HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Clanton, T. L. Articles by Klawitter, P. Search for Related Content PubMed PubMed Citation Articles by Clanton, T. L. Articles by Klawitter, P.

---

Original Article

Oxidants and Skeletal Muscle Function: Physiologic and Pathophysiologic Implications

Thomas L. Clanton^*,,1, Li Zuo^*, and Paul Klawitter^,

^* Department of Internal Medicine, Pulmonary and Critical Care Medicine;
Biophysics Program, and
Department of Emergency Medicine, The Ohio State University, Columbus, Ohio 43210

	Abstract

Top Abstract Introduction Evidence for ROS Production... Effects of AOXs on... AOXs and Skeletal Muscle... Muscle Conditioning and... ROS and Heat Stress Hypoxia as a Stimulant... Reactive Oxygen and Muscle... ROS-Induced Respiratory Muscle... Conclusions and Future... References

 
Previous studies have demonstrated that skeletal muscles generate considerable reactive oxygen during intense muscle contraction. However, the significance of this phenomenon and whether it represents normal physiology or pathology are poorly understood. Treatment with exogenous antioxidants suggests that normal redox tone during contraction is influencing ongoing contractile function, both at rest and during intense exercise. This could represent the influence of redox-sensitive proteins responsible for excitation-contraction coupling or redox-sensitive metabolic enzymes. Some conditions associated with intense exercise, such as local tissue hypoxia or elevated tissue temperatures, could also contribute to reactive oxygen production. Evidence that muscle conditioning results in upregulation of antioxidant defenses also suggests a close relationship between reactive oxygen and contractile activity. Therefore, there appears to be a significant role for reactive oxygen in normal muscle physiology. However, a number of conditions may lead to an imbalance of oxidant production and antioxidant defense, and these, presumably, do create conditions of oxidant stress. Ischemia-reperfusion, severe hypoxia, severe heat stress, septic shock, and stretch-induced injury may all lead to oxidant-mediated injury to myocytes, resulting in mechanical dysfunction.

	Introduction

Top Abstract Introduction Evidence for ROS Production... Effects of AOXs on... AOXs and Skeletal Muscle... Muscle Conditioning and... ROS and Heat Stress Hypoxia as a Stimulant... Reactive Oxygen and Muscle... ROS-Induced Respiratory Muscle... Conclusions and Future... References

 
Skeletal muscles are rarely thought of as primary targets of oxidative stress. In fact, they appear to be uniquely designed to withstand stresses of many kinds. During intense exercise they are exposed to levels of mechanical and metabolic insult that would seriously injure or kill most other cells. For example, no other tissue of the body undergoes such drastic incremental changes in O₂ metabolism during what would be considered "normal activity." O₂ flux through the mitochondria can increase 100 times, when going from rest to maximum exercise in highly trained oxidative muscle fibers (1). If we assume a fixed percentage of this O₂ [i.e., 1%–2% (2)] is reduced to superoxide (O₂^•-), then exercising muscles could be potent generators of reactive oxygen species (ROS). Many previous investigators have associated measurements of oxidative stress with exercise, but the biological implications of these measurements are unclear. Is this normal physiology or is it pathology?

	Evidence for ROS Production in Skeletal Muscle

Top Abstract Introduction Evidence for ROS Production... Effects of AOXs on... AOXs and Skeletal Muscle... Muscle Conditioning and... ROS and Heat Stress Hypoxia as a Stimulant... Reactive Oxygen and Muscle... ROS-Induced Respiratory Muscle... Conclusions and Future... References

 
Some of the earliest work suggesting that free radicals are produced in exercising muscle was reported by Davies et al. (3), who showed an electron paramagnetic resonance (EPR) signal consistent with free radical formation and evidence of lipid peroxidation. Our laboratory has demonstrated similar results in diaphragm, quickly frozen from animals undergoing mechanical loading of their respiratory system (4). These results are often quoted as direct evidence of free radical formation in exercise. However, it is just as likely that they reflect paramagnetic species generated from normal electron flow of mitochondrial electron transport (5). Nevertheless, many other forms of evidence suggest that skeletal muscles produce considerable quantities of ROS, as discussed below.

Resting muscles in vitro (6), or in situ (7) produce extracellular superoxide (O₂•-), as measured by the reduction of cytochrome-c. The significance of extracellular ROS production in muscle is not known, but it may reflect the myocyte's attempts to regulate intracellular ROS or to remove excessive reducing equivalents. Alternatively, it could represent ROS produced by the abundant capillary endothelium, which in vivo may involve responses to shear stress (8, 9). Resting muscle also generates substantial intracellular ROS, as evidenced by the use of various redox-sensitive fluorescent probes such as dichlorofluorescin (10) and hydroethidine (11).

The production of intracellular and extracellular ROS is markedly increased with muscle contraction (10, 11). In addition, our laboratory (12) and others (13) have shown that muscle contraction induces hydroxylation of aromatic compounds such as salicylate, as shown in Figure 1. For many years, this assay was thought to be specific for hydroxyl radical activity. However, recently we and others have shown that at physiologic pH, peroxynitrite may be as responsible for hydroxylation of aromatic compounds as is hydroxyl radical (14, 15). Nitric oxide (^·NO), which would be required for peroxynitrite formation, is also produced in significant quantities in contracting skeletal muscle (16). Substantial constitutive nitric oxide synthase (NOS) exists both in the myocytes and in the associated vascular endothelium (16). The roles of ^·NO production in muscle contractile function, metabolic regulation, and blood flow distribution are under intense investigation in many laboratories and though related to oxidant stress, for purposes of brevity, ^·NO is not discussed in detail in this review.

View larger version (31K): [in this window] [in a new window]   Figure 1.   The salicylate hydroxylation product (2,3-dihydroxybenzoic acid) taken from muscle after various degrees of nerve-stimulated activation. (Derived from Ref. 12).

An interesting new hypothesis regarding the source of ROS in contracting skeletal muscle has originated from the laboratory of Supinski et al. (11) who has shown that ROS may be formed as a consequence of phospholipase A2 activation. Relatively specific phospholipase A2 inhibitors nearly eliminate the intracellular ROS produced during muscle contraction (11).

