Nitric oxide, reactive oxygen species, and skeletal muscle contraction : Medicine & Science in Sports & Exercise

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BASIC SCIENCES: Reviews: Symposium: Antioxidant and Redox Regulation of Cellular Signaling

Nitric oxide, reactive oxygen species, and skeletal muscle contraction


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Medicine and Science in Sports and Exercise 33(3):p 371-376, March 2001.
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Skeletal muscle continually produces nitric oxide (abbreviated NO.NO) and reactive oxygen species (ROS) that exert complex effects on muscle contraction, metabolism, blood flow, and gene expression. From the outset, the research in our laboratory has focused on the functional importance of NO and ROS as modulators of the contractile process. This review outlines my current view of things from the perspective of data that we and our collaborators have generated. The text contains numerous references to the discoveries of other investigators—the concepts could not be developed otherwise—but such a focused article cannot do justice to the exciting and important research contributed by other laboratories. Readers who are interested in examining the topic more closely may wish to consult recent review articles by Supinski (73), Lawler and Powers (43), Wolin and coworkers (80), and myself (60–62).

NO Regulation of Muscle Contraction

NO production by muscle.

NO is produced in biological systems via the enzymatic action of NO synthase (NOS), a family of enzymes that are differentially expressed among mammalian cell types. Skeletal muscle constitutively expresses the type 1 isoform of NO synthase (NOS1), which is localized to the sarcolemma and is preferentially expressed by fast-type muscle fibers (36). Skeletal muscle fibers of rodents also constitutively express the type 3 isoform or NOS3 (37). Expression of NOS3 does not obviously correlate with myosin isoform expression or contractile velocity. Rather, NOS3 co-localizes with muscle mitochondria. To our knowledge, rodent skeletal muscle was the first mammalian cell type shown to constitutively co-express more than one NOS isoform. We have used several methods to assess NO production by skeletal muscle, including the chemiluminescence technique (36) and cytochrome c assay (7,36). Isolated rat diaphragm releases NO derivatives at slow rates under resting conditions and at higher rates during repetitive contraction (7,36,65). NO production by mouse diaphragm and mouse limb muscles exhibits similar characteristics.

NO depression of force.

Endogenous NO clearly modulates contractile function of mammalian skeletal muscle. Studies using excised diaphragm showed that the force of submaximal tetanic contractions was increased by each of several NOS inhibitors (7,36) and by hemoglobin (36), a NO scavenger. Conversely, submaximal force was depressed by NO donors (7,36). None of these interventions altered maximal tetanic force. These findings indicated that endogenous NO exerts a tonic effect in unfatigued muscle, shifting the force-frequency curve rightward. Such data indicate that NO effects on skeletal muscle force production are similar to those observed in cardiac and smooth muscle, i.e., NO inhibits contraction (61). Other experiments indicate that endogenous NO does not influence acute fatigue (see below) or the stability of excised muscle preparations in vitro(1,51,65) as neither is altered by NOS blockade.

NO/cGMP signaling.

NO modulates skeletal muscle contraction via multiple pathways. In cardiac and smooth muscle, NO effects are primarily mediated by cGMP as a second messenger. A similar NO/cGMP pathway exists in skeletal muscle (1,36). NO donors increase cGMP concentration in muscle whereas NOS blockade decreases cGMP levels. Immunohistochemical studies have demonstrated that cGMP content is not fiber type-specific and that cGMP staining preferentially localizes to the subsarcolemmal region in close approximation to NOS1. Force is diminished by interventions that increase cGMP signaling, i.e., NO donors, cGMP analogs, and phosphodiesterase inhibitors (1,36). Conversely, force is increased by probes that decrease cGMP signaling, i.e., NOS inhibitors and soluble guanylate cyclase inhibitors. These observations indicate that NO/cGMP signaling inhibits skeletal muscle contraction. However, the magnitude of cGMP-dependent changes are consistently less than the overall effect of NO on muscle. We therefore have concluded that NO depresses contractile function via both a cGMP-dependent mechanism and a cGMP-independent effect (61).

