Exercise-induced oxidative stress: past, present and future

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Exercise-induced oxidative stress: past, present and future

Scott K. Powers1, Zsolt Radak 2 and Li Li Ji3

1Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, FL 32608, USA 2 Research Institute of Sport Science, University of Physical Education, Budapest, Hungary 3School of Kinesiology, University of Minnesota, 111 Cooke Hall, 1900 University Avenue, Minneapolis, MN 55455, USA

Abstract The existence of free radicals in living cells was first reported in 1954 and this importantfinding helped launch the field of free radical biology. However, the discovery that muscularexercise is associated with increased biomarkers of oxidative stress did not occur until 1978.Following the initial report that exercise promotes oxidative stress in humans, many studies haveconfirmed that prolonged or short-duration high intensity exercise results in increased radicalproduction in active skeletal muscles resulting in the formation of oxidized lipids and proteins inthe working muscles. Since these early descriptive studies, the investigation of radicals and redox biology related to exercise and skeletal muscle has grown as a discipline and the importance of this research in the biomedical sciences is widely recognized. This review will briefly summarizethe history of research in exercise-induced oxidative stress and will discuss the major paradigmshifts that the field has undergone and continues to experience. We conclude with a discussion of future directions in the hope of stimulating additional research in this important field.

(Received 1 July 2015; accepted after revision 6 November 2015)

Corresponding author S. K. Powers: Department of Applied Physiology and Kinesiology, University of Florida, Room

25 Florida Gym, Gainesville, FL, 32611, USA. E-mail: [email protected]

Introduction

The finding that living cells contain free radicals(i.e. radicals) was first reported in 1954 (Commoneret al. 1954). This landmark study combined with work suggesting that ionizing radiation damages cells via freeradicals launched a new field of biological researchfocusing on free radicals and cellular oxidoreductive(redox) balance. The discovery that muscular exerciseincreases oxidant damage in humans and other animalsdid not occur until the late 1970s (Dillard et al. 1978;Brady et al. 1979). Although the biological significanceof this early finding was unclear, these pioneeringstudies generated interest for future investigations to

ScottK.Powersisa professorin theDepartmentof Applied PhysiologyandKinesiology

at the University of Florida. Research in the Powers laboratory is focused on the cell

signaling pathways responsible for disuse muscle atrophy. Li Li Ji is Professor and

Director in the School of Physiology, University of Minnesota Twin Cities.His research

focuses on the roles of reactive oxygen species and antioxidants in exercise-induced

adaptation and redox signaling in the skeletal muscle and heart and during aging.

ZsoltRadak is a professor and head of the Sport and Natural Science Research Center

at Physical Education University in Budapest, Hungary. Dr. Radak also serves as a

visiting professor at the Faculty of Sport Sciences of Waseda University, Japan. His

primary research interest is in the field of exercise physiology, and especially the role of reactive oxygen species on skeletal muscle and brain function.

Moreover, the Radak group is also studying epigenetics and ROS signaling in cells.

examine the important role that radicals, reactive nitrogenspecies (RNS), and reactive oxygen species (ROS) play

in skeletal muscle and other metabolically active organsduring exercise. Indeed, growing evidence reveals thatwhile uncontrolled production of RNS and ROS candamage cells, intracellular oxidants also play importantregulatory roles in the modulation of skeletal muscleforce production, regulation of cell signalling pathways,and control of gene expression (Droge, 2002; Powers &Jackson, 2008; McClung et al. 2009a ,b ; Powers et al. 2010).

Many advances in the field of exercise and oxidativestress have occurred during the past decades and thisspecial issue of The Journal of Physiology provides astate-of-the-art update on discoveries that have greatly

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2 S. K. Powers and others J Physiol 000.0

impacted our understanding of this rapidly changingfield. Key topics addressed in this special issue include aredox modulation of muscle force production, the impactof high altitude on exercise-induced oxidative stress,ageing-induced changes in muscle redox biology, anti-oxidant interventions to improve exercise performance,andother important topics germane to the field of exercise

and oxidative stress.The objective of the current paper is to provide a

prelude to this special edition of The Journal of Physio-logy by delivering a historical overview of the researchprogress related to exercise and oxidative stress. By necessity, this review is a ‘brief’ summary of key events inthis discipline, and therefore, we apologize to colleagueswho completed studies that contributed to this fieldbut have been omitted because of space limitations.Moreover, similar to other historical overviews of ascientific field, this account is written from the authors’personal points of view. We will begin with a historical

recap of the birth of free radical biology and will thenchronicle the research progress in the field of exerciseand oxidative stress. We will conclude by discussingfuture directions for the field and exposing unansweredquestions that hopefully will provide a stimulus for futureresearch.

