Michael B. Reid

TNF-α acts via p38 MAPK to stimulate expression of the ubiquitin ligase atrogin1/MAFbx in skeletal muscle

Atrogin1/MAFbx is an ubiquitin ligase that mediates muscle atrophy in a variety of catabolic states. We recently found that H2O2 stimulates atrogin1/MAFbx gene expression. Since the cytokine tumor necrosis factor‐α (TNF‐α) stimulates both reactive oxygen production and general activity of the ubiquitin conjugating pathway, we hypothesized that TNF‐α would also increase atrogin1/MAFbx gene expression. As with H2O2, we found that TNF‐α exposure up‐regulates atrogin1/MAFbx mRNA within2hin C2C12 myotubes. Intraperitoneal injection of TNF‐α increased atrogin1/MAFbx mRNA in skeletal muscle of adult mice within 4 h. Exposing myotubes to either TNF‐α or H2O2 also produced general activation of the mitogen‐activated protein kinases (MAPKs): p38, ERK1/2, and JNK. The increase in atrogin1/MAFbx gene expression induced by TNF‐α was not altered significantly by ERK inhibitor PD98059 or the JNK inhibitor SP600125. In contrast, atrogin1/MAFbx up‐regulation and the associated increase in ubiquitin conjugating activity were both blunted by p38 inhibitors, either SB203580 or curcumin. These data suggest that TNF‐α acts via p38 to increase atrogin1/MAFbx gene expression in skeletal muscle.—Li, Y.‐P., Chen, Y., John, J., Moylan, J., Jin, B., Mann, D. L., Reid, M. B. TNF‐α acts via p38 MAPK to stimulate expression of the ubiquitin ligase atrogin1/MAFbx in skeletal muscle. FASEB J. 19, 362–370 (2005)

EFFECT OF HYDROGEN PEROXIDE AND DITHIOTHREITOL ON CONTRACTILE FUNCTION OF SINGLE SKELETAL MUSCLE FIBRES FROM THE MOUSE

1 We used intact single fibres from a mouse foot muscle to study the role of oxidation‐reduction in the modulation of contractile function. 2 The oxidant hydrogen peroxide (H2O2, 100‐300 μM) for brief periods did not change myoplasmic Ca2+ concentrations ([Ca2+]i) during submaximal tetani. However, force increased by 27 % during the same contractions. 3 The effects of H2O2 were time dependent. Prolonged exposures resulted in increased resting and tetanic [Ca2+]i, while force was significantly diminished. The force decline was mainly due to reduced myofibrillar Ca2+ sensitivity. There was also evidence of altered sarcoplasmic reticulum (SR) function: passive Ca2+ leak was increased and Ca2+ uptake was decreased. 4 The reductant dithiothreitol (DTT, 0.5‐1 mM) did not change tetanic [Ca2+]i, but decreased force by over 40 %. This was completely reversed by subsequent incubations with H2O2. The force decline induced by prolonged exposure to H2O2 was reversed by subsequent exposure to DTT. 5 These results show that the elements of the contractile machinery are differentially responsive to changes in the oxidation‐reduction balance of the muscle fibres. Myofibrillar Ca2+ sensitivity appears to be especially susceptible, while the SR functions (Ca2+ leak and uptake) are less so.

Tumor necrosis factor-α and muscle wasting: a cellular perspective

Tumor necrosis factor-α (TNF-α) is a polypeptide cytokine that has been associated with muscle wasting and weakness in inflammatory disease. Despite its potential importance in muscle pathology, the direct effects of TNF-α on skeletal muscle have remained undefined until recently. Studies of cultured muscle cells indicate that TNF-α disrupts the differentiation process and can promote catabolism in mature cells. The latter response appears to be mediated by reactive oxygen species and nuclear factor-κB which upregulate ubiquitin/proteasome activity. This commentary outlines our current understanding of TNF-α effects on skeletal muscle and the mechanism of TNF-α action.

N-acetylcysteine inhibits muscle fatigue in humans.