Despite the strong evidence that low levels of ROS are produced during various forms of contractile behavior in skeletal muscle, there is little particularly convincing data that this results in substantial oxidant "stress" under normal conditions of exercise. In exhaustive exercise, there are numerous reports of small elevations in oxidized glutathione, as well as increases in lipid and protein oxidation products, but these changes are, in general, small (3, 23, 24). However, oxidant stress has been demonstrated under numerous pathological conditions in skeletal muscle, some of which are described later in this review.

	Effects of AOXs on Skeletal Muscle Function

Top Abstract Introduction Evidence for ROS Production... Effects of AOXs on... AOXs and Skeletal Muscle... Muscle Conditioning and... ROS and Heat Stress Hypoxia as a Stimulant... Reactive Oxygen and Muscle... ROS-Induced Respiratory Muscle... Conclusions and Future... References

 
A variety of exogenously applied intracellular and extracellular antioxidants (AOXs) cause alterations in the resting contractile characteristics of skeletal muscle. Maximum force development in response to high frequency tetanic stimulation is not particularly affected by AOXs, but responses to twitch stimulation and low frequencies of stimulation are changed appreciably. In general, a wide variety of exogenously applied AOXs, including n-acetylcysteine (25, 26), superoxide dismutase (SOD) (27), and catalase (27) cause faster twitch contractions, as illustrated in the top of Figure 2. As a consequence of these effects, AOX exposure also results in a net reduction in tetanic force development in response to low frequencies of stimulation (bottom of Fig. 2). On a theoretical basis, the reduction in twitch force and low frequency force probably reflects decreases in total Ca⁺² available for cross-bridge activation; however, this has never been tested directly in intact muscle. The effect of mild oxidant treatment with H₂O₂ is opposite that of AOXs, suggesting again that resting redox tone is somehow modulating contractile function at rest (27, 28). The molecular mechanisms of how endogenous redox tone affects Ca⁺² release and uptake are poorly understood, but represent an area under intense investigation in a number of laboratories. Interestingly, like AOXs, ^·NO also decreases low frequency force production (16), and inhibition of NOS has the opposite effect (16). However, higher levels of H₂O₂ decrease Ca⁺² release, presumably by direct oxidation of critical channel proteins (28, 29). Could ^·NO be acting as an AOX in this environment by scavaging O₂^•-? Probably not, since these effects appear to be related primarily to alterations in cyclic GMP activation (30).

View larger version (13K): [in this window] [in a new window]   Figure 2.   Schematic showing the influence of antioxidant (AOX) treatment on resting skeletal muscle force development. (Above) Most commonly seen effects of AOXs on single twitch contractions, causing lower twitch force. Note relative twitch force inset on the lower left of the bottom graph. (Below) The force response to increasing stimulation frequencies. AOX treatment lowers force development at low frequencies of stimulation, the range in which most contractions occur.

	AOXs and Skeletal Muscle Fatigue

Top Abstract Introduction Evidence for ROS Production... Effects of AOXs on... AOXs and Skeletal Muscle... Muscle Conditioning and... ROS and Heat Stress Hypoxia as a Stimulant... Reactive Oxygen and Muscle... ROS-Induced Respiratory Muscle... Conclusions and Future... References

View larger version (17K): [in this window] [in a new window]   Figure 3.   The influence of antioxidant treatment (tiron in this example) on the development of muscle fatigue in a 4-min in vitro fatigue protocol at 20 Hz stimulation; from Ref. 36). These results are typical of a wide range of different antioxidant treatments.

	Muscle Conditioning and Antioxidant Expression

Top Abstract Introduction Evidence for ROS Production... Effects of AOXs on... AOXs and Skeletal Muscle... Muscle Conditioning and... ROS and Heat Stress Hypoxia as a Stimulant... Reactive Oxygen and Muscle... ROS-Induced Respiratory Muscle... Conclusions and Future... References

A number of investigators have looked at changes in AOX activity secondary to exercise training and have reported varying results. It now appears that specific AOX responses depend on age (45, 46), muscle fiber type or muscle region (47, 48), exercise intensity or duration (47, 49), and probably species. Most investigators have observed increases in superoxide dismutase (SOD) activity in oxidative fibers in response to endurance training (47-51), and recent data suggest that both cytosolic (i.e., Cu/Zn SOD) and mitochondrial (MnSOD) isoforms are upregulated (51, 52). However, other investigators have shown little or no change in SOD activity (45, 53).

Hydrogen peroxide metabolism may have some unique features in skeletal muscle (e.g., in contrast to heart muscle) because catalase activity is not associated with isolated muscle mitochondria, and overall catalase activity is only about 1.4% of that in liver cells (54). Furthermore, in contrast to heart (55) or diaphragm muscle (51), catalase activity has not been shown to increase with exercise training in limb muscle (45, 47). In fact, several reports have demonstrated decreases in catalase activity in both oxidative and mixed fiber limb muscle (46, 53). Glutathione peroxidase enyzme is clearly upregulated in nearly every exercise paradigm (45-49, 51, 53) suggesting that the glutathione scavenging system may be primarily responsible for H₂O₂ metabolism in exercising muscle. Interestingly, Girten et al. (56) showed that with simulated weightlessness (limb suspension), AOX expression is decreased in rats. Furthermore, exercise conditioning prior to a period of simulated weightlessness increased levels of SOD and CAT activity in soleus muscle following weightlessness, and pharmacologic stimulation of metabolism with dobutamine during simulation also attenuated the subsequent decreases in AOX function (56).

	ROS and Heat Stress

Top Abstract Introduction Evidence for ROS Production... Effects of AOXs on... AOXs and Skeletal Muscle... Muscle Conditioning and... ROS and Heat Stress Hypoxia as a Stimulant... Reactive Oxygen and Muscle... ROS-Induced Respiratory Muscle... Conclusions and Future... References

 
Muscles are tremendous heat generators during exercise, a fact that is largely overlooked in the free radical biology literature. Shortly after exhaustive exercise, rat limb muscles average temperatures in excess of 43°C, and core temperatures can exceed 42°C (57). Many of the results for the effects of exhaustive exercise (e.g., on antioxidant responses) may in fact relate to the influence of heat as a stimulus rather than increases in metabolic activity, but this has not been studied in detail.

