Free radicals generated by contracting muscle: by-products of metabolism or key regulators of muscle function?

Skeletal muscle fibers generate reactive oxygen species (ROS) at a number of subcellular sites and this generation is increased by contractile activity. Early studies suggested that generation of superoxide as a by-product of mitochondrial oxygen consumption was the major source of muscle ROS generation and that the species produced were inevitably damaging to muscle, but recent data argue against both of these possibilities. Developments in analytical approaches have shown that specific ROS are generated in a controlled manner by skeletal muscle fibers in response to physiological stimuli and play important roles in the physiological adaptations of muscle to contractions. These include optimization of contractile performance and initiation of key adaptive changes in gene expression to the stresses of contractions. These positive benefits of the ROS that are induced by contractile activity contrast starkly with the increasing evidence that ROS-induced degenerative pathways are fundamental to aging processes in skeletal muscle. A fuller understanding of these contrasting roles is recognized to be important in the design of strategies to maintain and optimize skeletal muscle function during exercise and to help prevent the devastating effects of sarcopenia and other muscle-wasting conditions.     

Refractoriness of urethral striated muscle contractility to nitric oxide-dependent cyclic GMP production

The purpose of this study was to investigate the role of cyclic GMP (cGMP) in the effects of nitric oxide (NO) on urethral striated muscle and its involvement in contractile function. The localization of cGMP, neuronal NO synthase (nNOS), vimentin, and neuronal markers was assessed by immunofluorescence in the sheep and rat urethra and the expression of nNOS was determined in Western blots. Nerve-mediated contractile responses to electrical field stimulation (EFS) were recorded in the sheep urethra. The scant nitrergic innervation of the striated muscle layer suggests that autonomic control of its activity is unlikely. The striated fiber itself may be the source of high levels NO produced by sarcolemmal and/or cytosolic μ or α variant of nNOS. This endogenous NO may provoke high basal production of soluble guanylate cyclase (GC) dependent cGMP, mainly in non-NO producing muscle fibers, which is not further enhanced by NO donors. cGMP co-localizes with neurofilament and PGP 9.5 at muscle endplates. Modulators of the cGMP pathway did not affect nerve-mediated contractile activity induced by EFS, suggesting that cGMP is not a significant mediator of neuromuscular transmission. In addition, NO donors did increase the accumulation of cGMP in dense networks of vimentin immunoreactive interstitial cells of Cajal (ICC), whose function is not yet known. These data suggest that there is a strong but non-regulated production of cGMP under resting conditions, which does not seem to affect contractile function. Modulation of cholinergic neurotransmission by NO through cGMP-independent mechanisms cannot be discarded.     

Endothelium-derived reactive oxygen species: their relationship to endothelium-dependent hyperpolarization and vascular tone

Opinions on the role of reactive oxygen species (ROS) in the vasculature have shifted in recent years, such that they are no longer merely regarded as indicators of cellular damage or byproducts of metabolism--they may also be putative mediators of physiological functions. Hydrogen peroxide (H2O2), in particular, can initiate vascular myocyte proliferation (and, incongruously, apoptosis), hyperplasia, cell adhesion, migration, and the regulation of smooth muscle tone. Endothelial cells express enzymes that produce ROS in response to various stimuli, and H2O2 is a potent relaxant of vascular smooth muscle. H2O2 itself can mediate endothelium-dependent relaxations in some vascular beds. Although nitric oxide (NO) is well recognized as an endothelium-derived dilator, it is also well established, particularly in the microvasculature, that another factor, endothelium-derived hyperpolarizing factor (EDHF), is a significant determinant of vasodilatory tone. This review primarily focuses on the hypothesis that H2O2 is an EDHF in resistance arteries. Putative endothelial sources of H2O2 and the effects of H2O2 on potassium channels, calcium homeostasis, and vascular smooth muscle tone are discussed. Furthermore, given the perception that ROS can more likely elicit cytotoxic effects than perform signalling functions, the arguments for and against H2O2 being an endogenous vasodilator are assessed.     