N-acetylcysteine (NAC) is a nonspecific antioxidant that selectively inhibits acute fatigue of rodent skeletal muscle stimulated at low (but not high) tetanic frequencies and that decreases contractile function of unfatigued muscle in a dose-dependent manner. The present experiments test the hypothesis that NAC pretreatment can inhibit acute muscular fatigue in humans. Healthy volunteers were studied on two occasions each. Subjects were pretreated with NAC 150 mg/kg or 5% dextrose in water by intravenous infusion. The subject then sat in a chair with surface electrodes positioned over the motor point of tibialis anterior, an ankle dorsiflexor of mixed-fiber composition. The muscle was stimulated to contract electrically (40-55 mA, 0.2-ms pulses) and force production was measured. Function of the unfatigued muscle was assessed by measuring the forces produced during maximal voluntary contractions (MVC) of ankle dorsiflexor muscle groups and during electrical stimulation of tibialis anterior at 1, 10, 20, 40, 80, and 120 Hz (protocol 1). Fatigue was produced using repetitive tetanic stimulations at 10 Hz (protocol 1) or 40 Hz (protocol 2); intermittent stimulations subsequently were used to monitor recovery from fatigue. The contralateral leg then was studied using the same protocol. Pretreatment with NAC did not alter the function of unfatigued muscle; MVC performance and the force-frequency relationship of tibialis anterior were unchanged. During fatiguing contractions stimulated at 10 Hz, NAC increased force output by approximately 15% (P < 0.0001), an effect that was evident after 3 min of repetitive contraction (P < 0.0125) and persisted throughout the 30-min protocol. NAC had no effect on fatigue induced using 40 Hz stimuli or on recovery from fatigue. N-acetylcysteine pretreatment can improve performance of human limb muscle during fatiguing exercise, suggesting that oxidative stress plays a causal role in the fatigue process and identifying antioxidant therapy as a novel intervention that may be useful clinically.

Oxidative stress, chronic disease, and muscle wasting.

Underlying the pathogenesis of chronic disease is the state of oxidative stress. Oxidative stress is an imbalance in oxidant and antioxidant levels. If an overproduction of oxidants overwhelms the antioxidant defenses, oxidative damage of cells, tissues, and organs ensues. In some cases, oxidative stress is assigned a causal role in disease pathogenesis, whereas in others the link is less certain. Along with underlying oxidative stress, chronic disease is often accompanied by muscle wasting. It has been hypothesized that catabolic programs leading to muscle wasting are mediated by oxidative stress. In cases where disease is localized to the muscle, this concept is easy to appreciate. Transmission of oxidative stress from diseased remote organs to skeletal muscle is thought to be mediated by humoral factors such as inflammatory cytokines. This review examines the relationship between oxidative stress, chronic disease, and muscle wasting, and the mechanisms by which oxidative stress acts as a catabolic signal. Muscle Nerve, 2007

Free radicals and muscle fatigue: Of ROS, canaries, and the IOC.

Skeletal muscle fibers continually generate reactive oxygen species (ROS) at a slow rate that increases during muscle contraction. This activity-dependent increase in ROS production contributes to fatigue of skeletal muscle during strenuous exercise. Existing data suggest that muscle-derived ROS primarily act on myofibrillar proteins to inhibit calcium sensitivity and depress force. Decrements in calcium sensitivity and force are acutely reversible by dithiothreitol, a thiol-selective reducing agent. These observations suggest that thiol modifications on one or more regulatory proteins are responsible for oxidant-induced losses during fatigue. More intense ROS exposure leads to losses in calcium regulation that mimic pathologic changes and are not reversible. Studies in humans, quadrupeds, and isolated muscle preparations indicate that antioxidant pretreatment can delay muscle fatigue. In humans, this phenomenon is best defined for N-acetylcysteine (NAC), a reduced thiol donor that supports glutathione resynthesis. NAC has been shown to inhibit fatigue in healthy adults during electrical muscle activation, inspiratory resistive loading, handgrip exercise, and intense cycling. These findings identify ROS as endogenous mediators of muscle fatigue and highlight the importance of future research to (a) define the cellular mechanism of ROS action and (b) develop antioxidants as novel therapeutic interventions for treating fatigue.

TNF-α increases ubiquitin-conjugating activity in skeletal muscle by up-regulating UbcH2/E220k

In some inflammatory diseases, TNF‐α is thought to stimulate muscle catabolism via an NF‐κB‐dependent process that increases ubiquitin conjugation to muscle proteins. The transcriptional mechanism of this response has not been determined. Here we studied the potential role of UbcH2, a ubiquitin carrier protein and homologue of murine E220k. We find that UbcH2 is constitutively expressed by human skeletal and cardiac muscles, murine limb muscle, and cultured myotubes. TNF‐α stimulates UbcH2 expression in mouse limb muscles in vivo and in cultured myotubes. The UbcH2 promoter region contains a functional NF‐κB binding site;NF‐κB binding to this sequence is increased by TNF‐α stimulation. A dominant negative inhibitor of NF‐κB activation blocks both UbcH2 up‐regulation and the increase in ubiquitin‐conjugating activity stimulated by TNF‐α. In extracts from TNF‐α‐treated myotubes, ubiquitin‐conjugating activity is limited by UbcH2 availability; activity is inhibited by an antiserum to UbcH2 or a dominant negative mutant of UbcH2 and is enhanced by wild‐type UbcH2. Thus, UbcH2 up‐regulation is a novel response to TNF‐α/NF‐κB signaling in skeletal muscle that appears to be essential for the increased ubiquitin conjugation induced by this cytokine.—Y.‐P.Li, S. H.Lecker, Y.Chen, I. D.Waddell, A. L.Goldberg, M. B.Reid TNF‐α increases ubiquitin‐conjugating activity in skeletal muscle by up‐regulating UbcH2/E220k. FASEB J. 17, 1048–1057 (2003)