Recent preliminary evidence from our laboratory has shown marked increases in intracellular ROS production in rodent diaphragm during brief exposure to 42°C, using the fluorescent probe, hydroethidine, as shown in Figure 4 (58, 59). Other researchers have also suggested that hyperthermia leads to oxidant production in skeletal muscle, resulting in increased heat shock protein (HSP) expression, mitochondrial production of O₂^•-, progressive mitochondrial uncoupling, and increased mitchondrial ubisemiquinone concentration immediately following heat stress, induced by exercise (60). We have demonstrated that in response to a 15-min exposure to 42°C, heat shock proteins such as HSP₇₀ are upregulated, and this can be blocked with superoxide or hydroxyl scavengers such as Tiron or DMSO (61). Thus, ROS may be partly responsible for the induction of HSP in heat stress (62), and these results illustrate that heat stress and ROS production are closely related. However, though we are reasonably certain ROS production is increased in heat, we are uncertain regarding the biological site of ROS production and by what specific mechanisms heat and or ROS can induce HSP expression and muscle contractile inhibition.

View larger version (105K): [in this window] [in a new window]   Figure 4.   Confocal image of mouse diaphragm, before (A) and after (B) exposure to 42°C heat stress. Tissues preloaded with hydroethidine, a superoxide-sensitive fluorescent probe. The lighter areas are hydroethidine fluorescence which are diminished markedly after 25 min of heat stress. Ethidium formation, the product of hydroethidine-superoxide reactions was also increased in this experiment; however, it is not visible in this black and white image.

View larger version (101K): [in this window] [in a new window]   Figure 5.   Electron microscopy of diaphragm muscle in: (A) matched control conditions at 37°C and (B) 60 min at 42°C. Note the mitochondrial swelling in both subsacrolemmal (around the nucleus) and cytosolic (between the myofibrils). Electron micrographs prior to 60 min showed more subtle evidence of mitochondrial injury.

	Hypoxia as a Stimulant for Oxidant Production

Top Abstract Introduction Evidence for ROS Production... Effects of AOXs on... AOXs and Skeletal Muscle... Muscle Conditioning and... ROS and Heat Stress Hypoxia as a Stimulant... Reactive Oxygen and Muscle... ROS-Induced Respiratory Muscle... Conclusions and Future... References

 
It is a paradox of free radical biology that excessive O₂ and decreased O₂ both result in increased ROS production in some tissues and under some conditions. The latter is often referred to as reductive stress and is believed to reflect the influence of an overabundance of reducing equivalents in the cell, driving one electron reduction of O₂ (66, 67). However, other mechanisms have been implicated in various tissues (66). In reductive stress we usually associate oxidant production with subsequent conditions of reoxygenation or reperfusion. However, elevated ROS formation can also occur in hypoxia alone. The most convincing evidence for this phenomenon has been demonstrated by Schumaker et al. (68), where in cardiac myocytes, in vitro, intracellular ROS production is markedly increased at PO₂ values of around 5–10 Torr. These investigators also have hypothesized that ROS produced during hypoxia play an important role as a molecular signaling agent in the cardiac myocyte. Whether similar mechanisms appear in skeletal muscle is not known at this time.

Recently, our laboratory has demonstrated striking protective effects of a variety of AOXs on muscle during hypoxia, prior to reoxygenation (69). The effects on max force of the superoxide scavenger, Tiron, are shown in Figure 6. These data suggest that low levels of oxidant production in conditions of hypoxia or reductive stress are contributing to the reduction of force in hypoxia. The extent to which individual skeletal muscle cells are exposed to regional hypoxia in any normal condition of exercise is poorly understood (70). However, it is likely that in extreme exercise, the delicate balance between oxygen supply and demand can be compromised, regionally, and that tissue oxygen can fall below a critical level in some portions of the cell, resulting in a buildup of reducing equivalents and enhanced ROS formation.

View larger version (46K): [in this window] [in a new window]   Figure 6.   Effects of antioxidant (AOX) treatment in response to hypoxia and reoxygenation. All AOXs tested resulted in preservation of force production in hypoxia and following reoxygenation. (Redrawn from Ref. 69).

The potential role of oxidant stress in ischemia/reperfusion injury is more established but still somewhat controversial. Ischemia is a considerably different stimulus from hypoxia because it is characterized by acidosis, accumulation of ·NO and NO-metabolites, accumulation of adenosine and other energy metabolites, and presumably accumulation of oxidants and oxidation products. In pure hypoxia, these conditions are less likely to occur. During reperfusion, not only is the tissue suddenly capable of generating large levels of reactive oxygen, but the metabolites are transiently changed, prior to the ability of the tissue to re-establish equilibrium. Muscle injury due to ischemia-reperfusion is of some practical importance in emergency medicine and surgery (e.g., revascularlization of severely injured tissues following trauma, following "encapsulation syndrome," or in surgical reconstruction). Most experimental models point to a definite role of hydroxyl radical or other hydroxylating species in ischemia/reperfusion of skeletal muscle because the injury can be attenuated by providing chelators of iron such as desferroxamine (22) or scavengers of hydroxyl radical (72, 73). Although early studies in rats (21, 73) suggested that a large part of the source of oxidative damage came from xanthine oxidase mechanisms, this has been difficult to show in humans and other species (22). A large portion of the xanthine oxidase activity in ischemia/reperfusion or in other forms of injury could come from the activity within inflammatory neutrophils or within endothelium during inflammation (74).

