INTRODUCTION

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DURING RESPIRATORY FAILURE and associated cardiovascular collapse and resuscitation, conditions are appropriate for local tissue hypoxia and ischemia-reperfusion to occur. Ischemia-reperfusion has been shown to be associated with the production of reactive O₂ species (ROS) in different organ systems (16, 28, 34). It is generally believed that they are primarily produced during the reperfusion period, when high O₂ concentrations promote the formation of superoxide [superoxide anion radical (O₂·)] through one electron reduction of O₂. However, several studies have demonstrated that hypoxia alone, without subsequent reoxygenation or reperfusion, can result in the production of oxidants in some tissues (26, 27, 38). For example, Park and Kehrer (27) have shown that, in the heart, hypoxia results in both oxidant formation and oxidative stress, which are not exacerbated further by subsequent reoxygenation. The mechanism responsible for oxidant production in hypoxia is not well understood and has not previously been demonstrated in skeletal muscle. The influence of hypoxia on oxidant production may be clinically important, because the working diaphragm can be exposed to severe hypoxia during conditions of respiratory failure or cardiopulmonary arrest. Furthermore, in a previous study (5), we have shown increased free-radical generation in the diaphragm during conditions of severe respiratory failure.

Skeletal muscles have been shown to produce ROS both intracellularly and extracellularly at rest (31, 33). Production appears to go up in response to intense, fatiguing exercise (5, 12, 31). Furthermore, during fatigue, force production and endurance are markedly preserved by treatment with a wide variety of antioxidants (31, 33, 36), and treatment with dithiothreitol, a potent reducing agent, improves recovery of muscle function after diaphragmatic fatigue (11). These earlier experiments demonstrate that ROS or other oxidizing agents are generated in skeletal muscle and may contribute to the loss of force development in fatigue.

In this study, we explore the hypothesis that ROS, free radicals, or other oxidizing agents are produced in hypoxic skeletal muscle and that they contribute to the loss of mechanical function associated with severe hypoxia. We reasoned that, if oxidizing species are actively involved in contractile depression, then antioxidant treatment should preserve function, much as it is preserved during conditions of muscle fatigue. Alternatively, if oxidizing species are produced primarily during reoxygenation, then antioxidant treatment would not preserve function during hypoxia but rather during the reoxygenation and/or recovery period.

Our results demonstrate that hypoxia causes significant muscle dysfunction that is markedly attenuated by antioxidant treatment. This effect appears to be independent of whether the antioxidants work intracellularly or extracellularly. Furthermore, antioxidant treatment improves recovery on reoxygenation.

	METHODS

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Experiments with Antioxidants

Surgical procedure and muscle strip preparation. The experiments were conducted on diaphragm muscle strips taken from adult male Sprague-Dawley rats (300-500 g). The animals (n = 7) were anesthetized with an injection of pentobarbital sodium (40 mg/kg ip), tracheotomized, and mechanically ventilated. The phrenic nerve was severed, and four diaphragm strips (~0.5 cm) were dissected, with portions of rib and central tendon intact. The strips were divided into four groups. The control tissue was placed in a tissue bath containing ~125 ml of a physiological salt solution (in meq/l: 21 NaHCO₃, 0.9 NaSO₄, 1.2 NaHPO₄, 0.9 MgCl₂, 2.25 CaCl₂, 5.9 KCl, and 109 NaCl with 2.07 g/l glucose). The experimental tissue was placed in a similar bath containing physiological salt solution with N-acetyl-L-cysteine (NAC, 18 mM), a commonly used antioxidant. The baths were maintained at 37°C and were continuously bubbled with 95% O₂-5% CO₂ during normoxia and 95% N₂-5% CO₂ during the hypoxic phase. Two additional baths containing tissues with and without NAC served as normoxia time controls. Muscle function (twitch characteristics and stress-frequency relationships) was assessed at the end of each phase. To determine the kinetics of gas pressures in the tissue baths, we continuously monitored the PO₂ in the baths with a Clark electrode (BMS3 MK2 Blood Micro System, Radiometer America, Cleveland, OH). The PO₂ generally dropped rapidly in the tissue baths, reaching <50 Torr within 3 min. A minimum partial pressure, indistinguishable from zero, was reached within 15-20 min into the hypoxic period (Fig. 1).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Washout of O2 in tissue bath during hypoxic phase. A single, representative experiment is shown. It took ~20 min for O2 level to fall to <6 Torr from an initial normoxic phase.

Stimulation paradigm. After the strips were mounted in the tissue chamber, the maximum required stimulation current and the optimal length were determined by adjusting the micropositioner between intermittent stimulations. All stimulations were performed at optimal length and at supramaximal current.

Twitch characteristics. Single twitches (0.5-Hz stimulation) were recorded, and the peak-twitch tension, time-to-peak tension, and half time of relaxation were calculated.

Force-frequency relationships. The muscle strips were tetanically stimulated at 20, 30, 40, 50, 60, 80, and 100 Hz with an interval of ~30 s between each tetanic stimulation. Pulses were of 0.2-ms duration, with a train duration of 400 ms.

