Are antioxidants useful for treating skeletal muscle atrophy?

Free Radical Biology & Medicine 47 (2009) 906–916

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Review Article

Are antioxidants useful for treating skeletal muscle atrophy? Andrea Bonetto a,1, Fabio Penna a,1, Maurizio Muscaritoli b, Valerio G. Minero a, Filippo Rossi Fanelli b, Francesco M. Baccino a, Paola Costelli a,⁎ a b

Dipartimento di Medicina e Oncologia Sperimentale, Università di Torino, 10125 Torino, Italy Dipartimento di Medicina Clinica, Sapienza–Università di Roma, Roma, Italy

a r t i c l e

i n f o

Article history: Received 28 April 2009 Revised 26 June 2009 Accepted 2 July 2009 Available online 8 July 2009 Keywords: Skeletal muscle Atrophy Protein breakdown Oxidative stress Reactive oxygen species Antioxidants Free radicals

a b s t r a c t Changes in the skeletal muscle protein mass frequently occur in both physiological and pathological states. Muscle hypotrophy, in particular, is commonly observed during aging and is characteristic of several pathological conditions such as neurological diseases, cancer, diabetes, and sepsis. The skeletal muscle protein content depends on the relative rates of synthesis and degradation, which must be coordinately regulated to maintain the equilibrium. Pathological muscle depletion is characterized by a negative nitrogen balance, which results from disruption of this equilibrium due to reduced synthesis, increased breakdown, or both. The current view, mainly based on experimental data, considers hypercatabolism as the major cause of muscle protein depletion. Several signaling pathways that probably contribute to muscle atrophy have been identified, and there is increasing evidence that oxidative stress, due to reactive oxygen species production overwhelming the intracellular antioxidant systems, plays a role in causing muscle depletion both during aging and in chronic pathological states. In particular, oxidative stress has been proposed to enhance protein breakdown, directly or by interacting with other factors. This review focuses on the possibility of using antioxidant treatments to target molecular pathways involved in the pathogenesis of skeletal muscle wasting. © 2009 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . Skeletal muscle protein breakdown . . . . . . . . Activation of muscle protein hypercatabolism . . . . Oxidative stress and muscle atrophy . . . . . . . . Oxidant sources in the skeletal muscle . . . . . . . Antioxidant systems . . . . . . . . . . . . . . . . ROS intracellular targets . . . . . . . . . . . . . . Correction of skeletal muscle atrophy by antioxidant Aging-associated sarcopenia . . . . . . . . . . Diabetes . . . . . . . . . . . . . . . . . . . Cancer cachexia . . . . . . . . . . . . . . . Nonsystemic muscle depletion. . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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Abbreviations: CSA, cross-sectional area; IGF-1, insulin growth factor-1; TNF-α, tumor necrosis factor-α; IL-1, interleukin-1; IFN, interferon; PIF, proteolysis-inducing factor; ROS, U U reactive oxygen species; RNS, reactive nitrogen species; O2 −, superoxide anion; OH, hydroxyl radical; ONOO−, peroxynitrite; MnSOD, manganese superoxide dismutase; CuZnSOD, copper–zinc superoxide dismutase; NADPH, nicotinamide adenine dinucleotide phosphate; NOS, nitric oxide synthase; GSH, glutathione; GPX, GSH peroxidase; MAPK, mitogenactivated protein kinase; NF-κB, nuclear factor-κB; STZ, streptozotocin; AGE, advanced glycation end product; DHEA, dehydroepiandrosterone; ALS, amyotrophic lateral sclerosis; ERAD, endoplasmic reticulum-associated degradation; NAC, N-acetylcysteine; DGC, dystrophin–glycoprotein complex. ⁎ Corresponding author. Fax: +39 0116707753. E-mail address: [email protected] (P. Costelli). 1 These authors contributed equally to this review. 0891-5849/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2009.07.002

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Introduction

Skeletal muscle protein breakdown

Skeletal muscle, the largest organ in the body, accounts for approximately half of its total protein mass and is in charge of several body functions such as movement, stability, heat production, and cold tolerance, playing a pivotal role in the overall energy balance. Skeletal muscle mass may depend on both myofiber number and dimensions, the latter being usually defined in terms of crosssectional area (CSA). However, whereas modifications of myofiber number are rarely seen, CSA can change markedly, increasing with normal growth or hypertrophy induced by exercise training and decreasing with atrophy associated with immobilization, inactivity, injury, aging, or disease. Skeletal muscle hypertrophy results from increased size of the preexisting muscle fibers, due to accumulation of contractile proteins. By contrast, skeletal muscle atrophy is associated with reduced CSA and decreased content of contractile proteins. In other words, skeletal muscle mass is directly proportional to the protein content, which ultimately depends on the balance between synthesis and degradation. In healthy organisms such a balance allows the maintenance of skeletal muscle mass. Although modulations of both sides of turnover eventually converge to produce a new steady state, physiological increases in muscle protein mass (hypertrophy) seem primarily associated with enhanced protein synthesis, which responds earlier than degradation to the inducing stimuli. The situation appears quite different when taking into account the mechanisms underlying skeletal muscle atrophy. Indeed, a multifactorial pathogenesis is demonstrated, in which nutritional, endocrine, metabolic, and immunological components contribute differently to muscle depletion. In particular, the idea that a complex interplay among classical hormones, cytokines, and growth factors is crucially involved in the onset of skeletal muscle atrophy is now widely accepted. Most of these factors, directly or indirectly, impinge on muscle protein metabolism, altering the physiological balance between synthesis and breakdown, favoring the latter and possibly reducing the former, eventually resulting in protein waste (Fig. 1). In this regard, oxidative stress has been proposed to be involved in the pathogenesis of skeletal muscle wasting in multifaceted ways, among which is the stimulation of protein breakdown.