	Reactive Oxygen and Muscle Injury

Top Abstract Introduction Evidence for ROS Production... Effects of AOXs on... AOXs and Skeletal Muscle... Muscle Conditioning and... ROS and Heat Stress Hypoxia as a Stimulant... Reactive Oxygen and Muscle... ROS-Induced Respiratory Muscle... Conclusions and Future... References

 
Muscle injury can occur in response to numerous stimuli (75), including exhaustive endurance exercise (76, 77), but it is most commonly seen in response to lengthening (i.e., eccentric contractions). Examples of lengthening contractions include the action of the quadriceps muscle while running downhill. Lengthening contractions lead to greater muscle injury than isometric or shortening contractions (78, 79), and this is probably related directly to strain absorbed by individual myofibrils (80). In the rodent model, there is a delayed onset of injury to the muscle, which peaks approximately 3 days after the initial insult (81). This coincides with the timing of secondary injury (82) and soreness (83) seen in humans. Faulkner et al. have shown that maximal muscle tension initially falls following eccentric contractions, then rises to 70% over the first day and falls again to 48% of initial force by Day 3.

What role do oxidants play in muscle injury? Recent evidence suggests that significant free radical activity and oxidative injury may occur as early as 24 hr after injury (84). Furthermore, AOXs may attenuate the injury in some conditions. For example, Zerba et al. (81) pretreated young, adult, and old mice with intraperitoneal injections of polyethylene glycol-superoxide dismutase (PEG-SOD) prior to a protocol of lengthening contractions. Three days postcontraction, untreated mice developed a maximum force of 60% in adult mice and 44% in aged mice, whereas PEG-SOD-treated mice demonstrated forces of 80%–90% in young and 70% in old mice. SOD-treated old mice also showed significant preservation of max force after 10 min of contraction, compared to control mice. Non-aged mice (i.e.,young and adult) showed no change. In the same study, histologic examination of the muscles was undertaken to determine the percentage of uninjured fibers in the young and adult mice. Uninjured fibers increased from 77%–96% with SOD treatment. These results were taken as evidence of a significant role for ROS in the delayed onset of muscle injury.

A similar study in rats was undertaken by the same group using vitamin E as the antioxidant instead of PEG-SOD (85). In contrast to PEG-SOD, vitamin E showed no protective effect in the muscles with regard to changes in maximum force or signs of histologic damage. However, increases in serum enzyme activities of creatine kinase (CK) and pyruvate kinase (PK), considered markers of cell damage, were decreased in treated muscle compared to control or vehicle-treated muscle. Similar failures by vitamin E to protect muscle from delayed onset injury were seen in studies by Jakeman et al. (86) and Warren et al. (87). Differences in effects of PEG-SOD and vitamin E are possibly due to differences in antioxidant solubility or cellular location (85). The most likely mechanism for AOX protection of contractile fibers and membranes is an inhibition of oxidation during inflammatory cell recruitment and activation associated with delayed onset of soreness (84).

	ROS-Induced Respiratory Muscle Dysfunction and Respiratory Failure

Top Abstract Introduction Evidence for ROS Production... Effects of AOXs on... AOXs and Skeletal Muscle... Muscle Conditioning and... ROS and Heat Stress Hypoxia as a Stimulant... Reactive Oxygen and Muscle... ROS-Induced Respiratory Muscle... Conclusions and Future... References

 
Considerable interest has been shown in the potential role of oxidative stress in respiratory failure, particularly with respect to its influence on respiratory skeletal muscle. Respiratory arrest is a major cause of death and hospitalization. Conditions often associated with respiratory failure such as fever, hypoxia, respiratory acidosis, increased respiratory muscle mechanical loads, sepsis, inflammation, nutritional imbalance, and poor perfusion can all promote oxidative stress in some conditions. Anzuetto et al. (23, 88-90) have performed a series of studies in which anesthetized rats were brought to complete apnea by adding inspiratory resistive loads to the airway. Under these conditions increases in lipid peroxidation products and oxidized glutathione (GSSG) were seen in the diaphragm, particularly when the muscle's AOX defenses were compromised (89, 90). Our laboratory, using a similar model, but with supplemental O₂ and increased mechanical loads for more prolonged periods, has observed more modest evidence of oxidative stress, primarily a reduction in total GSH, with little or no increase in GSSG (91). We also saw no consistent change in lipid peroxidation. However, blood taken from animals undergoing prolonged resistive loading did show increases in carbon-based radical adducts from the circulation (92). Supinski et al. (93) have also done extensive work with these models, particularly using decerebrate, unanesthetized preparations. These investigators have shown that the amount of oxidative stress depends a great deal on supplemental O₂ and that treatment of the animals with antioxidants can, under some conditions, delay the onset of failure and protect the diaphragm from mechanical dysfunction.

One of the more common forms of respiratory failure is associated with septic shock, often leading to a condition referred to as acute respiratory distress syndrome (ARDS). Interestingly, septic shock results in diaphragm and other skeletal muscle dysfunction, with evidence of oxidative stress (94). The loss of diaphragm function in sepsis can be greatly attenuated by preadministration of AOXs (95, 96), suggesting a significant role of oxidant stress in this syndrome. Septic shock could influence respiratory muscle function through a number of pathways such as upregulation of inducible NOS activity (97), recruitment and activation of neutrophils into the capillary circulation, and direct or indirect effects of sepsis and various related cytokines on mitochondrial function.

	Conclusions and Future Directions

Top Abstract Introduction Evidence for ROS Production... Effects of AOXs on... AOXs and Skeletal Muscle... Muscle Conditioning and... ROS and Heat Stress Hypoxia as a Stimulant... Reactive Oxygen and Muscle... ROS-Induced Respiratory Muscle... Conclusions and Future... References

 
Do normal skeletal muscles undergo oxidative stress or are the oxidants produced during exercise simply a pattern within the normal homeostatic environment of the cell? The answer is perhaps semantic and depends on one's definition of stress. We prefer to think of stress as a condition that seriously threatens cell survival. Furthermore, from this viewpoint, the minimal alterations of oxidized glutathione and lipid and protein oxidation products seen during exercise would be consequences of normal oxidant production and perhaps be important in cell signaling. Whatever the definition, skeletal muscles appear to function quite well in what appears to be relatively high oxidizing environments. There are conditions in which the delicate balance between oxidant production and antioxidant defense may be compromised, and these could include severe heat stress, infection or sepsis, severe ischemia, nutritional deficiencies, or wide variations in muscle activation for the given level of conditioning of the muscle, resulting in injury.