Protein S-nitrosylation: a physiological signal for neuronal nitric oxide

Nitric oxide (NO) has been linked to numerous physiological and pathophysiological events that are not readily explained by the well established effects of NO on soluble guanylyl cyclase. Exogenous NO S-nitrosylates cysteine residues in proteins, but whether this is an important function of endogenous NO is unclear. Here, using a new proteomic approach, we identify a population of proteins that are endogenously S-nitrosylated, and demonstrate the loss of this modification in mice harbouring a genomic deletion of neuronal NO synthase (nNOS). Targets of NO include metabolic, structural and signalling proteins that may be effectors for neuronally generated NO. These findings establish protein S-nitrosylation as a physiological signalling mechanism for nNOS.     

A comparison of arteries and veins in oxidative stress: producers, destroyers, function, and disease

Reactive oxygen species (ROS) are by-products of oxygen metabolism, normally present in low levels inside cells, where they participate in signaling processes. The delicate balance in the continuous cycle of ROS generation and inactivation is maintained by enzymatic and nonenzymatic endogenous systems. Overwhelming production of ROS (by such sources as the mitochondrial electron transport chain, NADPH oxidase, xanthine oxidase, or uncoupled nitric oxide synthase), when inadequately counteracted by destruction through antioxidant systems (such as superoxide dismutase or catalase), leads to a prooxidant state also known as oxidative stress. Increased levels of ROS and markers of oxidative stress have been consistently found in such cardiovascular diseases as atherosclerosis or hypertension, although controversy still exists over the pathophysiological role of oxidative stress in these conditions. ROS can modulate vascular function either by direct oxidative damage or by activating cellular signaling pathways that lead to abnormal contractile, inflammatory, proliferative, or remodeling properties of the blood vessel. Most current research focuses on these processes in arteries, leaving veins, "the other side" of vascular biology, in obscurity. Veins are different structurally and functionally from arteries. Equipped with a smaller smooth muscle layer compared to arteries, but being able to accommodate 70% of the circulating blood volume, veins can modulate cardiovascular homeostasis and contribute significantly to hypertension pathogenesis. Although the reports on the quantitative differences in ROS production in veins compared to arteries had conflicting results, there is a clear qualitative difference in ROS metabolism and utilization between the two vessel types. This review will compare and contrast the current knowledge of ROS metabolism in arteries versus veins in both physiological and pathophysiological conditions. Our understanding of the mechanisms underlying vascular diseases would greatly benefit from a more thorough exploration of the role of veins and venous oxidative stress.     

Calcium-independent phospholipase A2 modulates cytosolic oxidant activity and contractile function in murine skeletal muscle cells.

Phospholipase A2 (PLA2) activity supports production of reactive oxygen species (ROS) by mammalian cells. In skeletal muscle, endogenous ROS modulate the force of muscle contraction. We tested the hypothesis that skeletal muscle cells constitutively express the calcium-independent PLA2 (iPLA2) isoform and that iPLA2 modulates both cytosolic oxidant activity and contractile function. Experiments utilized differentiated C2C12 myotubes and a panel of striated muscles isolated from adult mice. Muscle preparations were processed for measurement of mRNA by real-time PCR, protein by immunoblot, cytosolic oxidant activity by the dichlorofluorescein oxidation assay, and contractile function by in vitro testing. We found that iPLA2 was constitutively expressed by all muscles tested (myotubes, diaphragm, soleus, extensor digitorum longus, gastrocnemius, heart) and that mRNA and protein levels were generally similar among muscles. Selective iPLA2 blockade by use of bromoenol lactone (10 microM) decreased cytosolic oxidant activity in myotubes and intact soleus muscle fibers. iPLA2 blockade also inhibited contractile function of unfatigued soleus muscles, shifting the force-frequency relationship rightward and depressing force production during acute fatigue. Each of these changes could be reproduced by selective depletion of superoxide anions using superoxide dismutase (1 kU/ml). These findings suggest that constitutively expressed iPLA2 modulates oxidant activity in skeletal muscle fibers by supporting ROS production, thereby influencing contractile properties and fatigue characteristics.     

Membrane fission by dynamin: what we know and what we need to know

The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion.     