Protein catabolism is of crucial importance for muscle homeostasis, being responsible for the clearance of damaged or aged proteins as well as for the elimination of molecules endowed with regulatory functions, such as cyclins and their inhibitors. However, upregulation of protein breakdown exceeding protein synthesis rates may result in skeletal muscle wasting. In particular it should be stressed that, whereas protein synthesis is a zero-order process, degradation of the bulk cell protein is a first-order process described by a fractional rate constant. This means that, under a given set of regulations, the fraction of proteins degraded is not affected by the size of the protein pool. Therefore, if the degradation rate constant is poised higher than normal levels, protein loss occurs irrespective of the protein synthesis rate [see 1]. Four major proteolytic machineries exist in muscles. The lysosomal and the proteasomal systems lead to an exhaustive degradation of cell proteins into amino acids or small peptides, whereas the Ca2+dependent and the caspase systems can perform only a limited proteolysis, owing to their restricted specificity. The endosome–lysosome system is relatively nonselective and mostly involved in the degradation of long-lived proteins [reviewed in 2]. The lysosomal autophagic degradation has been reported to be induced in the skeletal muscle by a 24-h nutrient starvation [3], whereas autophagins, a class of cysteine proteases putatively involved in the formation of autophagosomes, are particularly abundant in the skeletal muscle [4]. Recently, autophagy-related genes have been shown to be hyperexpressed during fasting or denervation-induced muscle atrophy [5]. The ubiquitin–proteasome system, initially described as relevant to the catabolism of regulatory or damaged proteins, is also involved in bulk protein degradation, at least in the skeletal muscle. In particular, the identification of muscle-specific components of the so-called SCF ubiquitin–ligase complexes has improved the knowledge of the regulation of the ubiquitin–proteasome system in the skeletal muscle [6]. These complexes are involved in targeting proteins for proteasomal degradation. They are formed by two molecules, SKP-1 (S) and Cullin-1 (C), that may be associated with a large series of F-box subunits (F) responsible for substrate specificity [reviewed in 1].

Fig. 1. Multifactorial pathogenesis of skeletal muscle atrophy. Extracellular signals lead to activation or down-regulation of some intracellular pathways, leading to increased protein breakdown, reduced protein synthesis, and cell death. These events contribute differently to the onset of muscle atrophy in chronic pathologies.

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The Ca2+-dependent system comprises several cysteine proteases called calpains and a physiological inhibitor named calpastatin. Calpains effect only a limited proteolysis on their substrates, resulting in irreversible modifications that lead to activity changes or to degradation by other proteolytic pathways [7,8]. A number of proteins, among which are protein kinase C, Cdk5, Ca2+/calmodulin-dependent protein kinase IV, calcineurin, and titin, have been proposed as in vivo calpain substrates. Calpains have been involved in processes such as cell proliferation, differentiation, migration, apoptotic death, and gene expression [7 and references therein]. Finally, caspases, a family of cysteine proteases, are mostly known for their role in the execution of apoptosis. Whether these proteases are relevant to the regulation of skeletal muscle mass still remains to be elucidated. In this regard, caspase 3 has been proposed to trigger the initial proteolytic step that renders myofibrillar proteins available for degradation by the proteasome [9], although this hypothesis has not been confirmed by other reports [10,11]. Not only have the mechanisms that lead to hyperactive intracellular proteolysis not been completely elucidated, but the relative contributions of the different proteolytic systems are far from being clarified. The results in the literature point to the ubiquitin– proteasome pathway as mostly relevant to the onset of muscle protein hypercatabolism [reviewed in 1], although several studies report on the possible roles of other proteolytic systems in the pathogenesis of muscle wasting [see 7 and references therein]. Activation of muscle protein hypercatabolism The enhancement of muscle protein breakdown mainly arises from the complex interplay of classical hormones and other humoral factors. Molecules such as insulin, growth hormone, and IGF-1 reduce protein degradation rates, in addition to stimulating the rates of synthesis. Moreover, insulin administration to tumor-bearing rats and cancer patients has been shown to reverse the muscle wasting pattern and the increase in protein breakdown [12–14]. By contrast, glucocorticoids exert a clear catabolic effect, and their levels are frequently increased in pathologic states [15]. The hormonal milieu probably plays a role in the regulation of the ubiquitin–proteasome pathway. Indeed, acute glucocorticoid treatment increases the expression of ubiquitin, 14-kDa E2, and 20S proteasome subunit in rat skeletal muscle [16]. To account for the inhibition of protein degradation achieved by insulin and IGF-1, active Akt has been proposed to phosphorylate and sequester in the cytoplasm the transcription factors FoxO-1 and FoxO-3, thus inhibiting the transcription of several genes, among which is atrogin-1 [17,18]. Cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)1, IL-6, and interferons have been proposed as mediators of tissue depletion. Experimental animals treated with TNF-α or IL-1 have been shown to develop cachexia, whereas muscle wasting and ubiquitin hyperexpression in tumor-bearing animals is prevented by the administration of anti-TNF-α antibodies or pentoxifylline, an inhibitor of TNF-α synthesis [reviewed in 19]. In addition, cachexia induced by the Lewis lung carcinoma is antagonized by treatment with anti-IFN-γ antibodies [20]. The murine C-26 adenocarcinoma seems to produce muscle wasting mainly by releasing IL-6 [21], and IL-6 hyperproduction achieved in transgenic mice increases the expression of components of the ATP–ubiquitin-dependent proteolytic systems [22]. Such observations are not confined to experimental studies, because TNF-α plasma levels in prostate cancer patients positively correlate with the mortality rate, and the levels of soluble TNF-α receptor I increase with disease progression in gastric or colorectal cancer patients, being higher in severely cachectic patients [reviewed in 19]. Moreover, proinflammatory cytokines play a role in muscle wasting associated with chronic nonmalignant diseases. TNF-α, IL-1, and IL-6 are frequently elevated in sepsis, and cytokine levels positively correlate with its severity and lethality. Finally, muscle