It seems to us that the more important questions are related to what reactive oxygen and products of oxidation chemistry are doing in normal cell function. We hypothesize, as others have, that oxidants are playing important roles in the normal contracting myocyte to regulate Ca⁺² metabolism, contractile behavior, and perhaps utilization and control of energy substrates. Fatigue and hypoxia experiments suggest that they may be playing important roles as negative feedback signaling molecules, functioning to protect the muscle from overstimulation and subsequent injury. In many other systems growth factors induce reactive oxygen production (98, 99). Are ROS involved with muscle fiber adaptations and differentiation in response to exercise stimuli? How are antioxidant control enzymes orchestrated during conditioning to maximize cell function and minimize the potential damaging influences of ROS? These and many other related questions lie in the frontiers of future free radical biology as it applies to skeletal muscle function in health and disease.

	Acknowledgments

 
Special thanks to Valerie Wright for manuscript preparation, editing, and experimental results.

	Footnotes

 
Supported by NIH HLBI 53333, The Emergency Medicine Foundation, and the American Heart Association.

¹ To whom requests for reprints should be addressed at The Ohio State University, Pulmonary and Critical Care Medicine, 325N Means Hall, 1654 Upham Drive, Columbus, OH 43210. E-mail: clanton.1{at}osu.edu

	References

Top Abstract Introduction Evidence for ROS Production... Effects of AOXs on... AOXs and Skeletal Muscle... Muscle Conditioning and... ROS and Heat Stress Hypoxia as a Stimulant... Reactive Oxygen and Muscle... ROS-Induced Respiratory Muscle... Conclusions and Future... References

 