The target of NO/cGMP signaling has not been established. At the cellular level, we tested the effects of NO using intact single fibers isolated from mouse limb muscle (9). LY83583, an inhibitor of soluble guanylate cyclase, exaggerated tetanic calcium transients, suggesting that endogenous cGMP inhibits calcium transients. One potential target for such action is phospholamban, a protein that inhibits SR Ca2+ATPase activity. Phospholamban is inhibited by cGMP (10), which thereby disinhibits the calcium pump and accelerates calcium uptake by the SR. However, phospholamban is expressed only by slow skeletal and cardiac muscles (75) and cannot mediate the effects of NO/cGMP signaling observed in fast-twitch muscle (1,36). Other actions of cGMP in striated muscle include phosphorylation of SR calcium release channels (79) and phosphorylation of sarcolemmal slow-type calcium channels (38). However, these actions tend to increase force and cannot mediate contractile depression by cGMP. Thus, the mechanism whereby NO/cGMP signaling depresses force remains enigmatic.

cGMP-independent mechanisms.

NO also inhibits contraction via a cGMP-independent mechanism (36) that may involve direct modification of NO-sensitive regulatory proteins. The sarcoplasmic reticulum (SR) is an attractive site for such interactions. NO has complex effects on activity of the SR calcium release channel (2,47,72). The channel is a large homotetramer composed of 565-kDa subunits. Each subunit contains multiple classes of reactive sulfhydryls (81), including < 3 sulfhydryls that are highly sensitive to redox modulation (84). NO donors have dose-dependent effects on channel activity (2). At low concentrations, NO donors protect the channel against oxidation-induced activation without altering channel function directly. At high concentrations, the effects of NO donors resemble those of hydrogen peroxide or other oxidants: the channel is activated, ryanodine binding is enhanced, and channel subunits undergo covalent cross-linking. The latter effect represents a possible mechanism whereby excess NO might compromise muscle function, e.g., during pharmacologic challenge or inflammatory disease. NO does not appear to directly inhibit the SR calcium release channel. NO donors do not inhibit calcium transients in intact single fibers when cGMP signaling is blocked (9). Nor do NO donors inhibit activity of isolated calcium release channels (2).

In contrast, NO directly inhibits myofilament function. NO donors shift the calcium-force relationship of intact muscle fibers rightward with no change in slope, a reversible effect that appears to be cGMP-independent (9). At similar concentrations, NO donors do not alter maximum force or the velocity of unloaded shortening (9,50,51). In combination, these findings suggest that NO inhibits the calcium sensitivity of myofilaments via a cGMP-independent mechanism. Other investigators also have found that NO donors alter myofilament function, i.e., reducing the maximum velocity of shortening (25) and decreasing myosin ATPase activity (56).

NO and muscle metabolism.

In theory, NO could inhibit contractile function indirectly by limiting cellular energetics. The NOS3 isoform is closely associated with skeletal muscle mitochondria (37) and attenuates mitochondrial respiration by inhibiting cytochrome oxidase (18) or other mitochondrial enzymes (6). NO derivatives also inhibit glyceraldehyde-3-phosphate dehydrogenase (48) and creatine kinase (28), mediate exercise-stimulated glucose transport (68), and stimulate oxidation of glucose, pyruvate, palmitate, and leucine (83). These actions could limit ATP availability. Such limitation might indirectly inhibit contractile function when muscle is exposed to NO donors (4,28) or when tissue inflammation exaggerates NO production (13,23). However, existing data do not support this model. Inhibition of NO synthesis increases oxygen consumption of resting skeletal muscle by only 0.5 fold (35,69), a subtle adjustment for tissue that is capable of 20- to 500-fold increases during exercise (26). Also, energetic limitation is predicted to lessen the stability of excised muscle, to accelerate fatigue, or both. NOS blockade does neither.

Contractile Regulation by Endogenous ROS

ROS activity in unfatigued muscle.