wasting associated with increased levels of several mediators such as TNF-α, IL-1, and IL-6 is a common clinical problem in elderly patients [23]. Mediators other than classical hormones and cytokines have also been proposed to contribute to muscle wasting. Particular regard should be given to the proteolysis-inducing factor (PIF). Described about 10 years ago as “the” mediator responsible for cachexia [24], its ability to enhance protein breakdown is now well demonstrated. PIF has been proposed to signal via phospholipase A2 and phospholipase C, resulting in protein kinase C activation shown to activate transcription factors such as NF-κB and STAT3. PIF administration to healthy animals causes development of muscle atrophy, and urinary PIF levels have been correlated with the occurrence of weight loss in cancer patients [reviewed in 25]. Recent reports, however, have questioned the real significance of PIF in the progression of wasting in cancer patients [26,27]. Oxidative stress and muscle atrophy Oxidative stress occurs when the balance between oxidants and antioxidants is perturbed. In mammalian tissues molecular oxygen and nitric oxide are rapidly converted into a variety of reactive oxygen or nitrogen species (ROS and RNS, respectively) of radical and nonradical nature; the most important are superoxide anion (O2U−), U hydroxyl radical ( OH), hydrogen peroxide (H2O2), and peroxynitrite − (ONOO ). Target molecules of these reactive species can be membrane lipids, structural and regulatory proteins, or nucleic acids. These reactions could potentially damage cell structures and functions and are normally counteracted by the activity of the antioxidant enzymatic and nonenzymatic systems. A modest variation of this reductive–oxidative (redox) balance is compatible with cell physiology and it is involved in the regulation of signal transduction and protein functions [28]. However, if antioxidant defenses are overwhelmed by ROS and RNS production, an excess of oxidative reactions, i.e., an oxidative stress, with consequent damage of the interested tissue, occurs. Oxidant sources in the skeletal muscle Oxidant species in the skeletal muscle arise from various sources. The mitochondrial electron transport chain is one of the most important. It accounts for the production of a significant amount of O2U−, generated at complexes I, II, and III of the chain, after reaction with species generated by single-electron transfers, such as those between Fe–S clusters and ubiquinone/ubisemiquinone [29]. Of the oxygen consumed in the mitochondria, about 2–4% is estimated to be reduced to O2U− [155], although only a small amount of O2U− is U released from these organelles. Indeed, O2 − is dismutated to H2O2 by two enzymatic systems, the manganese superoxide dismutase (MnSOD), localized in the mitochondrial matrix, and the CuZnSOD, active in the intermembrane space. When released, H2O2 can be U converted to OH by nonenzymatic reactions such as Fenton's process [30]. Another mitochondrial system, the monoamine oxidase, participates in oxidant production, catalyzing oxidative deamination to produce both H2O2 and reactive aldehydes. Its expression is upregulated by glucocorticoids and has been involved in the pathogenesis of glucocorticoid-induced muscle wasting [31]. An additional source of oxidative species is xanthine dehydrogenase. This enzyme catalyzes the formation of uric acid from xanthine and hypoxanthine, concomitantly reducing NAD+to NADH. In pathological states, xanthine dehydrogenase is converted to xanthine oxidase by cysteine residue modification and/or partial proteolysis. Xanthine oxidase cannot utilize NAD+as an electron acceptor and preferentially reduces molecular oxygen to O2U− and H2O2. Enhanced oxidative damage associated with increased xanthine oxidase activity has been reported in skeletal muscle after contractile claudication

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[32], and glucocorticoid administration results in increased muscle xanthine oxidase activity [33]. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, an enzymatic complex characteristically active in phagocytes, produces O2U− by catalyzing a single-electron transfer from NADH or NADPH to molecular oxygen. In the skeletal muscle, NADPH oxidase activity is associated not only with the sarcolemma, where it seems to be involved in signal transduction, but also with the sarcoplasmic reticulum, regulating Ca2+release and muscle contraction [34]. Finally, nitric oxide synthase (NOS) is the enzyme responsible for NO production. Three isoforms have been described: inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). Individual muscle fibers constitutively express eNOS and nNOS [35]; an alternatively spliced isoform of the latter (nNOSμ) has been localized at the subsarcolemmal region, associated with the dystrophin complex [36]. NO has important regulatory functions in the skeletal muscle, affecting blood flow, excitation–contraction coupling, glucose metabolism, and mitochondrial energy production [35]. However, NO can also exert negative effects such as cytostatic or cytotoxic activities [37], mainly mediated by the production of ONOO−, owing to the very rapid reaction between NO and O2U− [38]. Additional sources of oxidants in the skeletal muscle may be represented by neutrophil infiltration. Situations leading to inflammatory infiltration, such as physical exercise or Duchenne-type muscle dystrophy, have been associated with increased ROS production. Indeed, active phagocytes release significant amount of ROS owing to the above cited NADPH oxidase and to myeloperoxidase, an enzyme able to produce HClO from H2O2 and Cl− [39]. Antioxidant systems Regulated expression of antioxidant enzymes and nonenzymatic antioxidants such as glutathione (GSH) is crucial to the maintenance of redox balance. In particular, antioxidant defenses are rapidly induced by organisms to cope with oxidative stress. Consistently, deficiency or depletion of various antioxidant systems has been shown to exacerbate oxidative tissue injury. Cellular antioxidant systems include enzymes, vitamins, glutathione, and other thiols. Antioxidant enzymes include SOD, catalase, and GSH peroxidase (GPX) [40]. Tissues with high oxygen consumption rates, such as liver, heart, and brain, constitutively express higher levels of antioxidant enzymes than those characterized by low oxygen consumption [41]. As for the skeletal muscle, fiber type significantly affects antioxidant enzyme activity, which is greater in slow-twitch oxidative fibers than in fast-twitch glycolytic fibers [42]. Three SOD isoforms have been detected in humans. CuZnSOD and extracellular SOD, both using Cu2+and Zn2+as cofactors, are found in the cytoplasm and the extracellular space, respectively [43]. The former is found in nuclei, peroxisomes, and the mitochondrial intermembrane space. Extracellular SOD, secreted from smooth muscle, is endowed with anti-inflammatory and ROS-scavenging activity [44]. The third SOD isoform, Mn-dependent, is localized in mitochondria. Its expression is proportional to aerobic activity and is enhanced by chronic hypoxia, cytotoxic drugs, and inflammatory cytokines [45]. A second antioxidant enzyme, catalase, reduces H2O2 to H2O and O2. It is localized in peroxisomes, wherein it reacts with H2O2 produced during fatty acid oxidation, limiting its cytosolic accumulation. Finally, GPX represents a family of tetrameric selenoenzymes, among which GPX1 catalyzes the reduction of H2O2 to water and molecular oxygen with concomitant GSH oxidation. Other GPX isozymes induce the reduction of different hydroperoxides, including lipid peroxides. This reaction contributes to cell membrane protection against damage because it breaks the peroxidative chain reaction involving fatty acids and other organic hydroperoxides. Despite the crucial role played by antioxidant enzymes in maintaining the redox balance, endogenous antioxidants are mainly