1. Milnor WR. Regional circulations. In: Mountcastle VB, Ed. Medical Physiology (14th ed). St. Louis, MO: C.V. Mosby, pp1102–1103, 1980.
2. Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 59:527–605, 1979.[Free Full Text]
3. Davies KJ, Quintanilha TA, Brooks GA, Packer L. Free radicals and tissue damage produced by exercise. Biochem Biophys Res Commun 107:1198–1205, 1982.[Medline]
4. Rink TJ, Tsien RY, Pozzan T. Cytoplasmic pH and free Mg²⁺ in lymphocytes. J Cell Biol 95:189–196, 1982.[Abstract/Free Full Text]
5. Jackson MJ, Johnson K. Application of electron spin resonance techniques to the detection of free radicals in muscle tissue. In: Quintanilha TA, Ed. CRC Handbook of Free Radicals and Anitoxidants in Biomedicine. Boca Raton, FL: CRC, pp209–213, 1989.
6. Reid MB, Soji T, Moody MR, Entman ML. Reactive oxygen in skeletal muscle II: Extracellular release of free radicals. J Appl Physiol 73:1805–1809, 1992.[Abstract/Free Full Text]
7. Kolbeck RC, She Z-W, Callahan LA, Nosek TM. Increased superoxide production during fatigue in the perfused rat diaphragm. Am J Respir Crit Care Med 156:140–145, 1997.[Abstract/Free Full Text]
8. Zulueta JJ, Yu F-S, Hertig IA, Thannickal VJ, Hassoun PM. Release of hydrogen peroxide in response to hypoxia-reoxygenation: Role of NAD(P)H oxidase-like enzyme in endothelial cell plasma membrane. Am J Respir Cell Mol Biol 12:41–49, 1995.[Abstract]
9. De Keulenaer GW, Chappell DC, Ishizaka N, Nerem RM, Alexander RW, Griendling KK. Oscillatory and steady laminar shear stress differentially affect human endothelial redox state: Role of a superoxide-producing NADH oxidase. Circ Res 82:1094–1101, 1998.[Abstract/Free Full Text]
10. Reid MB, Haak KE, Francik KM, Volbertg PA, Kabzik PA, West MA. Reactive oxygen in skeletal muscle I. Intracellular oxidant kinetics and fatigue in vitro. J Appl Physiol 73:1797–1804, 1992.[Abstract/Free Full Text]
11. Supinski GS, Nethery D, Stofan D, Dimarco A. Superoxide generation by the contracting diaphragm is PLA2-dependent. Am J Respir Crit Care Med 155:A925, 1997.
12. Diaz PT, She ZW, Davis WB, Clanton TL. Hydroxylation of salicylate by the in vitro diaphragm: Evidence for hydroxyl radical production during fatigue. J Appl Physiol 75:540–545, 1993.[Abstract/Free Full Text]
13. O'neill CA, Stebbins CL, Bonigut S, Halliwell B, Longhurst JC. Production of hydroxyl radicals in contracting skeletal muscle in cats. J Appl Physiol 81:1206, 1996.
14. Narayan M, Wright VP, Berliner LJ et al. Do nitric oxide and peroxynitrite contribute to hydroxylation reactions in fatigued diaphragm? (abstract). FASEB J 11:A72, 1997.
15. Ramezanian MS, Padmaja S, Koppenol WH. Nitration and hydroxylation of phenolic compounds by peroxynitrite. Chem Res Toxicol 9:232–240, 1996.[Medline]
16. Kobzik L, Reid MB, Bredt DS, Stamler JS. Nitric oxide in skeletal muscle. Nature 372:546–548, 1994.[Medline]
17. Nohl H, Jordan W. The mitochondrial site of superoxide formation. Biochem Biophys Res Commun 138:533–539, 1986.[Medline]
18. Nohl H, Stolze K. Hypothesis: Ubisemiquinones of the mitochondrial respiratory chain do not interact with molecular oxygen. Free Rad Res Comms 16:409–419, 1992.[Medline]
19. Nohl H. A novel superoxide radical generator in heart mitochondria. FEBS Lett 214:269–273, 1987.[Medline]
20. Sjodin B, Westing YH, Apple FS. Biochemical mechanisms for oxygen free radical formation during exercise. Sports Med 10:236–254, 1990.[Medline]
21. Smith JK, Carden DL, Korthius RJ. Role of xanthine oxidase in postischemic microvascular injury in skeletal muscle. Am J Physiol 68:387–392, 1989.
22. Dorion D, Zhong A, Chiu C, Forrest CR, Boyd B. Role of xanthine oxidase in reperfusion injury of ischemic skeletal muscles in the pig and human. J Appl Physiol 75:246–255, 1993.[Abstract/Free Full Text]
23. Anzueto A, Andrade FH, Maxwell LC, Levine SM, Lawrence RA, Gibbons WJ, Jenkinson SJ. Resistive breathing activates the glutathione redox cycle and impairs performance of rat diaphragm. J Appl Physiol 72:529–534, 1992.[Abstract/Free Full Text]
24. Bejma J, Ji LL. Aging and acute exercise enhance free radical generation in rat skeletal muscle. J Appl Physiol 87:465–470, 1999.[Abstract/Free Full Text]
25. Diaz PT, Brownstein E, Clanton TL. Effects of N-acetylcysteine on in vitro diaphragm function are temperature dependent. J Appl Physiol 77:2434–2439, 1994.[Abstract/Free Full Text]
26. Khawli FA, Reid MB. N-acetylcysteine depresses contractility and inhibits fatigue of diaphragm in vitro. J Appl Physiol 77:317–324, 1994.[Abstract/Free Full Text]
27. Reid MB, Kwali F, Moody MR. Reactive oxygen in skeletal muscle III. Contractility of unfatigued muscle. J Appl Physiol 75:1081–1087, 1993.[Abstract/Free Full Text]
28. Andrade FH, Reid MB, Allen DG, 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.[Abstract/Free Full Text]
29. Brotto MA, Nosek TM. Hydrogen peroxide disrupts Ca²⁺ release from the sarcoplasmic reticulum of rat skeletal muscle fibers. J Appl Physiol 81:731–737, 1996.[Abstract/Free Full Text]
30. Abraham RZ, Kobzik L, Moddy MR, Reid MB, Stamler JS. Cyclic GMP is a second messenger by which nitric oxide inhibits diaphragm contraction. Comp Biochem Physiol 119A:177–183, 1998.
31. Shindoh CA, Dimarco A, Thomas A, Manubay P, Supinski G. Effect of N-acetylcysteine on diaphragm fatigue. J Appl Physiol 68:2107–2113, 1990.[Abstract/Free Full Text]
32. Barclay JM, Hansel M. Free radicals may contribute to oxidative skeletal muscle fatigue. Can J Physiol Pharmacol 69:279–284, 1991.[Medline]
33. Reid MB, Stokic DS, Koch SM, Khawli FA, Leis AA. N-Acetylcysteine inhibits muscle fatigue in humans. J Clin Invest 94:2468–2474, 1994.
34. Travaline JM, Sudarshan S, Roy BG, Cordova F, Leyenson V, Criner GJ. Effect of N-acetylcysteine on human diaphragm strength and fatigability. Am J Respir Crit Care Med 156:1567–1571, 1997.[Abstract/Free Full Text]
35. Kwali FA, Reid MB. N-Acetylcysteine depressed contractile function and inhibits fatigue of diaphragm in vitro. J Appl Physiol 77:317–324, 1994.
36. Mohanraj P, Wright V, Clanton TL. Tiron (1,2 Dihydroxybenzene-3,5-disulfonate) inhibits diaphragmatic muscle fatigue. Am J Respir Crit Care Med 155:A923, 1997.