Data Analysis

	RESULTS

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Twitch Characteristics

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Twitch characteristics of muscle in normoxia (Initial), in hypoxia, and on reoxygenation (Recovery) in control (open bars) and N-acetyl-L-cysteine-treated (hatched bars) tissues. A: peak-twitch tension. B: half time of relaxation. C: time-to-peak tension or contraction time. Data are means ± SE; n = 7 rats.

Stress-Frequency Relationships

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Stress-frequency relationships of control and N-acetyl-L-cysteine (NAC)-treated tissues during 3 phases of stimulation: baseline/initial normoxic phase (A); hypoxic phase (B); and reoxygenation/recovery phase (C). Data are means ± SE; n = 7 rats. * Individual post hoc contrasts.  Effect from initial with use of repeated-measures ANOVA. § Effect of treatment with NAC compared with control tissue.

The effects of other antioxidants (DMSO, SOD, and Tiron) on muscle function were similar to that of NAC (Table 1). Peak-twitch stress and the stress-frequency relationship during hypoxia and on reoxygenation in muscles treated with these antioxidants were similar to those in NAC-treated tissues. Similar to NAC, Tiron significantly lowered baseline stress at low frequencies (30 Hz, Table 1). Also similar to NAC, these antioxidants protected muscle function during hypoxia as well as on reoxygenation. Although the responses did not reach statistical significance at 30 Hz during hypoxia, they were significant at higher frequencies of stimulation (e.g., 50 and 100 Hz, Table 1). Furthermore, antioxidant-treated tissues also showed significantly increased recovery at nearly all frequencies of stimulation.

	DISCUSSION

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To the best of our knowledge, this is the first study demonstrating the effect of antioxidants on muscle function in hypoxic skeletal muscle. Our results show that there is significant impairment of skeletal muscle function during hypoxia, characterized by a reduction in force at all frequencies, as well as a decrease of both twitch contraction and relaxation time. The changes in tetanic force can be ameliorated by coincubation with antioxidants. Furthermore, antioxidants improve recovery of hypoxic muscles on reoxygenation. These findings suggest that oxidants, oxidizing agents, or free radicals are produced in skeletal muscle during hypoxia and contribute to decreased force production.

Potential Sources of ROS During Hypoxia

It has been shown that, even at a tissue PO₂ of 1 Torr (normal, 35 Torr), there is a significant production of free radicals despite the low Michaelis constant (K_m) of cytochrome oxidase complex in the mitochondria (30). Our tissues were subjected to hypoxic levels comparable to other studies in hypoxia (22, 26, 28). In our model, there was an exponential washout of O₂ over time. It took ~20 min for O₂ to fall to <6 Torr (Fig. 1). It is conceivable that this initial period of relative hypoxia could result in the formation of O₂ free radicals by direct single-electron reduction of the available molecular O₂ to form O₂· (9). These ROS, on reaction with cell membranes and polyunsaturated fatty acids, could propagate into a self-perpetuating chain reaction, resulting in a significant accumulation of free radicals (6).

Other possible sources of ROS include activation of certain enzymatic pathways during hypoxia. Xanthine dehydrogenase is known to be present in a reasonably high concentration in rat diaphragm (1). Its exact location within skeletal muscles is not clear, but it seems to be predominantly located within endothelial cells in the vasculature (1); the latter is abundant in diaphragm. During hypoxia, an insufficient supply of ATP can result in the malfunction of ATP-dependent Ca²⁺ pumps of the cell. The excess Ca²⁺ stimulates the transformation of xanthine dehydrogenase to xanthine oxidase by the action of Ca²⁺-activated proteases. Xanthine oxidase catalyzes the breakdown of hypoxanthine (formed as a catabolic product of adenine nucleotides in increased amounts during hypoxia) into xanthine and uric acid, releasing O₂· (15). ROS may also be formed by cytosolic enzymes such as cyclooxygenase. Under hypoxic conditions, there is an increased Ca²⁺-dependent activation of the enzyme phospholipase A₂, which releases arachidonic acid from phospholipids. Cyclooxygenase reacts with this accumulated fatty acid, simultaneously generating singlet O₂ and hydroxyl radical (· OH) (7). Finally, hypoxia can also cause increased nitric oxide radical (· NO) production. Recent studies on endothelial cells showed that · NO production may be stimulated by elevated levels of cytosolic Ca²⁺ (20) or by increased activity of constitutive NO synthase (39) occurring during hypoxia. · NO is known to be toxic to the cell by its direct interaction with iron-sulfur proteins, inhibition of ribonucleotide reductase and other enzyme systems, nitrosylation of thiols, as well as inhibition of protein synthesis (4). It can also exert indirect toxic effects via peroxynitrite formed by its combination with O₂·. Peroxynitrite is a potent oxidant with "· OH-like activity" and has been shown to oxidize thiols, sulfides, lipids, and deoxyribose (4). In this study, antioxidants may have functioned in part by preventing peroxynitrite formation during hypoxia, i.e., by scavenging superoxide or perhaps by direct antioxidant activity on peroxynitrite itself.