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represented by small molecules [46]. Most of them can be divided into hydrophobic and hydrophilic antioxidants. The former, mainly involved in the protection against lipid peroxidation, include αtocopherol, β-carotene, flavonoids, and coenzyme Q10. Among others, GSH and its oxidized form GSSG, ascorbic acid, and thiols belong to the hydrophilic group. In addition to protecting lipids against peroxidation, they also exert their action toward protein, carbohydrate, and nucleic acid oxidation. Vitamins A, C, and E function as scavengers, molecules able to neutralize free radicals by donating an electron, but that are subsequently destroyed. Alternatively, thiol-containing compounds such as GSH, thioredoxin, and cysteine are rapidly regenerated after oxidation by the action of GSH reductase, thioredoxin reductase, or cystine reductase [47]. Both GSH and thioredoxin can form disulfides also with cellular proteins, a process that has been proposed to serve regulatory purposes [48,49]. In addition to its oxidant activity, reflected by ONOO− production, NO seems also to be endowed with antioxidant properties. Indeed, U NO-releasing compounds have been shown to protect against O2 − toxicity in both fibroblasts and neuron primary cultures [50]. In U addition, lipid peroxidation and the conversion of H2O2 to OH seem to be prevented by NO, which has also been shown to promote the expression of antioxidant enzymes and to enhance GSH and thioredoxin antioxidant activity [reviewed in 51]. ROS intracellular targets Whereas there are several reports in the literature describing ROS production in skeletal muscle in various physiological and pathological states, a role for ROS in regulating intracellular signal transduction pathways has emerged in the past few years (Fig. 2). Muscle transcriptional activity has been demonstrated to be redoxregulated. Indeed, up-regulation of both protective enzymes and stress proteins after contraction results from activation of transcription factors such as NF-κB and AP-1 [52,53]. These are activated in the cytosol by oxidation, whereas both DNA-binding and transcriptional activity require a reducing environment [52,54]. Skeletal muscle force production has been shown to be influenced by both ROS and RNS [55]. Indeed, muscle ROS levels are crucial to regulate contraction, being low under basal conditions and increased when force hyperproduction is required, although high ROS concentrations result in inhibition of contraction [56]. In addition, ROS are involved in the generation of muscle fatigue in repeated or sustained contractions. ROS and NO effects on muscle force production seem to derive from changes in intracellular Ca2+or from modulations of the myofilament sensitivity to Ca2+[57,58]. The balance between protein degradation and synthesis rates, which ensures maintenance of the skeletal muscle mass in adult individuals, has been proposed to be redox-sensitive. Some reports in the literature suggest that pro-oxidant exposure reduces protein synthesis. In this regard, H2O2 treatment has been shown to decrease translational activity in CHO cells by interfering with p70S6K and eIF4E [59]. Similar observations have been reported in experimental models of muscle atrophy such as hind-limb suspension or denervation [60,61], as well as in experimental animals treated with TNF-α [62]. However, although reduced protein synthesis has been reported in both experimental and clinical muscle depletion, the relevance of oxidative stress in this regard is still unclear. Oxidative stress has also been suggested to drive muscle protein hypercatabolism, contributing to skeletal muscle dysfunction. In this regard, ROS are involved in the activation of the ubiquitin– proteasome-dependent proteolytic pathway, either by marking myofibrillar proteins for degradation [63–65] or by inducing both expression and activity of components of this proteolytic machinery [66]. The link between ROS and activation of the ubiquitin– proteasome degradative pathway has been stressed by both in vivo [64] and in vitro studies [67,68]. C2C12 murine myoblasts exposed to

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Fig. 2. Effects of ROS production on signaling pathways in skeletal muscle. Mitochondrial release of ROS, induced by humoral mediators or metabolic alterations, results in activation of various intracellular signaling pathways that work to increase both protein breakdown and cytotoxicity in the skeletal muscle.