In the beginning – the birth of free radical biology

The existence of the electron (originally called acorpuscle) was first reported by J. J. Thomson in1897 (Griffiths, 1997). The discovery of electrons pavedthe way for Moses Gomberg to successfully synthesizetriphenylmethyl, which was the first moderately stableradical (Gomberg, 1900). This work was importantbecause most chemists of the day did not believe thatradicals could exist independently (Tomioka, 1997).Unfortunately, Gomberg’s report about the existence of free radicals was not widely accepted for 30 years followinghis initial publication. During the decade of the 1930s,Haber and Willstatter first proposed the existence of hydroxyl radicals and this suggestion was followed by theHaber and Weiss finding that hydroxyl radicals can begenerated by the interaction between hydrogen peroxideand superoxide (Hensley & Floyd, 2002). The discovery of

this now well-known chemical reaction (the Haber–Weissreaction) brought additional acceptance to the conceptthat free radicals can exist in solution.

By the early 1950s, radicals were accepted asindependent chemical entities in scientific fields outsideof biology (Hensley & Floyd, 2002). For example, duringthe 1940s, most scientists who studied free radicals werechemists interested in the organic synthesis of novelcompounds. This interestwas driven by global demandforrubber products, and because free radical chain reactionswere important to synthesize polyethylene, radicals were

studied for their potential to produce rubber (Hensley &Floyd, 2002).

The birth of free radical biology did not occur until1954 when two important and independent reportssuggested that radicals can be important in biology.Together, these studies stimulated interest in radicals andlaunched free radical biology as a field of study. Note that

the events of World War II played an important role inthe birth of free radical biology (Hensley & Floyd, 2002).Indeed, the radiation poisoning and radiation-inducedmutations resulting from the explosion of atomic bombsduring World War II stimulated scientific attentiontoward understanding the mechanisms responsible forthe illness associated with exposure to high levels of radiation. In this regard, Gershman and colleagues werethe first to hypothesize that the cellular damage causedby exposure to ionizing radiation was due to free radicals(Gerschman et al. 1954). Moreover, they also predictedthat the biological injury that occurs when mice are

exposed to hyperoxia is due to the formation of radicals.Without question, this key report inspired scientificinterest in the concept that free radicals play importantroles in cellular injury in response to both ionizingradiation and hyperoxia.

The second report that played a major role in the originof free radical biology was published by Barry Commonerand colleagues in 1954. At this time, it was establishedthat ionizing radiation could generate free radicals inaqueous solution but it was unknown that living cellsproduced radicals. Using the newly developed techniqueof electron paramagnetic resonance, Commoner andcolleagues demonstrated that living cells contain freeradicals (Commoner et al. 1954). This seminal paperprovided the first proof that free radicals exist in livingorganisms.

After the reports by Gerschman et al. and Commoneret al. launched free radical biology as a field of study,several additional events occurred in the 1950s and1960s to solidify the importance of free radicals inbiology. For example, Britton Chance and colleagues firstreported that respiring mitochondria produce hydrogenperoxide (H2O2) (Chance & Williams, 1956). This findingestablished that mitochondria are a source of oxidants inthe cell and that ROS (i.e. H2O2) are likely to be diffusible

within cells. Another report that accelerated the interest infree radicalbiology wasthe introduction of the‘free radicaltheory of ageing’ (Harman, 1956). This landmark paperby Denham Harman proposed that the rate of cellularageing is directly related to radical-mediated damage tocellular components. Without question, the free radicaltheory of ageing provided significant motivation for many life scientists to investigate the effects of radicals on livingorganisms.

