Author’s Accepted Manuscript Muscle Redox Signalling Pathways in Exercise. Role of Antioxidants Shaun A Mason, Dale Morrison, Glenn K McConell, Glenn D Wadley www.elsevier.com

PII: DOI: Reference:

S0891-5849(16)00073-3 http://dx.doi.org/10.1016/j.freeradbiomed.2016.02.022 FRB12762

To appear in: Free Radical Biology and Medicine Received date: 1 November 2015 Revised date: 5 February 2016 Accepted date: 17 February 2016 Cite this article as: Shaun A Mason, Dale Morrison, Glenn K McConell and Glenn D Wadley, Muscle Redox Signalling Pathways in Exercise. Role of A n t i o x i d a n t s , Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2016.02.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

MUSCLE REDOX SIGNALLING PATHWAYS IN EXERCISE. ROLE OF ANTIOXIDANTS

2

Shaun A Mason1, Dale Morrison1, Glenn K McConell2, & Glenn D Wadley1

3 4

1

5 6

2

Deakin University, Geelong, Victoria, Australia. Centre for Physical Activity and Nutrition (CPAN) Research, School of Exercise and Nutrition Sciences. Victoria University, Melbourne, Victoria, Australia Clinical Exercise Science Research Program, Institute for Sport, Exercise and Active Living (ISEAL)

7 8 9

Corresponding author/ requests for reprints:

10 11

Glenn Wadley

12

Centre for Physical Activity and Nutrition Research

13

School of Exercise and Nutrition Sciences, Deakin University

14

221 Burwood Highway

15

Burwood, Australia

16

3125

17

Phone: +61 3 92446018; Fax: +61 3 92446017; Email: [email protected]

18 19

ABSTRACT

20 21 22 23 24 25 26 27 28 29 30 31

Recent research highlights the importance of redox signalling pathway activation by contraction-induced reactive oxygen species (ROS) and nitric oxide (NO) in normal exerciserelated cellular and molecular adaptations in skeletal muscle. In this review, we discuss some potentially important redox signalling pathways in skeletal muscle that are involved in acute and chronic responses to contraction and exercise. Specifically, we discuss redox signalling implicated in skeletal muscle contraction force, mitochondrial biogenesis and antioxidant enzyme induction, glucose uptake and muscle hypertrophy. Furthermore, we review evidence investigating the impact of major exogenous antioxidants on these acute and chronic responses to exercise. Redox signalling pathways involved in adaptive responses in skeletal muscle to exercise are not clearly elucidated at present, and further research is required to better define important signalling pathways involved. Evidence of beneficial or detrimental effects of specific antioxidant compounds on exercise adaptations in muscle is 1

32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

similarly limited, particularly in human subjects. Future research is required to not only investigate effects of specific antioxidant compounds on skeletal muscle exercise adaptations, but also to better establish mechanisms of action of specific antioxidants in vivo. Although we feel it remains somewhat premature to make clear recommendations in relation to application of specific antioxidant compounds in different exercise settings, a bulk of evidence suggests that N-acetylcysteine (NAC) is ergogenic through its effects on maintenance of muscle force production during sustained fatiguing events. Nevertheless, a current lack of evidence from studies using performance tests representative of athletic competition and a potential for adverse effects with high doses (>70 mg/kg body mass) warrants caution in its use for performance enhancement. In addition, evidence implicates high dose vitamin C (1g/day) and E (≥260 IU/day) supplementation in impairments to some skeletal muscle cellular adaptations to chronic exercise training. Thus, determining the utility of antioxidant supplementation in athletes likely requires a consideration of training and competition periodization cycles of athletes in addition to type, dose and duration of antioxidant supplementation.

47 48 49

Key words:

50

reactive oxygen species, nitric oxide, antioxidants, skeletal muscle, exercise

51 52

ABBREVIATIONS

53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71

Akt – Protein kinase B; AMPK –adenosine monophosphate-activated protein kinase; COX – cytochrome oxidase; DTT – dithiothreitol; ERK1/2 – Extracellular signal-regulated kinases 1/ 2; Gpx – glutathione peroxidase; Grx – glutaredoxins; GSH – glutathione; GSSG – glutathione disulphide; H2O2 – hydrogen peroxide; IGF-1 – insulin-like growth factor 1; JNK – c-Jun Nterminal kinase; L-NAME – N G-nitro-l-arginine methyl ester; mTOR – mammalian target of rapamycin; NAC – N-acetyl cysteine; NADPH – nicotinamide adenine dinucleotide phosphate; NFκB – nuclear factor kappa B; nNOS – neuronal nitric oxide synthase; NO – nitric oxide; NOS- nitric oxide synthase, NRF – nuclear respiratory factor; O2●─ – superoxide; ● OH – hydroxyl radical; ONOO—– peroxynitrite; p38 MAPK – p38 Mitogen-activated protein kinase; p70S6K – ribosomal protein S6 kinase; PGC-1α – peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1-alpha; PI3K – Phosphoinositide 3-kinase; PKA – protein kinase A; pO2 – partial pressure of oxygen; Prx – peroxiredoxins; ROS – reactive oxygen species; RyR1 – ryanodine receptor/Ca2+ release channel; SERCA1 – sarco(endo)plasmic reticulum Ca2+-dependent ATPase; -2; SH – thiol; SIRT-1 – sirtuin 1; SIRT3 – sirtuin 3; SOD – superoxide dismutase; Srx – sulfiredoxins; SSG –glutathionylation; TFAM – mitochondrial transcription factor A; TnIf – fast twitch skeletal muscle-specific troponin I isoform; TRIM – 1-(2-trifluoromethyl-phenyl)-imidazole; TRPV1 – transient receptor potential cation channel, subfamily V, member 1; Trx – Thioredoxins; VO2 – volume of oxygen consumed 2

72

INTRODUCTION

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

It is well established that muscular contraction and intense exercise generate an increased production of reactive oxygen species (ROS) and nitric oxide (NO) and promote oxidative stress in skeletal muscle [1-8]. While the principal cellular and tissue sites of ROS production during exercise have been a matter of debate [9], recent evidence appears to indicate that exercise-induced ROS is primarily of non-mitochondrial origin, particularly from nicotinamide adenine dinucleotide phosphate (NADPH) oxidases [10-12]. Historically, an increased level of ROS has been regarded as deleterious to cells [13, 14]. Exposure to increased levels of ROS has been implicated in damage and modifications to cellular lipids, DNA and proteins [15] . ROS has also been implicated in chronic conditions such as cardiovascular disease [16-18] and type 2 diabetes [19]. Such negative effects of ROS might be related to an excessive level and/or duration of ROS exposure and to the cellular origin of ROS produced [11]. On the other hand, evidence has emerged over the past two decades showing that ROS and NO produced physiologically by cells are important signalling molecules, acting through mechanisms such as post-translational redox modifications of cysteine thiols on proteins [20, 21]. Such signalling can regulate diverse biological functions such as the maintenance of tissue homeostasis, regulation of transcriptional activity, cell proliferation and differentiation, and cell migration [15, 22-26]. Recent research has also highlighted the potential importance of ROS and NO-mediated signalling in normal exerciserelated molecular and cellular responses [14]. In particular, redox-signalling pathways have been implicated in several acute and chronic responses of skeletal muscle to exercise, including skeletal muscle glucose uptake and muscle insulin sensitivity [27, 28]; modulation of endogenous antioxidant enzyme levels [6, 29, 30]; mitochondrial biogenesis [31-33]; muscle contraction force [34-36] and muscle hypertrophy [37, 38].

97 98 99 100 101 102 103 104 105 106 107 108 109 110 111

Antioxidants play an important role in regulating tissue levels of ROS through free radical scavenging and adaptive electrophilic-like mechanisms (interested readers are referred to ref. [13] for a comprehensive review). Acute and chronic exercise tends to upregulate endogenous antioxidant enzyme abundances and activities in skeletal muscle [6, 29, 30], therefore enabling an improved capacity to decrease adverse effects of increased ROS production. Moreover, the common supplementation of antioxidants by elite and recreational athletes [39, 40] may also enhance the capacity of skeletal muscle to neutralize ROS produced during exercise. Benefits might relate to an improvement in cellular redox state and decreased oxidative modifications to DNA, lipids and proteins. Some evidence shows an ameliorating effect of antioxidant supplementation on muscle damage associated with delayed onset muscle soreness [41], although other evidence does not support a protective effect of supplementary antioxidants [42-44]. ROS has also been implicated in premature muscular fatigue during sustained submaximal muscle contraction and exercise [45-47]. Therefore, the use of supplementary antioxidants might help to delay muscular fatigue and improve exercise performance.

112 113

Despite the aforementioned potential benefits of antioxidant supplementation in exercising humans, recent research has implicated the use of antioxidants in impairments rather than 3

114 115 116 117 118 119 120 121

improvements in some acute and chronic responses of skeletal muscle to exercise [31, 33, 35, 48, 49]. These impairments in adaptive changes within skeletal muscle are presumably a result of an attenuation of normal redox-signalling pathways in muscle by antioxidants [14]. In particular, antioxidant supplementation has been found in some studies to impair some adaptive responses to endurance exercise training [33, 48, 49] and resistance exercise training [35, 38]. Nonetheless, study findings overall remain equivocal in human participants in relation to effects of antioxidants on skeletal muscle adaptations and performance outcomes following exercise training [50-52].

122 123 124 125 126 127 128 129 130 131 132 133

The present review aims to firstly present a discussion of some important redox-signallingrelated pathways implicated in acute and chronic responses of skeletal muscle to muscle contraction and exercise; and secondly, to discuss the impact of antioxidants on these redox-signalling-related pathways. Where possible, we have focussed on evidence arising from studies using healthy human participants, given the potential applicability of such findings to human athletic endeavours. However, considering existing ethical and methodological limitations in human-based studies, a vast amount of important mechanistic information can only currently be gathered using in vitro models, ex vivo models, in-situ models and in vivo animal models. Additionally, a wealth of information exists in studies concerned with elderly or infirm populations. Thus, we wish for readers to bear in mind the inherent limitations of translating findings from discrete populations, or from non-in vivo, non-human studies directly to human athletes.

134 135 136

EXERCISE-RELATED REDOX SIGNALING PATHWAYS IN SKELETAL MUSCLE

137 138 139 140 141 142 143 144 145 146 147 148 149

ROS including superoxide (O2●─) and hydrogen peroxide (H2O2), NO, and reactive NO derivatives including peroxynitrite (ONOO─) have been implicated in redox signalling in cells either directly or indirectly [53]. Key sites of ROS production during exercise include NADPH oxidase enzymes (which are associated with the sarcoplasmic reticulum [SR], transverse tubules and plasma membrane), phospholipase A2 and xanthine oxidase [12, 46]. Skeletal muscle mitochondria are also important biological generators of ROS, however in vitro and ex vivo evidence suggests that they are not likely to be key contributors to the increased muscle ROS production during exercise given higher mitochondrial ROS production at rest compared with exercise [9, 54, 55]. In terms of NO, neuronal nitric oxide synthase (nNOS) is the likely key generator of NO in skeletal muscle during contraction [46, 56]. NADPH oxidase production of ROS and nNOS production of NO appears to be especially important for redox signalling in muscle during exercise [57-61].

150 151 152 153

Transient and reversible post translational chemical modifications of reactive cysteine thiol residues on cell proteins, such as through processes including S-nitrosylation, Sglutathionylation, sulfenylation and disulphide formation, likely constitute important redox modifications through which cells respond to altered levels of ROS and NO [21, 36, 62]. S4

154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173

nitrosylation of proteins involves the coupling of a NO group to a reactive cysteine thiol to form an S-nitrosylated protein [63]. Protein S-nitrosylation can produce diverse cellular effects, including altered regulation of enzyme activities, altered receptor and transporter activities, altered gene transcription and translation, and protein-protein interactions [63]. S-nitrosylation has been observed in numerous proteins associated with skeletal muscle contraction and exercise, including the skeletal muscle ryanodine receptor/Ca 2+ release channel (RyR1), myosin, cAMP response binding protein (CREB), calpain-2, caspase-3, sarco(endo)plasmic reticulum Ca2+-dependent ATPase (SERCA1a), histone deacetylase-2 (HDAC2), plasma membrane Ca2+-ATPase type-1 (PMCA1), and specific insulin-signalling proteins [64-73]. S-glutathionylation involves the formation of mixed disulphides between GSH and cysteine thiol groups of proteins [74]. S-glutathionylation of a protein can result in its activation or deactivation, which may be important in the regulation of cell signalling mediators [21, 74]. S-glutathionylation is known to interface with protein phosphorylation via modulation of cellular kinases and phosphatases, including protein kinase A (PKA), creatine kinase, mitogen-activated protein kinase kinase kinase 1 (MEKK1), phosphatase and tensin homologue deleted from chromosome 10 (PTEN), adenosine monophosphateactivated protein kinase (AMPK), and protein tyrosine phosphatase 1B (PTP1B) [75-81]. Post-translational redox modifications such as S-nitrosylation and S-glutathionylation may therefore play important molecular signalling roles in skeletal muscle given their intricate associations with key proteins linked to muscle contraction and exercise adaptations (Fig. 1).

174 175 176 177 178 179 180 181 182 183 184 185 186

In addition to laying the foundation for cellular redox signalling [62], reversible redox modifications of cysteine thiols appear to protect proteins against further irreversible oxidation of critical cysteine residues [21]. Reactive methionine thiol residues on proteins are also prone to reversible redox modifications by ROS [82-84]. However, the relevance of methionine redox modifications to cellular redox signalling is presently unclear due to its relatively slow rates of oxidation, but relatively rapid rates of reduction [82, 85]. In addition to ROS and NO, antioxidants are intricately involved in redox signalling in cells. Antioxidants can play key roles in the regulation of thiol redox state and thus regulate redox signalling pathways [86]. Endogenous antioxidants, which include enzymes and small molecules such as thioredoxins, sulfiredoxins, glutathione reductases, peroxiredoxins and glutathione (GSH) are considered fundamental to the control of redox signalling networks [87]. Additionally, exogenous antioxidants such as vitamin C and vitamin E consumed in the diet or as dietary supplements, may also participate in and interact with these redox signalling networks.

187 188 189 190 191 192 193 194 195 196

Additional complexities of redox signalling that are mostly beyond the scope of this review include considerations of the subcellular location of redox alterations and the proximity of reactive protein thiols to sites of ROS/NO production [88] (such as NADPH oxidases and nNOS during exercise). Redox signalling is compartmentalized in cells [89]. Therefore, the global redox status of cells and tissues may not necessarily reflect important changes in subcellular location-specific redox signalling [53, 89]. Furthermore, the intrinsic chemical reactivities of specific ROS/NO-derived species and specific antioxidants/reducing compounds with respect to other competitive redox reactants present physiologically will affect how each can mediate redox signalling in vivo [24, 53, 82, 85, 90]. For example, exogenous antioxidants including NAC, vitamin C and vitamin E react relatively poorly with 5

197 198 199

H2O2 when compared to several endogenously generated antioxidants at physiologically relevant levels [53, 88]. Thus, it is unlikely that these exogenous antioxidants will impact on redox signalling in skeletal muscle via direct scavenging of H2O2 in vivo.

200 201 202 203 204 205 206 207

Effects of exercise-related redox signalling in skeletal muscle might include acute and/or chronic alterations in gene expression, modified kinase and phosphatase activities, and altered levels of molecular chaperones, transporters, proteasomes, and transcription factors [25, 36]. We now focus our discussion on redox signalling pathways implicated in the acute and chronic effects of exercise. In particular, we aim to discuss potential redox signalling pathways in skeletal muscle and their modulation by antioxidants during (a) muscle contraction; (b) exercise-related antioxidant enzyme induction and mitochondrial biogenesis; (c) exercise-related glucose uptake; and (d) skeletal muscle hypertrophy.

208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237

A) REDOX SIGNALLING AND SKELETAL MUSCLE CONTRACTION FORCE Optimal muscle contractile function depends on the cellular redox state [91]. In intact, unfatigued muscle, transient or low level exposure to H2O2 increases myofibrillar Ca2+ sensitivity and muscle force production; while prolonged or high level H 2O2 exposure decreases myofibrillar Ca2+ sensitivity and muscle force production [36, 91-93]. In fatiguing muscle, removal of ROS through antioxidant treatments improves contractile function and attenuates the loss of muscle force production [94]. Andrade et al. [92] demonstrated that force loss in intact single muscle fibres due to prolonged exposure to H2O2 could be reversed with the antioxidant dithiothreitol (DTT). Conversely, brief exposure of unfatigued muscle fibres to DTT decreased submaximal force production, although force production was restored after the addition of H2O2 [92]. These and similar findings in relation to ROS and antioxidants [45, 94, 95] imply that both a reduced redox state (i.e. during resting, unfatigued conditions) and a highly oxidized state (i.e. during prolonged fatiguing exercise) will impair muscle contractile force. Reid et al. proposed a homeostatic model of biphasic ROS effects on isometric force production [91] that essentially reflects the above observations and implicates an optimal intermediate muscle redox environment for optimal muscle contraction force (interested readers may refer to Fig. 2 of ref. [91]). Caveats in interpreting the aforementioned study findings are that some studies used nonphysiological H2O2 concentrations [96] and antioxidants typically unavailable for human use. Nonetheless, as discussed in later sections, studies investigating effects of antioxidants such as NAC appear to support these experimental observations in human participants. Reid et al. more recently proposed a ‘redox brake’ hypothesis [97], which proposes that high ROS exposure-induced force decrements and muscle fatigue are an intrinsic negative feedbacktype mechanism to minimize ROS-induced damage to contracting muscle. Such damage, however, might be facilitated by the concomitant use of antioxidants via a prolongation of force output and onset of fatigue in the presence of high ROS levels. Some evidence, at least in relation to NAC supplementation, would appear to support this hypothesis as

6

238 239

reflected by findings of increased levels of muscle damage markers in association with improved exercise performance in participants supplementing with NAC [98].

240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256

ROS-mediated impairments of muscle contractile function during repetitive exhaustive contractions could potentially occur at multiple steps and sites during the excitationcontraction coupling process. However, evidence suggests that a decrease in SR Ca 2+ release and/or a decrease in myofibrillar Ca2+ sensitivity are more likely causes of ROS-mediated fatigue than alterations in membrane excitability, action potential generation capacity or cross-bridge cycling (see [47] for review) [92, 99, 100]. Interestingly, the type of ROS produced might promote specific effects in relation to Ca2+ handling and Ca2+ sensitivity in intact fibres. H2O2 appears to preferentially affect myofibrillar Ca2+ sensitivity [101], while O2●─ appears to target SR Ca2+ release [100, 102]. Exposure of intact fast-twitch muscle fibres to exogenous NO donors was found to decrease Ca2+ sensitivity and increase SR Ca2+ release [103]. However, findings of effects of NO and NO donors on Ca 2+ handling and Ca2+ sensitivity are inconsistent in the literature [104] and likely depend on their concentration [47, 104, 105]. Moreover, effects of NO and NO donors on contractile force production and fatigue are unclear [46, 47, 57, 91]. We now briefly discuss contraction-related proteins RyR1 and Troponin I and their redox modulation by ROS and NO. Understanding the modulation of such proteins may help to better define underlying redox signalling in relation to skeletal muscle contraction force [97].