H2O2 show increased ubiquitinating-conjugating activity and altered expression of genes encoding components of the ubiquitin–proteasome pathway, such as polyubiquitin, E214k protein, and the musclespecific ubiquitin ligases atrogin1/MAFbx and MuRF1 [66]. Such response is mirrored by TNF both in vivo and in vitro, suggesting that ROS may act as second messengers in TNF-induced muscle catabolism [6,69]. Consistent with this hypothesis, TNF has been shown to induce an oxidative burst in C2C12 myotube cultures [65]. Finally, TNF, in the presence of interferon-γ, has been shown to stimulate iNOS expression in C2C12 myocytes [70]. TNF and ROS share the possibility of interfering with molecules involved in the activation of protein catabolism, such as the transcription factors NF-κB and AP-1 and the p38 MAPK. The involvement of ROS in NF-κB activation in the skeletal muscle is demonstrated by the observation that its DNA-binding activity is enhanced in C2C12 myotubes exposed to H2O2 or TNF [65,66], whereas pretreatment with catalase inhibits such activation [65]. p38 MAPK has been proposed to play a role in the pathogenesis of muscle atrophy in type 2 diabetes, aging, or TNF exposure [71]. In particular, this kinase seems required for muscle-specific ubiquitin ligase hyperexpression and increased ubiquitin-conjugating activity [72]. p38 activation by ROS seems to involve thioredoxin: when oxidized, thioredoxin dissociates from apoptosis-stimulating kinase 1, allowing p38 phosphorylation and activation [73]. Muscle-specific ubiquitin ligase hyperexpression is generally considered the hallmark of enhanced proteolysis by the ubiquitin– proteasome pathway, although contrasting data are emerging in the literature. In many experimental models of muscle atrophy, atrogin-1 hyperexpression seems to depend on activation (dephosphorylation) of transcription factors of the FoxO family, FoxO-1 and-3, in particular [17,18]. FoxO factors have been shown to be activated by H2O2, TNF, and menadione-or heat-shock-induced oxidative stress in C2C12 myoblasts [74]. Oxidative stress has also been proposed to disrupt the negative regulation exerted on FoxO by the PI3K/Akt signaling pathway [74]. Correction of skeletal muscle atrophy by antioxidant treatments The first indication that oxidative stress occurs in the skeletal muscle derives from the observation that physical exercise is

associated with increased production of both ROS and RNS [75]. Since then, a lot of reports in the literature have described the occurrence of oxidative stress, due to enhanced ROS production, impaired antioxidant systems, or both, in physiological as well as pathological states associated with muscle atrophy. Keeping this in mind, several antioxidant approaches have been proposed to prevent skeletal muscle depletion (Table 1). Aging-associated sarcopenia Skeletal muscle is particularly affected by the age-related loss of function, whether directly or also because of aging of other organs and systems that support its functionality, such as the neural, endocrine, and cardiovascular systems [76,77]. ROS production has been shown to increase in the skeletal muscle during aging [52,78], and oxidative stress has been claimed to be relevant to age-related cell damage [79]. ROS in aged muscle are probably mitochondria-derived. In addition, these organelles have been proposed to be the prime target of ROS, leading to the accumulation of oxidatively damaged components [80] and to skeletal muscle mitochondrial dysfunction [79]. The inability of mitochondria to meet ATP demand will result in reduced myofiber oxidative capacity, disrupted cellular energetics, and compromised ability to adapt to physiological stress [78]. In this regard, the aged skeletal muscle is less prone to activate the regenerative response Table 1 Effectiveness of antioxidant treatments on skeletal muscle wasting Antioxidant treatment

Effective

Ineffective or uncertain

Vitamin E Vitamin C Resveratrol Dehydroepiandrosterone Ornithine, cysteine, N-acetylcysteine Carnitine

Diabetes Diabetes Aging, diabetes Diabetes, cancer cachexia Cancer cachexia, DMD

Aging, ALS ALS, aging Cancer cachexia ALS ALS

Cancer cachexia, aging, ALS, diabetes DMD Aging, diabetes, cancer cachexia, DMD

-

Epigallocatechin gallate Low-intensity training

-

ALS, amyotrophic lateral sclerosis ; DMD, Duchenne muscle dystrophy.