Hypoxia-induced reactive oxygen species formation in skeletal muscle.

The existence of hypoxia-induced reactive oxygen species (ROS) production remains controversial. However, numerous observations with a variety of methods and in many cells and tissue types are supportive of this idea. Skeletal muscle appears to behave much like heart in that in the early stages of hypoxia there is a transient elevation in ROS, whereas in chronic exposure to very severe hypoxia there is evidence of ongoing oxidative stress. Important remaining questions that are addressed in this review include the following. Are there levels of PO2 in skeletal muscle, typical of physiological or mildly pathophysiological conditions, that are low enough to induce significant ROS production? Does the ROS associated with muscle contractile activity reflect imbalances in oxygen uptake and demand that drive the cell to a more reduced state? What are the possible molecular mechanisms by which ROS may be elevated in hypoxic skeletal muscle? Is the production of ROS in hypoxia of physiological significance, both with respect to cell signaling pathways promoting cell function and with respect to damaging effects of long-term exposure? Discussion of these and other topics leads to general conclusions that hypoxia-induced ROS may be a normal physiological response to imbalance in oxygen supply and demand or environmental stress and may play a yet undefined role in normal response mechanisms to these stimuli. However, in chronic and extreme hypoxic exposure, muscles may fail to maintain a normal redox homeostasis, resulting in cell injury or dysfunction.     

Redox signaling in macrophages.

Macrophages are phagocytic cells that produce and release reactive oxygen species (ROS) in response to phagocytosis or stimulation with various agents. The enzyme responsible for the production of superoxide and hydrogen peroxide is a multi-component NADPH oxidase that requires assembly at the plasma membrane to function as an oxidase. In addition to participating in bacterial killing, ROS, which have recently been shown to be produced enzymatically by non-phagocytic cells, have been implicated in inflammation and tissue injury. These toxic effects have been largely explored over the years and these studies have overshadowed initial observations supporting a role for ROS in modulating cellular function. In recent years, it has become increasingly evident that ROS can function as second messengers and, at low levels, can activate signaling pathways resulting in a broad array of physiological responses from cell proliferation to gene expression and apoptosis. Macrophages can also produce large amounts of nitric oxide (nitrogen monoxide, *NO). *NO was first identified as the endothelial-derived relaxing factor, EDRF and its role in the signaling pathway leading to its physiological effect was rapidly established. The ability of *NO to react with O(2)(*-) to produce peroxynitrite (ONOO(-)) was later recognized. As it is diffusion-limited, this reaction is more likely to occur in cells like macrophages that produce both ROS and RNS. In this review, we will summarize the current knowledge in redox signaling, and describe more specifically studies that are particular to macrophages.     

Effects of modulation of nitric oxide on rat diaphragm isotonic contractility during hypoxia.

Nitric oxide (NO) is essential for optimal myofilament function of the rat diaphragm in vitro during active shortening. Little is known about the role of NO in muscle contraction under hypoxic conditions. Hypoxia might increase the NO synthase (NOS) activity within the rat diaphragm. We hypothesized that NO plays a protective role in isotonic contractile and fatigue properties during hypoxia in vitro. The effects of the NOS inhibitor N(G)-monomethyl-l-arginine (l-NMMA), the NO scavenger hemoglobin, and the NO donor spermine NONOate on shortening velocity, power generation, and isotonic fatigability during hypoxia were evaluated (Po(2) approximately 7 kPa). l-NMMA and hemoglobin slowed the shortening velocity, depressed power generation, and increased isotonic fatigability during hypoxia. The effects of l-NMMA were prevented by coadministration with the NOS substrate l-arginine. Spermine NONOate did not alter isotonic contractile and fatigue properties during hypoxia. These results indicate that endogenous NO is needed for optimal muscle contraction of the rat diaphragm in vitro during hypoxia.     