257 258

RyR1

259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274

Ca2+ release from the SR stimulates skeletal muscle contraction via RyR1. Activation of RyR1 is further regulated by endogenously produced ROS/NO [57, 59, 61] (Fig. 2), and is known to undergo both S-nitrosylation and S-glutathionylation at different cysteine sites [64, 106]. RyR1 activation is sensitive to NO concentration [105, 107] and oxygen (O2) tension [57, 108]. The presence of physiological (submicromolar) NO concentrations can potentiate RyR1 S-nitrosylation at a single cysteine residue (Cys3635), increase RyR1 activation and increase isometric twitch tension at physiological O2 tensions (1% O2) in situ and in vitro [57, 64, 108]. On the other hand, physiological levels of NO were found to impair twitch tension and tetanic force at higher, ambient oxygen tension (20% O2) in situ [57]. It was therefore proposed that low O2 tension in active muscle might promote S-nitrosylation of Cys3635 (and/or other reactive cysteines), leading to increased RyR1 activation and increased muscle contraction; while rises in O2 tension in resting muscle might attenuate these changes [57]. However, findings of Eu et al. were unable to be replicated in a study by Cheong et al. [109]. Furthermore, the above studies were conducted using isolated muscle. Therefore, the critical role of O2 tension on NO-induced RyR1 nitrosylation and activation in vivo remains uncertain.

275 276 277 278

Redox signalling might be important in over-training-induced impairments in muscle force following an abrupt increase in training volume and/or intensity [110]. RyR1 S-nitrosylation increased significantly in mice undertaking three weeks of twice-daily high intensity swimming exercise and in trained human cyclists performing three consecutive days of 7

279 280 281 282 283 284 285 286 287 288 289 290 291 292 293

prolonged fatiguing cycling (3h at 70% VO2 max) [111]. Although force was not measured in the human participants, a progressive decrease in maximal force production occurred in mice during the training period [111]. RyR1 remodelling, including an increase in RyR1 PKA phosphorylation and decreases in levels of phosphodiesterase 4D3 (PDE4D3) and the stabilizing binding protein calstabin1 occurred in both mice and humans. These changes corresponded with higher open RyR1 channel probabilities, suggestive of “leaky” RyR1 channels [111]. SR Ca2+ leak can potentially deplete SR Ca2+ stores, leading to decreased tetanic SR Ca2+ release and impaired muscle contraction [112]. Moreover, increased Snitrosylation of RyR1 has been shown to deplete calstabin1 from RyR1 [113], leading to “leaky” RyR1 channels and impaired muscle function [65, 112]. Conversely, increased Ca2+ leak and muscle contractile force impairments were reversed in rodents treated with a drug specifically targeting the binding of calstabin1 to RyR1 [65, 111, 112]. Therefore, increased SR leak and contractile force impairments occurring in response to chronic, fatiguing exercise bouts might in part result from excessive S-nitrosylation of RyR1 and oxidativestress-induced remodelling of RyR1.

294 295

Troponin I

296 297 298 299 300 301 302 303 304 305 306 307

Troponin is a redox-sensitive three-globular protein complex that constitutes part of the thin filaments involved in sarcomeric contractions [91, 114]. In particular, the protein Troponin I binds to the protein actin and promotes stabilization of the troponintropomyosin complex [114]. The fast-twitch skeletal muscle Troponin I isoform (TnIf) is susceptible to S-glutathionylation in human and rodent muscles [115, 116]. Sglutathionylation of TnIf was found to promote increased Ca2+ sensitivity of the skeletal muscle contractile apparatus, which paralleled increases in peak twitch force and rate of force production in response to action potential stimulation [115]. Additionally, moderate intensity aerobic exercise (60% VO2 peak) increased levels of S-glutathionylation of TnIf by around 4-fold in exercising humans [116]. Thus, redox-signalling via S-glutathionlyation of contractile proteins such as Troponin I could be a significant factor influencing muscle contraction in exercising humans [116].

308 309 310 311 312 313 314 315 316 317 318 319 320

ROS have also been implicated in the force depression that can occur following fatiguing muscle contractions [47, 100]. Recovery of submaximal force in particular can be delayed following fatiguing contractions, taking hours to days to recover [117, 118]. Watanabe et al. [119] found the depression in low frequency force following fatiguing contractions to correspond to a decrease in SR Ca2+ release and increases in myofibrillar Ca2+ sensitivity and TnIf S-glutathionylation in whole and skinned rodent muscle in situ. It was consequently suggested that TnIf S-glutathionylation contributed to increased myofibrillar Ca2+ sensitivity to help offset the depression in contractile force after fatiguing contractions caused primarily by a decrease in SR Ca2+ release [119]. Other studies have produced discordant findings to Watanabe et al. in relation to the relative contribution of altered Ca 2+ release versus altered Ca2+ sensitivity in the causation of submaximal force depression following fatiguing contractions [100, 120]. However, it is likely that factors including varying model species used (rat vs. mouse), use of isolated vs. non-isolated fibres [119], different 8

321 322

experimental temperatures [93] and different relative levels of O2●─ complicate comparisons of different study findings.

323 324 325 326 327 328 329

While the number of proteins identified to undergo specific molecular redox modifications that are of potential importance to skeletal muscle contractile function is growing, effects of specific antioxidant compounds on these redox modifications and the significance of such changes for muscle contraction are much less understood. Thus, our discussion of effects of antioxidants on skeletal muscle contraction and adaptations in subsequent sections of the review will be mostly limited to effects of antioxidants on downstream skeletal muscle redox signalling and muscle-related performance effects.

and H2O2 [100]

330 331 332

EFFECTS OF ANTIOXIDANTS ON SKELETAL MUSCLE CONTRACTION FORCE

333 334 335 336 337 338 339 340 341

In this section, we discuss findings of studies that investigated effects of exogenous antioxidant supplementation on muscle contractile force and fatigue, with a priority on studies with human participants. Currently, limited evidence appears to exist in relation to isolated effects of antioxidants β-carotene and α-lipoic acid [121] on muscle contraction force in humans. Other compounds with antioxidant properties such as taurine [122], melatonin [123], quercetin [124] and resveratrol [125] either have insufficient evidence from studies using human participants or have not been found to be effective at enhancing muscle contraction function in humans. Therefore, in this section we focus our discussion on antioxidants that have been most commonly investigated.

342 343

Vitamin C and E

344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360

Vitamin C is a water-soluble antioxidant, capable of reacting with numerous ROS and NO derivatives [126] including O2●─ [127] and ONOO─-derived radicals [53, 128]. Vitamin C also assists in recycling of cellular vitamin E [129]. Although vitamin C does not react appreciably with H2O2 [53], its physiologically relevant reaction with O2●─ [126] might indirectly affect H2O2-based redox signalling through its competition with SODs for O2●─. Furthermore, given (a) the high second-order reaction rates of vitamin C with numerous ROS [126]; and (b) the fact that chronic oral vitamin C supplementation can augment vitamin C concentration in skeletal muscle [130, 131], vitamin C supplementation may have effects on redox signalling pathways in skeletal muscle. Vitamin C can also denitrosylate S-nitrosylated proteins via copper-dependent and copper-independent mechanisms, at least in vitro [132]. Vitamin C treatment was shown to denitrosylate RyR1 in SR membranes of transgenic mice (RyR1Y522S/wt) who express leaky RyR1 channels and malignant hypothermia [133]. Vitamin C treatment prior to muscle activation was also shown to partially attenuate a ONOO─induced maximal force decrement in skinned rodent soleus muscle fibres in situ, suggesting attenuation of S-nitrosylation by vitamin C [134, 135]. However, the in vivo physiological relevance of vitamin C-based denitrosylation has been questioned due to likely metal ion chelation in vivo and non-physiological high levels of vitamin C required for the copper9

361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379

independent mechanism to proceed [53]. Unfortunately, there is a paucity of studies specifically investigating effects of chronic vitamin C supplementation in isolation of other antioxidants on force production during exercise in healthy individuals. Moreover, given that it might take up to seven days of supplementation with high dose oral vitamin C (1g/day) to significantly increase muscle vitamin C concentrations [131], it is unclear what effect an acute dose would have in muscle. Acute vitamin C infusion (2g) was shown to improve knee extensor fatigue resistance during repetitive exhaustive knee extensions in patients with chronic obstructive pulmonary disease (COPD), as demonstrated by improved maintenance of maximal and magnetic femoral nerve stimulated knee extensor force production [136]. However, acute intake of oral vitamin C (2 x 500mg, in combination with vitamin E and α-lipoic acid) could not recapitulate these effects in the same population group [137]. The effect of oral vitamin C supplementation on muscle force recovery following a bout of intense fatiguing exercise has been investigated in several studies. An improved rate of contractile force recovery would be theoretically beneficial to athletes when recovery periods between exercise or competition bouts are limited. At least two studies [138, 139] demonstrated a more rapid recovery of maximal force production after chronic vitamin C supplementation when compared to placebo. On the other hand, other studies involving chronic vitamin C supplementation found either no effect [43, 140, 141] or impaired [44] maximal muscle force recovery after an intense bout of exercise.

380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403

Vitamin E is a primary lipid-soluble chain-breaking antioxidant in tissues and cell membranes [46, 142]. Importantly, vitamin E is capable of directly scavenging lipid-derived peroxyl radicals in vivo [13, 143]. However, similar to vitamin C, vitamin E appears to react relatively poorly with H2O2 and NO in physiologically relevant environments [53]. It is unclear how Vitamin E might affect redox signalling in muscle, however cell signalling pathways might be regulated by changes in lipid peroxidation in bioactive lipids of cell membranes [143]. Further, vitamin E appears to affect gene expression [46, 144]. How these changes in gene expression integrate into redox signalling pathways is unclear however. Coombes et al. [145] found combined vitamin E and α-lipoic acid supplementation in rodents to impair low stimulation frequency (but not high frequency) muscle contractile force in unfatigued, but not fatigued skeletal muscle in situ. Antioxidant supplementation also improved lipid peroxidation measures in muscle. It was established in a subsequent in vitro experiment that the impairment in force production was primarily mediated by vitamin E supplementation [145]. This finding lends support to earlier in vitro and in situ studies discussed earlier [45, 92, 94, 120], in that unfatigued muscle will have impaired submaximal force production when redox state is more reduced after treatment with an antioxidant. However, in contrast to some other study findings of antioxidant treatments [45, 92, 94, 95], vitamin E was unable to improve low frequency contraction force during (or after) fatiguing contractions. There is some evidence in humans that chronic vitamin E supplementation increases muscle α-tocopherol concentration and tends to reduce levels of conjugated dienes (a markers of lipid peroxidation) in muscle post intense eccentric exercise [146]. Studies investigating effects of vitamin E supplementation on muscle contraction force during contraction/exercise in humans are lacking. On the other hand, some studies have investigated effects of vitamin E on recovery of muscle contraction force following fatiguing 10

404 405

exercise in humans. Overall, these studies are not supportive of any benefit of vitamin E supplementation on rate of recovery of muscle contraction force [138, 147-149].

406 407 408 409 410 411 412 413 414 415 416 417 418 419 420

A combination of both vitamin C (500 mg/day) and vitamin E (1200 IU/day) was found to enhance the rate of recovery of maximal knee extensor voluntary isometric contraction force after intense repetitive fatiguing eccentric knee extension exercises in humans [150]. In contrast, another study in young healthy adults found that combined vitamin C (1g/day) and vitamin E (260 IU/day) did not alter maximal voluntary knee extensor force recovery following an acute exercise bout [35]. In addition, they found that the combined supplementation with vitamin C and vitamin E supplementation impaired maximal strength development during 10 weeks of resistance training in the biceps muscle group and attenuated exercise-induced activation of p70S6k and MAP kinases p38 MAPK and ERK1/2 in skeletal muscle [35]. Other recent studies involving combined vitamin C and vitamin E supplementation and resistance training in young and elderly individuals over 3-6 months, have observed no impairment in strength performance of any measured muscle group following supplementation [38, 151, 152]. Overall, effects of vitamin C and vitamin E supplementation on skeletal muscle contractile function and force production in humans are equivocal.

421 422

NAC

423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445

NAC is a reduced thiol donor and redox modulator that has been shown to enhance availability of cysteine and GSH in muscle [153, 154]. Direct reactions of NAC with H2O2 and O2●─ are likely inefficient under physiological conditions due to competition for these ROS with endogenous antioxidants [155, 156]. Therefore, redox modulation of skeletal muscle contraction by NAC might relate to its capacity to replenish depleted intracellular GSH levels (which can occur during prolonged exhaustive exercise [157, 158]); establish a less oxidized cellular redox state; maintain Na2+/K+ pump activity [153]; and to reduce disulphide bonds [159] and promote denitrosylation and deglutathionylation [133, 160]. It should be noted that GSH synthesis is subject to feedback inhibition based on GSH concentration [161]. Thus, effects of NAC relating to increased provision of GSH might be limited if GSH levels are not impaired. Intravenous supplementation of NAC in humans has been shown to delay fatigue and maintain higher muscle force/power production during sustained fatiguing low frequency contractions and prolonged submaximal exercise followed by a maximal effort bout [45, 153, 154, 162]. Orally-administered NAC enhanced knee extensor endurance performance during repetitive sub-maximal contractions in patients with chronic obstructive pulmonary disease [163], delayed fatigue during repetitive submaximal isometric hand grip exercise in healthy adults [164] and improved the maintenance of respiratory muscle strength during discontinuous sustained high intensity (85% VO 2 max) cycling in health males [165]. In contrast to submaximal exercise, NAC supplementation does not appear to attenuate fatigue at very high/supra-maximal intensities [45, 46, 166]. Corn & Barstow [167] found maintenance of power output and time to fatigue during cycling to be improved at 80% peak power output, but not at 90, 100 or 110% peak power outputs in young males. While factors such as training status, NAC dose and 11

446 447 448 449 450 451 452

supplementation duration might complicate direct study comparisons [168], findings appear to be consistent with the notion that ROS mediate muscle contractility and promote fatigue during sustained fatiguing submaximal contractions, but have limited effects on muscle contraction at maximal/supra-maximal intensities [45, 46, 120]. The amelioration of submaximal force/power loss by NAC during fatiguing exercise suggests NAC supplementation establishes a more optimal redox state for muscle contractile function during sustained oxidative stress.

453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483

Study findings therefore appear to implicate use of NAC supplementation prior to and/or during competition events that are sustained and fatiguing to help maintain muscle contractile force/power production and attenuate fatigue. However, a number of factors might limit its practical efficacy. According to the 2015 World Anti-Doping Agency Prohibited List, methods of intravenous infusion >50ml per 6h period are banned, therefore restricting intravenous NAC use in athletes. There is also a relative lack of studies that have used performance tests directly relevant to athletic competition such as time trials or variable and intermittent intensity physical bouts to investigate the effects of oral NAC supplementation. One study [169] found trained cyclists who consumed five moderately high oral doses of NAC (100mg/kg body mass per dose) over 48 h had a 4.9% impairment in power output during a 10-minute self-paced cycling bout following interval cycling exercise (6 x 5 mins at ~80% peak power output). In contrast, a recent study by Slattery et al. [170] used a lower chronic loading NAC dose (2 x 600mg per day for 9 days) prior to exercise and reported an improvement in mean power output during a prolonged simulated cycle ergometer race involving fatiguing repetitive maximal sprints in well trained triathletes. Furthermore, a recent placebo-controlled study in recreationally trained males [98] found performance in a high intensity maximal exercise bout undertaken following a 60 minute bout of intermittent exercise to significantly improve during a 6 day period of supplementation with 2 x 50 mg/kg NAC per day. Given these contrasting findings and methodological variations in NAC dose and performance tests used, findings remain equivocal in relation to performance benefits of NAC in tasks representative of athletic events. Moreover, no redox measures in muscle were measured in these studies, thus limiting insights into redox effects of NAC specifically in skeletal muscle. Potential side effects of oral NAC ingestion are dose-related [171, 172] and mostly include gastrointestinal disturbances and bad taste [98, 171]; although anaphylactoid reactions have been reported rarely [173]. Such adverse effects might interfere with exercise performance and could be exacerbated by exercise [172]. Findings of Ferreira et al [172] implicate an acute oral dose of 70mg/kg body mass as a threshold of intake to avoid adverse effects in individuals undertaking fatiguing exercise. Future investigations should therefore focus on studies using representative performance tests of athletic events, whilst limiting acute oral intakes to 70mg NAC/kg body mass [172].

484 485

Coenzyme Q10

486 487

Coenzyme Q10 is a lipid-soluble vitamin-like quinone with vital functional roles in energy metabolism and mitochondrial oxidative phosphorylation [174]. It also possesses 12

488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530

antioxidant properties and can assist in recycling of other cellular antioxidants [174, 175]. Improvements in some aspects of exercise performance, including maintenance of maximal cycling velocity during fatiguing exercise in healthy individuals [174] and attainment of a higher cycling power output at fatigue in trained cyclists [176] have been reported. Another study [177] found a small but significant improvement in cycling peak power output in elite athletes after coenzyme Q10 supplementation when compared to placebo supplementation, however the study lacked stringent control of diet and extraneous lifestyle factors in order to mimic real-world circumstances of athletes. A few studies reported no effect of coenzyme Q10 supplementation on muscle function [178-180]. Conversely, impairments in time to exhaustion of a maximal cycling bout following prolonged submaximal cycling [181] and decreased mean power output during repetitive high intensity sprints [182] were found following coenzyme Q10 supplementation. It is difficult to clearly establish reasons for the different findings of these studies. However, study heterogeneity is a likely complicating factor. Studies employed different dosage regimens (ranging from 100mg/day to 300mg/day for 8 days to 6 months), used different training regimens, included untrained and/or trained athletes and included different measures of performance. No clear pattern in relation to dose, performance measure or training status is obvious from findings. Given the focus of this review is skeletal muscle, it must be noted that it is difficult to clearly reconcile beneficial effects of coenzyme Q10 supplementation on skeletal muscle contractile function, given that supplementation appears ineffective at enhancing total muscle and muscle mitochondrial coenzyme Q10 concentrations in humans [180, 182-184]. Nevertheless, additional well controlled dosetitrated studies in athletes are required to better evaluate the potential of coenzyme Q10 as an ergogenic supplement.