Although Harman’s free radical theory of ageing wasan attractive concept to explain cellular ageing, the lack of


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experimental techniques available to measure radicals andoxidative damage delayed scientific progress in free radicalbiology in the 1950s and 1960s because it was difficultto prove that radicals were responsible for ageing anddiseases. Hence, many biologists remained skeptical of thecausal role of radicals in natural disease processes becauseof the persistent doubt that radicals exist in solution

without the extreme conditions imposed by ionizingradiation (Hensley & Floyd, 2002). Nonetheless, a majorbreakthrough occurred in 1969 when Joe McCord andIrwin Fridovich discovered that the previously discoveredproteinerythrocuprein catalyses the dismutation of super-oxide radicals (McCord & Fridovich, 1969a ,b ). Followingthis important discovery, McCord and Fridovich namedthe enzyme superoxide dismutase (SOD) (McCord &Fridovich, 1969a ). This milestone discovery of SOD1(i.e. copper, zinc-superoxide dismutase) is credited withproviding the first convincing evidence that biologicalROS exist in solution and that oxygen radicals are likely

to play an important role in cell biology (Bannister, 1988;Bannister & Bannister, 1988). Figure 1 summarizes themajor historical steps leading to the birth of free radicalbiology during the 1950s and 1960s.

Exercise and oxidative stress – four decades

of research progress

Almost four decades have passed since the discovery that muscular exercise promotes an increase in oxidativebiomarkers in humans. In the following segments weprovide a summary of key scientific discoveries in this

field beginning with the finding that exercise is associatedwith oxidative stress.

Discovery that exercise is associated with biomarkers

of oxidation. The discovery that muscular exercise isassociated with oxidant stress in humans was reportedby Dillard et al. (1978). This landmark study revealedthat 60 min of endurance exercise at 50% of V̇ O2max results in increased expired pentane (a biomarkerof lipid peroxidation) and that supplementation with

the antioxidant vitamin E reduced both resting andexercise-induced pentane production. These investigatorsconcluded that muscular exercise promotes increasedoxidant production but the organs responsible for oxidantproduction remained unknown. One year later, Brady et al. confirmed that swimming exercise is also associatedwith increased lipid peroxidation in rats (Brady et al.1979). Since these early studies, many investigatorshave demonstrated that prolonged endurance exercise orshort-duration, high intensity exercise results in increasedbiomarkers of oxidative stress in both blood and skeletalmuscle in humans and other animals. The remainder

of this report will summarize key research findings intothe mechanisms and consequences of exercise-inducedoxidative stress in the decades following the discovery of exercise-induced oxidative damage.

Decade of the 1980s – discovery that skeletal muscles

produce oxidants and more. The finding that exerciseis associated with an increase in biomarkers of lipidperoxidation stimulated further curiosity in the fieldof exercise and oxidative stress. This increased interestresulted in the discovery that contracting skeletal musclesproduce oxidants and the first definition of the term‘oxidative stress’. Moreover, groundbreaking studies also

revealed that regular bouts of exercise result in adaptivechanges in cellular antioxidants and that vitamin E plays

Key steps leading to the birth and earlyevolution of free radical biology

Gerschman et al.

Radicals responsiblefor cell injury during

exposure to hyperoxiaand ionizing radiation

1954

1954 1956

Chance et al.

Mitochondriaproduce ROS

19561969

McCord and Fridovich

Discovery of superoxidedismutase 1-provides

evidence that ROS existin solution

Commoner et al.

Radicals existin living cells

Harman

Free radicaltheory of aging

1950 1970

Figure 1. Key steps leading to the birth

and early development of the discipline of

free radical biology




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an important role in protecting muscle membranes fromoxidant damage. A brief historical overview of these key findings follows.

Discovery that contracting skeletal muscle generates

radicals. Four years following the finding that exerciseis associated with increased biomarkers of lipid

peroxidation, Kelvin Davies and colleagues, using electronspin resonance, madethe importantdiscovery thatcontra-cting skeletal muscles produce ROS and that vitamin Edeficiency exacerbates ROS production in both liver andmuscle (Davies et al. 1982). These results were confirmedby Malcolm Jackson and colleagues (Jackson et al. 1985).

Exercise, vitamin E and endogenous antioxidant systems.

