration, which occur in the early stages of fatigue, affect isometric force generation but have little or no effect on shortening velocity (for a review, see Ref. 1). It has been shown (4) that the shortening velocity correlates with myosin ATPase activity. Apparently, ATPase activity was not reduced in the hypoxic diaphragm during nonfatiguing contractions. The curvature of the force-velocity relationship (derived from a/Po) was not altered by hypoxia, indicating that hypoxia did not affect the efficacy of contraction in the unfatigued muscle.However, hypoxia had detrimental effects on the isotonic fatigue rate as indicated by a progressive decline in power output during the fatigue protocol (Fig. 6A). Power is the product of force and the shortening velocity. Because force was clamped at 33.3% of Po, force generation was constant during the entire fatigue protocol. The reduction in power is the result of a reduction in the shortening velocity. As previously mentioned, the shortening velocity is correlated with myosin ATPase activity (4); thus it is likely that ATPase activity decreased during fatigue. Several studies suggested that an elevated concentration of ADP inhibits the rate of cross-bridge detachment and, consequently, Vmax (for a review, see Ref. 1). The increase in inosine and xanthine in diaphragm muscle bundles after fatiguing contractions is consistent with such a hypothesis. Because hypoxia will slow ATP regeneration, ADP accumulation will occur sooner under hypoxic than hyperoxic conditions. Consequently, the fatigue rate will be higher during hypoxia, as shown in the present study. Although we did not measure ATP degradation products after fatiguing contractions under hyperoxic conditions, it is likely that the response will be similar as under hypoxia because both groups of bundles fatigued to the same level, i.e., inability of the muscle to shorten at 33.3% of Po.
Free Radicals in Skeletal Muscle During Hypoxia
Several studies have demonstrated that hypoxia enhances the generation of oxidants (8) and that these free radicals might act as second messengers in the adaptive response to hypoxia (6, 26). For instance, Park et al. (26) showed that in a perfused rat heart model, 10 min of hypoxia (PO2 ~0.4 kPa) resulted in a significant increase in markers for the generation of free radicals, such as lipid-peroxides, carbonyls, and oxidized glutathione. Generation of free radicals in cultured cardiac myocytes starts within several minutes after the initiation of hypoxia, and the rate of generation is more pronounced in severe hypoxia (8). No direct evidence exists for the elevated generation of free radials in the diaphragm during hypoxia.In the present study, the effects of NAC and Tiron on contractility of the rat diaphragm were tested during hypoxia. Surprisingly, Tiron increased Pt, whereas NAC tended to decrease Pt (P < 0.05 and P < 0.07, respectively, by one-way ANOVA and SNK post hoc testing). We do not have a clear explanation for this discrepancy. However, both antioxidants significantly reduced Po, indicating a reduction in the number of available cross bridges or a reduction in force per cross bridge. The reduction in the shortening velocity induced by both NAC and Tiron indicates that oxidants play a role in cross-bridge cycling. In addition, the normalized force-velocity relationship in the Tiron-treated bundles was less curved (lower a/Po) compared with hypoxia alone, indicating that Tiron impaired the efficacy of contraction (40). Power is the product of force and velocity and is considered as a more physiological estimation of muscle performance in vivo than either force or velocity alone. Because both NAC and Tiron reduced power generation over a wide range of loads, it could be expected that antioxidants will impair the performance of the hypoxic unfatigued muscle in vivo. Based on a previous study showing that hypoxia enhances generation of oxidants (26), we hypothesized that this would contribute to impaired contractile performance of the diaphragm under hypoxic conditions. Our findings partly refute this hypothesis. NAC and Tiron impaired contractile performance of the unfatigued diaphragm. It could be argued that the degree of hypoxia was not sufficient to enhance the generation of free radicals. However, as previously discussed (37), the degree of hypoxia should be considered as severe. Nevertheless, the amount of oxidants generated depends on the severity of hypoxia (8); thus further reducing PO2 might have changed the response of antioxidants on contractility in the present study. However, the clinical and physiological relevance of such experiments would be limited. The study by Mohanraj et al. (21) supports these speculations. In their study, both NAC (18 mM) and Tiron (10 mM) increased Pt and Po in the hypoxic/anoxic rat diaphragm (PO2 <0.08 kPa). Because the concentrations of NAC and Tiron used were similar between their study and the present study and the differences cannot be explained by time-dependent effects of NAC (Fig. 8), it is likely that the difference in PO2 contributed to the differences in outcome. Indeed, in their study, hypoxia decreased the maximal force until it was ~25% of initial value (Table 1 in Ref. 21), whereas in the present study, Po decreased until it was ~90% of baseline value. Another explanation for rejection of our hypothesis is that the relatively high concentration of the antioxidants shifted the redox balance from slightly oxidized to the reduced state, which will also compromise muscle performance. Reid et al. (29) proposed a model of force production as a function of the cellular redox balance. Both oxidant and reductant stresses will impair muscle performance. Such a model would fit well with the data in the present study. Adding free radical scavengers to the tissue bath placed the unfatigued muscle in a reduced state, which compromised muscle performance. This study is the first to demonstrate that antioxidants modulate isotonic fatigue in skeletal muscle. During isotonic fatiguing contractions, when the generation of free radicals is enhanced (19, 28), these antioxidants exerted beneficial effects on muscle performance as indicated by the reduced fatigue rate and increased endurance time. As mentioned above, the reduction in power is the result of a reduction in the shortening velocity. Therefore, these antioxidants modulate cross-bridge cycling.