Reactive oxygen species and redox-regulation of skeletal muscle adaptations to exercise

Skeletal muscle has been shown to generate a complex set of reactive oxygen and nitrogen species (ROS) both at rest and during contractile activity. The primary ROS generated are superoxide and nitric oxide and the pattern and magnitude of their generation is influenced by the nature of the contractile activity. It is increasingly clear that the ROS generated by skeletal muscle play an important role in influencing redox-regulated processes that control, at least some of, the adaptive responses to contractile activity. These processes are also recognized to be modified during ageing and in some disease states, providing the potential that interventions affecting ROS activity may influence muscle function or viability in these situations.     

Hydrogen peroxide as a paracrine vascular mediator: regulation and signaling leading to dysfunction

Numerous studies have demonstrated the ability of a variety of vascular cells, including endothelial cells, smooth muscle cells, and fibroblasts, to produce reactive oxygen species (ROS). Until recently, major emphasis was placed on the production of superoxide anion (O(2)(-)) in the vasculature as a result of its ability to directly attenuate the biological activity of endothelium-derived nitric oxide (NO). The short half-life and radius of diffusion of O(2)(-) drastically limit the role of this ROS as an important paracrine hormone in vascular biology. On the contrary, in recent years, the O(2)(-) metabolite hydrogen peroxide (H(2)O(2)) has increasingly been viewed as an important cellular signaling agent in its own right, capable of modulating both contractile and growth-promoting pathways with more far-reaching effects. In this review, we will assess the vascular production of H(2)O(2), its regulation by endogenous scavenger systems, and its ability to activate a variety of vascular signaling pathways, thereby leading to vascular contraction and growth. This discussion will include the ability of H(2)O(2) to (i) initiate calcium flux as well as (ii) stimulate pathways leading to sensitization of contractile elements to calcium. The latter involves a variety of protein kinases that have also been strongly implicated in vascular hypertrophy. Previous intensive study has emphasized the ability of NADPH oxidase-derived O(2)(-) and H(2)O(2) to activate these pathways in cultured smooth muscle cells. However, growing evidence indicates a considerably more complex array of unique oxidase systems in the endothelium, media, and adventitia that appear to participate in these deleterious effects in a sequential and temporal manner. Taken together, these findings seem consistent with a paracrine effect of H(2)O(2) across the vascular wall.     

Mitochondrial functional specialization in glycolytic and oxidative muscle fibers: tailoring the organelle for optimal function

In skeletal muscle, two major types of muscle fibers exist: slow-twitch oxidative (type I) fibers designed for low-intensity long-lasting contractions, and fast-twitch glycolytic (type II) fibers designed for high-intensity short-duration contractions. Such a wide range of capabilities has emerged through the selection across fiber types of a narrow set of molecular characteristics suitable to achieve a specific contractile phenotype. In this article we review evidence supporting the existence of distinct functional phenotypes in mitochondria from slow and fast fibers that may be required to ensure optimal muscle function. This includes differences with respect to energy substrate preferences, regulation of oxidative phosphorylation, dynamics of reactive oxygen species, handling of Ca2+, and regulation of cell death. The potential physiological implications on muscle function and the putative mechanisms responsible for establishing and maintaining distinct mitochondrial phenotype across fiber types are also discussed.     

Functional deficits in nNOSmu-deficient skeletal muscle: myopathy in nNOS knockout mice

Skeletal muscle nNOSmu (neuronal nitric oxide synthase mu) localizes to the sarcolemma through interaction with the dystrophin-associated glycoprotein (DAG) complex, where it synthesizes nitric oxide (NO). Disruption of the DAG complex occurs in dystrophinopathies and sarcoglycanopathies, two genetically distinct classes of muscular dystrophy characterized by progressive loss of muscle mass, muscle weakness and increased fatigability. DAG complex instability leads to mislocalization and downregulation of nNOSmu; but this is thought to play a minor role in disease pathogenesis. This view persists without knowledge of the role of nNOS in skeletal muscle contractile function in vivo and has influenced gene therapy approaches to dystrophinopathy, the majority of which do not restore sarcolemmal nNOSmu. We address this knowledge gap by evaluating skeletal muscle function in nNOS knockout (KN1) mice using an in situ approach, in which the muscle is maintained in its normal physiological environment. nNOS-deficiency caused reductions in skeletal muscle bulk and maximum tetanic force production in male mice only. Furthermore, nNOS-deficient muscles from both male and female mice exhibited increased susceptibility to contraction-induced fatigue. These data suggest that aberrant nNOSmu signaling can negatively impact three important clinical features of dystrophinopathies and sarcoglycanopathies: maintenance of muscle bulk, force generation and fatigability. Our study suggests that restoration of sarcolemmal nNOSmu expression in dystrophic muscles may be more important than previously appreciated and that it should be a feature of any fully effective gene therapy-based intervention.     