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after injury. This has been related to reduction of anabolic signal transduction pathways such as that regulated by IGF-1 [81], but also to the observation that the redox balance is altered in satellite cells, impairing their myogenic properties [47]. Oxidative stress has been proposed to mediate the increased apoptotic rate that characterizes various cell populations during aging or age-related neurodegenerative diseases [82,83]. The intrinsic/ mitochondrial mechanism of apoptosis has been shown to be activated by oxidative stress, although evidence is emerging that ROS also promote caspase 2 activation in various cell systems [84]. In this regard, the increased oxidative stress in aging could result in caspase 2 activation and muscle cell death. In addition to derangements directly occurring at the skeletal muscle level, sarcopenia in aged individuals is also associated with reduced antioxidant dietary intake [85]. In this regard, poor muscle strength and low physical performance have been associated with low levels of circulating carotenoids [86,87]. The lack of physical activity, which has been proposed to induce an antioxidant response in itself [88], may also contribute to the onset of sarcopenia. Although a lot of studies have investigated the possibility of delaying the aging process by enhancing, endogenously or exogenously, the organism antioxidant capacity [89–91], very little is known about the effectiveness of such strategies on the development of sarcopenia. Some studies have shown that the treatment of aged individuals with high-dose β-carotene or vitamin E does not seem to replace the effects of diets high in fruits, vegetables, and whole grains and low in saturated fats [reviewed in 92 and 93]. In addition, a recent trial shows that zinc supplementation does not affect oxidative stress status in elderly people [94]. In addition, several reports have shown deleterious effects of antioxidant treatment on muscle function and redox state. Indeed, supplementation with vitamin E or ubiquinone-10 has been shown to unfavorably affect endurance performance in training subjects [95,96]. Finally, supplementation with vitamin C has been shown to significantly lower both training efficiency and the expression of antioxidant enzymes in the skeletal muscle [97]. Diabetes The persistent hyperglycemia that characterizes diabetes significantly impairs the pro-oxidant/antioxidant balance, both increasing ROS/RNS and reducing antioxidant levels. There is now clear evidence that oxygen radicals contribute to the progression of diabetes and its complications, and promising strategies based on antioxidant compounds have been proposed [98–100]. As far as skeletal muscle metabolism is concerned, oxidative stress has been shown to affect the expression of redox-sensitive genes involved in protein synthesis [65], and in vitro H2O2 has been demonstrated to inhibit myogenesis at the level of muscle-specific protein expression [101]. Muscle atrophy in animals rendered diabetic by treatment with streptozotocin (STZ) is associated with reduced levels of the myogenic regulatory factors MyoD, Myf5, and myogenin; decreased MEF-1 DNA-binding activity; and impaired synthesis of both myosin chains and muscle creatine phosphokinase [102]. Another report is in line with these observations, showing also that the activity of the calcineurin/NF-AT pathway is reduced in diabetic rats [103]. In addition, evidence has been provided that mitochondrial dysfunction is also relevant to the development of insulin resistance [104]. Indeed, the oxidative capacity of the skeletal muscle, mostly dependent on mitochondrial function, is directly correlated with insulin sensitivity [105], and reduced mitochondrial oxidative phosphorylation is associated with insulin resistance [106]. Vitamin C supplementation has been shown to improve muscle glycogen content and to counteract oxidative events in STZ-induced diabetes [107], and treatment of diabetic ob/ob mice with the ROS scavenger probucol decreases both plasma and muscle oxidative stress markers [108]. Along the same line, administration of α-lipoic

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acid to obese Zucker rats results in reduced oxidative stress and decreased formation of advanced glycation end products (AGEs), together with improved peripheral insulin sensitivity. Similarly, the AGE inhibitor pyridoxamine prevents irreversible protein glycation, reducing the onset of diabetes-related complications [109]. Moreover, administration of resveratrol, an antioxidant present in red wine, to rats with STZ-induced diabetes exerts hypoglycemic and hypolipidemic effects, also resulting in increased glucose uptake in the skeletal muscle [110]. Finally, skeletal muscle wasting in diabetic rats can be significantly improved by treatment with the antioxidant dehydroepiandrosterone (DHEA) [102,111]. This pattern is associated with restoration of physiological levels of myogenic regulatory factors and of myosin chains [102]. DHEA administration, in addition, partially restores normal levels of calcineurin expression and normalizes the DNA-binding activity of the NF-AT transcription factor [104]. As for human pathology, a few studies are available in the literature showing that skeletal muscle insulin resistance is improved in type 2 diabetes patients administered α-lipoic acid [112], vitamin E [113], or glutathione [114]; by contrast, such effect is not observed after treatment with vitamin C [115; reviewed in 116]. In addition to the administration of compounds directly endowed with antioxidant properties, some studies in the literature report on the attempt to indirectly interfere with the occurrence of oxidative stress. In this regard, exercise has been shown to improve muscle mitochondria dysfunction in patients with diabetes mellitus, increasing mitochondria biogenesis and restoring to about normal UCP3 expression levels [117]. In addition, supplementation of STZ-treated mice with a specific commercially available amino acid mixture has been shown to increase SOD expression, improving the muscle antioxidant capacity [118]. Cancer cachexia ROS and increased oxidative stress have been proposed to play a significant role in activating at least some of the mechanisms leading to cancer cachexia [reviewed in 119]. Consistent evidence indicates that increased oxidative stress is involved in muscle wasting in experimental models of cancer cachexia. In this regard, increased levels of ROS, and related oxidation and nitration end products, together with an impaired ability to upregulate antioxidant enzymes, have been reported in tumor-bearing animals [67,111,120]. In addition, mild oxidative stress has been shown to increase protein degradation in skeletal muscle by enhancing the expression of components of the ubiquitin–proteasome degradative pathway [67]. Increased oxidative stress is a frequent feature in cancer patients. High ROS levels, associated with reduced GPX and MnSOD activity, have been reported in patients with advanced cancer [121,122]. In addition, the occurrence of oxidative stress in cachectic individuals is highly predictive of both clinical outcome and survival [123]. The mechanisms leading to increased oxidative stress in cancer cachexia are still unclear. In addition to the pro-oxidant effects exerted by proinflammatory cytokines, ROS themselves seem to stimulate an excessive production of proinflammatory cytokines, probably by activating the NF-κB transcription factor, in turn resulting in further increased ROS production [124]. At the same time, some antineoplastic regimens (alkylating agents and cisplatin) induce ROS excess leading to oxidative stress [125]. Recently a brain–muscle axis has also been proposed to contribute to ROS production. In particular, increased malonyl-CoA levels in hypothalamic neurons rapidly enhance fatty acid oxidation and increase the expression of UCP3 and other key oxidative mitochondrial enzymes in the skeletal muscle [126]. A number of studies in the literature report on antioxidant treatments in experimental cancer cachexia. In this regard, tumorbearing rats fed an antioxidant-enriched diet increased their food