B) REDOX SIGNALLING IN MITOCHONDRIAL BIOGENESIS AND ANTIOXIDANT ENZYME INDUCTION Major beneficial adaptions of skeletal muscle to endurance training include increased mitochondrial content and improved antioxidant defenses. Furthermore, the content of mitochondria within the skeletal muscle cell at any one time is a balance between its degradation via mitophagy and mitochondrial biogenesis (synthesis) [185]. During contraction there is an increase in several stress signals in skeletal muscle that are responsible, at least in part, for the initial activation of mitochondrial biogenesis after exercise. These molecular signals include increased ROS [186], but also elevated levels of AMP [187, 188], cytosolic Ca2+ [187, 189] and possibly NAD+ [190]. The increase in ROS during contraction is known to activate several redox-sensitive kinases including AMPK, activating transcription factor-2 (ATF-2), the MAP kinases p38 MAPK, JNK and ERK1/2 [186, 191-193] and the transcription factor nuclear factor B (NF B) [194]. Importantly, AMPK, and the MAP kinases have all been implicated in the regulation of mitochondrial biogenesis [195, 196] at least partly through peroxisome proliferatoractivated receptor (PPAR)- coactivator-1 (PGC-1 ), a key regulator of mitochondrial 13

531 532 533 534 535 536 537 538 539 540 541

biogenesis [197, 198]. Indeed, we have shown that exacerbating skeletal muscle oxidative stress during exercise by depleting endogenous antioxidant glutathione content results in a higher PGC-1 mRNA response following exercise [199]. Key steps in mitochondrial biogenesis following an acute bout of exercise, also involve PGC-1 coactivating nuclear respiratory factor (NRF) -1 and NRF-2, and increasing their skeletal muscle DNA binding [200-202]. These transcription factors then activate genes encoding oxidative enzymes (i.e. cytochrome oxidase, COX) as well as mitochondrial transcription factor A (TFAM), which activates mitochondrial DNA transcription, thus increasing mitochondrial synthesis. Furthermore, this activation of mitochondrial biogenesis is involved in the regulation of antioxidant enzymes, since PGC-1 is required for the induction of SOD, glutathione peroxidase (GPx) and catalase [203], with NF B also regulating SOD2 transcription [194].

542 543 544 545 546 547 548

NO increases skeletal muscle mitochondrial biogenesis [187, 188, 204] and this is mediated at least partly by AMPK [187, 188] and perhaps Ca2+ [187]. We have previously used pharmacological [205] and also gene knockout [206] approaches to inhibit NOS and thus NO production during exercise in rodents. Neither of these aforementioned approaches prevents the increases in markers of mitochondrial biogenesis following acute exercise [205] or training [206]. Therefore, NO appears to play a role in the regulation of skeletal muscle mitochondrial biogenesis under basal (non-contraction) but not exercise conditions.

549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565

The role of ROS in the signaling pathways that activate mitochondrial biogenesis following muscle contraction has yet to be fully defined. However, there is strong evidence to implicate ROS in the activation of mitochondrial biogenesis following skeletal muscle contraction via p38 MAPK. Recent work from David Hoods laboratory show that NAC treatment of muscle cells in vitro prevents the contractile activity induced increase in p38 MAPK phosphorylation and the promoter activity and co-activation activity of PGC-1 [207]. However, although ROS activates AMPK [186, 192] it remains unclear if the ROS produced during muscle contraction is required by AMPK to increase mitochondrial biogenesis [207]. Furthermore, evidence suggests there is considerable redundancy within these signaling pathways to compensate for inhibition of a single stress signal. For example, we [193] and others [191, 208] have found in rodents that p38 MAPK phosphorylation in skeletal muscle is blocked during acute exercise with the xanthine oxidase inhibitor, allopurinol, along with the attenuation of mitochondrial biogenesis markers such as TFAM mRNA [193] and protein abundance of TFAM and PGC-1 [208]. However, the inhibition of these cellular processes with allopurinol following acute exercise is not sufficient to blunt the increase in mitochondria proteins and antioxidant enzymes following several weeks of endurance training when combined with allopurinol treatment (see Fig. 3).

566 567 568

MITOCHONDRIAL BIOGENESIS, ANTIOXIDANT ENZYME INDUCTION AND THE EFFECTS OF ANTIOXIDANTS

569 570 571

Research on the effects of antioxidant supplementation on mitochondrial biogenesis and antioxidant defences has used vitamins C and E (alone or in combination), coenzyme Q10, NAC, resveratrol, allopurinol, -carotene and α-lipoic acid in rats [31, 32, 189, 199, 206, 20914

572 573

212] and humans [33, 49, 50, 125, 213-215]. The following section will discuss the impact of the most commonly researched antioxidants, with a particular focus on human research.

574 575

Vitamin C and E

576 577 578 579 580 581 582 583 584

The most prevalent vitamin supplements are vitamins C and E, with ~20% of the population reported to use them [216]. With regards to the antioxidant effects of vitamin C and E in humans, Yfanti et al have convincingly shown that there are no negative effects using doses of vitamin C (500 mg/day) and E (400 IU/day) on the adaptive responses of skeletal muscle to endurance training, such as increased mitochondrial proteins and antioxidant enzymes [50, 51, 217]. The findings in rodents are mixed, with several studies reporting vitamin C or E alone, or in combination attenuate skeletal muscle markers of mitochondrial biogenesis or antioxidant enzymes following endurance training [31, 210], whilst others report no attenuation of the endurance training response [32, 209, 218].

585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607

However, there are several human studies that have reported blunting of skeletal muscle adaptations to endurance training using doses of 1g/day vitamin C in combination with vitamin E [33, 48, 49]. Ristow et al. [49] were the first to report 1g/day vitamin C in combination with 400 IU/day vitamin E in humans attenuated mRNA responses in several markers of mitochondrial biogenesis and antioxidant enzymes following endurance training. Paulsen et al. [33] have since observed in humans that following training, the increase in COX IV protein abundance and the cytosolic (but not whole cell) levels of PGC-1α are attenuated with antioxidant 1g/day vitamin C and 260 IU/day E supplementation. Recently, our group has also found that 1g/day vitamin C in combination with 400 IU/day vitamin E in humans attenuates the increase in skeletal muscle TFAM protein abundance and SOD enzyme activity [48]. However, there were other skeletal muscle adaptations that were not attenuated, such as increased citrate synthase activity [48], which strongly reflect mitochondrial content levels [219]. Therefore, collectively, there is now strong evidence that 1g/day vitamin C in combination with vitamin 400 IU/d E in humans hampers some, but not all of the skeletal muscle adaptations to endurance training. However, much of the blunting effect of the antioxidants from these studies also appears related to the higher dose of 1g/day vitamin C, rather than any effect of the vitamin E in these studies [33, 48-51, 217]. Therefore, further studies are now required to confirm if this hampering of some training adaptations is also seen with 1g/day vitamin C along. Importantly, there is also no evidence from these studies that combined 1g/day vitamin C and 400 IU/day E supplementation prevents improvements in VO2max or endurance performance in humans, despite the potential for some cellular adaptations involved in mitochondrial biogenesis and antioxidant defences to be impaired.

608 609

NAC

610 611

NAC is a strong scavenger of ROS and prevents PGC-1 promoter and co-activation activity following contractile activity in skeletal muscle in vitro [207]. However, there is currently 15

612 613 614 615 616 617 618 619

limited evidence available that suggests NAC hampers skeletal muscle adaptations to endurance training in humans. Infusion of NAC during acute endurance exercise in humans does attenuate skeletal muscle JNK phosphorylation, but not phosphorylation of p38 MAPK, ERK1/2 or AMPK [220]. However, the increase in mRNA levels of SOD2, but not PGC-1α are attenuated in skeletal muscle by NAC infusion during acute exercise [28, 220]. The impact of NAC on skeletal muscle training adaptations, particularly using oral supplementation has yet to be examined. Therefore, further human exercise studies are needed to examine if NAC supplementation hampers skeletal muscle adaptations to endurance exercise in humans.

620 621

Resveratrol

622 623

Resveratrol is a polyphenol that has antioxidant properties [221, 222] and is also known to activate sirtuin1 (SIRT1) and AMPK [221]. Because of its purported ability to stimulate SIRT1 and AMPK [221] it has been shown in rodents to act via this pathway to increase PGC-1 and thus skeletal muscle mitochondrial biogenesis [211, 212]. However, supplementing healthy humans with resveratrol (250mg/day) under basal (non-exercised) conditions for 8 weeks does not increase phosphorylation of the stress signalling kinases AMPK, p38 MAPK, JNK or alter PGC-1 mRNA levels in skeletal muscle [215]. Furthermore, supplementing even higher doses (3 x 500mg/day) to obese males also does not activate skeletal muscle AMPK or increase PGC-1 mRNA levels [223].

624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651

Several rodent studies have investigated the impact of resveratrol supplementation during endurance training. The impact of resveratrol on training adaptations such as increased mitochondrial biogenesis, antioxidant defences and endurance performance are mixed in rodent studies and are thoroughly discussed by Mankowski et al. [224]. However, in humans, there is no effect of resveratrol supplementation (150-250 mg/day) during endurance training on the increased skeletal muscle abundance or enzyme activity of mitochondrial proteins or antioxidant enzymes [214, 215]. Thus, increased skeletal muscle mitochondrial content following training doesn’t appear hampered by resveratrol. However, the impact of resveratrol at the level of gene expression in skeletal muscle and at the wholebody performance level is equivocal. In young (22 year old) [214], but not older (65 year old) [215] males, resveratrol prevents increases in gene expression of PGC-1 , SIRT1 and SOD2 following four weeks of endurance training. Given these findings don’t appear to translate to attenuated muscle oxidative capacity [214, 215], the significance of these outcomes at the level of the whole-muscle is probably minor. In addition, the impact of resveratrol at the whole body level in terms of exercise performance is also equivocal in humans. The increase in maximal oxygen uptake following endurance training has been reported to be blunted by resveratrol in old [225], but not young males [214]. Thus, despite some conflicting reports of attenuation of skeletal muscle gene expression and performance, it doesn’t appear that increases in mitochondrial content are hampered by resveratrol, although further exercise studies in humans are required to confirm this and clarify its impact on endurance performance.

16

652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683

Potential reasons for interspecies discrepancies of resveratrol supplementation study findings may include dosing differences in human vs rodent studies [226] and/or a relatively poor bioavailability of resveratrol in humans, particularly after oral administration [227, 228]. While the majority of orally ingested resveratrol is absorbed by humans, it is extensively metabolized through processes of sulfation and glucuronidation in intestinal cells and the liver [229]. Thus, only a small proportion of the resveratrol parent compound appears in peripheral plasma [230]. Concentrations reached in animal models/animal studies may therefore exceed the level attainable in humans/human studies, subsequently resulting in attenuated biological effects of resveratrol supplementation in humans. Limitations of the current research in humans include use of generally low-moderate dose supplementation only (2g trans-resveratrol/day) supplementation yields higher plasma concentrations, but is associated with a potentially increased risk of adverse effects [231]. Secondly, the metabolites of resveratrol metabolism, which accumulate at much higher levels in plasma than resveratrol itself [232], might produce meaningful in vivo biological effects that have not been well investigated [230]. Thirdly, effects of resveratrol supplementation on skeletal muscle concentrations of resveratrol metabolites have not been measured in humans. Data from rodent studies suggest that resveratrol metabolites only accumulate to relatively low levels in skeletal muscle compared with liver and adipose tissue; albeit in a dose-dependent manner [230]. Thus, whether dosing of resveratrol at safe levels is capable of sufficiently increasing levels in human skeletal muscle and modulating local redox signalling pathways during muscle contraction and exercise remains to established. In addition to activation of SIRT1 and AMPK [221, 233-235], resveratrol has been shown to induce antioxidant enzyme activities and improve oxidative stress in rodent skeletal muscle [236]. Findings in young adult mice [236] who undertook repeated fatiguing maximal isometric contractions with resveratrol supplementation (156mg/kg/day for 10 days) show an attenuated exerciseinduced activation of xanthine oxidase, improved oxidative stress (increased GSH/GSSG), increased activity of SOD2 and improved maintenance of muscle contractile force during exercise. However, it remains to be confirmed whether such findings are also reproducible in skeletal muscle of human subjects. Considering the above limitations, more research is required before any convincing conclusions can be made in relation to effects of resveratrol supplementation on human skeletal muscle performance enhancement.

684 685 686 687 688 689 690 691 692 693 694

C) REDOX SIGNALLING IN GLUCOSE UPTAKE DURING EXERCISE As outlined in previous reviews [27, 237, 238], there is evidence that ROS and NO play a role in skeletal muscle glucose uptake during contraction and exercise. It is naturally difficult to discern if the observed effects are due to NO, ROS or a combination of both given that these entities are so closely interrelated. Indeed, in mouse skeletal muscle bundles, NO donors, the NOS substrate L-arginine and the ROS H2O2 each individually increase net oxidant activity [239]. Furthermore, anti- ROS enzymes, NOS inhibitors and NO scavengers each individually decrease net oxidant activity [239]. ROS and NO signalling are simultaneously 17

695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717

active in the cytosol of muscle fibres [239] and ONOO─ is increased during contraction of cells [8]. Indeed, Merry et al. [240] found that NOS inhibition and NAC both separately attenuated the increase of glucose uptake in isolated skeletal muscle during ex vivo contraction with no additive effect when both agents were applied together. This suggests that ONOO─ is playing a role but it should be kept in mind that when contractions are of a high intensity, such as during ex vivo rodent muscle contractions, large amounts of ROS and NO are produced [7, 27]. However, during more physiological situations such as rat in situ contractions [241] and during exercise at ~60% VO2 max in humans [242] there is no effect of NAC on skeletal muscle glucose uptake. The amount of ROS produced during moderate intensity exercise in humans is not large and any effect of antioxidants on glucose uptake is likely only minimal. Therefore, exercise studies using higher intensities need to be conducted. It should also be considered that as exercise intensity increases the reliance on muscle glycogen is greatly increased compared with the reliance on blood glucose. That is, even though the use of both muscle glycogen and blood glucose increase with exercise intensity, the relative contribution of glucose is reduced [243]. Therefore, if antioxidants did reduced glucose uptake during intense exercise the absolute effect on carbohydrate oxidation would likely be small. Given that any potential effect of antioxidants during exercise would be a reduction in skeletal muscle glucose uptake, not an increase, antioxidants would be unlikely to be ergogenic in relation to their effects on glucose uptake. In theory, increasing skeletal muscle ROS levels during contraction above the normal increase with contraction could increase skeletal muscle glucose uptake during exercise [192]; but as mentioned earlier, this may also have detrimental effects on muscle contraction.

718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736

In contrast to the limited research on ROS and glucose uptake, more research has been undertaken in regards to NO and skeletal muscle glucose uptake during exercise. On balance, studies have shown that NOS inhibition attenuates increases in skeletal muscle glucose uptake during contraction and during exercise [237, 238]. Unlike NAC infusion, where no effects on glucose uptake during human exercise were observed [242], infusion of a NOS inhibitor attenuates the normal increases in skeletal muscle glucose uptake during exercise at 60% VO2 max in healthy individuals [244]. Even greater attenuations in glucose uptake during exercise with NOS inhibition were observed in people with type 2 diabetes [245]. However, studies that attempted to increase glucose uptake during contractions with infusion of the NOS substrate L-arginine [246] provided no evidence of beneficial effects on glucose uptake or exercise performance during exercise in humans. Moreover, other studies have been unable to detect any effect of NOS inhibition on muscle glucose uptake during contraction [247, 248], including our own recent studies with in situ contraction in SpragueDawley rats [249]. There appear to be strain differences between hooded Wistar and Sprague-Dawley rats that may explain these rat study discrepancies [249] Given that nNOSµ is considered the main isoform of NOS activated during skeletal muscle contraction [250], we were surprised that skeletal muscle glucose uptake was normal during ex vivo contraction in nNOSµ knockout mouse muscle [251] and that NOS inhibitors attenuated skeletal muscle glucose uptake normally in nNOSµ knockout mouse muscle. We are

18

737 738

currently examining if other isoforms of nNOS in addition to nNOSµ may be important in the regulation of muscle metabolism [251, 252].

739 740 741 742

Taken together, there is evidence that both ROS and NO play a role in skeletal muscle glucose uptake but these findings are not universal. In addition, the mechanisms by which NO [240, 250] and ROS [192, 253] regulate skeletal muscle glucose uptake during exercise are unclear and any role of antioxidants is unlikely to improve glucose uptake.

743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764

D) REDOX SIGNALLING IN SKELETAL MUSCLE HYPERTROPHY

765 766 767 768 769 770 771 772

The potentially important role of ROS in IGF-1-mediated muscle hypertrophy was shown in a recent investigation in vitro. Exogenous ROS treatment and endogenous ROS production via NADPH oxidases increased IGF-1-induced tyrosine phosphorylation of the IGF-I receptor in C2C12 myocytes [255]. Increased IGF-I promoted increased phosphorylation of Akt, mTOR, p70S6k and ERK1/2; while NAC treatment reversed these changes in C2C12 myocytes. In addition, IGF-1 increased C2C12 myocyte hypertrophy, while antioxidant treatments reversed this effect [255]. Thus, ROS appear to be important for skeletal muscle hypertrophy mediated via IGF-1 signalling, at least in vitro.

773 774 775 776 777

Several studies in rodents that have investigated the relationship between NO and skeletal muscle hypertrophy utilized synergistic ablation of the gastrocnemius and soleus muscles to cause compensatory muscle hypertrophy of the plantaris muscle [37, 58, 60, 265, 266]. Although this represents a rather extreme model of overload-induced muscle hypertrophy, it may nonetheless help to identify NO-mediated effects on skeletal muscle hypertrophy.

Skeletal muscle hypertrophy involves mechanically- and chemically-transduced cellular and molecular responses in muscle fibres and satellite cells through load-induced disturbances in skeletal muscle integrity [254]. Skeletal muscle hypertrophy essentially reflects a net increase in protein synthesis relative to protein degradation, and involves the integration of numerous anabolic and catabolic signalling pathways. Among the pathways promoting protein synthesis is the insulin/IGF-1-PI3K signalling pathway, which involves activation of kinases Akt and mammalian target of rapamycin (mTOR) and their downstream effectors including ribosomal protein S6 kinase (p70S6k) and eukaryotic translation initiation factor 4E (eIF4E) [255] MAP kinase (p38, ERK 1/2) signalling and Ca2+/calmodulin signalling have also been implicated as important pathways in the regulation of skeletal muscle hypertrophy [254, 256, 257]. Currently there is a paucity of data in relation to the importance of resistance exercise-induced ROS/NO production [258, 259] for muscle hypertrophic growth in either young or elderly human populations. Elevated levels of oxidative stress and ROS/NO in skeletal muscle has been associated with muscle atrophy and sarcopenia in sedentary rodents and humans [260-264], suggesting that excessive muscle ROS/NO levels are detrimental to the maintenance of skeletal muscle size. However, recent studies also indicate that ROS/NO might mediate important signalling pathways involved in skeletal muscle hypertrophy [60, 255, 265] (see Fig. 4).