nNOS regulation of skeletal muscle fatigue and exercise performance

Neuronal nitric oxide synthases (nNOS) are Ca²⁺/calmodulin-activated enzymes that synthesize the gaseous messenger nitric oxide (NO). nNOSμ and the recently described nNOSβ, both spliced nNOS isoforms, are important enzymatic sources of NO in skeletal muscle, a tissue long considered to be a paradigmatic system for studying NO-dependent redox signaling. nNOS is indispensable for skeletal muscle integrity and contractile performance, and deregulation of nNOSμ signaling is a common pathogenic feature of many neuromuscular diseases. Recent evidence suggests that both nNOSμ and nNOSβ regulate skeletal muscle size, strength, and fatigue resistance, making them important players in exercise performance. nNOSμ acts as an activity sensor and appears to assist skeletal muscle adaptation to new functional demands, particularly those of endurance exercise. Prolonged inactivity leads to nNOS-mediated muscle atrophy through a FoxO-dependent pathway. nNOS also plays a role in modulating exercise performance in neuromuscular disease. In the mdx mouse model of Duchenne muscular dystrophy, defective nNOS signaling is thought to restrict contractile capacity of working muscle in two ways: loss of sarcolemmal nNOSμ causes excessive ischemic damage while residual cytosolic nNOSμ contributes to hypernitrosylation of the ryanodine receptor, causing pathogenic Ca²⁺ leak. This defect in Ca²⁺ handling promotes muscle damage, weakness, and fatigue. This review addresses these recent advances in the understanding of nNOS-dependent redox regulation of skeletal muscle function and exercise performance under physiological and neuromuscular disease conditions.     

NO and ROS implications in the organization of root system architecture

Over the past decades the role of nitric oxide (NO) and reactive oxygen species (ROS) in signaling and cellular responses to stress has witnessed an exponential trend line. Despite advances in the subject, our knowledge of the role of NO and ROS as regulators of stress and plant growth and their implication in signaling pathways is still partial. The crosstalk between NO and ROS during root formation offers new domains to be explored, as it regulates several plant functions. Previous findings indicate that plants utilize these signaling molecules for regulating physiological responses and development. Depending upon cellular concentration, NO either can stimulate or impede root system architecture (RSA) by modulating enzymes through post-translational modifications. Similarly, the ROS signaling molecule network, in association with other hormonal signaling pathways, control the RSA. The spatial regulation of ROS controls cell growth and ROS determine primary root and act in concert with NO to promote lateral root primordia. NO and ROS are two central messenger molecules which act differentially to upregulate or downregulate the expression of genes pertaining to auxin synthesis and to the configuration of root architecture. The investigation concerning the contribution of donors and inhibitors of NO and ROS can further aid in deciphering their role in root development. With this background, this review provides comprehensive details about the effect and function of NO and ROS in the development of RSA.     

ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells

During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y₁ receptor agonist, induced a large signal while UTPyS (P2Y₂ agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y₁. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.     

Chemical regulation of nitric oxide: a role for intracellular myoglobin?