Under hyperoxic conditions, NAC did not affect isometric or isotonic contractility of the diaphragm. These findings are in apparent contrast with previous studies (18, 33) showing that NAC attenuates diaphragm fatigability under hyperoxic conditions. However, in those studies, the effects of NAC on isometric fatigability were assessed. In contrast, in the present study, the effect of NAC on isotonic fatigability (force clamped at 33% of Po) was determined. Thus different physiological parameters were studied. As discussed above, these parameters (i.e., force and velocity) are not dependent; thus these previous studies and the present study are not necessarily conflicting and suggest that under hyperoxic conditions, free radicals affect cross-bridge recruitment or force per cross bridge but not cross-bridge cycling kinetics.
XO Inhibition and Skeletal Muscle Function
We hypothesized that XO is a prominent source for free radicals in the diaphragm under hypoxic conditions. Although xanthine concentration in the diaphragm bundle was significantly increased after strenuous contractions, blocking XO activity did not alter the contractile properties at rest or during fatiguing contractions. Thus our original hypothesis must be rejected. Allopurinol effectively blocked XO as indicated by the accumulation of hypoxanthine and a sharp decrease in the xanthine concentration in bundles incubated in allopurinol. Apparently, free radicals generated by XO do not affect contractility in the rat diaphragm. Our findings are in line with a recent study by Supinski et al. (35). Oxypurinol administration before inspiratory resistive breathing in rats failed to prolong task endurance and did not prevent lipid peroxidation in the diaphragm. This indicates that XO-dependent pathways do not affect diaphragm contractility in vivo. Chandel and Schumacker (6) recently proposed that the hypoxia-induced reduction in mitochondrial cytochrome oxidase activity results in an increase in the mitochondrial redox state, which, in turn, accelerates free radical formation during hypoxia. Whether free radicals generated via this pathway affect excitation-contraction coupling under hypoxic conditions remains to be investigated.Clinical Relevance
Respiratory muscle dysfunction frequently occurs in patients with severe COPD (27). Free radicals may be involved in the pathogenesis of respiratory muscle dysfunction in these patients (14). First, in patients with COPD, especially emphysema, the altered geometry of the thorax puts the diaphragm on a disadvantageous position of the length-tension curve (7), thereby increasing the load on the diaphragm. It has been shown that elevated loading enhances the generation of free radicals in skeletal muscle (19). Second, it has been shown that hypoxia enhances the generation of free radicals in muscle (26). Because hypoxia frequently occurs in severe COPD, investigating the role of free radicals in the contractile performance of the diaphragm is important. In addition, identifying the pathways for free radical generation is important to effectively inhibit the generation of free radicals from specific pathways. ![]() |
ACKNOWLEDGEMENTS |
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Karina Wethly and Anneke Stegemans are gratefully acknowledged for allopurinol and purine analysis and Kay Poelen and Leo Ennen for biotechnical assistance.
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FOOTNOTES |
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This study was financially supported by Dutch Asthma Foundation Grant 97.34.
Address for reprint requests and other correspondence: P. N. R. Dekhuijzen, Dept. of Pulmonary Diseases, Univ. Medical Centre Nijmegen, Postbox 9101, 6500 HB Nijmegen, The Netherlands (E-mail: R.Dekhuijzen{at}long.azn.nl).
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. Section 1734 solely to indicate this fact.
Received 8 September 2000; accepted in final form 2 August 2001.
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