37. Nohl H, Gille L, Schonheit K, Liu Y. Conditions allowing redox-cycling ubisemiquinone in mitochondria to establish a direct redox couple with molecular oxygen. Free Radic Biol Med 20:207–213, 1996.[Medline]
38. Piantadosi CA, Zhang J. Mitochondrial generation of reactive oxygen species after brain ischemia in the rat. Stroke 27:327–332, 1996.[Abstract/Free Full Text]
39. Andersson U, Leighton B, Young ME, Blomstrand E, Newsholme EA. Inactivation of aconitase and oxoglutarate dehydrogenase in skeletal muscle in vitro by superoxide anions and/or nitric oxide. Biochem Biophys Res Commun 249:512–516, 1998.[Medline]
40. Gardner PR, Nguyen D-DH, White CW. Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs. Proc Natl Acad Sci U S A 91:12248–12252, 1994.[Abstract/Free Full Text]
41. Stachowiak O, Dolder M, Wallimann T, Richter C. Mitochondrial creatine kinase is a prime target of peroxynitrite-induced modification and inactivation. J Biol Chem 273:16694–16699, 1998.[Abstract/Free Full Text]
42. Mekhfi H, Veksler V, Mateo P, Maupoil V, Rochette L, Ventura-Clapier R. Creatine kinase is the main target of reactive oxygen species in cardiac myofibrils. Circ Res 78:1016–1027, 1996.[Abstract/Free Full Text]
43. Corretti MC, Koretsune Y, Kusuoka H, Chacko VP, Zweier JL, Marban E. Glycolytic inhibition and calcium overload as consequences of exogenously generated free radicals in rabbit hearts. J Biol Chem 257:12086–12091, 1991.[Abstract/Free Full Text]
44. Gilbert HF. Biological disulfides: The third messenger? J Biol Chem 257:12086–12091, 1982.
45. Ji LL, Wu E, Thomas DP. Effect of exercise training on antioxidant and metabolic function in senescent rat skeletal muscle. Gerontology 37:317–325, 1991.[Medline]
46. Leeuwenburgh C, Frebig R, Chandwancey R, Ji LL. Aging and exercise training in skeletal muscle: Responses of glutathione and antioxidant enzyme systems. Am J Physiol 267:R439–R443, 1994.[Abstract/Free Full Text]
47. Powers SK, Criswell D, Lawler J, Martin D. Influence of exercise and fiber type on antioxidant enzyme activity in rat skeletal muscle. Am J Physiol 266:R375–R380, 1994.[Abstract/Free Full Text]
48. Powers SK, Criswell D, Lawler J, Martin D, Ji LL. Regional training-induced alterations in diaphragmatic oxidative and antioxidant enzymes. Respir Physiol 95:227–237, 1999.
49. Criswell D, Powers SK, Dodd S, Lawler J, Edwards W. High intensity training–induced changes in skeletal muscle antioxidant enzyme activity. Med Sci Sports Sci 25:1135–1140, 1993.
50. Higuchi M, Cartier LJ, Chen M, Holloszy J. Superoxide dismutase and catalase in skeletal muscle: Adaptive response to exercise. J Gerontol 40:281–286, 1985.[Medline]
51. Oh-ishi S, Kizaki T, Ookawara T, Sakurai T, Izawa T, Nagata N, Ohno H. Endurance training improves the resistance of rat diaphragm to exercise-induced oxidative stress. Am J Respir Crit Care Med 156:1579–1585, 1997.[Abstract/Free Full Text]
52. Oh-ishi S, Kizaki T, Nagasawa J, Izawa T. Effects of endurance training on superoxide dismutase activity, content, and mRNA expression in rat muscle. Clin Exp Pharmacol Physiol 24:326–332, 1997.[Medline]
53. Laughlin MH, Simpson T, Sexton WL, Brown OR, Smith JK, Korthius RJ. Skeletal muscle oxidative capacity, antioxidant enzymes, and exercise training. J Appl Physiol 68:2337–2343, 1990.[Abstract/Free Full Text]
54. Phung CD, Ezieme JA, Turrens JP. Hydrogen peroxide metabolism in skeletal muscle mitochondria. Arch Biochem Biophys 315:479–482, 1994.[Medline]
55. Kanter MM, Hamlin RL, Unverferth DV, Davis HW, Merola AJ. Effect of exercise training on antioxidant enzymes and cardiotoxicity of doxorubicin. J Appl Physiol 59:1298–1303, 1985.[Abstract/Free Full Text]
56. Girten B, Oloff C, Plato P, Eveland E, Merola AJ, Kazarian L. Skeletal muscle antioxidant enzyme levels in rats after simulated weightlessness, exercise, and dobutamine. Physiologist 32:S59–S60, 1989.[Medline]
57. Brooks GA, Hittelman AK, Faulkner JA, Beryer RE. Tissue temperatures and whole animal oxygen consumption after exercise. Am J Physiol 221:427–431, 1971.[Free Full Text]
58. Zuo L, Berliner LJ, Clanton TL. Detection of reactive oxygen produced by heat stress using a novel surface fluorometry technique. Free Radic Biol Med 25:S25, 1998.
59. Zuo L, Liu CY, Berliner LJ, Wright VP, Clanton TL. Detection of free radicals during heat stress in mouse diaphragm using laser scan confocal microscopy. Biophys J 76:A358, 1999.
60. Salo DC, Donovan CM, Davies KJ. HSP70 and other possible heat shock or oxidative stress proteins are induced in skeletal muscle, heart, and liver during exercise. Free Radic Biol Med 11:239–249, 1991.[Medline]
61. Andersen KA, Clanton TL. Redox modulation of contractile function and shock protein expression in skeletal muscle following heat (program and abstracts). Free Radic Biol Med 39, Abstract No. 1-40, 1996.
62. Gorman AM, Heavey B, Creagh E, Cotter TG, Samali A. Antioxidant-mediated inhibition of the heat shock response leads to apoptosis. FEBS Lett 445:98–102, 1999.[Medline]
63. Brooks GA, Hittelman KJ, Faulkner JA, Beyer RE. Temperature, skeletal muscle mitochondrial functions, and oxygen debt. Am J Physiol 220:1053–1059, 1971.[Free Full Text]
64. Febbraio MA, Snow RJ, Stathis CG, Hargreaves. M, Carey MF. Effect of heat stress on muscle energy metabolism during exercise. J Appl Physiol 77:2827–2831, 1994.[Abstract/Free Full Text]
65. Ranatunga KW. Thermal stress and Ca-independent contractile activation in mammalian skeletal muscle fibers at high temperatures. Biophys J 66:1531–1541, 1994.[Abstract/Free Full Text]
66. Dawson TL, Gores GJ, Nieminen A-L, Herman B, Lemasters JJ. Mitochondria as a source of reactive oxygen species during reductive stress in rat hepatocytes. Am J Physiol 264:C961–C967, 1993.[Abstract/Free Full Text]
67. Kehrer JP, Lund LG. Cellular reducing equivalents and oxidative stress. Free Radic Biol Med 17:65–75, 1994.[Medline]
68. Vanden Hoek TL, Li C, Shao Z, Shumacker PT, Becker LB. Significant levels of oxidants are generated by isolated cardiomyocytes during ischemia prior to reperfusion. J Mol Cell Cardiol 29:2571–2583, 1997.[Medline]
