Oxidants and Skeletal Muscle Function: Physiologic and Pathophysiologic Implications (44450)

Previous studies havedemonstrated thatskeletal 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 withexogenous antioxidants suggests that normal redox tone during contraction Is Influencing ongoing contractile function, bothat 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 alsosuggests a closerelationship between reactive oxygen andcontractile activity. Therefore, thereappears to bea significant rolefor 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. Ischemla-reperfuslon, severe hypoxia, severe heat stress, septic shock, and stretch-Induced Injury may all lead to oxidant-mediated Injury to myocytes, resulting In mechanical dysfunction. [P,S.E. B.M. 1999, Vol 222l 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 Oz metabolism during what would be considered "normal activity. " Oz 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 Oz [i.e., 1%-2% (2)] is reduced to superoxide (OZO_), then exercising muscles could be potent genSupported by NIH HLBI 53333. The Emergency Medicine Foundation. and the American Heart Association. I 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. @osu.edu 0037-9727/99/2223·0253$14,00/0 Copyright © 1999 by the Society for Experimental Biology and Medicine erators 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 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 OXIDANTS AND SKELETAL MUSCLE FUNCTION 253 extracellular superoxide (02) , 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 redoxsensitive 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 I. 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 . ~re 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. The molecular sources of ROS in skeletal muscle are poorly understood, and they probably differ depending on the physiologic state of the tissue and possibly the fiber type of the muscle. One likely candidate is mitochondrial electron transport. For example, ROS may be formed at several sites (including complex I, II, and III) that are capable of single reduction of O2 to O2' . , depending on a number of Level of Muscle Stimulation Figure 1. The salicylate hydroxylation product (2.3dihydroxybenzoicacid) taken from muscle after various degrees of nerve-stimulatedactivation. (Derived from Ref. 12). 254 OXIDANTS AND SKELETAL MUSCLE FUNCTION conditions (17, 18). In heart mitochondria, exogenous NADH oxidoreductase may be another source of O2•• production under conditions of high NADH (19). Whether this source of ROS is common to mammalian skeletal muscle is not yet known. Other possibilities include various poorly understood oxidoreductases on the membrane and cytosol. It has also been suggested that xanthine oxidase is involved in ROS formation in skeletal muscle (20), particularly in the vascular compartment. Interestingly, substrates for its activity (i.e, hypoxanthine and xanthine) can be delivered to a muscle capillary bed via the circulation from other tissues, However, its role as a source of ROS in skeletal muscle remains unclear. Even in conditions of ischemia reperfusion injury, there appears to be species-specific and conditionspecific aspects to its involvement (21, 22). 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 (II). 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 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+ available for cross-bridge activation; however, this has never been tested directly in intact muscle. The effect of mild oxidant treatment with H20 2 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+