19

778 779 780 781 782 783 784 785 786 787 788

Some of these studies have investigated the impact of NO signalling on muscle hypertrophy using NOS inhibitor compounds. Two studies treated rodents with N G-nitro-l-arginine methyl ester (L-NAME), an inhibitor of NOS, followed by synergistic ablation [60, 265]. After 12-14 days of overload in these studies, plantaris muscle mass was found to be decreased by ~50% after L-NAME treatment. Despite the impaired hypertrophic response to L-NAME treatment, Sellman et al. [265] found that the overload-induced increase in active satellite cells was unaffected by L-NAME treatment. Sellman et al. [265] additionally treated rodents with 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM), a specific blocker of nNOS activity. After five days of overload, plantaris hypertrophy was significantly increased with TRIM treatment compared with non-loading; although 12-day overload measurements were not taken to better evaluate the effect of nNOS blockade on muscle hypertrophy [265].

789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808

To better investigate the role of nNOS on the regulation of muscle hypertrophy, Ito et al. [58] undertook synergistic ablation in nNOS null (nNOS-/-) rodents. nNOS null mice experienced an attenuated gain in plantaris mass (24% vs. 42%) and muscle fibre crosssectional area when compared to wild type mice after 7 days of overload, but no differences in overload-induced satellite cell activation. Skeletal muscle NOS activity increased more than two-fold 3 minutes after the onset of overload in wild type but not in nNOS null mice. Ito et al. further implicated NADPH-generated O2●─ and subsequent ONOO─ produced as likely pivotal in the hypertrophic response [58]. Administration of ONOO─ scavengers to wild type mice attenuated plantaris hypertrophy in response to overload, while administration of ONOO─ donors promoted a normal hypertrophic responses to overload in nNOS null mice. Synergistic ablation also activated mTOR signalling and increased phosphorylation of p70S6k (at Thr389) in wild type mice 3 minutes after initiation of overload, although at no time point after 1 day [58]. This response was absent in nNOS null mice and mice treated with ONOO scavengers. Through a series of insightful experiments, Ito et al. identified Ca 2+-mediated activation of mTOR via transient receptor potential cation channel V1 (TRPV1) as a likely mechanism of NO/ONOO─-induced muscle hypertrophy [58]. This mechanism appears to be independent of the insulin/IGF-1-P13K pathway, since IGF-1-induced phosphorylation of Akt and p70S6k was unaffected in nNOS null and TRPV1 null mice. Moreover, treatment with NOS inhibitors L-NAME or TRIM was found to increase IGF-1 mRNA and relative p70S6k phosphorylation after synergistic ablation, despite impaired muscle hypertrophy [265].

809 810

EFFECTS OF ANTIOXIDANTS ON SKELETAL MUSCLE HYPERTROPHY

811

Vitamin C and E

812 813 814 815 816 817 818

In a study investigating effects of oral vitamin C supplementation (500mg/kg for 14 days) on skeletal muscle hypertrophy in rodents [37], it was found that vitamin C supplementation decreased plantaris hypertrophy by approximately 11% after synergistic ablation. Despite these findings, vitamin C supplementation did not significantly affect muscle redox status or phosphorylated levels of p70S6k, ERK1/2, Murf or atrogin-1 following ablation when compared with placebo. However, when compared with the sham treatment group, the placebo but not antioxidant group had significantly elevated phosphorylation of p70 S6k and 20

819 820 821

ERK1/2 and decreased levels of atrogin-1 [37]. Therefore, impairments in muscle hypertrophy due to vitamin C supplementation may be reflected in relatively attenuated changes in both anabolic and catabolic signalling pathways.

822 823 824 825 826 827 828 829 830 831 832 833 834 835 836

Recent human studies have demonstrated mixed findings in relation to alterations in skeletal muscle hypertrophy following a resistance exercise training regimen combined with antioxidant supplementation. BjØrnsen et al. [38] investigated effects of combined vitamin C (500mg/day) and vitamin E (175 IU/day) supplementation during 12 weeks of resistance exercise training on lean muscle mass and muscle thickness in elderly males. Antioxidant supplementation attenuated the gains in total lean mass, leg lean mass and rectus femoris thickness when compared with placebo. Other body segment masses and muscle thicknesses did not vary between groups after training [38]. In contrast, Bobeuf et al. [151] observed significant increases in lean mass in elderly individuals after 6 months of resistance training only when training was combined with vitamin C (1g/day) and vitamin E (400 IU/day) supplementation. However, in the same study, antioxidant supplementation had no effect on systemic markers of oxidative stress, despite improving systemic antioxidant levels [151]. It should be re-emphasized to the readers that the interpretation of results related to ROS/antioxidants in ageing or elderly subjects may not be representative of an athletic population, and therefore translation into advice for elite athletes is difficult.

837 838 839 840 841 842 843 844 845 846 847 848 849 850

Paulsen et al. [35] reported no significant difference in the improvement in lean body mass accretion or muscle group cross-sectional areas in young healthy adults in response to supplementation with vitamin C (1g/day) and vitamin E (350 IU/day) during 10 weeks of resistance exercise training. Fractional protein synthetic rate was also unaltered with antioxidant supplementation following an acute exercise bout. Despite these findings, antioxidant supplementation attenuated the acute exercise-induced activation of p70S6k and MAP kinases p38 MAPK and ERK1/2. Furthermore, antioxidant supplementation attenuated the post-exercise increase in activation of the ubiquitin proteasome pathway observed in the placebo supplementation group. This latter finding is intriguing, given that it implies protein degradation might have been attenuated with antioxidant supplementation. The somewhat contrasting findings of Paulsen et al. [35] with rodent studies [37, 58, 266] investigating effects of antioxidants on overload-induced activation of kinases involved in protein synthesis and protein degradation suggests that redox-related signalling pathways in human skeletal muscle hypertrophy remain to be clearly established in future investigations.

851 852

APPLICATION TO ATHLETES

853 854 855 856 857 858 859

The efficacy of antioxidants (particularly NAC) for enhancement of athletic performance is probably most apparent through acute or short-term use prior to a competition event when skeletal muscle adaptation is not generally required [98]. Conversely, supplementation with antioxidants (particularly vitamin C and E) has been cautioned in athletes while undertaking chronic training cycles during which adaptations in skeletal muscle are desired [14]. The implication of these contrasting positions is to cycle between use and non-use of 21

860 861 862 863 864 865 866 867 868

antioxidants, depending on the stage of an athlete’s training and competition periodization cycle. Thus, use of antioxidants (particularly NAC) prior to and/or during a prolonged competition bout of fatiguing exercise may be advantageous in offsetting muscle force and power decrements and fatigue. On the other hand, use of antioxidant supplements (particularly vitamin C and/or E) during training periods involving a chronic overload of training volume and/or intensity, should be used sparingly if at all. Further research establishing different dose-related effects and different redox-modulating properties of specific antioxidant compounds will likely further strengthen this approach to antioxidant supplementation in order to optimize training and performance in athletes.

869 870

CONCLUSION

871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895

In this review, we have discussed some potentially important redox signalling pathways in skeletal muscle that are involved in acute and chronic adaptive responses to contraction and exercise. Furthermore, we have reviewed evidence investigating the impact of major exogenous antioxidants on these acute and chronic responses to exercise. The potential impact of these antioxidants on exercise responses is summarized in Table 1. A bulk of evidence suggests that NAC could be ergogenic through its effects on the maintenance of muscle force production during sustained fatiguing events. However, potential safety risks with higher intakes and a current lack of supportive evidence from studies using performance tests representative of typical athletic events currently warrants a conservative approach by athletes. Evidence also shows that high dose vitamin C (1 g) and E (≥260 IU) supplementation can impair some of the skeletal muscle adaptations to both endurance and resistance exercise training. Thus, while NAC might be beneficial acutely in relation to maintenance of redox state and improved muscle contraction force during a strenuous performance event, the prolonged supplementation of high doses of vitamin C and E during exercise training might promote a less oxidative redox state in muscle, thus facilitating hampered adaptive responses. Additional research is required to better establish effects of antioxidants such as α-lipoic acid, β-carotene and resveratrol on acute and chronic skeletal muscle responses to contraction and exercise. Future research should also focus on establishing a better understanding of mechanisms of action of specific antioxidants in vivo. We feel that this is critical to establishing the utility of antioxidant supplementation in athletes, since evidence suggests that different antioxidant compounds have different doserelated effects and different redox-modulating biological properties that may affect the optimum timing of their use by athletes looking to maximize their training and competition performance.

896 897 898

REFERENCES

22

899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949

[1] Alessio, H. M.; Goldfarb, A. H.; Cutler, R. G. MDA content increases in fast- and slow-twitch skeletal muscle with intensity of exercise in a rat. The American journal of physiology 255:C874-877; 1988. [2] Davies, K. J.; Quintanilha, A. T.; Brooks, G. A.; Packer, L. Free radicals and tissue damage produced by exercise. Biochemical and biophysical research communications 107:1198-1205; 1982. [3] Reid, M. B.; Haack, K. E.; Franchek, K. M.; Valberg, P. A.; Kobzik, L.; West, M. S. Reactive oxygen in skeletal muscle. I. Intracellular oxidant kinetics and fatigue in vitro. Journal of applied physiology (Bethesda, Md. : 1985) 73:1797-1804; 1992. [4] Reid, M. B.; Shoji, T.; Moody, M. R.; Entman, M. L. Reactive oxygen in skeletal muscle. II. Extracellular release of free radicals. Journal of applied physiology (Bethesda, Md. : 1985) 73:18051809; 1992. [5] Jackson, M. J.; Edwards, R. H.; Symons, M. C. Electron spin resonance studies of intact mammalian skeletal muscle. Biochimica et biophysica acta 847:185-190; 1985. [6] McArdle, A.; Pattwell, D.; Vasilaki, A.; Griffiths, R. D.; Jackson, M. J. Contractile activityinduced oxidative stress: cellular origin and adaptive responses. American journal of physiology. Cell physiology 280:C621-627; 2001. [7] Silveira, L. R.; Pereira-Da-Silva, L.; Juel, C.; Hellsten, Y. Formation of hydrogen peroxide and nitric oxide in rat skeletal muscle cells during contractions. Free radical biology & medicine 35:455464; 2003. [8] Pattwell, D. M.; McArdle, A.; Morgan, J. E.; Patridge, T. A.; Jackson, M. J. Release of reactive oxygen and nitrogen species from contracting skeletal muscle cells. Free radical biology & medicine 37:1064-1072; 2004. [9] Powers, S. K.; Nelson, W. B.; Hudson, M. B. Exercise-induced oxidative stress in humans: cause and consequences. Free radical biology & medicine 51:942-950; 2011. [10] Sakellariou, G. K.; Jackson, M. J.; Vasilaki, A. Redefining the major contributors to superoxide production in contracting skeletal muscle. The role of NAD(P)H oxidases. Free radical research 48:1229; 2014. [11] Mason, S.; Wadley, G. D. Skeletal muscle reactive oxygen species: a target of good cop/bad cop for exercise and disease. Redox report : communications in free radical research 19:97-106; 2014. [12] Sakellariou, G. K.; Vasilaki, A.; Palomero, J.; Kayani, A.; Zibrik, L.; McArdle, A.; Jackson, M. J. Studies of mitochondrial and nonmitochondrial sources implicate nicotinamide adenine dinucleotide phosphate oxidase(s) in the increased skeletal muscle superoxide generation that occurs during contractile activity. Antioxidants & redox signaling 18:603-621; 2013. [13] Forman, H. J.; Davies, K. J.; Ursini, F. How do nutritional antioxidants really work: nucleophilic tone and para-hormesis versus free radical scavenging in vivo. Free radical biology & medicine 66:24-35; 2014. [14] Gomez-Cabrera, M. C.; Salvador-Pascual, A.; Cabo, H.; Ferrando, B.; Vina, J. Redox modulation of mitochondriogenesis in exercise. Does antioxidant supplementation blunt the benefits of exercise training? Free radical biology & medicine 86:37-46; 2015. [15] Nordberg, J.; Arner, E. S. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free radical biology & medicine 31:1287-1312; 2001. [16] Vendrov, A. E.; Vendrov, K. C.; Smith, A.; Yuan, J.; Sumida, A.; Robidoux, J.; Runge, M. S.; Madamanchi, N. R. NOX4 NADPH Oxidase-Dependent Mitochondrial Oxidative Stress in AgingAssociated Cardiovascular Disease. Antioxidants & redox signaling; 2015. [17] Brown, D. I.; Griendling, K. K. Regulation of signal transduction by reactive oxygen species in the cardiovascular system. Circulation research 116:531-549; 2015. [18] Youn, J. Y.; Siu, K. L.; Lob, H. E.; Itani, H.; Harrison, D. G.; Cai, H. Role of vascular oxidative stress in obesity and metabolic syndrome. Diabetes 63:2344-2355; 2014. [19] Folli, F.; Corradi, D.; Fanti, P.; Davalli, A.; Paez, A.; Giaccari, A.; Perego, C.; Muscogiuri, G. The role of oxidative stress in the pathogenesis of type 2 diabetes mellitus micro- and macrovascular 23

950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000

complications: avenues for a mechanistic-based therapeutic approach. Current diabetes reviews 7:313-324; 2011. [20] Powers, S. K.; Talbert, E. E.; Adhihetty, P. J. Reactive oxygen and nitrogen species as intracellular signals in skeletal muscle. The Journal of physiology 589:2129-2138; 2011. [21] Biswas, S.; Chida, A. S.; Rahman, I. Redox modifications of protein–thiols: Emerging roles in cell signaling. Biochemical Pharmacology 71:551-564; 2006. [22] Sundaresan, M.; Yu, Z. X.; Ferrans, V. J.; Irani, K.; Finkel, T. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science (New York, N.Y.) 270:296-299; 1995. [23] Rhee, S. G.; Bae, Y. S.; Lee, S. R.; Kwon, J. Hydrogen peroxide: a key messenger that modulates protein phosphorylation through cysteine oxidation. Science's STKE : signal transduction knowledge environment 2000:pe1; 2000. [24] Hess, D. T.; Matsumoto, A.; Kim, S. O.; Marshall, H. E.; Stamler, J. S. Protein S-nitrosylation: purview and parameters. Nature reviews. Molecular cell biology 6:150-166; 2005. [25] Janssen-Heininger, Y. M.; Mossman, B. T.; Heintz, N. H.; Forman, H. J.; Kalyanaraman, B.; Finkel, T.; Stamler, J. S.; Rhee, S. G.; van der Vliet, A. Redox-based regulation of signal transduction: principles, pitfalls, and promises. Free radical biology & medicine 45:1-17; 2008. [26] Finkel, T. Signal transduction by reactive oxygen species. The Journal of cell biology 194:7-15; 2011. [27] Merry, T. L.; McConell, G. K. Do reactive oxygen species regulate skeletal muscle glucose uptake during contraction? Exercise and sport sciences reviews 40:102-105; 2012. [28] Trewin, A. J.; Lundell, L. S.; Perry, B. D.; Patil, K. V.; Chibalin, A. V.; Levinger, I.; McQuade, L. R.; Stepto, N. K. Effect of N-acetylcysteine infusion on exercise-induced modulation of insulin sensitivity and signaling pathways in human skeletal muscle. American journal of physiology. Endocrinology and metabolism 309:E388-397; 2015. [29] Alessio, H. M.; Goldfarb, A. H. Lipid peroxidation and scavenger enzymes during exercise: adaptive response to training. Journal of applied physiology (Bethesda, Md. : 1985) 64:1333-1336; 1988. [30] Vincent, H. K.; Powers, S. K.; Stewart, D. J.; Demirel, H. A.; Shanely, R. A.; Naito, H. Shortterm exercise training improves diaphragm antioxidant capacity and endurance. European journal of applied physiology 81:67-74; 2000. [31] Gomez-Cabrera, M. C.; Domenech, E.; Romagnoli, M.; Arduini, A.; Borras, C.; Pallardo, F. V.; Sastre, J.; Vina, J. Oral administration of vitamin C decreases muscle mitochondrial biogenesis and hampers training-induced adaptations in endurance performance. The American journal of clinical nutrition 87:142-149; 2008. [32] Strobel, N. A.; Peake, J. M.; Matsumoto, A.; Marsh, S. A.; Coombes, J. S.; Wadley, G. D. Antioxidant supplementation reduces skeletal muscle mitochondrial biogenesis. Medicine and science in sports and exercise 43:1017-1024; 2011. [33] Paulsen, G.; Cumming, K. T.; Holden, G.; Hallen, J.; Ronnestad, B. R.; Sveen, O.; Skaug, A.; Paur, I.; Bastani, N. E.; Ostgaard, H. N.; Buer, C.; Midttun, M.; Freuchen, F.; Wiig, H.; Ulseth, E. T.; Garthe, I.; Blomhoff, R.; Benestad, H. B.; Raastad, T. Vitamin C and E supplementation hampers cellular adaptation to endurance training in humans: a double-blind, randomised, controlled trial. The Journal of physiology 592:1887-1901; 2014. [34] Reid, M. B.; Moody, M. R. Dimethyl sulfoxide depresses skeletal muscle contractility. Journal of applied physiology (Bethesda, Md. : 1985) 76:2186-2190; 1994. [35] Paulsen, G.; Hamarsland, H.; Cumming, K. T.; Johansen, R. E.; Hulmi, J. J.; Borsheim, E.; Wiig, H.; Garthe, I.; Raastad, T. Vitamin C and E supplementation alters protein signalling after a strength training session, but not muscle growth during 10 weeks of training. The Journal of physiology 592:5391-5408; 2014. [36] Jackson, M. J. Redox regulation of adaptive responses in skeletal muscle to contractile activity. Free radical biology & medicine 47:1267-1275; 2009. 24