69. Mohanraj P, Merola AJ, Wright VP, Clanton TL. Antioxidants protect rat diaphragmatic muscle function under hypoxic conditions. J Appl Physiol 84:1960–1966, 1998.[Abstract/Free Full Text]
70. Jones DP. Intracellular diffusion gradients of O₂ and ATP. Am J Physiol 250:C663–C675, 1986.[Abstract/Free Full Text]
71. Clanton TL, Wright VP, Merola AJ. Antioxidants preserve creatine phosphate in hypoxic diaphragm. Am J Respir Crit Care Med 159:A719, 1999.
72. Supinski G, Stofan D, Dimarco A. Effect of ischemia-reperfusion on diaphragm strength and fatigability. J Appl Physiol 75:2180–2187, 1993.[Abstract/Free Full Text]
73. McCutchan HJ, Schwappach JR, Enquist EG, Walden DL, Terada LS, Reiss OK, Leff JA, Repine JE. Xanthine oxidase–derived H₂O₂ contributes to reperfusion injury of ischemic skeletal muscle. Am J Physiol 258:H1415–H1419, 1990.[Abstract/Free Full Text]
74. Hellsten Y, Frandsen U, Orthenblad N, Sjodin B. Xanthine oxidase in human skeletal muscle following eccentric exercise: A role in inflammation. J Physiol (Lond) 498:239–248, 1997.[Medline]
75. Faulkner JA, Brooks SV, Opiteck JA. Injury to skeletal muscle fibers during contractions: Conditions of occurrence and prevention. Phys Ther 73:911–921, 1993.[Abstract/Free Full Text]
76. Peeze Binkhorst FM, Kuipers H, Heymans J, Frederik PM, Slaaf DW, Tangelder G-J, Reneman RS. Exercise-induced focal skeletal muscle fiber degeneration and capillary morphology. J Appl Physiol 66:2857–2865, 1989.[Abstract/Free Full Text]
77. Byrd SK. Alterations in sarcoplasmic reticulum: A possible link to exercise-induced muscle damage. Med Sci Sports Sci 24:531–536, 1992.
78. Armstrong RG, Ogilvie RW, Schwane JA. Eccentric exercise–induced injury to rat skeletal muscle. J Appl Physiol 54:80–93, 1983.[Abstract/Free Full Text]
79. Faulkner JA, Jones DA, Round JM. Injury to skeletal muscles of mice by forced lengthening during contractions. Q J Exp Physiol 74:661–670, 1989.[Abstract/Free Full Text]
80. Brooks SV, Zerba E, Faulkner JA. Injury to muscle fibres after single stretches of passive and maximally stimulated muscles in mice. J Physiol 488:459–469, 1995.[Medline]
81. Zerba E, Komorowske TE, Faulkner JA. Free radical injury to skeletal muscles of young, adult, and old mice. Am J Physiol 258:C429–C435, 1990.[Abstract/Free Full Text]
82. Jones DA, Newham DJ, Round JM, Tolfree SE. Experimental human muscle damage: Morphological changes in relation to other indices of damage. J Physiol (Lond) 375:435–448, 1986.[Abstract/Free Full Text]
83. Armstrong RG. Mechanisms of exercise-induced delayed onset muscular soreness: A brief review. Med Sci Sports Exerc 16:529–538, 1984.[Medline]
84. Best TM, Fiebig R, Corr DT, Brickson S, Ji LL. Free radical activity, antioxidant enzyme and glutathione changes with muscle stretch injury in rabbits. J Appl Physiol 87:74–82, 1999.[Abstract/Free Full Text]
85. Van Der Meulen JH, McArdle A, Jackson MJ, Faulkner JA. Contraction-induced injury to the extensor digitorum longus muscles of rats: The role of vitamin E. J Appl Physiol 83:817–823, 1997.[Abstract/Free Full Text]
86. Jakeman P, Maxwell S. Effect of antioxidant supplementation on muscle function after eccentric exercise. Eur J Appl Physiol 67:430, 1993.
87. Warren JA, Jenkins RR, Packer L, Witt EH, Armstrong RG. Elevated muscle vitamin E does not attenuate eccentric exercise-induced muscle injury. J Appl Physiol 72:2168–2175, 1992.[Abstract/Free Full Text]
88. Anzueto A, Supinski GS, Levine SM, Jenkinson SG. Mechanisms of disease: Are oxygen-derived free radicals involved in diaphragmatic dysfunction? Am J Respir Crit Care Med 149:1048–1052, 1994.[Medline]
89. Morales CF, Anzueto A, Andrade F, Levine SM, Maxwell LC, Lawrence RA, Jenkinson SG. Diethylmaleate produces diaphragmatic impairment after resistive breathing. J Appl Physiol 75:2406–2411, 1993.[Abstract/Free Full Text]
90. Andrade F, Anzueto A, Napier W, Levine S, Lawrence RA, Jenkinson SG, Maxwell LC. Effects of selenium deficiency on diaphragmatic function after resistive loading. Acta Physiol Scand 162:141–148, 1998.[Medline]
91. Borzone G, Julian M, Merola AJ, Clanton TL. Loss of diaphragm glutathione is associated with respiratory failure induced by resistive breathing. J Appl Physiol 76:2825–2831, 1994.[Abstract/Free Full Text]
92. Hartell MG, Borzone G, Clanton TL, Berliner LJ. Detection of free radicals in blood by electron spin resonance in a model of respiratory failure in the rat. Free Radic Biol Med 17:467–472, 1994.[Medline]
93. Supinski GS, Stofan D, Ciufo R, Dimarco A. N-acetylcysteine administration alters the response to inspiratory resistive loading in oxygen supplemented rats. J Appl Physiol 82:1119–1125, 1997.[Abstract/Free Full Text]
94. Supinski G, Nethery D, Stofan D, Dimarco A. Comparison of the effects of endotoxin on limb, respiratory, and cardiac muscles. J Appl Physiol 81:1370–1378, 1996.[Abstract/Free Full Text]
95. Van Surell C, Boczkowski J, Pasquier C, Du Y, Franzini E, Aubier M. Effects of N-acetylcysteine on diaphragmatic function and malondialdehyde content in Escherichia coli endotoxemic rats. Am Rev Respir Dis 146:730–734, 1992.[Medline]
96. Supinski G, Nethery D, Dimarco A. Effect of free radical scavengers on endotoxin-induced respiratory muscle dysfunction. Am Rev Respir Dis 148:1318–1324, 1993.[Medline]
97. Boczkowski J, Lanone S, Ungureanu-Longrois D, Danialou G, Fournier T, Aubier M. Induction of diaphragmatic nitric oxide synthase after endotoxin administration in rats: Role on diaphragmatic contractile dysfunction. J Clin Invest 98:1550–1559, 1996.[Medline]
98. Sundaresan M, Yu ZX, Ferrans VJ, Sulciner DJ, Gutkind JS, Irani K, Goldschmidt-Clermont PJ, Finkel T. Regulation of reactive-oxygen-species generation in fibroblasts by Rac1. Biochem J 318:379–382, 1996.
99. Irani K, Xia Y, Zweier JL, Sollott SJ, Der CJ, Fearon ER, Sundaresan M, Finkel T, Goldschmidt-Clermont PJ. Mitogenic signaling mediated by oxidants in ras-transformed fibroblasts. Science 275:1649–1652, 1997.[Abstract/Free Full Text]</