Shortly after the discovery that contracting musclesproduce radicals, Lester Packer and colleagues from theUnited States and a research team from the UnitedKingdom reported two important observations that

generated further interest in the field of exercise andoxidative stress (Jacksonet al. 1983; Quintanilha & Packer,1983). Specifically, this work revealed that vitamin Edeficiency in rats results in a greater susceptibility of cellular membranesto exercise-induced oxidative damage.Moreover, Quintanilha and Packer discovered that end-urance exercise training increases the levels of anti-oxidant enzymes in both cardiac and skeletal muscles(Quintanilha & Packer, 1983). This investigation alsorevealed the importance of vitamin E in protectingbiological membranes from exercise-induced oxidativedamage and provided the first evidence that the end-ogenous antioxidant systems of both cardiac and skeletal

muscle are plastic and adapt in response to exercisetraining. Numerous studies quickly confirmed thatexercise training promotes an increase in key antioxidant

enzymes in skeletal muscles and that exercise-inducedoxidant production is likely to contribute to the allostericdown-regulation of theactivitiesof keymetabolicenzymes(e.g. citrate synthase and malate dehydrogenase) (Higuchiet al. 1985; Kanter et al. 1985; Ji et al. 1988a ,b ).

‘Oxidative stress’ defined. The term oxidative stress

was first defined in 1985 as ‘a disturbance in thepro-oxidant–antioxidant balance in favor of the former’(Sies & Cadenas, 1985). Although this definition waswidely accepted for over two decades, this description of oxidative stress has undergone scrutiny, and changes tothe definition of oxidative stress have been proposed (Azziet al. 2004; Jones, 2006). Subsequently, Dean Jones andHelmut Sies collaborated to provide a refined definitionof oxidative stress as ‘an imbalance between oxidantsand antioxidants in favor of the oxidants, leading toa disruption of redox signalling and control and/ormolecular damage’ (Sies & Jones, 2007). A summary of

the key discoveries in this field of exercise and oxidativestress during1980–1989 are summarized in Fig. 2.

Decade of the 1990s – discovery that redox balance

regulates muscle force production and much more.

Research during the 1980s established a foundation of basic knowledge about exercise and oxidative stress. Thisearly work provided the basis for studies during the 1990sthat greatly expanded our understanding of the role thatradicals play in cardiac and skeletal muscle biology. Majordiscoveries related to muscle and oxidants that occurredduring decade of the 1990s are highlighted in the nextsegments.

Radicals contribute to muscle fatigue and modulate

muscle force production. The discovery that radicals

Davies et al.

Contracting skeletalmuscles produce

radicals

Progress in exercise and oxidativestress research: 1980-1989

Quintanilha and

Packer

Exercise increasesantioxidant enzymes

Quintanilha & Packerand Jackson et al.

Vitamin E deficiencypromotes

exercise-inducedoxidative damage

Sies

Oxidative stressdefined

1980 1989

1982 1983

1983 1985

Figure 2. Progress in exercise and oxidative

stress research: decade of the 1980s




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contribute to muscle fatigue in animals (mice and rabbits)was first reported by two independent investigationsin 1990 (Novelli et al. 1990; Shindoh et al. 1990).These initial findings were rapidly supported in a variety of experimental models using non-human mammalianskeletal muscle (Barclay & Hansel, 1991; Reid et al.1992a ; Diaz et al. 1994; Khawli & Reid, 1994). Four

years after the first report that radicals promote musclefatigue in animals, Reid and colleagues confirmed thatoxidants contribute to muscle fatigue in humans duringprolonged, electrically stimulated leg exercise (Reid et al.1994). Further, numerous studies using isolated musclepreparations also confirmed that exposure of skeletalmuscle fibres to oxidants results in impaired muscle forceproduction (Andrade et al. 1998; Clanton et al. 1999).Together, these studies stimulated further interest in therole that radicals play in skeletal muscle fatigue and many reports on this topic have emerged during the past twodecades.

Shortly after the discovery that radicals contribute tomuscle fatigue, Michael Reid and colleagues proposedthat the redox status of muscle fibres plays an importantrole in the modulation of muscle force production (Reidet al. 1993). Specifically, this prediction was based onexperiments indicating that maximal force productionin skeletal muscle fibres occurs at an optimal redox state. It follows that movement away from this optimalredox condition results in lower muscle force production.These studies led to the development of the now well-known ‘inverted U’ curve describing the relationshipbetween redox status and muscle force generation.Together, the discoveries thatradicals contribute to musclefatigue and that muscle redox balance modulates muscleforce production served as a major stimulant for new investigators to enter the field of exercise and oxidativestress.