In unfatigued muscle, ROS are produced at low rates (7,63) and are essential for normal force production. Selective depletion of endogenous ROS by antioxidant enzymes causes a reduction in force that reverses with enzyme washout (59,64). Exogenous ROS have biphasic effects on unfatigued muscle. Exposure to low levels of ROS increases force (8,54,64), whereas higher concentrations depress force (8,52).

Mechanisms of ROS action.

The literature suggests several molecular targets whereby ROS could increase force. ROS increase the open probability of SR calcium release channels (3,24,54) and inhibit the calcium-dependent ATPase (29,31,44,49,82). Either action would tend to increase calcium transients in contracting muscle and thereby increase force. The t-tubule voltage sensor also has been suggested as a molecular target whereby ROS might influence SR calcium release (16,54,58). However, tetanic calcium transients are relatively insensitive to hydrogen peroxide challenge (8), suggesting the low level of ROS in rested muscle does not exert a large effect on SR function. ROS may also act downstream of the SR. In unfatigued muscle fibers, brief exposure to hydrogen peroxide shifts the force-calcium relationship leftward, increasing the force developed at a given level of cytosolic calcium (8). Thus, low levels of ROS appear to increase calcium sensitivity of the myofilaments. It should be noted that most studies of ROS effects on muscle contraction have used exogenous hydrogen peroxide as the ROS stimulus (8,16,54,58,64). Exogenous sources of other ROS, e.g., systems that generate superoxide anions or hydroxyl radicals, might yield different results and have not been tested systematically.

Antioxidant effects of NO.

The net effect of endogenous ROS on unfatigued muscle—to increase force—is opposed by NO which depresses force. These actions may be directly linked. NO has antioxidant properties in a number of biological systems (33) and could have similar actions in skeletal muscle. For example, endogenous NO inhibits the release of muscle-derived ROS (7), suggesting that NO limits either synthesis or diffusion of ROS within the tissue. Such “antioxidant” actions might restrict the effects of endogenous ROS, thereby depressing force indirectly.

ROS activity in fatigue.

During strenuous contractions, skeletal muscle produces free radicals at accelerated rates (14,19,32,40,41). These include both NO derivatives (11,36) and ROS (22,30,39,53,63,66). Oxidant accumulation within exercising muscle causes oxidative stress (5) and contributes to the development of acute fatigue (60). It does not appear that NO and ROS are equally involved in this process.

In principle, NO could promote fatigue. It can be argued that NO might limit muscle energetics (an argument at odds with the data; see above) and that NO diminishes the calcium sensitivity of muscle myofilaments (25,56), a hallmark of fatigue (78). However, endogenous NO does not appear to directly mediate fatigue. NOS blockade does not inhibit fatigue of excised muscle in vitro(60). Systemic administration of NOS inhibitors accelerates fatigue (4) and paradoxically depresses oxygen consumption of intact muscle (17); these appear to be indirect effects caused by loss of vascular regulation.

In contrast, endogenous ROS appear to play a causal role in the fatigue process. Strenuous exercise increases ROS levels in the cytosol (63), extracellular space (66), and vascular compartment (39,53) of exercising muscle. This elevates biochemical indices of oxidative stress including glutathione oxidation and malondialdehyde production (5,55). Pharmacologic antioxidants inhibit fatigue of excised rodent muscle (20,34,63), perfused mammalian muscle in situ(12,70,74), and intact human muscle (67,76). These observations strongly implicate ROS as mediators of fatigue (60).

Mechanisms of ROS action in fatiguing muscle.

In skinned fibers, exogenous oxidants preferentially inhibit voltage-dependent calcium release, an effect attributed to the voltage sensor, the SR calcium release channel, or associated proteins (16,58). In intact fibers, exogenous ROS compromise calcium sensitivity of the myofilaments (8). Exposure to higher ROS concentrations alters calcium regulation by the SR; calcium leak is increased and calcium pump activity is diminished (8). Thus, ROS could accelerate fatigue by several possible mechanisms. However, the physiologic importance of these mechanisms remains the subject of conjecture.