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intake [127], whereas carnitine supplementation resulted in improvement of anorexia, reduced wasting of the lean body mass, and decreased systemic inflammation [128]. Treatment of tumor-bearing mice with ornithine, a precursor of the radical scavenger spermine, has been shown to reverse both the reduced mitochondrial respiratory chain activity and the increased oxidative stress, whereas treatment with cysteine, a GSH precursor, normalized only the latter. In addition, ornithine, cysteine, and N-acetylcysteine are able to restore normal levels of the GSH precursor glutamate in the skeletal muscle of tumor-bearing mice [129]. Finally, a recent report shows that muscle wasting in rats bearing the Yoshida AH-130 hepatoma can be slightly, but significantly, attenuated by treatment with the antioxidant DHEA. Such protection is associated with partial restoration of the normal oxidative environment, MuRF1 mRNA levels, and proteasome enzymatic activity [111]. In contrast to these observations, treatment of tumor-bearing animals with the antioxidant resveratrol has proven ineffective in preventing muscle wasting [130]. As for human cancer cachexia, a phase III trial aimed at assessing the efficacy of an integrated approach, consisting of antioxidants, pharmaconutritional support, progestogens, and cyclo-oxygenase-2 inhibitors, has been recently concluded [131]. This treatment significantly improved body weight, lean body mass, appetite, and quality of life, while decreasing systemic inflammation. This approach, however, does not allow one to dissect the real role played by antioxidants in exerting such beneficial effects. A recent report shows that patients treated with carnitine display reduced fatigue and increased appetite and lean body mass with respect to nontreated patients. In addition, ROS levels decreased and GPX increased [132]. These data suggest that reduction of oxidative stress in cancer patients is associated with improved muscle mass and function. Nonsystemic muscle depletion Atrophy of the skeletal muscle may result from local factors or systemic effects. On one hand, it may occur as a significant component of an organismal wasting syndrome (cachexia), such as is observed in diabetes, cancer, chronic obstructive pulmonary disease, or autoimmune disorders. On the other hand, it may depend on molecular alterations that directly affect the muscle or reflect disturbances in its nerve supply, such as occur in muscular dystrophies, neurological myopathies, disuse, and denervation. Amyotrophic lateral sclerosis (ALS), one of the most common neuromuscular diseases due to degeneration of motor neurons, is characterized by progressive muscle wasting, inanition, and respiratory failure, leading to death in a few years from onset [133]. ALS is associated with a high production of ROS, such as peroxynitrite and hydroxyl radicals, which inactivate essential molecular components. Consistently, G93A mice, which lack the CuZnSOD and are a wellknown experimental model of ALS, show high levels of oxidative damage to lipid, protein, and DNA in several tissues, including skeletal muscle [134]. ROS are produced to some extent by mitochondria, and when the electron transport is inhibited, they accumulate to abnormal levels [135], eventually exacerbating mitochondrial damage, an important feature of ALS. Recently SOD1 mutants have been shown to specifically interact with Derlin-1, a component of the endoplasmic reticulum-associated degradation (ERAD) machinery, triggering ER stress through ERAD dysfunction and leading to apoptotic motor neuron death [136]. In addition, MnSOD has also been proposed to produce ROS, by catalyzing the conversion of peroxide to hydroxyl radical [137] or, through a reverse catalysis, oxygen to superoxide, which then can combine with NO to form peroxynitrite [138]. In particular, MnSOD mutants have been shown to possess an increased ability to produce hydroxyl or peroxynitrite radicals [reviewed in 139]. ROS production may inhibit glutamate uptake, stimulate the release of proinflammatory mediators by the microglia, induce apoptosis, and mediate growth factor signaling; most of these alterations in turn can

determine in a further increase in ROS production [139], suggesting that ALS progression results from a network of abnormal processes, all of which may make a substantial contribution to the ultimate outcome of motor neuron degeneration. Common antioxidant interventions in ALS include vitamins C and E, selegiline, N-acetylcysteine (NAC), DHEA, and various combined treatments [140]. Oral administration of L-carnitine or melatonin has been shown to delay the onset of disease signs and of motor activity deterioration and to extend life span in G93A transgenic mice [141]. Similarly, the free radical scavenger edaravone effectively slows symptom progression and motor neuron degeneration in an experimental ALS model [142]. However, despite the amount of reports showing the rationale for antioxidant regimens in ALS patients, and their widespread use, no significant evidence of their beneficial effect is available [reviewed in 143]. Duchenne and Becker-type muscle dystrophies are characterized by muscle damage (milder in the latter than in the former) that derives from mutations of the gene coding for dystrophin. Oxidative damage has been proposed to play a role in the pathogenesis of muscle degeneration in both forms of dystrophy. Indeed, oxidative stress has been shown to occur in dystrophic patients, as demonstrated by the observation that their circulating levels of F2-isoprostanes are significantly higher than those evaluated in healthy subjects [144]. Consistently, increased protein and DNA oxidation has been observed in the muscle of patients with Duchenne-type muscle dystrophy, and lipid peroxidation is increased in mdx mice [145]. In dystrophic muscles, ROS may derive from several sources such as mitochondria, phagocyte infiltration, and activation of the arachidonic acid cascade. In addition, the skeletal muscle isoform of nNOS is associated with the dystrophin–glycoprotein complex (DGC), and loss of dystrophin or other DGC components also leads to nNOS depletion from the sarcolemma [146,147]. DGC-associated nNOS seems to be the major source of NO release from skeletal muscle cells at rest and during contractile activity. When nNOS activity is reduced, extracellular NO release declines, whereas superoxide release increases. Although a direct correlation has not been demonstrated, it is possible that this pattern results in dysregulation of NF-κB signaling, leading to muscle degeneration. Consistent with this hypothesis, treatment with antioxidants, or muscle nNOS overexpression, results in muscle damage improvement, whereas muscle dystrophy may be induced in animals fed a diet depleted of both vitamin E and selenium, two well-known antioxidants [reviewed in 145]. Antioxidants have been shown to reduce muscle necrosis in mdx dystrophic mice [148,149]. Recent reports have shown that in mdx mice the green tea compound epigallocatechin gallate is effective in both preventing disease onset and improving muscle function [150,151], whereas NAC administration results in reduced ROS levels, decreased number of fibers with centrally located nuclei (regenerating fibers), and caveolin-3 expression [152]. In addition, glutamine supplementation has proven effective in improving muscle protein catabolism in children affected by Duchenne-type muscle dystrophy [153] and in reducing both oxidized glutathione and MAPK signaling in dystrophic mdx mice [154]. As previously reported for diabetes (see above), also in mdx mice, markers of oxidative stress can be reduced by low-intensity training [151]. Conclusions The hypothesis that perturbations in the production, distribution, or interactions of ROS/RNS can contribute to the pathogenesis of skeletal muscle atrophy, in both physiological and pathological states, is supported by experimental and clinical evidence. Indeed, at least some of the mechanisms involved in muscle depletion, such as enhanced proteolysis or reduced protein synthesis, may also depend on an altered redox balance, supporting the adoption of antioxidant therapeutic regimens. However, despite the observation that oxidative