1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050

[37] Makanae, Y.; Kawada, S.; Sasaki, K.; Nakazato, K.; Ishii, N. Vitamin C administration attenuates overload-induced skeletal muscle hypertrophy in rats. Acta physiologica (Oxford, England) 208:57-65; 2013. [38] Bjornsen, T.; Salvesen, S.; Berntsen, S.; Hetlelid, K. J.; Stea, T. H.; Lohne-Seiler, H.; Rohde, G.; Haraldstad, K.; Raastad, T.; Kopp, U.; Haugeberg, G.; Mansoor, M. A.; Bastani, N. E.; Blomhoff, R.; Stolevik, S. B.; Seynnes, O. R.; Paulsen, G. Vitamin C and E supplementation blunts increases in total lean body mass in elderly men after strength training. Scandinavian journal of medicine & science in sports; 2015. [39] Braun, H.; Koehler, K.; Geyer, H.; Kleiner, J.; Mester, J.; Schanzer, W. Dietary supplement use among elite young German athletes. International journal of sport nutrition and exercise metabolism 19:97-109; 2009. [40] Petroczi, A.; Naughton, D. P.; Pearce, G.; Bailey, R.; Bloodworth, A.; McNamee, M. Nutritional supplement use by elite young UK athletes: fallacies of advice regarding efficacy. Journal of the International Society of Sports Nutrition 5:22; 2008. [41] He, F.; Hockemeyer, J. A.; Sedlock, D. Does combined antioxidant vitamin supplementation blunt repeated bout effect? International journal of sports medicine 36:407-413; 2015. [42] Bloomer, R. J.; Falvo, M. J.; Schilling, B. K.; Smith, W. A. Prior exercise and antioxidant supplementation: effect on oxidative stress and muscle injury. Journal of the International Society of Sports Nutrition 4:9; 2007. [43] Connolly, D. A.; Lauzon, C.; Agnew, J.; Dunn, M.; Reed, B. The effects of vitamin C supplementation on symptoms of delayed onset muscle soreness. The Journal of sports medicine and physical fitness 46:462-467; 2006. [44] Close, G. L.; Ashton, T.; Cable, T.; Doran, D.; Holloway, C.; McArdle, F.; MacLaren, D. P. Ascorbic acid supplementation does not attenuate post-exercise muscle soreness following muscledamaging exercise but may delay the recovery process. The British journal of nutrition 95:976-981; 2006. [45] Reid, M. B.; Stokic, D. S.; Koch, S. M.; Khawli, F. A.; Leis, A. A. N-acetylcysteine inhibits muscle fatigue in humans. The Journal of clinical investigation 94:2468-2474; 1994. [46] Powers, S. K.; Jackson, M. J. Exercise-induced oxidative stress: cellular mechanisms and impact on muscle force production. Physiological reviews 88:1243-1276; 2008. [47] Lamb, G. D.; Westerblad, H. Acute effects of reactive oxygen and nitrogen species on the contractile function of skeletal muscle. The Journal of physiology 589:2119-2127; 2011. [48] Morrison, D.; Hughes, J.; Della Gatta, P. A.; Mason, S.; Lamon, S.; Russell, A. P.; Wadley, G. D. Vitamin C and E supplementation prevents some of the cellular adaptations to endurance-training in humans. Free radical biology & medicine; 2015. [49] Ristow, M.; Zarse, K.; Oberbach, A.; Kloting, N.; Birringer, M.; Kiehntopf, M.; Stumvoll, M.; Kahn, C. R.; Bluher, M. Antioxidants prevent health-promoting effects of physical exercise in humans. Proceedings of the National Academy of Sciences of the United States of America 106:8665-8670; 2009. [50] Yfanti, C.; Akerstrom, T.; Nielsen, S.; Nielsen, A. R.; Mounier, R.; Mortensen, O. H.; Lykkesfeldt, J.; Rose, A. J.; Fischer, C. P.; Pedersen, B. K. Antioxidant supplementation does not alter endurance training adaptation. Medicine and science in sports and exercise 42:1388-1395; 2010. [51] Yfanti, C.; Nielsen, A. R.; Akerstrom, T.; Nielsen, S.; Rose, A. J.; Richter, E. A.; Lykkesfeldt, J.; Fischer, C. P.; Pedersen, B. K. Effect of antioxidant supplementation on insulin sensitivity in response to endurance exercise training. American journal of physiology. Endocrinology and metabolism 300:E761-770; 2011. [52] Cumming, K. T.; Raastad, T.; Holden, G.; Bastani, N. E.; Schneeberger, D.; Paronetto, M. P.; Mercatelli, N.; Ostgaard, H. N.; Ugelstad, I.; Caporossi, D.; Blomhoff, R.; Paulsen, G. Effects of vitamin C and E supplementation on endogenous antioxidant systems and heat shock proteins in response to endurance training. Physiological reports 2; 2014.

25

1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100

[53] Cobley, J. N.; McHardy, H.; Morton, J. P.; Nikolaidis, M. G.; Close, G. L. Influence of vitamin C and vitamin E on redox signaling: Implications for exercise adaptations. Free radical biology & medicine 84:65-76; 2015. [54] Goncalves, R. L.; Quinlan, C. L.; Perevoshchikova, I. V.; Hey-Mogensen, M.; Brand, M. D. Sites of superoxide and hydrogen peroxide production by muscle mitochondria assessed ex vivo under conditions mimicking rest and exercise. The Journal of biological chemistry 290:209-227; 2015. [55] Di Meo, S.; Venditti, P. Mitochondria in exercise-induced oxidative stress. Biological signals and receptors 10:125-140; 2001. [56] Hirschfield, W.; Moody, M. R.; O'Brien, W. E.; Gregg, A. R.; Bryan, R. M., Jr.; Reid, M. B. Nitric oxide release and contractile properties of skeletal muscles from mice deficient in type III NOS. American journal of physiology. Regulatory, integrative and comparative physiology 278:R95-r100; 2000. [57] Eu, J. P.; Hare, J. M.; Hess, D. T.; Skaf, M.; Sun, J.; Cardenas-Navina, I.; Sun, Q. A.; Dewhirst, M.; Meissner, G.; Stamler, J. S. Concerted regulation of skeletal muscle contractility by oxygen tension and endogenous nitric oxide. Proceedings of the National Academy of Sciences of the United States of America 100:15229-15234; 2003. [58] Ito, N.; Ruegg, U. T.; Kudo, A.; Miyagoe-Suzuki, Y.; Takeda, S. i. Activation of calcium signaling through Trpv1 by nNOS and peroxynitrite as a key trigger of skeletal muscle hypertrophy. Nature medicine 19:101-106; 2013. [59] Espinosa, A.; Leiva, A.; Pena, M.; Muller, M.; Debandi, A.; Hidalgo, C.; Carrasco, M. A.; Jaimovich, E. Myotube depolarization generates reactive oxygen species through NAD(P)H oxidase; ROS-elicited Ca2+ stimulates ERK, CREB, early genes. Journal of cellular physiology 209:379-388; 2006. [60] Smith, L. W.; Smith, J. D.; Criswell, D. S. Involvement of nitric oxide synthase in skeletal muscle adaptation to chronic overload. Journal of applied physiology (Bethesda, Md. : 1985) 92:2005-2011; 2002. [61] Sun, Q. A.; Hess, D. T.; Nogueira, L.; Yong, S.; Bowles, D. E.; Eu, J.; Laurita, K. R.; Meissner, G.; Stamler, J. S. Oxygen-coupled redox regulation of the skeletal muscle ryanodine receptor-Ca2+ release channel by NADPH oxidase 4. Proceedings of the National Academy of Sciences of the United States of America 108:16098-16103; 2011. [62] Lo Conte, M.; Carroll, K. S. The redox biochemistry of protein sulfenylation and sulfinylation. The Journal of biological chemistry 288:26480-26488; 2013. [63] Marozkina, N. V.; Gaston, B. S-Nitrosylation signaling regulates cellular protein interactions. Biochimica et biophysica acta 1820:722-729; 2012. [64] Sun, J.; Xin, C.; Eu, J. P.; Stamler, J. S.; Meissner, G. Cysteine-3635 is responsible for skeletal muscle ryanodine receptor modulation by NO. Proceedings of the National Academy of Sciences of the United States of America 98:11158-11162; 2001. [65] Bellinger, A. M.; Reiken, S.; Carlson, C.; Mongillo, M.; Liu, X.; Rothman, L.; Matecki, S.; Lacampagne, A.; Marks, A. R. Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle. Nature medicine 15:325-330; 2009. [66] Yasukawa, T.; Tokunaga, E.; Ota, H.; Sugita, H.; Martyn, J. A.; Kaneki, M. S-nitrosylationdependent inactivation of Akt/protein kinase B in insulin resistance. The Journal of biological chemistry 280:7511-7518; 2005. [67] Carvalho-Filho, M. A.; Ueno, M.; Hirabara, S. M.; Seabra, A. B.; Carvalheira, J. B.; de Oliveira, M. G.; Velloso, L. A.; Curi, R.; Saad, M. J. S-nitrosation of the insulin receptor, insulin receptor substrate 1, and protein kinase B/Akt: a novel mechanism of insulin resistance. Diabetes 54:959-967; 2005. [68] Nogueira, L.; Figueiredo-Freitas, C.; Casimiro-Lopes, G.; Magdesian, M. H.; Assreuy, J.; Sorenson, M. M. Myosin is reversibly inhibited by S-nitrosylation. The Biochemical journal 424:221231; 2009.

26

1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151

[69] Aquilano, K.; Baldelli, S.; Ciriolo, M. R. Nuclear recruitment of neuronal nitric-oxide synthase by alpha-syntrophin is crucial for the induction of mitochondrial biogenesis. The Journal of biological chemistry 289:365-378; 2014. [70] Samengo, G.; Avik, A.; Fedor, B.; Whittaker, D.; Myung, K. H.; Wehling-Henricks, M.; Tidball, J. G. Age-related loss of nitric oxide synthase in skeletal muscle causes reductions in calpain Snitrosylation that increase myofibril degradation and sarcopenia. Aging cell 11:1036-1045; 2012. [71] Salanova, M.; Schiffl, G.; Blottner, D. Atypical fast SERCA1a protein expression in slow myofibers and differential S-nitrosylation prevented by exercise during long term bed rest. Histochemistry and cell biology 132:383-394; 2009. [72] Salanova, M.; Schiffl, G.; Gutsmann, M.; Felsenberg, D.; Furlan, S.; Volpe, P.; Clarke, A.; Blottner, D. Nitrosative stress in human skeletal muscle attenuated by exercise countermeasure after chronic disuse. Redox biology 1:514-526; 2013. [73] Colussi, C.; Mozzetta, C.; Gurtner, A.; Illi, B.; Rosati, J.; Straino, S.; Ragone, G.; Pescatori, M.; Zaccagnini, G.; Antonini, A.; Minetti, G.; Martelli, F.; Piaggio, G.; Gallinari, P.; Steinkuhler, C.; Clementi, E.; Dell'Aversana, C.; Altucci, L.; Mai, A.; Capogrossi, M. C.; Puri, P. L.; Gaetano, C. HDAC2 blockade by nitric oxide and histone deacetylase inhibitors reveals a common target in Duchenne muscular dystrophy treatment. Proceedings of the National Academy of Sciences of the United States of America 105:19183-19187; 2008. [74] Dalle-Donne, I.; Rossi, R.; Colombo, G.; Giustarini, D.; Milzani, A. Protein S-glutathionylation: a regulatory device from bacteria to humans. Trends in biochemical sciences 34:85-96; 2009. [75] Humphries, K. M.; Deal, M. S.; Taylor, S. S. Enhanced dephosphorylation of cAMP-dependent protein kinase by oxidation and thiol modification. The Journal of biological chemistry 280:27502758; 2005. [76] Reddy, S.; Jones, A. D.; Cross, C. E.; Wong, P. S.; Van Der Vliet, A. Inactivation of creatine kinase by S-glutathionylation of the active-site cysteine residue. The Biochemical journal 347 Pt 3:821-827; 2000. [77] Anselmo, A. N.; Cobb, M. H. Protein kinase function and glutathionylation. The Biochemical journal 381:e1-2; 2004. [78] Cruz, C. M.; Rinna, A.; Forman, H. J.; Ventura, A. L.; Persechini, P. M.; Ojcius, D. M. ATP activates a reactive oxygen species-dependent oxidative stress response and secretion of proinflammatory cytokines in macrophages. The Journal of biological chemistry 282:2871-2879; 2007. [79] Zmijewski, J. W.; Banerjee, S.; Bae, H.; Friggeri, A.; Lazarowski, E. R.; Abraham, E. Exposure to hydrogen peroxide induces oxidation and activation of AMP-activated protein kinase. The Journal of biological chemistry 285:33154-33164; 2010. [80] Salsman, S. J.; Hensley, K.; Floyd, R. A. Sensitivity of protein tyrosine phosphatase activity to the redox environment, cytochrome C, and microperoxidase. Antioxidants & redox signaling 7:10781088; 2005. [81] Pastore, A.; Piemonte, F. S-Glutathionylation signaling in cell biology: progress and prospects. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences 46:279-292; 2012. [82] Wani, R.; Nagata, A.; Murray, B. W. Protein redox chemistry: post-translational cysteine modifications that regulate signal transduction and drug pharmacology. Frontiers in Pharmacology 5:224; 2014. [83] Rao, R. S. P.; Xu, D.; Thelen, J. J.; Miernyk, J. A. Circles within circles: crosstalk between protein Ser/Thr/Tyr-phosphorylation and Met oxidation. BMC Bioinformatics 14:S14-S14; 2013. [84] Bigelow, D. J.; Squier, T. C. Redox modulation of cellular signaling and metabolism through reversible oxidation of methionine sensors in calcium regulatory proteins. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1703:121-134; 2005. [85] D'Autreaux, B.; Toledano, M. B. ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nature reviews. Molecular cell biology 8:813-824; 2007. 27

1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201

[86] Bindoli, A.; Fukuto, J. M.; Forman, H. J. Thiol Chemistry in Peroxidase Catalysis and Redox Signaling. Antioxidants & redox signaling 10:1549-1564; 2008. [87] Levonen, A.-L.; Hill, B. G.; Kansanen, E.; Zhang, J.; Darley-Usmar, V. M. Redox regulation of antioxidants, autophagy, and the response to stress: Implications for electrophile therapeutics. Free Radical Biology and Medicine 71:196-207; 2014. [88] Winterbourn, C. C.; Hampton, M. B. Thiol chemistry and specificity in redox signaling. Free radical biology & medicine 45:549-561; 2008. [89] Jones, D. P.; Go, Y. M. Redox compartmentalization and cellular stress. Diabetes, obesity & metabolism 12 Suppl 2:116-125; 2010. [90] Forman, H. J.; Ursini, F.; Maiorino, M. An overview of mechanisms of redox signaling. Journal of molecular and cellular cardiology 73:2-9; 2014. [91] Reid, M. B. Invited Review: redox modulation of skeletal muscle contraction: what we know and what we don't. Journal of applied physiology (Bethesda, Md. : 1985) 90:724-731; 2001. [92] Andrade, F. H.; Reid, M. B.; Allen, D. G.; Westerblad, H. Effect of hydrogen peroxide and dithiothreitol on contractile function of single skeletal muscle fibres from the mouse. The Journal of physiology 509 ( Pt 2):565-575; 1998. [93] Moopanar, T. R.; Allen, D. G. Reactive oxygen species reduce myofibrillar Ca(2+) sensitivity in fatiguing mouse skeletal muscle at 37°C. The Journal of physiology 564:189-199; 2005. [94] Reid, M. B.; Khawli, F. A.; Moody, M. R. Reactive oxygen in skeletal muscle. III. Contractility of unfatigued muscle. Journal of applied physiology (Bethesda, Md. : 1985) 75:1081-1087; 1993. [95] Novelli, G. P.; Bracciotti, G.; Falsini, S. Spin-trappers and vitamin E prolong endurance to muscle fatigue in mice. Free radical biology & medicine 8:9-13; 1990. [96] Forman, H. J. Use and abuse of exogenous H(2)O(2) in studies of signal transduction. Free radical biology & medicine 42:926-932; 2007. [97] Reid, M. B. Free radicals and muscle fatigue: Of ROS, canaries, and the IOC. Free Radical Biology and Medicine 44:169-179; 2008. [98] Cobley, J. N.; McGlory, C.; Morton, J. P.; Close, G. L. N-Acetylcysteine's attenuation of fatigue after repeated bouts of intermittent exercise: practical implications for tournament situations. International journal of sport nutrition and exercise metabolism 21:451-461; 2011. [99] Allen, D. G.; Lannergren, J.; Westerblad, H. Muscle cell function during prolonged activity: cellular mechanisms of fatigue. Experimental physiology 80:497-527; 1995. [100] Bruton, J. D.; Place, N.; Yamada, T.; Silva, J. P.; Andrade, F. H.; Dahlstedt, A. J.; Zhang, S. J.; Katz, A.; Larsson, N. G.; Westerblad, H. Reactive oxygen species and fatigue-induced prolonged lowfrequency force depression in skeletal muscle fibres of rats, mice and SOD2 overexpressing mice. The Journal of physiology 586:175-184; 2008. [101] Andrade, F. H.; Reid, M. B.; Westerblad, H. Contractile response of skeletal muscle to low peroxide concentrations: myofibrillar calcium sensitivity as a likely target for redox-modulation. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 15:309-311; 2001. [102] Kawakami, M.; Okabe, E. Superoxide anion radical-triggered Ca2+ release from cardiac sarcoplasmic reticulum through ryanodine receptor Ca2+ channel. Molecular pharmacology 53:497503; 1998. [103] Andrade, F. H.; Reid, M. B.; Allen, D. G.; Westerblad, H. Effect of nitric oxide on single skeletal muscle fibres from the mouse. The Journal of physiology 509 ( Pt 2):577-586; 1998. [104] Pouvreau, S.; Allard, B.; Berthier, C.; Jacquemond, V. Control of intracellular calcium in the presence of nitric oxide donors in isolated skeletal muscle fibres from mouse. The Journal of physiology 560:779-794; 2004. [105] Aghdasi, B.; Reid, M. B.; Hamilton, S. L. Nitric oxide protects the skeletal muscle Ca2+ release channel from oxidation induced activation. The Journal of biological chemistry 272:25462-25467; 1997.