Much of what we currently know about the intracellular regulation of fatigue has been established using fixed-frequency stimulation of excised muscle preparations. Within this setting, there are several processes that are possible targets of ROS action. The initial drop in force observed within the first few min of a fixed-frequency fatigue protocol is caused by a fall in calcium sensitivity of the myofilaments (77). Antioxidants attenuate this early force decrement (36,63,70), suggesting ROS involvement. Consistent with this logic, loss of calcium sensitivity is the first event to limit force production by isolated single fibers during prolonged exposure to hydrogen peroxide (8). This response to exogenous hydrogen peroxide appears to mimic the action of endogenous ROS during the early phase of fatiguing exercise. Late in the fatigue process, skeletal muscle loses the capacity to regulate intracellular calcium (77). Exaggerated ROS production could mediate this event. Prolonged exposure of intact muscle fibers to hydrogen peroxide causes loss of SR function: a rise in intracellular calcium and slowing of calcium recovery after tetanic contractions (8). Prolonged exposure to hydrogen peroxide thus appears to increase SR calcium leak, slow calcium reuptake by the SR, or both. Accelerated ROS production by fatiguing muscle could have similar effects.

Reversibility of ROS effects.

Oxidative alterations in fatigued muscle appear to be partially reversible. Incubation with the reducing agent dithiothreitol has been shown to accelerate recovery of force after fatigue (21) although force did not return to prefatigue levels. The component of fatigue that is dithiothreitol-sensitive represents a process that is capable of reversible, redox modulation. This process has not yet been identified. An analogous response occurs in intact single fibers after prolonged exposure to hydrogen peroxide; dithiothreitol treatment increases force production, increases calcium sensitivity of the myofilaments, and restores SR function (8). Perhaps reducing agents cause similar changes within fatigued muscle fibers.


Studies from our laboratory and from other investigators have identified regulatory proteins in skeletal muscle that are sensitive to NO and ROS. These include the dihydropyridine-sensitive voltage sensor, the ryanodine-sensitive calcium release channel of the SR, the SR calcium-dependent ATPase, troponin, and myosin. Nitrosative or oxidative modification of these proteins suggest plausible mechanisms whereby NO and ROS might regulate muscle contraction. At present, however, a fundamental question persists: Which mechanisms predominate under physiologic conditions? There are few tools available to address this issue. Studies of intact muscle or excised tissue provide limited information about intracellular mechanisms. The relevance of data from subcellular systems is restricted by our inability to reproduce the redox environment of the cell. The predominant sources and intracellular concentrations of NO and ROS are not known. Nor can we duplicate the molecular localizations that appear to influence signaling events. NOS isoforms are associated with specific structures in the muscle fiber including the dystrophin complex (15), costomeres (27), the motor end-plate (42), and mitochondria (37). ROS also are produced at specific intracellular sites (57,71) and antioxidant buffers are compartmentalized within the cell (45,46). Finally, NO and ROS signaling are strongly influenced by second messenger systems that are poorly characterized in skeletal muscle. At present, we believe the most rapid progress can be made by studying intact muscle fibers. This preparation has its limitations, lacking adjacent fibers that may exert paracrine effects and cytoskeletal attachments that could influence NO or ROS production. However, important regulatory features remain. Intracellular sources of NO and ROS production are preserved, antioxidant buffers are retained, and downstream signaling pathways are relatively unperturbed. Systematic use of intact fibers should establish the relative importance of NO/ROS effects on SR calcium release, calcium sensitivity of the myofilaments, and SR calcium reuptake. Such knowledge would resolve persistent questions about redox regulation at the cellular level and should lay the groundwork for targeted investigation of molecular mechanisms.

Our research in this field has been supported by National Institutes of Health grant #HL45721.

Address for correspondence: Michael B. Reid, Ph.D., Pulmonary Medicine, Suite 520B, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030; E-mail: [email protected]


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