Contracting skeletal muscle fibres release superoxide

radicals. The discovery that contracting rodent skeletalmuscle releases superoxide radicals into the interstitialspace was first reported in 1992 (Reid et al. 1992b ). Theseprovocative observations stimulated numerous questionsabout the site of superoxide generation in contractingmuscle and raised the question of whether superoxide

radicals are capable of crossing cell membranes. Anotherlandmark study published in the decade of the 1990srevealedthat hydroxyl radicals are producedin contractingskeletal muscles and that the rate of radical productionincreased in proportion to thepercentage of maximal forceproduction (O’Neill et al. 1996). Experimental interest inthe site(s) of radical production in contracting musclefibres continues to this day.

Impact of acute and chronic exercise on muscle anti-

oxidant capacity. Although studies on the impact of

exercise on skeletal muscle antioxidant systems beganin the1980s, research in the early 1990s produced new findings that detailed the exercise dose–response effecton the levels of endogenous antioxidant enzymes in bothcardiac and skeletal muscle. Specifically, numerous studiesconfirmed that exercise training promotes an increasedin primary antioxidant enzymes in cardiac and skeletal

muscle and that this adaptation increases as a functionof exercise intensity and duration (Hammeren et al.1992; Criswell et al. 1993; Powers et al. 1993,1994a ,b ).Studies also began to unravel the impact of exerciseon the glutathione redox system (Sen et al. 1992, 1994;Leeuwenburgh et al. 1994, 1997; Ji, 1995). Moreover, it wasdiscovered that an acute bout of intense exercise resultedin a depression in the activities of several antioxidantenzymes (Lawler et al. 1993,1994). These discoveriesfuelled further interest in understanding those factors thatregulate redox balance in contracting skeletal muscles.

Exercise-induced increases in superoxide dismutase

2 contributes to cardioprotection. Numerous studiesduring the 1990s established that endurance exercisetraining results in a cardiac myoctye phenotypethat resists ischaemia–reperfusion injury (i.e. cardio-protection; reviewed in Powers et al. 2014). Nonethelessthe mechanism(s) responsible for this exercise-inducedcardioprotection remained unknown until 1999.Specifically, Yamashita et al. demonstrated anexercise-induced increase in SOD2 in cardiac myo-cytes is required to achieve optimal cardioprotectionagainst ischaemia–reperfusion injury (Yamashita et al.

1999). This finding was later confirmedby an independentlaboratory (French et al. 2008). Together, these resultsconfirm that an exercise-induced change in a key anti-oxidant enzyme plays a required role in exercise-inducedcardioprotection. Figure 3 illustrates research progressin the field of exercise and oxidative stress during1990–1999.

Detection of NOS in muscle and NO production in muscle.

Another important discovery that occurred during thedecade of the 1990s was the finding that skeletal muscleexpresses two isoforms of nitric oxide synthase (NOS)

and that contracting muscles produce nitric oxide (NO)(Balon & Nadler, 1994; Kobzik et al. 1994; Radak et al.1999). The discovery that muscle fibres contain more thanone isoform of NOS and that these isoforms are located indifferent regions of the muscle fuelled speculation aboutthe role that NO plays in muscle biology. Moreover, thediscovery that NOSwas localized near thesarcolemmaanddystrophin complex would later prove to be an importantfinding with implications for some forms of musculardystrophy (reviewed in Tidball & Wehling-Henricks,2014).




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Paradigm shift – radicals recognized as signalling

molecules in skeletal muscle. From the initial discovery of exercise-induced oxidative stress (1978) through mostof the 1980s, the radicals produced in contracting musclewere widely considered to be damaging molecules.However, this concept began to change with the discovery that NO is the signalling molecule responsible for vaso-

dilatation (Palmer et al. 1987). Although this signallingrole for NO became recognized in 1987, the concept thatselect ROS participate as signal transduction messengersand regulate gene expression was not fully accepted untilthe decade of the 1990s. This paradigm change in theconcept that ROS are only damaging agents was discussedin a classic review that summarized the evidence that bothROS and RNS are important agents in the regulation of gene expression and cellular homeostasis (Sen & Packer,1996).

Decade of 2000–2009 – research on radicals as signalling

molecules matures. The decade of 2000–2009 broughtseveral new and important findings to the field of exerciseand oxidative stress. A few of the major discoveries arehighlighted in the next segment.