28

1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251

[106] Aracena-Parks, P.; Goonasekera, S. A.; Gilman, C. P.; Dirksen, R. T.; Hidalgo, C.; Hamilton, S. L. Identification of cysteines involved in S-nitrosylation, S-glutathionylation, and oxidation to disulfides in ryanodine receptor type 1. The Journal of biological chemistry 281:40354-40368; 2006. [107] Hart, J. D.; Dulhunty, A. F. Nitric oxide activates or inhibits skeletal muscle ryanodine receptors depending on its concentration, membrane potential and ligand binding. The Journal of membrane biology 173:227-236; 2000. [108] Eu, J. P.; Sun, J.; Xu, L.; Stamler, J. S.; Meissner, G. The skeletal muscle calcium release channel: coupled O2 sensor and NO signaling functions. Cell 102:499-509; 2000. [109] Cheong, E.; Tumbev, V.; Stoyanovsky, D.; Salama, G. Effects of pO2 on the activation of skeletal muscle ryanodine receptors by NO: a cautionary note. Cell calcium 38:481-488; 2005. [110] Margonis, K.; Fatouros, I. G.; Jamurtas, A. Z.; Nikolaidis, M. G.; Douroudos, I.; Chatzinikolaou, A.; Mitrakou, A.; Mastorakos, G.; Papassotiriou, I.; Taxildaris, K.; Kouretas, D. Oxidative stress biomarkers responses to physical overtraining: Implications for diagnosis. Free Radical Biology and Medicine 43:901-910; 2007. [111] Bellinger, A. M.; Reiken, S.; Dura, M.; Murphy, P. W.; Deng, S. X.; Landry, D. W.; Nieman, D.; Lehnart, S. E.; Samaru, M.; LaCampagne, A.; Marks, A. R. Remodeling of ryanodine receptor complex causes "leaky" channels: a molecular mechanism for decreased exercise capacity. Proceedings of the National Academy of Sciences of the United States of America 105:2198-2202; 2008. [112] Andersson, D. C.; Betzenhauser, M. J.; Reiken, S.; Meli, A. C.; Umanskaya, A.; Xie, W.; Shiomi, T.; Zalk, R.; Lacampagne, A.; Marks, A. R. Ryanodine Receptor Oxidation Causes Intracellular Calcium Leak and Muscle Weakness in Aging. Cell metabolism 14:196-207; 2011. [113] Aracena, P.; Tang, W.; Hamilton, S. L.; Hidalgo, C. Effects of S-glutathionylation and Snitrosylation on calmodulin binding to triads and FKBP12 binding to type 1 calcium release channels. Antioxidants & redox signaling 7:870-881; 2005. [114] Martini, F.; Nath, J. L.; Bartholomew, E. F. Fundamentals of anatomy & physiology. San Francisco: Benjamin Cummings; 2012. [115] Dutka, T. L.; Mollica, J. P.; Posterino, G. S.; Lamb, G. D. Modulation of contractile apparatus Ca2+ sensitivity and disruption of excitation-contraction coupling by S-nitrosoglutathione in rat muscle fibres. The Journal of physiology 589:2181-2196; 2011. [116] Mollica, J. P.; Dutka, T. L.; Merry, T. L.; Lamboley, C. R.; McConell, G. K.; McKenna, M. J.; Murphy, R. M.; Lamb, G. D. S-glutathionylation of troponin I (fast) increases contractile apparatus Ca2+ sensitivity in fast-twitch muscle fibres of rats and humans. The Journal of physiology 590:14431463; 2012. [117] Edwards, R. H.; Hill, D. K.; Jones, D. A.; Merton, P. A. Fatigue of long duration in human skeletal muscle after exercise. The Journal of physiology 272:769-778; 1977. [118] Allen, D. G.; Lamb, G. D.; Westerblad, H. Skeletal Muscle Fatigue: Cellular Mechanisms. Physiological reviews 88:287-332; 2008. [119] Watanabe, D.; Kanzaki, K.; Kuratani, M.; Matsunaga, S.; Yanaka, N.; Wada, M. Contribution of impaired myofibril and ryanodine receptor function to prolonged low-frequency force depression after in situ stimulation in rat skeletal muscle. Journal of muscle research and cell motility 36:275286; 2015. [120] Cheng, A. J.; Bruton, J. D.; Lanner, J. T.; Westerblad, H. Antioxidant treatments do not improve force recovery after fatiguing stimulation of mouse skeletal muscle fibres. The Journal of physiology; 2014. [121] Zembron-Lacny, A.; Slowinska-Lisowska, M.; Szygula, Z.; Witkowski, K.; Stefaniak, T.; Dziubek, W. Assessment of the antioxidant effectiveness of alpha-lipoic acid in healthy men exposed to muscle-damaging exercise. Journal of physiology and pharmacology : an official journal of the Polish Physiological Society 60:139-143; 2009. [122] Spriet, L. L.; Whitfield, J. Taurine and skeletal muscle function. Current opinion in clinical nutrition and metabolic care 18:96-101; 2015.

29

1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270

[123] Mero, A. A.; Vahalummukka, M.; Hulmi, J. J.; Kallio, P.; von Wright, A. Effects of resistance exercise session after oral ingestion of melatonin on physiological and performance responses of adult men. European journal of applied physiology 96:729-739; 2006. [124] Cureton, K. J.; Tomporowski, P. D.; Singhal, A.; Pasley, J. D.; Bigelman, K. A.; Lambourne, K.; Trilk, J. L.; McCully, K. K.; Arnaud, M. J.; Zhao, Q. Dietary quercetin supplementation is not ergogenic in untrained men. Journal of applied physiology (Bethesda, Md. : 1985) 107:1095-1104; 2009. [125] Gliemann, L.; Schmidt, J. F.; Olesen, J.; Bienso, R. S.; Peronard, S. L.; Grandjean, S. U.; Mortensen, S. P.; Nyberg, M.; Bangsbo, J.; Pilegaard, H.; Hellsten, Y. Resveratrol blunts the positive effects of exercise training on cardiovascular health in aged men. The Journal of physiology 591:5047-5059; 2013. [126] Packer, L. Antioxidants: Vitamins C and E for Health. Taylor & Francis; 2002. [127] Som, S.; Raha, C.; Chatterjee, I. B. Ascorbic acid: a scavenger of superoxide radical. Acta vitaminologica et enzymologica 5:243-250; 1983. [128] Augusto, O.; Bonini, M. G.; Amanso, A. M.; Linares, E.; Santos, C. C.; De Menezes, S. L. Nitrogen dioxide and carbonate radical anion: two emerging radicals in biology. Free radical biology & medicine 32:841-859; 2002. [129] Bentley, D. J.; Ackerman, J.; Clifford, T.; Slattery, K. S. Acute and Chronic Effects of Antioxidant Supplementation on Exercise Performance. In: Lamprecht, M., ed. Antioxidants in Sport Nutrition. Boca Raton (FL): CRC Press

1271 1272 1273 1274 1275 1276 1277 1278 1279 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296 1297 1298 1299 1300 1301

(c) 2015 by Taylor & Francis Group, LLC.; 2015. [130] Carr, A. C.; Bozonet, S. M.; Pullar, J. M.; Simcock, J. W.; Vissers, M. C. Human skeletal muscle ascorbate is highly responsive to changes in vitamin C intake and plasma concentrations. The American journal of clinical nutrition 97:800-807; 2013. [131] Mason, S. A.; Baptista, R.; Della Gatta, P. A.; Yousif, A.; Russell, A. P.; Wadley, G. D. High-dose vitamin C supplementation increases skeletal muscle vitamin C concentration and SVCT2 transporter expression but does not alter redox status in healthy males. Free radical biology & medicine 77:130138; 2014. [132] Holmes, A. J.; Williams, D. L. H. Reaction of ascorbic acid with S-nitrosothiols: clear evidence for two distinct reaction pathways. Journal of the Chemical Society, Perkin Transactions 2:1639-1644; 2000. [133] Durham, W. J.; Aracena-Parks, P.; Long, C.; Rossi, A. E.; Goonasekera, S. A.; Boncompagni, S.; Galvan, D. L.; Gilman, C. P.; Baker, M. R.; Shirokova, N.; Protasi, F.; Dirksen, R.; Hamilton, S. L. RyR1 Snitrosylation underlies environmental heat stroke and sudden death in Y522S RyR1 knockin mice. Cell 133:53-65; 2008. [134] Dutka, T. L.; Mollica, J. P.; Lamb, G. D. Differential effects of peroxynitrite on contractile protein properties in fast- and slow-twitch skeletal muscle fibers of rat. Journal of applied physiology (Bethesda, Md. : 1985) 110:705-716; 2011. [135] Burgoyne, J. R.; Eaton, P. A rapid approach for the detection, quantification, and discovery of novel sulfenic acid or S-nitrosothiol modified proteins using a biotin-switch method. Methods in enzymology 473:281-303; 2010. [136] Rossman, M. J.; Garten, R. S.; Groot, H. J.; Reese, V.; Zhao, J.; Amann, M.; Richardson, R. S. Ascorbate infusion increases skeletal muscle fatigue resistance in patients with chronic obstructive pulmonary disease. American journal of physiology. Regulatory, integrative and comparative physiology 305:R1163-1170; 2013. [137] ROSSMAN, M. J.; GROOT, H. J.; REESE, V.; ZHAO, J.; AMANN, M.; RICHARDSON, R. S. Oxidative Stress and COPD: The Effect of Oral Antioxidants on Skeletal Muscle Fatigue. Medicine & Science in Sports & Exercise 45:1235-1243; 2013. [138] Jakeman, P.; Maxwell, S. Effect of antioxidant vitamin supplementation on muscle function after eccentric exercise. European journal of applied physiology and occupational physiology 67:426430; 1993.

30

1302 1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 1342 1343 1344 1345 1346 1347 1348 1349 1350 1351 1352

[139] Thompson, D.; Williams, C.; McGregor, S. J.; Nicholas, C. W.; McArdle, F.; Jackson, M. J.; Powell, J. R. Prolonged vitamin C supplementation and recovery from demanding exercise. International journal of sport nutrition and exercise metabolism 11:466-481; 2001. [140] Thompson, D.; Bailey, D. M.; Hill, J.; Hurst, T.; Powell, J. R.; Williams, C. Prolonged vitamin C supplementation and recovery from eccentric exercise. European journal of applied physiology 92:133-138; 2004. [141] Bryer, S. C.; Goldfarb, A. H. Effect of high dose vitamin C supplementation on muscle soreness, damage, function, and oxidative stress to eccentric exercise. International journal of sport nutrition and exercise metabolism 16:270-280; 2006. [142] Janero, D. R. Therapeutic potential of vitamin E in the pathogenesis of spontaneous atherosclerosis. Free radical biology & medicine 11:129-144; 1991. [143] Traber, M. G.; Atkinson, J. Vitamin E, antioxidant and nothing more. Free radical biology & medicine 43:4-15; 2007. [144] Azzi, A.; Gysin, R.; Kempna, P.; Munteanu, A.; Villacorta, L.; Visarius, T.; Zingg, J. M. Regulation of gene expression by alpha-tocopherol. Biological chemistry 385:585-591; 2004. [145] Coombes, J. S.; Powers, S. K.; Rowell, B.; Hamilton, K. L.; Dodd, S. L.; Shanely, R. A.; Sen, C. K.; Packer, L. Effects of vitamin E and alpha-lipoic acid on skeletal muscle contractile properties. Journal of applied physiology (Bethesda, Md. : 1985) 90:1424-1430; 2001. [146] Meydani, M.; Evans, W. J.; Handelman, G.; Biddle, L.; Fielding, R. A.; Meydani, S. N.; Burrill, J.; Fiatarone, M. A.; Blumberg, J. B.; Cannon, J. G. Protective effect of vitamin E on exercise-induced oxidative damage in young and older adults. The American journal of physiology 264:R992-998; 1993. [147] Beaton, L. J.; Allan, D. A.; Tarnopolsky, M. A.; Tiidus, P. M.; Phillips, S. M. Contractioninduced muscle damage is unaffected by vitamin E supplementation. Medicine and science in sports and exercise 34:798-805; 2002. [148] Bailey, D. M.; Williams, C.; Betts, J. A.; Thompson, D.; Hurst, T. L. Oxidative stress, inflammation and recovery of muscle function after damaging exercise: effect of 6-week mixed antioxidant supplementation. European journal of applied physiology 111:925-936; 2011. [149] Bloomer, R. J.; Goldfarb, A. H.; McKenzie, M. J.; You, T.; Nguyen, L. Effects of antioxidant therapy in women exposed to eccentric exercise. International journal of sport nutrition and exercise metabolism 14:377-388; 2004. [150] Shafat, A.; Butler, P.; Jensen, R. L.; Donnelly, A. E. Effects of dietary supplementation with vitamins C and E on muscle function during and after eccentric contractions in humans. European journal of applied physiology 93:196-202; 2004. [151] Bobeuf, F.; Labonte, M.; Dionne, I. J.; Khalil, A. Combined effect of antioxidant supplementation and resistance training on oxidative stress markers, muscle and body composition in an elderly population. The journal of nutrition, health & aging 15:883-889; 2011. [152] Theodorou, A. A.; Nikolaidis, M. G.; Paschalis, V.; Koutsias, S.; Panayiotou, G.; Fatouros, I. G.; Koutedakis, Y.; Jamurtas, A. Z. No effect of antioxidant supplementation on muscle performance and blood redox status adaptations to eccentric training. The American journal of clinical nutrition 93:1373-1383; 2011. [153] McKenna, M. J.; Medved, I.; Goodman, C. A.; Brown, M. J.; Bjorksten, A. R.; Murphy, K. T.; Petersen, A. C.; Sostaric, S.; Gong, X. N-acetylcysteine attenuates the decline in muscle Na+,K+-pump activity and delays fatigue during prolonged exercise in humans. The Journal of physiology 576:279288; 2006. [154] Medved, I.; Brown, M. J.; Bjorksten, A. R.; Murphy, K. T.; Petersen, A. C.; Sostaric, S.; Gong, X.; McKenna, M. J. N-acetylcysteine enhances muscle cysteine and glutathione availability and attenuates fatigue during prolonged exercise in endurance-trained individuals. Journal of applied physiology (Bethesda, Md. : 1985) 97:1477-1485; 2004. [155] Murphy, Michael P.; Holmgren, A.; Larsson, N.-G.; Halliwell, B.; Chang, Christopher J.; Kalyanaraman, B.; Rhee, Sue G.; Thornalley, Paul J.; Partridge, L.; Gems, D.; Nyström, T.; Belousov, V.; 31

1353 1354 1355 1356 1357 1358 1359 1360 1361 1362 1363 1364 1365 1366 1367 1368 1369 1370 1371 1372 1373 1374 1375 1376 1377 1378 1379 1380 1381 1382 1383 1384 1385 1386 1387 1388 1389 1390 1391 1392 1393 1394 1395 1396 1397 1398 1399 1400 1401 1402 1403

Schumacker, Paul T.; Winterbourn, Christine C. Unraveling the Biological Roles of Reactive Oxygen Species. Cell Metabolism 13:361-366; 2011. [156] Winterbourn, C. C.; Metodiewa, D. Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide. Free Radical Biology and Medicine 27:322-328; 1999. [157] Lew, H.; Pyke, S.; Quintanilha, A. Changes in the glutathione status of plasma, liver and muscle following exhaustive exercise in rats. FEBS letters 185:262-266; 1985. [158] Pyke, S.; Lew, H.; Quintanilha, A. Severe depletion in liver glutathione during physical exercise. Biochemical and biophysical research communications 139:926-931; 1986. [159] Ullian, M. E.; Gelasco, A. K.; Fitzgibbon, W. R.; Beck, C. N.; Morinelli, T. A. N-acetylcysteine decreases angiotensin II receptor binding in vascular smooth muscle cells. Journal of the American Society of Nephrology : JASN 16:2346-2353; 2005. [160] Finn, N. A.; Kemp, M. L. Pro-oxidant and antioxidant effects of N-acetylcysteine regulate doxorubicin-induced NF-kappa B activity in leukemic cells(). Molecular Biosystems 8:650-662; 2012. [161] Deneke, S. M.; Fanburg, B. L. Regulation of cellular glutathione. The American journal of physiology 257:L163-173; 1989. [162] Travaline, J. M.; Sudarshan, S.; Roy, B. G.; Cordova, F.; Leyenson, V.; Criner, G. J. Effect of Nacetylcysteine on human diaphragm strength and fatigability. American journal of respiratory and critical care medicine 156:1567-1571; 1997. [163] Koechlin, C.; Couillard, A.; Simar, D.; Cristol, J. P.; Bellet, H.; Hayot, M.; Prefaut, C. Does oxidative stress alter quadriceps endurance in chronic obstructive pulmonary disease? American journal of respiratory and critical care medicine 169:1022-1027; 2004. [164] Matuszczak, Y.; Farid, M.; Jones, J.; Lansdowne, S.; Smith, M. A.; Taylor, A. A.; Reid, M. B. Effects of N-acetylcysteine on glutathione oxidation and fatigue during handgrip exercise. Muscle & nerve 32:633-638; 2005. [165] Kelly, M. K.; Wicker, R. J.; Barstow, T. J.; Harms, C. A. Effects of N-acetylcysteine on respiratory muscle fatigue during heavy exercise. Respiratory physiology & neurobiology 165:67-72; 2009. [166] Medved, I.; Brown, M. J.; Bjorksten, A. R.; Leppik, J. A.; Sostaric, S.; McKenna, M. J. Nacetylcysteine infusion alters blood redox status but not time to fatigue during intense exercise in humans. Journal of applied physiology (Bethesda, Md. : 1985) 94:1572-1582; 2003. [167] Corn, S. D.; Barstow, T. J. Effects of oral N-acetylcysteine on fatigue, critical power, and W' in exercising humans. Respiratory physiology & neurobiology 178:261-268; 2011. [168] Medved, I.; Brown, M. J.; Bjorksten, A. R.; McKenna, M. J. Effects of intravenous Nacetylcysteine infusion on time to fatigue and potassium regulation during prolonged cycling exercise. Journal of applied physiology (Bethesda, Md. : 1985) 96:211-217; 2004. [169] Trewin, A. J.; Petersen, A. C.; Billaut, F.; McQuade, L. R.; McInerney, B. V.; Stepto, N. K. Nacetylcysteine alters substrate metabolism during high-intensity cycle exercise in well-trained humans. Applied physiology, nutrition, and metabolism = Physiologie appliquee, nutrition et metabolisme 38:1217-1227; 2013. [170] Slattery, K. M.; Dascombe, B.; Wallace, L. K.; Bentley, D. J.; Coutts, A. J. Effect of Nacetylcysteine on cycling performance after intensified training. Medicine and science in sports and exercise 46:1114-1123; 2014. [171] Pendyala, L.; Creaven, P. J. Pharmacokinetic and pharmacodynamic studies of Nacetylcysteine, a potential chemopreventive agent during a phase I trial. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 4:245-251; 1995. [172] Ferreira, L. F.; Campbell, K. S.; Reid, M. B. N-acetylcysteine in handgrip exercise: plasma thiols and adverse reactions. International journal of sport nutrition and exercise metabolism 21:146154; 2011. [173] Sandilands, E. A.; Bateman, D. N. Adverse reactions associated with acetylcysteine. Clinical toxicology (Philadelphia, Pa.) 47:81-88; 2009. 32

1404 1405 1406 1407 1408 1409 1410 1411 1412 1413 1414 1415 1416 1417 1418 1419 1420 1421 1422 1423 1424 1425 1426 1427 1428 1429 1430 1431 1432 1433 1434 1435 1436 1437 1438 1439 1440 1441 1442 1443 1444 1445 1446 1447 1448 1449 1450 1451 1452