The detailed chemistry of nitric oxide (*NO) and regulation of this potent signal molecule through interactions with cellular components are complex and not clearly understood. In the vasculature, *NO plays a crucial role in vessel dilation by activating soluble guanylyl cyclase (sGC) in vascular smooth muscle cells (VSMC). *NO is responsible for maintaining coronary blood flow and normal cardiac function. However, *NO is a highly reactive molecule and this reactivity toward a range of alternate substrates may interfere with the activation of its preferred molecular target within VSMC. Interestingly, marked changes to *NO homeostasis are linked to disease progression. Thus, the physiological concentration of *NO is carefully regulated. Myoglobin is a haem-containing protein that is present in relatively high concentration in cardiac and skeletal muscle. Recently, the presence of myoglobin has been confirmed in human smooth muscle. The role of intracellular myoglobin is generally accepted as that of a passive di-oxygen storage protein. However, oxygenated myoglobin readily reacts with *NO to yield higher order N-oxides such as nitrate, while both the ferrous and ferric forms of the protein form a stable complex with *NO. Together, these two reactions effectively eliminate *NO on the physiological time-scale and strongly support the idea that myoglobin plays a role in maintaining *NO homeostasis in tissues that contain the protein. Interestingly, human myoglobin contains a sulfhydryl group and forms an S-nitroso-adduct similar to haemoglobin. In this article we discuss the potential for human myoglobin to actively participate in the regulation of *NO by three distinct mechanisms, namely oxidation, ligand binding, and through formation of biologically active S-nitroso-myoglobin.     

Nitric oxide availability is increased in contracting skeletal muscle from aged mice,but does not differentially decrease muscle superoxide

Reactive oxygen and nitrogen species have been implicated in the loss of skeletal muscle mass and function that occurs during aging. Nitric oxide (NO) and superoxide are generated by skeletal muscle and where these are generated in proximity their chemical reaction to form peroxynitrite can compete with the superoxide dismutation to hydrogen peroxide. Changes in NO availability may therefore theoretically modify superoxide and peroxynitrite activities in tissues, but published data are contradictory regarding aging effects on muscle NO availability. We hypothesised that an age-related increase in NO generation might increase peroxynitrite generation in muscles from old mice, leading to an increased nitration of muscle proteins and decreased superoxide availability. This was examined using fluorescent probes and an isolated fiber preparation to examine NO content and superoxide in the cytosol and mitochondria of muscle fibers from adult and old mice both at rest and following contractile activity. We also examined the 3-nitrotyrosine (3-NT) and peroxiredoxin 5 (Prx5) content of muscles from mice as markers of peroxynitrite activity. Data indicate that a substantial age-related increase in NO levels occurred in muscle fibers during contractile activity and this was associated with an increase in muscle eNOS. Muscle proteins from old mice also showed an increased 3-NT content. Inhibition of NOS indicated that NO decreased superoxide bioavailability in muscle mitochondria, although this effect was not age related. Thus increased NO in muscles of old mice was associated with an increased 3-NT content that may potentially contribute to age-related degenerative changes in skeletal muscle.     

Reactive oxygen species formation during tetanic contractions in single isolated Xenopus myofibers

Contracting skeletal muscle produces reactive oxygen species (ROS) that have been shown to affect muscle function and adaptation. However, real-time measurement of ROS in contracting myofibers has proven to be difficult. We used amphibian (Xenopus laevis) muscle to test the hypothesis that ROS are formed during contractile activity in isolated single skeletal muscle fibers and that this contraction-induced ROS formation affects fatigue development. Single myofibers were loaded with 5 μM dihydrofluorescein-DA (Hfluor-DA), a fluorescent probe that reacts with ROS and results in the formation of fluorescein (Fluor) to precisely monitor ROS generation within single myofibers in real time using confocal miscroscopy. Three identical periods of maximal tetanic contractions (1 contraction/3 s for 2 min, separated by 60 min of rest) were conducted by each myofiber (n = 6) at 20°C. Ebselen (an antioxidant) was present in the perfusate (10 μM) during the second contractile period. Force was reduced by ～30% during each of the three contraction periods, with no significant difference in fatigue development among the three periods. The Fluor signal, indicative of ROS generation, increased significantly above baseline in both the first (42 ± 14%) and third periods (39 ± 10%), with no significant difference in the increase in fluorescence between the first and third periods. There was no increase of Fluor in the presence of ebselen during the second contractile period. These results demonstrated that, in isolated intact Xenopus myofibers, 1) ROS can be measured in real time during tetanic contractions, 2) contractile activity induced a significant increase above resting levels of ROS production, and 3) ebselen treatment reduced ROS generation to baseline levels but had no effect on myofiber contractility and fatigue development.