Role of reactive oxygen species in contraction-mediated glucose transport in mouse skeletal muscle

Exercise increases glucose transport into skeletal muscle via a pathway that is poorly understood. We investigated the role of endogenously produced reactive oxygen species (ROS) in contraction‐mediated glucose transport. Repeated contractions increased 2‐deoxyglucose (2‐DG) uptake roughly threefold in isolated, mouse extensor digitorum longus (fast‐twitch) muscle. N‐Acetylcysteine (NAC), a non‐specific antioxidant, inhibited contraction‐mediated 2‐DG uptake by ∼50% (P < 0.05 versus control values), but did not significantly affect basal 2‐DG uptake or the uptake induced by insulin, hypoxia or 5‐aminoimidazole‐4‐carboxamide‐1‐β‐d‐ribofuranoside (AICAR, which mimics AMP‐mediated activation of AMP‐activated protein kinase, AMPK). Ebselen, a glutathione peroxidase mimetic, also inhibited contraction‐mediated 2‐DG uptake (by almost 60%, P < 0.001 versus control values). Muscles from mice overexpressing Mn2+‐dependent superoxide dismutase, which catalyses H2O2 production from superoxide anions, exhibited a ∼25% higher rate of contraction‐mediated 2‐DG uptake versus muscles from wild‐type control mice (P < 0.05). Exogenous H2O2 induced oxidative stress, as judged by an increase in the [GSSG]/[GSH + GSSG] (reduced glutathione + oxidized glutathione) ratio to 2.5 times control values, and this increase was substantially blocked by NAC. Similarly, NAC significantly attenuated contraction‐mediated oxidative stress as judged by measurements of glutathione status and the intracellular ROS level with the fluorescent indicator 5‐(and‐6)‐chloromethyl‐2′,7′‐dichlorodihydrofluorescein (P < 0.05). Finally, contraction increased AMPK activity and phosphorylation ∼10‐fold, and NAC blocked ∼50% of these changes. These data indicate that endogenously produced ROS, possibly H2O2 or its derivatives, play an important role in contraction‐mediated activation of glucose transport in fast‐twitch muscle.

Cardiac-Specific Overexpression of Tumor Necrosis Factor-α Causes Oxidative Stress and Contractile Dysfunction in Mouse Diaphragm

BackgroundWe have developed a transgenic mouse with cardiac-restricted overexpression of tumor necrosis factor-&agr; (TNF-&agr;). These mice develop a heart failure phenotype characterized by left ventricular dysfunction and remodeling, pulmonary edema, and elevated levels of TNF-&agr; in the peripheral circulation from cardiac spillover. Given that TNF-&agr; causes atrophy and loss of function in respiratory muscle, we asked whether transgenic mice developed diaphragm dysfunction and whether contractile losses were caused by oxidative stress or tissue remodeling. Methods and ResultsMuscles excised from transgenic mice and littermate controls were studied in vitro with direct electrical stimulation. Cytosolic oxidant levels were measured with 2′,7′-dichlorofluorescin diacetate; emissions of the oxidized product were detected by fluorescence microscopy. Force generation by the diaphragm of transgenic animals was 47% less than control (13.2±0.8 [±SEM] versus 25.1±0.6 N/cm2;P <0.001); this weakness was associated with greater intracellular oxidant levels (P <0.025) and was partially reversed by 30-minute incubation with the antioxidant N-acetylcysteine 10 mmol/L (P <0.01). Exogenous TNF-&agr; 500 &mgr;mol/L increased oxidant production in diaphragm of wild-type mice and caused weakness that was inhibited by N-acetylcysteine, suggesting that changes observed in the diaphragm of transgenic animals were mediated by TNF-&agr;. There were no differences in body or diaphragm weights between transgenic and control animals, nor was there evidence of muscle injury or apoptosis. ConclusionsElevated circulating levels of TNF-&agr; provoke contractile dysfunction in the diaphragm through an endocrine mechanism thought to be mediated by oxidative stress.

Nitric oxide, reactive oxygen species, and skeletal muscle contraction

Nitric oxide (NO) derivatives and reactive oxygen species (ROS) modulate contractile function of respiratory and limb skeletal muscle. The intracellular processes regulated by NO and ROS remain enigmatic, however. Studies of reduced preparations have identified a number of regulatory proteins that exhibit altered function when exposed to exogenous NO or ROS donors ex vivo. The relative importance of these targets in the intact cell is not known and conflicting theories abound regarding the mechanism(s) whereby NO and ROS regulate contraction. This review article provides a personal perspective on the processes regulated by NO and ROS by addressing three major topics: 1) the regulatory mechanisms by which endogenous NO depresses force production, 2) the processes whereby endogenous ROS modulate contraction of unfatigued muscle, and 3) the site(s) of action and reversibility of ROS effects in muscle fatigue.