[174] Mizuno, K.; Tanaka, M.; Nozaki, S.; Mizuma, H.; Ataka, S.; Tahara, T.; Sugino, T.; Shirai, T.; Kajimoto, Y.; Kuratsune, H.; Kajimoto, O.; Watanabe, Y. Antifatigue effects of coenzyme Q10 during physical fatigue. Nutrition (Burbank, Los Angeles County, Calif.) 24:293-299; 2008. [175] Crane, F. L. Biochemical functions of coenzyme Q10. Journal of the American College of Nutrition 20:591-598; 2001. [176] Bonetti, A.; Solito, F.; Carmosino, G.; Bargossi, A. M.; Fiorella, P. L. Effect of ubidecarenone oral treatment on aerobic power in middle-aged trained subjects. The Journal of sports medicine and physical fitness 40:51-57; 2000. [177] Alf, D.; Schmidt, M. E.; Siebrecht, S. C. Ubiquinol supplementation enhances peak power production in trained athletes: a double-blind, placebo controlled study. Journal of the International Society of Sports Nutrition 10:24-24; 2013. [178] Mizuno, M.; Quistorff, B.; Theorell, H.; Theorell, M.; Chance, B. Effects of oral supplementation of coenzyme Q10 on 31P-NMR detected skeletal muscle energy metabolism in middle-aged post-polio subjects and normal volunteers. Molecular aspects of medicine 18 Suppl:S291-298; 1997. [179] Bloomer, R. J.; Canale, R. E.; McCarthy, C. G.; Farney, T. M. Impact of oral ubiquinol on blood oxidative stress and exercise performance. Oxid Med Cell Longev 2012:465020; 2012. [180] Cooke, M.; Iosia, M.; Buford, T.; Shelmadine, B.; Hudson, G.; Kerksick, C.; Rasmussen, C.; Greenwood, M.; Leutholtz, B.; Willoughby, D.; Kreider, R. Effects of acute and 14-day coenzyme Q10 supplementation on exercise performance in both trained and untrained individuals. Journal of the International Society of Sports Nutrition 5:8-8; 2008. [181] Laaksonen, R.; Fogelholm, M.; Himberg, J. J.; Laakso, J.; Salorinne, Y. Ubiquinone supplementation and exercise capacity in trained young and older men. European journal of applied physiology and occupational physiology 72:95-100; 1995. [182] Malm, C.; Svensson, M.; Ekblom, B.; Sjodin, B. Effects of ubiquinone-10 supplementation and high intensity training on physical performance in humans. Acta physiologica Scandinavica 161:379384; 1997. [183] Zhou, S.; Zhang, Y.; Davie, A.; Marshall-Gradisnik, S.; Hu, H.; Wang, J.; Brushett, D. Muscle and plasma coenzyme Q10 concentration, aerobic power and exercise economy of healthy men in response to four weeks of supplementation. The Journal of sports medicine and physical fitness 45:337-346; 2005. [184] Svensson, M.; Malm, C.; Tonkonogi, M.; Ekblom, B.; Sjodin, B.; Sahlin, K. Effect of Q10 supplementation on tissue Q10 levels and adenine nucleotide catabolism during high-intensity exercise. International journal of sport nutrition 9:166-180; 1999. [185] Yan, Z.; Lira, V. A.; Greene, N. P. Exercise training-induced regulation of mitochondrial quality. Exerc Sport Sci Rev 40:159-164; 2012. [186] Irrcher, I.; Ljubicic, V.; Hood, D. A. Interactions between ROS and AMP kinase activity in the regulation of PGC-1{alpha} transcription in skeletal muscle cells. Am J Physiol Cell Physiol 296:C116123; 2009. [187] McConell, G. K.; Ng, G. P.; Phillips, M.; Ruan, Z.; Macaulay, S. L.; Wadley, G. D. Central role of nitric oxide synthase in AICAR and caffeine-induced mitochondrial biogenesis in L6 myocytes. Journal of applied physiology (Bethesda, Md. : 1985) 108:589-595; 2010. [188] Lira, V. A.; Brown, D. L.; Lira, A. K.; Kavazis, A. N.; Soltow, Q. A.; Zeanah, E. H.; Criswell, D. S. Nitric oxide and AMPK cooperatively regulate PGC-1 in skeletal muscle cells. The Journal of physiology 588:3551-3566; 2010. [189] Baar, K.; Song, Z.; Semenkovich, C. F.; Jones, T. E.; Han, D. H.; Nolte, L. A.; Ojuka, E. O.; Chen, M.; Holloszy, J. O. Skeletal muscle overexpression of nuclear respiratory factor 1 increases glucose transport capacity. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 17:1666-1673; 2003.

33

1453 1454 1455 1456 1457 1458 1459 1460 1461 1462 1463 1464 1465 1466 1467 1468 1469 1470 1471 1472 1473 1474 1475 1476 1477 1478 1479 1480 1481 1482 1483 1484 1485 1486 1487 1488 1489 1490 1491 1492 1493 1494 1495 1496 1497 1498 1499 1500 1501 1502 1503

[190] Gerhart-Hines, Z.; Rodgers, J. T.; Bare, O.; Lerin, C.; Kim, S. H.; Mostoslavsky, R.; Alt, F. W.; Wu, Z.; Puigserver, P. Metabolic control of muscle mitochondrial function and fatty acid oxidation through SIRT1/PGC-1alpha. Embo J 26:1913-1923; 2007. [191] Gomez-Cabrera, M. C.; Borras, C.; Pallardo, F. V.; Sastre, J.; Ji, L. L.; Vina, J. Decreasing xanthine oxidase-mediated oxidative stress prevents useful cellular adaptations to exercise in rats. J Physiol 567:113-120; 2005. [192] Sandstrom, M. E.; Zhang, S. J.; Bruton, J.; Silva, J. P.; Reid, M. B.; Westerblad, H.; Katz, A. Role of reactive oxygen species in contraction-mediated glucose transport in mouse skeletal muscle. The Journal of physiology 575:251-262; 2006. [193] Wadley, G. D.; Nicolas, M. A.; Hiam, D.; McConell, G. K. Xanthine oxidase inhibition attenuates skeletal muscle signaling following acute exercise but does not impair mitochondrial adaptations to endurance training. American Journal of Physiology, Endocrinology and Metabolism 304:E853–E862; 2013. [194] Ji, L. L.; Gomez-Cabrera, M. C.; Vina, J. Role of nuclear factor kappaB and mitogen-activated protein kinase signaling in exercise-induced antioxidant enzyme adaptation. Appl Physiol Nutr Metab 32:930-935; 2007. [195] Wright, D. C.; Geiger, P. C.; Han, D. H.; Jones, T. E.; Holloszy, J. O. Calcium induces increases in peroxisome proliferator-activated receptor gamma coactivator-1alpha and mitochondrial biogenesis by a pathway leading to p38 mitogen activated protein kinase activation. J Biol Chem; 2007. [196] Jager, S.; Handschin, C.; St-Pierre, J.; Spiegelman, B. M. AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of PGC-1alpha. Proc Natl Acad Sci U S A 104:12017-12022; 2007. [197] Puigserver, P.; Spiegelman, B. M. Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1 alpha): transcriptional coactivator and metabolic regulator. Endocrine reviews 24:78-90; 2003. [198] Wu, Z.; Puigserver, P.; Andersson, U.; Zhang, C.; Adelmant, G.; Mootha, V.; Troy, A.; Cinti, S.; Lowell, B.; Scarpulla, R. C.; Spiegelman, B. M. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 98:115-124; 1999. [199] Strobel, N. A.; Matsumoto, A.; Peake, J. M.; Marsh, S. A.; Peternelj, T. T.; Briskey, D.; Fassett, R. G.; Coombes, J. S.; Wadley, G. D. Altering the redox state of skeletal muscle by glutathione depletion increases the exercise-activation of PGC-1alpha. Physiological reports 2; 2014. [200] Murakami, T.; Shimomura, Y.; Yoshimura, A.; Sokabe, M.; Fujitsuka, N. Induction of nuclear respiratory factor-1 expression by an acute bout of exercise in rat muscle. Biochimica et biophysica acta 1381:113-122; 1998. [201] Wright, D. C.; Han, D. H.; Garcia-Roves, P. M.; Geiger, P. C.; Jones, T. E.; Holloszy, J. O. Exercise-induced mitochondrial biogenesis begins before the increase in muscle PGC-1alpha expression. J Biol Chem 282:194-199; 2007. [202] Wadley, G. D.; Choate, J.; McConell, G. K. NOS isoform-specific regulation of basal but not exercise-induced mitochondrial biogenesis in mouse skeletal muscle. The Journal of Physiology 585:253-262; 2007. [203] St-Pierre, J.; Drori, S.; Uldry, M.; Silvaggi, J. M.; Rhee, J.; Jager, S.; Handschin, C.; Zheng, K.; Lin, J.; Yang, W.; Simon, D. K.; Bachoo, R.; Spiegelman, B. M. Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators. Cell 127:397-408; 2006. [204] Nisoli, E.; Falcone, S.; Tonello, C.; Cozzi, V.; Palomba, L.; Fiorani, M.; Pisconti, A.; Brunelli, S.; Cardile, A.; Francolini, M.; Cantoni, O.; Carruba, M. O.; Moncada, S.; Clementi, E. Mitochondrial biogenesis by NO yields functionally active mitochondria in mammals. Proceedings of the National Academy of Sciences of the United States of America 101:16507-16512; 2004. [205] Wadley, G. D.; McConell, G. K. Effect of nitric oxide synthase inhibition on mitochondrial biogenesis in rat skeletal muscle. Journal of applied physiology (Bethesda, Md. : 1985) 102:314-320; 2007. 34

1504 1505 1506 1507 1508 1509 1510 1511 1512 1513 1514 1515 1516 1517 1518 1519 1520 1521 1522 1523 1524 1525 1526 1527 1528 1529 1530 1531 1532 1533 1534 1535 1536 1537 1538 1539 1540 1541 1542 1543 1544 1545 1546 1547 1548 1549 1550 1551 1552 1553

[206] Wadley, G. D.; Choate, J.; McConell, G. K. NOS isoform-specific regulation of basal but not exercise-induced mitochondrial biogenesis in mouse skeletal muscle. The Journal of physiology 585:253-262; 2007. [207] Zhang, Y.; Uguccioni, G.; Ljubicic, V.; Irrcher, I.; Iqbal, S.; Singh, K.; Ding, S.; Hood, D. A. Multiple signaling pathways regulate contractile activity-mediated PGC-1alpha gene expression and activity in skeletal muscle cells. Physiological reports 2; 2014. [208] Kang, C.; O'Moore, K. M.; Dickman, J. R.; Ji, L. L. Exercise activation of muscle peroxisome proliferator-activated receptor-gamma coactivator-1alpha signaling is redox sensitive. Free radical biology & medicine 47:1394-1400; 2009. [209] Higashida, K.; Kim, S. H.; Higuchi, M.; Holloszy, J. O.; Han, D. H. Normal adaptations to exercise despite protection against oxidative stress. American journal of physiology. Endocrinology and metabolism 301:E779-784; 2011. [210] Venditti, P.; Napolitano, G.; Barone, D.; Di Meo, S. Vitamin E supplementation modifies adaptive responses to training in rat skeletal muscle. Free radical research 48:1179-1189; 2014. [211] Price, Nathan L.; Gomes, Ana P.; Ling, Alvin J. Y.; Duarte, Filipe V.; Martin-Montalvo, A.; North, Brian J.; Agarwal, B.; Ye, L.; Ramadori, G.; Teodoro, Joao S.; Hubbard, Basil P.; Varela, Ana T.; Davis, James G.; Varamini, B.; Hafner, A.; Moaddel, R.; Rolo, Anabela P.; Coppari, R.; Palmeira, Carlos M.; de Cabo, R.; Baur, Joseph A.; Sinclair, David A. SIRT1 Is Required for AMPK Activation and the Beneficial Effects of Resveratrol on Mitochondrial Function. Cell Metab 15:675-690; 2012. [212] Lagouge, M.; Argmann, C.; Gerhart-Hines, Z.; Meziane, H.; Lerin, C.; Daussin, F.; Messadeq, N.; Milne, J.; Lambert, P.; Elliott, P.; Geny, B.; Laakso, M.; Puigserver, P.; Auwerx, J. Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha. Cell 127:1109-1122; 2006. [213] Petersen, A. C.; McKenna, M. J.; Medved, I.; Murphy, K. T.; Brown, M. J.; Della Gatta, P.; Cameron-Smith, D. Infusion with the antioxidant N-acetylcysteine attenuates early adaptive responses to exercise in human skeletal muscle. Acta physiologica (Oxford, England) 204:382-392; 2012. [214] Scribbans, T. D.; Ma, J. K.; Edgett, B. A.; Vorobej, K. A.; Mitchell, A. S.; Zelt, J. G.; Simpson, C. A.; Quadrilatero, J.; Gurd, B. J. Resveratrol supplementation does not augment performance adaptations or fibre-type-specific responses to high-intensity interval training in humans. Applied physiology, nutrition, and metabolism = Physiologie appliquee, nutrition et metabolisme 39:13051313; 2014. [215] Olesen, J.; Gliemann, L.; Bienso, R.; Schmidt, J.; Hellsten, Y.; Pilegaard, H. Exercise training, but not resveratrol, improves metabolic and inflammatory status in skeletal muscle of aged men. The Journal of physiology 592:1873-1886; 2014. [216] Rock, C. L.; Newman, V. A.; Neuhouser, M. L.; Major, J.; Barnett, M. J. Antioxidant Supplement Use in Cancer Survivors and the General Population. The Journal of Nutrition 134:3194S3195S; 2004. [217] Yfanti, C.; Fischer, C. P.; Nielsen, S.; Akerstrom, T.; Nielsen, A. R.; Veskoukis, A. S.; Kouretas, D.; Lykkesfeldt, J.; Pilegaard, H.; Pedersen, B. K. Role of vitamin C and E supplementation on IL-6 in response to training. Journal of applied physiology (Bethesda, Md. : 1985) 112:990-1000; 2012. [218] Leeuwenburgh, C.; Hansen, P. A.; Holloszy, J. O.; Heinecke, J. W. Oxidized amino acids in the urine of aging rats: potential markers for assessing oxidative stress in vivo. The American journal of physiology 276:R128-135; 1999. [219] Larsen, S.; Nielsen, J.; Hansen, C. N.; Nielsen, L. B.; Wibrand, F.; Stride, N.; Schroder, H. D.; Boushel, R.; Helge, J. W.; Dela, F.; Hey-Mogensen, M. Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects. The Journal of Physiology 590:3349-3360; 2012. [220] Petersen, A. C.; McKenna, M. J.; Medved, I.; Murphy, K. T.; Brown, M. J.; Della Gatta, P.; Cameron-Smith, D. Infusion with the antioxidant N-acetylcysteine attenuates early adaptive responses to exercise in human skeletal muscle. Acta Physiol (Oxf) 204:382-392; 2012.

35

1554 1555 1556 1557 1558 1559 1560 1561 1562 1563 1564 1565 1566 1567 1568 1569 1570 1571 1572 1573 1574 1575 1576 1577 1578 1579 1580 1581 1582 1583 1584 1585 1586 1587 1588 1589 1590 1591 1592 1593 1594 1595 1596 1597 1598 1599 1600 1601 1602 1603 1604

[221] Baur, J. A. Biochemical effects of SIRT1 activators. Biochimica et biophysica acta 1804:16261634; 2010. [222] Selvaraj, S.; Mohan, A.; Narayanan, S.; Sethuraman, S.; Krishnan, U. M. Dose-dependent interaction of trans-resveratrol with biomembranes: effects on antioxidant property. J Med Chem 56:970-981; 2013. [223] Poulsen, M. M.; Vestergaard, P. F.; Clasen, B. F.; Radko, Y.; Christensen, L. P.; StodkildeJorgensen, H.; Moller, N.; Jessen, N.; Pedersen, S. B.; Jorgensen, J. O. High-dose resveratrol supplementation in obese men: an investigator-initiated, randomized, placebo-controlled clinical trial of substrate metabolism, insulin sensitivity, and body composition. Diabetes 62:1186-1195; 2013. [224] Mankowski, R. T.; Anton, S. D.; Buford, T. W.; Leeuwenburgh, C. Dietary Antioxidants as Modifiers of Physiologic Adaptations to Exercise. Medicine & Science in Sports & Exercise 47:18571868; 2015. [225] Gliemann, L.; Schmidt, J. F.; Olesen, J.; Bienso, R. S.; Peronard, S. L.; Grandjean, S. U.; Mortensen, S. P.; Nyberg, M.; Bangsbo, J.; Pilegaard, H.; Hellsten, Y. Resveratrol blunts the positive effects of exercise training on cardiovascular health in aged men. The Journal of Physiology 591:5047-5059; 2013. [226] Smoliga, J. M.; Blanchard, O. L. Recent data do not provide evidence that resveratrol causes 'mainly negative' or 'adverse' effects on exercise training in humans. The Journal of physiology 591:5251-5252; 2013. [227] Walle, T.; Hsieh, F.; DeLegge, M. H.; Oatis, J. E., Jr.; Walle, U. K. High absorption but very low bioavailability of oral resveratrol in humans. Drug metabolism and disposition: the biological fate of chemicals 32:1377-1382; 2004. [228] Smoliga, J. M.; Baur, J. A.; Hausenblas, H. A. Resveratrol and health--a comprehensive review of human clinical trials. Molecular nutrition & food research 55:1129-1141; 2011. [229] Wenzel, E.; Somoza, V. Metabolism and bioavailability of trans-resveratrol. Molecular nutrition & food research 49:472-481; 2005. [230] Andres-Lacueva, C.; Macarulla, M. T.; Rotches-Ribalta, M.; Boto-Ordonez, M.; Urpi-Sarda, M.; Rodriguez, V. M.; Portillo, M. P. Distribution of resveratrol metabolites in liver, adipose tissue, and skeletal muscle in rats fed different doses of this polyphenol. Journal of agricultural and food chemistry 60:4833-4840; 2012. [231] Brown, V. A.; Patel, K. R.; Viskaduraki, M.; Crowell, J. A.; Perloff, M.; Booth, T. D.; Vasilinin, G.; Sen, A.; Schinas, A. M.; Piccirilli, G.; Brown, K.; Steward, W. P.; Gescher, A. J.; Brenner, D. E. Repeat dose study of the cancer chemopreventive agent resveratrol in healthy volunteers: safety, pharmacokinetics, and effect on the insulin-like growth factor axis. Cancer research 70:9003-9011; 2010. [232] Boocock, D. J.; Faust, G. E.; Patel, K. R.; Schinas, A. M.; Brown, V. A.; Ducharme, M. P.; Booth, T. D.; Crowell, J. A.; Perloff, M.; Gescher, A. J.; Steward, W. P.; Brenner, D. E. Phase I dose escalation pharmacokinetic study in healthy volunteers of resveratrol, a potential cancer chemopreventive agent. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 16:1246-1252; 2007. [233] Yun, H.; Park, S.; Kim, M. J.; Yang, W. K.; Im, D. U.; Yang, K. R.; Hong, J.; Choe, W.; Kang, I.; Kim, S. S.; Ha, J. AMP-activated protein kinase mediates the antioxidant effects of resveratrol through regulation of the transcription factor FoxO1. The FEBS journal 281:4421-4438; 2014. [234] Menzies, K. J.; Singh, K.; Saleem, A.; Hood, D. A. Sirtuin 1-mediated effects of exercise and resveratrol on mitochondrial biogenesis. The Journal of biological chemistry 288:6968-6979; 2013. [235] Price, N. L.; Gomes, A. P.; Ling, A. J. Y.; Duarte, F. V.; Martin-Montalvo, A.; North, B. J.; Agarwal, B.; Ye, L.; Ramadori, G.; Teodoro, J. S.; Hubbard, B. P.; Varela, A. T.; Davis, J. G.; Varamini, B.; Hafner, A.; Moaddel, R.; Rolo, A. P.; Coppari, R.; Palmeira, C. M.; De Cabo, R.; Baur, J. A.; Sinclair, D. A. SIRT1 is required for AMPK activation and the beneficial effects of resveratrol on mitochondrial function. Cell Metabolism 15:675-690; 2012. 36