Mechanisms contributing to redox modulation of muscle

force production. The fact that oxidants play animportant role in the modulation of skeletal muscle forceproduction was established in the 1990s and progresstoward understanding the mechanisms responsible foroxidant-mediated control of muscle force productioncontinued during the decade of 2000–2009. For example,using single intact skeletal muscle fibres, research in the

early 2000s revealed that relatively high levels of H2O2do not impair calcium release from the sarcoplasmicreticulum but decrease myofibrillar calcium sensitivity

and depress the force generated per cross-bridge (Plantet al. 2000; Andrade et al. 2001). Nonetheless, the questionof whether H2O2 impacts calcium release from thesarcoplasmic reticulum remains a topic of debate (Brotto& Nosek, 1996).

Discovery that N -acetylcysteine improves human exercise

performance. As discussed earlier, the fact that radicalscontribute to skeletal muscle fatigue was confirmed in theearly1990s. Nonetheless, numerousstudies duringthe late1990s and early 2000s concluded that supplementationwith common dietary antioxidants (e.g. vitamins C orE) does not improve exercise performance (Powers et al.2004). Although the observation that infusion with theantioxidant N -acetylcysteine (NAC) can improve humanrespiratory muscle performance was reported in 1997(Travaline et al. 1997), the first studies to confirm thatNAC can improve voluntary exercise performance inhuman limb muscles did not appear until 2004–2006

(Medved et al. 2004; McKenna et al. 2006). These studiesrevealed that although NAC can improve human exerciseperformance during submaximal exercise (e.g. 60–80%V̇ O2peak ), NAC does not improve exercise tolerance duringhigh intensity exercise (i.e. 90% V̇ O2peak ) (Medved et al.2003; Matuszczak et al. 2005).

ROS is required to promote exercise training response

in muscles. Davies and colleagues first suggested thatROS production could be a stimulus for skeletal muscleadaptation to exercise (Davies et al. 1982). Since thisearly prognostication, many investigations have providedevidence to support this forecast. For instance, inhibiting

xanthine oxidase activity in vivo during acute exercise inrats prevents the exercise-induced activation of signallingpathways leading to exercise-induced adaptations in the


Radicals contributeto muscle fatigue

1990

Detection of nitric oxideproduction in

skeletal muscles1994

Paradigm shift: radicals

recognized as signallingmolecules

1995-1999

Contracting musclesrelease superoxide

radicals

Exercise dose-responseeffect on muscle

antioxidant systems

1993-1999

SOD2 is keyplayer mediatingexercise-inducedcardioprotection

1999

1999

1992

1990


stress research: 1990–1999




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maintain cellular oxidant–antioxidant homeostasisduringexercise (Radak et al. 2005; Ji et al. 2006). For example, anacute bout of exercise promotes expression of superoxidedismutase 2 (SOD2) in rat skeletal muscle mitochondriaresulting in increased SOD2 activity following exercisetraining (Hollander et al. 2001). Given that SOD2 playsa key role in prevention of superoxide accumulation

in the cell, the exercise-induced upregulation of SOD2in skeletal muscle illustrates the hormetic principleof adaptation. The concept of exercise and hormesiscontinues to receive experimental attention by numerousinvestigative groups. Figure 4 summarizes the researchprogress in the field of exercise and oxidative stress during2000–2009.

Decade of 2010 to the present – research in the redox

biology of skeletal muscle reaches new heights. As wereach themidway point of the2010–2019 decade, advancesin the field of redox biology are providing new tools and

ideas for researchers in the field of exercise and oxidativestress. Key recent developments are highlighted in thenextsections.

Advances in understanding redox control systems.

Improving our understanding of the balance betweenexercise-induced oxidative damage and ROS-dependentadaptive signalling in cardiac and skeletal muscle remainsan important topic in the field of exercise and oxidativestress (Radak et al. 2013). Indeed, interest in redox controlsystems has rapidly grown during the past 5 years andhas led to the concept that redox switches regulate proteinfunction by cysteine modifications (Go & Jones, 2013a ,b ;

Jones& Sies, 2015). In this regard, recent advances in redox proteomics and the understanding of oxidizable thiols willundoubtedly present new opportunities to appreciate the

role of ROS in mediating exercise-induced adaptations incardiac andskeletalmuscles fibres (McDonaghetal. 2014).