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stress generally occurs in the atrophying muscle, irrespective of the causative mechanisms, the extent of its contribution to tissue wasting is still unclear. As an example, the effectiveness of DHEA treatment in correcting/preventing muscle atrophy in experimental diabetes compared to that in cancer cachexia is quite different, being very marked in the former and just partial, although significant, in the latter [111]. In addition, the relevance of antioxidant treatments in protecting the skeletal muscle against oxidative stress has been questioned, at least under some experimental conditions [97]. On the whole, these observations suggest that caution should be used in generalizing, in terms of both mechanisms and treatments, among the various types of muscle atrophies. In this regard, in some cases a strong contribution of redox imbalance to the pathogenesis of muscle depletion can be recognized and even inferred from the effectiveness of antioxidant therapies. This is particularly evident in diabetes, in which antioxidants such as vitamins C and E, resveratrol, DHEA, and others have been shown to significantly protect from muscle wasting (see Table 1). In other cases, the results obtained with antioxidant treatments are contrasting, although some studies demonstrated a certain degree of protection against muscle atrophy in cancer cachexia, Duchenne muscle dystrophy, aging, and ALS. In all these conditions, oxidative stress probably behaves as an additional factor that certainly amplifies the wasting stimuli, but probably does not play a leading role. Taking this in mind, a comprehensive therapeutic approach to muscle atrophy should take into account the relative contribution of oxidative stress. In this regard, the data actually available seem to support antioxidant treatments, with vitamins and DHEA in particular, to prevent diabetes-associated muscle atrophy, whereas further studies are needed to warrant their use in other wasting conditions. Acknowledgments The authors thank Professor Fiorella Biasi for critical discussion. P.C. is supported by MIUR, University of Torino, Compagnia di San Paolo, and Regione Piemonte. References [1] Costelli, P.; Baccino, F. M. Mechanisms of skeletal muscle depletion in wasting syndromes: role of ATP–ubiquitin-dependent proteolysis. Curr. Opin. Clin. Nutr. Metab. Care 6:407–412; 2003. [2] Kadowaki, M.; Kanazawa, T. Amino acids as regulators of proteolysis. J. Nutr. 133: 2052S–2056S; 2003. [3] Mizushima, N.; Yamamoto, A.; Matsui, M.; Yoshimori, T.; Ohsumi, Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol. Biol. Cell 15:1101–1111; 2004. [4] Marino, G.; Uria, J. A.; Puente, X. S.; Quesada, V.; Bordallo, J.; Lopez-Otin, C. Human autophagins, a family of cysteine proteinases potentially implicated in cell degradation by autophagy. J. Biol. Chem. 278:3671–3678; 2003. [5] Mammucari, C.; Milan, G.; Romanello, V.; Masiero, E.; Rudolf, R.; Del Piccolo, P.; Burden, S. J.; Di Lisi, R.; Sandri, C.; Zhao, J.; Goldberg, A. L.; Schiaffino, S.; Sandri, M. FoxO3 controls autophagy in skeletal muscle in vivo. Cell Metab. 6:458–471; 2007. [6] Bodine, S. C.; Latres, E.; Baumhueter, S.; Lai, V. K.; Nunez, L.; Clarke, B. A.; Poueymirou, W. T.; Panaro, F. J.; Na, E.; Dharmarajan, K.; Pan, Z. Q.; Valenzuela, D. M.; DeChiara, T. M.; Stitt, T. N.; Yancopoulos, G. D.; Glass, D. J. Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 294:1704–1708; 2001. [7] Costelli, P.; Reffo, P.; Penna, F.; Autelli, R.; Bonelli, G.; Baccino, F. M. Ca2+-dependent proteolysis in muscle wasting. Int. J. Biochem. Cell Biol. 37: 2134–2146; 2005. [8] Smith, I. J.; Dodd, S. L. Calpain activation causes a proteasome-dependent increase in protein degradation and inhibits the Akt signalling pathway in rat diaphragm muscle. Exp. Physiol. 92:561–573; 2007. [9] Du, J.; Wang, X.; Miereles, C.; Bailey, J. L.; Debigare, R.; Zheng, B. Activation of caspase-3 is an initial step triggering accelerated muscle proteolysis in catabolic conditions. J. Clin. Invest. 113:115–123; 2004. [10] Wei, W.; Fareed, M. U.; Evenson, A.; Menconi, M. J.; Yang, H.; Petkova, V.; Hasselgren, P. O. Sepsis stimulates calpain activity in skeletal muscle by decreasing calpastatin activity but does not activate caspase-3. Am. J. Physiol. Regul. Integr. Comp. Physiol. 288:R580–R590; 2005.

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