1605 1606 1607 1608 1609 1610 1611 1612 1613 1614 1615 1616 1617 1618 1619 1620 1621 1622 1623 1624 1625 1626 1627 1628 1629 1630 1631 1632 1633 1634 1635 1636 1637 1638 1639 1640 1641 1642 1643 1644 1645 1646 1647 1648 1649 1650 1651 1652 1653 1654

[236] Ryan, M. J.; Jackson, J. R.; Hao, Y.; Williamson, C. L.; Dabkowski, E. R.; Hollander, J. M.; Alway, S. E. Suppression of oxidative stress by resveratrol after isometric contractions in gastrocnemius muscles of aged mice. The journals of gerontology. Series A, Biological sciences and medical sciences 65:815-831; 2010. [237] McConell, G. K.; Rattigan, S.; Lee-Young, R. S.; Wadley, G. D.; Merry, T. L. Skeletal muscle nitric oxide signaling and exercise: a focus on glucose metabolism. American journal of physiology. Endocrinology and metabolism 303:E301-307; 2012. [238] Richter, E. A.; Hargreaves, M. Exercise, GLUT4, and skeletal muscle glucose uptake. Physiological reviews 93:993-1017; 2013. [239] Arbogast, S.; Reid, M. B. Oxidant activity in skeletal muscle fibers is influenced by temperature, CO2 level, and muscle-derived nitric oxide. American journal of physiology. Regulatory, integrative and comparative physiology 287:R698-705; 2004. [240] Merry, T. L.; Lynch, G. S.; McConell, G. K. Downstream mechanisms of nitric oxide-mediated skeletal muscle glucose uptake during contraction. American journal of physiology. Regulatory, integrative and comparative physiology 299:R1656-1665; 2010. [241] Merry, T. L.; Dywer, R. M.; Bradley, E. A.; Rattigan, S.; McConell, G. K. Local hindlimb antioxidant infusion does not affect muscle glucose uptake during in situ contractions in rat. Journal of applied physiology (Bethesda, Md. : 1985) 108:1275-1283; 2010. [242] Merry, T. L.; Wadley, G. D.; Stathis, C. G.; Garnham, A. P.; Rattigan, S.; Hargreaves, M.; McConell, G. K. N-Acetylcysteine infusion does not affect glucose disposal during prolonged moderate-intensity exercise in humans. The Journal of physiology 588:1623-1634; 2010. [243] Romijn, J. A.; Coyle, E. F.; Sidossis, L. S.; Gastaldelli, A.; Horowitz, J. F.; Endert, E.; Wolfe, R. R. Regulation of endogenous fat and carbohydrate metabolism in relation to exercise intensity and duration. The American journal of physiology 265:E380-391; 1993. [244] Bradley, S. J.; Kingwell, B. A.; McConell, G. K. Nitric oxide synthase inhibition reduces leg glucose uptake but not blood flow during dynamic exercise in humans. Diabetes 48:1815-1821; 1999. [245] Kingwell, B. A.; Formosa, M.; Muhlmann, M.; Bradley, S. J.; McConell, G. K. Nitric oxide synthase inhibition reduces glucose uptake during exercise in individuals with type 2 diabetes more than in control subjects. Diabetes 51:2572-2580; 2002. [246] Linden, K. C.; Wadley, G. D.; Garnham, A. P.; McConell, G. K. Effect of l-arginine infusion on glucose disposal during exercise in humans. Medicine and science in sports and exercise 43:16261634; 2011. [247] Etgen, G. J., Jr.; Fryburg, D. A.; Gibbs, E. M. Nitric oxide stimulates skeletal muscle glucose transport through a calcium/contraction- and phosphatidylinositol-3-kinase-independent pathway. Diabetes 46:1915-1919; 1997. [248] Higaki, Y.; Hirshman, M. F.; Fujii, N.; Goodyear, L. J. Nitric oxide increases glucose uptake through a mechanism that is distinct from the insulin and contraction pathways in rat skeletal muscle. Diabetes 50:241-247; 2001. [249] Hong, Y. H.; Betik, A. C.; Premilovac, D.; Dwyer, R. M.; Keske, M. A.; Rattigan, S.; McConell, G. K. No effect of NOS inhibition on skeletal muscle glucose uptake during in situ hindlimb contraction in healthy and diabetic Sprague-Dawley rats. American journal of physiology. Regulatory, integrative and comparative physiology 308:R862-871; 2015. [250] Lau, K. S.; Grange, R. W.; Isotani, E.; Sarelius, I. H.; Kamm, K. E.; Huang, P. L.; Stull, J. T. nNOS and eNOS modulate cGMP formation and vascular response in contracting fast-twitch skeletal muscle. Physiological genomics 2:21-27; 2000. [251] Hong, Y. H.; Frugier, T.; Zhang, X.; Murphy, R. M.; Lynch, G. S.; Betik, A. C.; Rattigan, S.; McConell, G. K. Glucose uptake during contraction in isolated skeletal muscles from neuronal nitric oxide synthase mu knockout mice. Journal of applied physiology (Bethesda, Md. : 1985) 118:11131121; 2015.

37

1655 1656 1657 1658 1659 1660 1661 1662 1663 1664 1665 1666 1667 1668 1669 1670 1671 1672 1673 1674 1675 1676 1677 1678 1679 1680 1681 1682 1683 1684 1685 1686 1687 1688 1689 1690 1691 1692 1693 1694 1695 1696 1697 1698 1699 1700 1701 1702 1703 1704

[252] Percival, J. M.; Anderson, K. N.; Huang, P.; Adams, M. E.; Froehner, S. C. Golgi and sarcolemmal neuronal NOS differentially regulate contraction-induced fatigue and vasoconstriction in exercising mouse skeletal muscle. The Journal of clinical investigation 120:816-826; 2010. [253] Merry, T. L.; Steinberg, G. R.; Lynch, G. S.; McConell, G. K. Skeletal muscle glucose uptake during contraction is regulated by nitric oxide and ROS independently of AMPK. American journal of physiology. Endocrinology and metabolism 298:E577-585; 2010. [254] Schoenfeld, B. J. Potential mechanisms for a role of metabolic stress in hypertrophic adaptations to resistance training. Sports medicine (Auckland, N.Z.) 43:179-194; 2013. [255] Handayaningsih, A. E.; Iguchi, G.; Fukuoka, H.; Nishizawa, H.; Takahashi, M.; Yamamoto, M.; Herningtyas, E. H.; Okimura, Y.; Kaji, H.; Chihara, K.; Seino, S.; Takahashi, Y. Reactive oxygen species play an essential role in IGF-I signaling and IGF-I-induced myocyte hypertrophy in C2C12 myocytes. Endocrinology 152:912-921; 2011. [256] Shi, H.; Scheffler, J. M.; Zeng, C.; Pleitner, J. M.; Hannon, K. M.; Grant, A. L.; Gerrard, D. E. Mitogen-activated protein kinase signaling is necessary for the maintenance of skeletal muscle mass. American Journal of Physiology - Cell Physiology 296:C1040-C1048; 2009. [257] Al-Shanti, N.; Stewart, C. E. Ca2+/calmodulin-dependent transcriptional pathways: potential mediators of skeletal muscle growth and development. Biological reviews of the Cambridge Philosophical Society 84:637-652; 2009. [258] Goldfarb, A. H.; Bloomer, R. J.; McKenzie, M. J. Combined antioxidant treatment effects on blood oxidative stress after eccentric exercise. Medicine and science in sports and exercise 37:234239; 2005. [259] Lee, J.; Goldfarb, A. H.; Rescino, M. H.; Hegde, S.; Patrick, S.; Apperson, K. Eccentric exercise effect on blood oxidative-stress markers and delayed onset of muscle soreness. Medicine and science in sports and exercise 34:443-448; 2002. [260] Kondo, H.; Miura, M.; Itokawa, Y. Oxidative stress in skeletal muscle atrophied by immobilization. Acta physiologica Scandinavica 142:527-528; 1991. [261] Moylan, J. S.; Reid, M. B. Oxidative stress, chronic disease, and muscle wasting. Muscle & nerve 35:411-429; 2007. [262] Rando, T. A. Oxidative stress and the pathogenesis of muscular dystrophies. American journal of physical medicine & rehabilitation / Association of Academic Physiatrists 81:S175-186; 2002. [263] Kondo, H.; Nishino, K.; Itokawa, Y. Hydroxyl radical generation in skeletal muscle atrophied by immobilization. FEBS letters 349:169-172; 1994. [264] Powers, S. K.; Kavazis, A. N.; DeRuisseau, K. C. Mechanisms of disuse muscle atrophy: role of oxidative stress. American journal of physiology. Regulatory, integrative and comparative physiology 288:R337-344; 2005. [265] Sellman, J. E.; DeRuisseau, K. C.; Betters, J. L.; Lira, V. A.; Soltow, Q. A.; Selsby, J. T.; Criswell, D. S. In vivo inhibition of nitric oxide synthase impairs upregulation of contractile protein mRNA in overloaded plantaris muscle. Journal of applied physiology (Bethesda, Md. : 1985) 100:258-265; 2006. [266] Hornberger, T. A.; McLoughlin, T. J.; Leszczynski, J. K.; Armstrong, D. D.; Jameson, R. R.; Bowen, P. E.; Hwang, E. S.; Hou, H.; Moustafa, M. E.; Carlson, B. A.; Hatfield, D. L.; Diamond, A. M.; Esser, K. A. Selenoprotein-deficient transgenic mice exhibit enhanced exercise-induced muscle growth. Journal of Nutrition 133:3091-3097; 2003. [267] Zizkova, P.; Blaskovic, D.; Majekova, M.; Svorc, L.; Rackova, L.; Ratkovska, L.; Veverka, M.; Horakova, L. Novel quercetin derivatives in treatment of peroxynitrite-oxidized SERCA1. Molecular and cellular biochemistry 386:1-14; 2014. [268] Adachi, T.; Weisbrod, R. M.; Pimentel, D. R.; Ying, J.; Sharov, V. S.; Schoneich, C.; Cohen, R. A. S-Glutathiolation by peroxynitrite activates SERCA during arterial relaxation by nitric oxide. Nature medicine 10:1200-1207; 2004.

38

1705 1706 1707 1708 1709 1710

[269] Murphy, R. M.; Dutka, T. L.; Lamb, G. D. Hydroxyl radical and glutathione interactions alter calcium sensitivity and maximum force of the contractile apparatus in rat skeletal muscle fibres. The Journal of physiology 586:2203-2216; 2008. [270] Viner, R. I.; Williams, T. D.; Schoneich, C. Peroxynitrite modification of protein thiols: oxidation, nitrosylation, and S-glutathiolation of functionally important cysteine residue(s) in the sarcoplasmic reticulum Ca-ATPase. Biochemistry 38:12408-12415; 1999.

1711 1712 1713

FIGURE LEGENDS

1714 1715

Fig 1. Overview of exercise-induced redox signalling pathways in skeletal muscle

1716 1717 1718 1719 1720 1721 1722 1723

Skeletal muscle contraction during exercise produces increased production of ROS and NO. These reactive species promote redox signalling in part through post-translational modifications (including S-nitrosylation, S-glutathionylation, sulfenylation and disulphide formation) of reactive cysteine thiol groups of target proteins. Redox signalling involves activation of key kinases and phosphatases that are pivotal to acute and chronic responses to exercise. Grx – glutaredoxins; GSH – glutathione; H2O2 – hydrogen peroxide; NO – nitric oxide; O2●─ – superoxide; ONOO─ – peroxynitrite; ;Prx – peroxiredoxins; SH – reactive thiol group; Srx – sulfiredoxins; Trx – thioredoxins.

1724 1725 1726 1727 1728 1729 1730 1731 1732 1733 1734 1735 1736 1737 1738 1739 1740 1741 1742 1743

Fig 2. Proposed effects of exercise-induced ROS and NO on muscle force and power production The left hand side of the figure shows how transient or low-level contraction-induced ROS (mainly via NADPH oxidases) and NO (mainly via nNOS) can increase contractile force/power in skeletal muscle via S-nitrosylation and S-glutathionylation of contraction-related proteins such as RyR1 and Troponin I. Myofibrillar calcium sensitivity is likely increased under these conditions. The right hand side of the figure shows how excessive and/or prolonged exercise-induced ROS/NO exposure can decrease contractile force/power in muscle via hyper-S-nitrosylation, RyR1 remodelling, SR Ca2+ leak and decreased stimulated SR Ca2+ release. Myofibrillar calcium sensitivity is likely decreased under these conditions. Thicker arrows and marks indicate increased levels. Broken arrows from ·OH and ONOO - indicate some uncertainty in relation to contractile force/power production [267-270]. Grx – glutaredoxins; GSH – glutathione; ; H2O2 – hydrogen peroxide; NADPH – nicotinamide adenine dinucleotide phosphate; nNOS – neuronal nitric oxide synthase; NO – nitric oxide, O2●─– superoxide; ●OH – hydroxyl radical; ONOO─ – peroxynitrite; pO2 – partial pressure of oxygen; RyR1 – ryanodine receptor/Ca2+ release channel; SERCA1 – sarco(endo)plasmic reticulum Ca2+-dependent ATPase; SNO – S-nitrosylation; SOD – superoxide dismutase; SSG –S-glutathionylation; Trx – thioredoxins.

1744 39

1745 1746 1747

Fig 3. Proposed redox signaling pathways involved in exercise-induced mitochondrial biogenesis and endogenous antioxidant enzyme induction.

1748 1749 1750 1751 1752 1753

Skeletal muscle contraction during exercise increases production of ROS, AMP and NAD+ and there is an increase in cytosolic Ca2+ levels. Redox signalling involves activation of key kinases such as NFkB, p38 MAPK and possibly AMPK that are pivotal to acute and chronic responses to endurance exercise. AMPK – adenosine monophosphate-activated protein kinase; CAMK II – Ca2+/calmodulin-dependent protein kinase; CAT – catalase; GPx1 – glutathione peroxidase 1; NFκB – nuclear factor kappa B; NRF – nuclear respiratory factor. p38 MAPK – p38 Mitogen-activated protein kinase; PGC-1 – PPAR- coactivator-1 ; ROS – reactive oxygen species; SIRT-3 – sirtuin 3; SOD – superoxide dismutase.

1754 1755 1756 1757

Fig 4. Candidate redox signalling pathways involved in skeletal muscle hypertrophy

1758 1759 1760 1761 1762 1763 1764 1765 1766 1767 1768

ROS and NO produced by muscle contraction, such as via NADPH oxidases and nNOS, have been implicated in signalling pathways involved in skeletal muscle hypertrophy and protein synthesis, including the IGF-1/Insulin/PI3K pathway, MAPK signalling pathways and Ca2+ signalling via activation of TRPV1. 4E-BP – eukaryotic translation initiation factor 4E binding protein 1; Akt – Protein kinase B; ERK1/2 – Extracellular signal-regulated kinases 1/ 2; H2O2 – hydrogen peroxide; IGF-1 –insulin-like growth factor 1; MAPK – Mitogen-activated protein kinase; mTOR – mammalian target of rapamycin; NADPH – nicotinamide adenine dinucleotide phosphate; nNOS – neuronal nitric oxide synthase; NO – nitric oxide; O2●─– superoxide; ONOO─ – peroxynitrite; p70S6K – ribosomal protein S6 kinase; PI3K – Phosphoinositide 3-kinase; TRPV1 – transient receptor potential cation channel, subfamily V, member 1.

1769 1770

Table 1. Summary of effects of discussed antioxidants in skeletal muscle during exercise Antioxidant NAC

Key findings in relation to exercise Human studies using both oral and intravenous NAC show improvements in time tofatigue and improved maintenance of muscle contractile force/power during exercise, particularly during sustained submaximal bouts. Findings of studies using performance tests more representative of athletic competition are conflicting and require additional research. The most optimal NAC dosing prior to a competitive athletic bout is currently unclear. Currently no evidence indicates that NAC infusion impairs muscle glucose uptake, although studies using oral supplementation and higher exercise intensities are warranted. There are potential adverse effects such as gastrointestinal distress with NAC supplementation. However, in the context of fatiguing exercise, acute oral intakes of 70mg/kg body mass or less appear to minimize adverse effects [172].

40

Vitamin C and E

Coenzyme Q10

Resveratrol

Evidence is equivocal for use of vitamin C in relation to muscle contractile force and its recovery after fatiguing exercise. Studies of vitamin E supplementation currently show no evidence of benefit in relation to muscle contractile force in humans. Use of high dose vitamin C (1g/day) alone or in combination with at least 260 IU/day vitamin E impairs some molecular markers of mitochondrial biogenesis and antioxidant enzyme induction. However, doses of 500 mg/day vitamin C do not appear to hamper these measures. Limited evidence in humans and rodents suggest a potential impairment in muscle hypertrophy and associated markers of redox signalling during overload, although additional research is warranted. Despite potential cellular impairments, exercise performance outcomes with supplementation are equivocal. Findings are currently not convincing in terms of improvement in performance and maintenance of power during sustained intense and intermittent exercise with oral supplementation in humans. Well-controlled dose-titrated studies in athletes are required to better evaluate its ergogenic potential. Effects in skeletal muscle are unclear given the apparent poor bioavailability of oral supplementation in human skeletal muscle. Limited evidence suggests that supplementation might impair expression of key genes involved in mitochondrial biogenesis and antioxidant enzyme induction. However, it is less likely that these changes translate into impairments in whole muscle function and whole body performance in humans. Additional studies are required in humans to more thoroughly investigate effects of resveratrol on muscle function with exercise. While systemic bioavailability of oral supplements appears poor, studies using higher but relatively safe doses (i.e. 1-2 g/day) are scant in relation to effects on human muscle performance during exercise Studies are required to investigate the bioavailability and antioxidant actions of resveratrol and its metabolites in human skeletal muscle

1771 1772 1773 1774 1775 1776 1777 1778 1779 1780 1781

41

1782

Figure 1

1783 1784 1785 1786

Figure 2

1787 1788 1789 1790 1791 1792 1793

42

1794

Figure 3

1795 1796 1797

Figure 4

1798 1799 1800 1801 1802 43

1803 1804 1805

Highlights Redox signalling in muscle is implicated in acute and chronic responses to exercise

1806

Some antioxidants can modulate redox state and thus some of the responses to exercise

1807

Clear evidence of effects in human muscle are lacking for most exogenous antioxidants

1808

NAC (≤70 mg/kg) might be ergogenic & safe for a fatiguing bout of prolonged exercise

1809

Doses of ≥ 1g/d vitamin C with ≥ 260IU/d vitamin E hampers some training effects

1810 1811 1812

44

Muscle redox signalling pathways in exercise. Role of antioxidants.

Recent research highlights the importance of redox signalling pathway activation by contraction-induced reactive oxygen species (ROS) and nitric oxide...
566B Sizes 0 Downloads 8 Views