Emerging field of exercise epigenomics. Interest in theimpact of exercise epigenetics has grown rapidly duringthe past 5 years (Denham et al. 2014; Pareja-Galeanoet al. 2014). Moreover, the fact that redox regulation of

epigenetic pathways can occur by DNA methylation andby post-translational modification of histones suggeststhat exercise-induced ROS production is likely to bean important player in epigenetic events (Mikhed et al.2015). Nonetheless, the intricate mechanism(s) linkingexercise-induced ROS production to epigenetic eventsin skeletal muscle and other tissues remains largely unresolved.

Site of ROS production in contracting skeletal muscles.

The sites of ROS production in contracting skeletalmuscles have remained a controversial topic for over three

decades. During the 1980s–90s it was widely assumed thatmitochondria were the dominant site of ROS productionin contracting muscles (Pearson et al. 2014; Sakellariouet al. 2014). In the past, it has been difficult to discernthe sub-cellular sources of ROS in contracting musclesbecause of inadequate analytical techniques available tostudy this problem (Pearson et al. 2014). Nonetheless,recent advances in the field indicate that mitochondria arenot the dominant source of ROS in contracting skeletalmuscle (Sakellariou et al. 2013; Goncalves et al. 2015)and that NADPH oxidase is likely to play a key rolein contraction-induced production of ROS (Sakellariouet al. 2013). Indeed, mitochondria produce more ROS in

state 4 (i.e. the resting state) than in the ADP-stimulatedstate 3 respiration (Powers et al. 2011; Goncalves et al.2015); this finding alone suggests that mitochondria are


Advances inunderstanding redox

control systems

Sites of ROSproduction incontracting

skeletal muscle

Emergence ofexercise

epigenomics

2010 2015

2010-2015

2010-2015 2013-2015


stress research: 2010–2015




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not the primary source for ROS production in contra-cting skeletal muscles. Figure 5 illustrates the key stepsin research progress in the field of exercise and oxidativestress during 2010–2015.

Future directions

Clearly, research during the past 30+ years has advancedour understanding of exercise-induced oxidative stress.Nonetheless, significant gaps in our knowledge of thisfield remain and several roadblocks to progress exist.For example, the lack of precision tools to study theevents that connect ROS production and redox signallingin the cell remains a major barrier for advancementin this field. Although numerous redox-sensitive probescurrently exist, further development of quantitative andsensitive ROS probes is needed to monitor the sites of ROS production in cells. Further, although progress hasbeen made in tools to study redox signalling, additionaladvances in methodology for assessing components of

redox signalling is essential to identify the precise redox signalling pathways that contribute to skeletal muscleadaptation in response to exercise training.

As discussed earlier in this report, interest in the impactof exercise on epigenetic events has increased in recent years. Nonetheless, many unanswered questions remainand the field of exercise epigenomics is ripe for new andimportant discoveries related to how exercise-inducedincreases in ROS production contribute to epigenomicevents in both cardiac and skeletal muscle as well as othertissues of the body.

As mentioned earlier, the ongoing advances in

redox proteomics will provide new opportunitiesfor investigators to explore the specific role of ROS in mediating post-translational modifications of redox-sensitive proteins. Indeed, as methods becomemore robust, the field of redox proteomics has thepotential to greatly improve our understanding of how exercise-induced post-translational modification of redox-sensitive molecules contribute to cell signallingpathways.

Most of the previous research on redox signallingin skeletal muscle has used one of two experimentalapproaches: (1) Inhibition of a redox-sensitive pathway using a chemical (usually an antioxidant or reductant); or2) deletion (knockout) of a gene that presumably playsa critical role in the signalling pathway. Each of theseapproaches has limitations that have hampered progressin this field. Clearly, new and innovative approaches arerequired to improve our knowledge in this arena. In thisregard, recent advances in gene transfection techniqueswill provide opportunities for investigators to explorecomplex questions in the field of redox signalling in cells.Indeed, the ability to transfect the gene of interest in aspecific tissue provides significant prospects for future

advances in our understanding in the redox signallingfield. Clearly, there is much more to be learned in theexciting field of exercise and oxidative stress.

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Exercise-induced oxidative stress: past, present and future

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