Muscle Function During Hypoxia

The cause of acceleration of twitch kinetics during hypoxia is unclear. Green et al. (17) showed an increased activity of sarcoplasmic reticulum (SR) ATPase in both slow- and fast-twitch skeletal muscle during ischemia. This could result in increased reuptake of Ca²⁺ by the SR ATPase, resulting in faster relaxation. Alternatively, during hypoxia there may be a reduction in the contribution from slow-twitch oxidative fibers, which depend more on O₂ for energy metabolism. In contrast, fast-twitch glycolytic fibers would be expected to be more preserved in hypoxic muscle, as glycolytic pathways could continue to provide energy. This could result in a predominant fast-twitch fiber influence on force. Interestingly, slow-twitch oxidative fibers are rich in mitochondria, which may, as mentioned, represent a source of ROS in hypoxia.

Effect of Specific Antioxidants on Muscle Function

NAC, a well-known antioxidant in ischemia-reperfusion models, is a low-molecular-weight intracellular antioxidant with a nucleophilic sulfhydryl group that can interact with and detoxify reactive electrophiles and free radicals directly via conjugation or reduction reactions. NAC has been shown to scavenge · OH and hypochlorous acid; its antioxidant effect on other ROS is unlikely. Its indirect effects involve raising the intracellular concentration of cysteine, thereby supporting glutathione (an endogenous antioxidant) biosynthesis (2, 35). In addition, NAC has been shown to increase the activity of other endogenous intracellular antioxidant enzymes such as SOD (25). Recent in vivo and in vitro studies on skeletal muscle (10, 23, 36) have shown that NAC inhibits skeletal muscle fatigue in both human and animal models. The exact mechanism by which NAC influences the contractile process in muscle is unknown. Khawli and Reid (23) proposed that improved contractility and inhibition of fatigue seen with NAC treatment could be due to its effect on SR. During oxidative stress, the sulfhydryl groups on the Ca²⁺ release channels and SR ATPase can be oxidized, thereby causing loss of Ca²⁺ homeostasis in the contractile apparatus. This may be prevented or reversed by treatment with a free radical scavenger and thiol donor such as NAC (23).

Previous studies (31, 33, 37) on the diaphragm have shown both DMSO and SOD to be effective antioxidants under conditions of diaphragmatic fatigue. DMSO is a commonly used, highly permeable, nonenzymatic, sulfur-containing antioxidant and is primarily a · OH scavenger, although it can also scavenge other ROS (3). Its mechanism of action on the contractile process is poorly understood (32). SOD, on the other hand, primarily catalyzes the dismutation of O₂· to hydrogen peroxide and O₂. It is a high-molecular-weight metalloenzyme, and so it may be restricted to an extracellular distribution (31). Radak et al. (29) demonstrated that administration of SM-SOD [poly-(stylene-co-maleic acid) butyl ester-linked derivative of SOD] to rats resulted in reduced oxidative damage to skeletal muscles during exhaustive exercise. They showed a significant reduction in plasma and skeletal muscle thiobarbituric acid-reactive substances and reduced plasma xanthine oxidase activity after exercise in the treated group. In our model, it is likely that SOD prevented a decline in muscle function during hypoxia by a combination of the above effects.

Tiron is a less well-known antioxidant, and to our knowledge its effects on skeletal muscle have so far not been studied. It is a low-molecular-weight phenolic compound and has been shown to scavenge · OH, O₂·, and hydrogen peroxide (18, 24). It has been used for in vivo animal studies as a nontoxic metal chelator (24). In addition, it can be used as a spin trap. Combined with free radicals, it is easily oxidized to its semiquinone radical, which can be detected by electron paramagnetic resonance imaging (24). We have also shown that Tiron inhibits diaphragmatic fatigue similar to other antioxidants (unpublished data). In this study, the improvement in muscle function on coincubation with Tiron is perhaps due to its free-radical scavenging and metal-chelating properties.

In conclusion, hypoxia results in skeletal muscle dysfunction, which can be partially prevented by treatment with antioxidants. We speculate that hypoxia results in the production of reactive O₂ or other oxidizing species through a mechanism initiated by reductive stress. These findings suggest a new role for antioxidants in providing protection during conditions of reductive stress, much as they have been shown to provide protection in oxidative stress. This may apply to the proven effectiveness of antioxidant treatment in many other tissue beds and clinical conditions in which problems with O₂ delivery and utilization are present. For example, cell dysfunctions caused by ischemia, sepsis, or intense metabolic activity seen in fatiguing skeletal muscle have all been shown to be partially prevented by treatment with antioxidants and are likely to represent conditions of a high-reducing environment within the cells. Therefore, the traditional view that antioxidants protect tissue from reactive O₂ produced during reperfusion or reoxygenation must be expanded to include the concept of protection during the preceding period of hypoxia or ischemia.