1.1. INTRODUCTION

An atom or a group of atoms that contains one or more unpaired electron(s) is termed a free radical, which is often a highly reactive and unstable molecule. Two groups of these radical molecules are often classified as reactive oxygen and reactive nitrogen species, respectively. Stabilisation of these radicals requires electron donation from proteins, lipids and DNA which oftentimes leads to degradation and damage to these molecules. Owing to the potential for cellular damage, much controversy was created by initial reports that indicated that physical exercise increased the production of reactive oxygen species (ROS) (Dillard et al. 1978). This initial work did not reveal the specific location, but later work revealed the contracting skeletal muscle to be a prominent source of ROS (Davies et al. 1982). Years later, it was also revealed that contracting muscles also produced nitric oxide (NO), the predominant parent molecule of reactive nitrogen species (Balon and Nadler 1994), and a number of well-constructed review articles since then have confirmed the contribution of skeletal muscle to the production of both ROS and reactive nitrogen species (Powers and Jackson 2008, Jackson 2009, Powers et al. 2011).

The most abundant biological free radicals are formed when oxygen or nitrogen is incompletely reduced, leading to the production of superoxide () and NO, processes which will be explained in greater detail later in the chapter. The superoxide parent molecule can subsequently be converted into other ‘radicals’, namely hydrogen peroxide (H₂O₂) and the hydroxyl radical (•OH). Removal of ROS is managed by a host of antioxidant systems (e.g. catalase, glutathione/thiol regulation) in the body, and the balance of oxygen species to antioxidants is termed the ‘redox state’. As mentioned previously, dysregulation of the redox state results in radical scavenging of key biomolecules such as proteins, lipids (cell membranes are a common target) and DNA, a process which can leave them damaged and unable to function. For these reasons, early theories in the 1980s and 1990s led to the belief that ROS production was mostly a negative consequence of physical exercise. Furthermore, evidence began to mount that a number of clinical situations such as heart disease, amyotrophic lateral sclerosis, irritable bowel disease, diabetes and ageing were a consequence of excessive ROS production and free radical damage (Sies 1985, Powers and Jackson 2008, Jackson 2009, Tsutsui et al. 2011).

Recent perspectives, however, have begun to highlight the fact that both oxygen and nitrogen species exert a key role in the regulation of many intracellular mechanisms and also contribute significantly to various cellular signalling pathways involved with muscle adaptation. For example, several studies and review articles have highlighted the fact that controlled production of both reactive species contribute to mitochondrial biogenesis, angiogenesis, skeletal muscle hypertrophy and proper immune function (Ji et al. 2006, Jackson 2009, Powers et al. 2010, 2011). In this respect, it appears that maintaining a proper balance between radical production and removal is a vital physiological process in the body. The purpose of this chapter is first to briefly explain the main pathways in the human body, which lead to free radical production, and then to highlight the impact of free radical regulation in both cardiac and skeletal muscle tissues. It is these pathways upon which many of the proposed theories for antioxidant regulation occur through manipulation of training, environment, diet or supplementation of the diet with ingredients purported to favourably alter the cellular antioxidant milieu.

1.2. PRIMARY CELLULAR SYSTEMS OF RADICAL GENERATION

1.2.1. Mitochondrial Electron Transport Chain Leaking

The electron transport chain is a four protein complex that uses the reduction potential of molecular oxygen to create an electrical gradient to drive ATP regeneration. Electrons are delivered to complexes I and II by NADH and FADH₂, respectively, and the movement of electrons down the chain is controlled by the reduction potential of each successive complex. Molecular oxygen has the highest reduction potential, and is the final electron acceptor in the chain, combining with two protons to create water.

Oxygen consumed by the electron transport chain may undergo one electron reduction, mainly during the corresponding transport of electrons through components I, II and III. NADH dehydrogenase-coenzyme Q (complex I) accepts electrons from NADH where coenzyme Q is reduced and serves as an electron carrier and transports electrons to cytochrome reductase (complex III). Succinate dehydrogenase-coenzyme Q (complex II) accepts electrons from FADH₂ where another coenzyme Q is reduced and transports the electrons to complex III. The cytochrome reductase complex contains cytochrome-b, and -c, along with an iron-containing protein. The cytochromes function as electron-transferring proteins that oxidise coenzyme Q, thus advancing the electrons to cytochrome oxidase (complex IV). Cytochrome oxidase removes the electrons from cytochrome-c, and transfers them to molecular oxygen along with two protons to create water molecules. Evidence suggests that when the reduced form of coenzyme Q (UQH₂) delivers electrons to complex III and is reconverted to the oxidised form (UQ), it passes through a semiquinone anion free radical state (UQ^•-) (Boss et al. 1998, Becker et al. 1999, Ascensao et al. 2005). Oxygen will accept an electron from the unstable UQ^•-, and become partially reduced forming a superoxide anion (). In addition, generation has been shown to occur at high levels in complexes I and III (Ide et al. 1999, Drose and Brandt 2012). The inadequate transfer of electrons from complexes I and III requires oxygen reduction mainly through the coenzyme Q redox state as previously reviewed (Muller et al. 2004, Xu et al. 2009, Gomes et al. 2012). It is thought that superoxide generation from complex I migrates towards the mitochondrial matrix, where it is released from complex III and moves into the matrix and inner membrane space (Muller et al. 2004).

In eukaryotic cells, superoxide () production is mainly controlled at the site of the mitochondria by a well-equipped antioxidant system (Ascensao et al. 2005). It is important to remember, however, that the contribution of this system is somewhat dependent on the tissue(s) involved, as recent evidence seems to indicate that superoxide production inside the mitochondria of skeletal muscle cells is limited (Powers et al. 2010, 2011). Dismutation of superoxide occurs spontaneously or through catalytic conversion into H₂O₂ using the superoxide dismutase (Cu- or MnSOD) enzyme (Figure 1.1) (Halliwell and Gutteridge 2008). H₂O₂ is considered to be a non-radical and a weak oxidant with a relatively long half-life; a characteristic that sufficiently allows for it to readily diffuse throughout cells and across cell membranes (Halliwell and Gutteridge 2008, Powers et al. 2010). H₂O₂ is further scavenged by the enzymes glutathione peroxidase (GPX) and catalase to produces water (Figure 1.2). Glutathione holds a higher affinity for H₂O₂ than catalase and thus exerts a greater antioxidant effect (Le et al. 1993, Tsutsui et al. 2011). However, when superoxide is produced at high levels, which may occur under conditions of accelerated respiratory chain activity (i.e. exercise), levels may exceed the antioxidant capacity of these enzymes. When this occurs, high levels of H₂O₂ are formed and can be further reduced to a hydroxyl radical (•OH) either by the Fenton reaction in the presence of iron or the Haber–Weiss reaction (Tsutsui et al. 2011). The hydroxyl radical is the most potent ROS, and is capable of damaging carbohydrates, lipids and DNA (Lipinski 2011).

FIGURE 1.1

Superoxide dismutase reaction showing the two-step dismutation of the superoxide anion to hydrogen peroxide and oxygen.

FIGURE 1.2

Glutathione and catalase reactions resulting in the scavenging of H₂O₂.

The production of the superoxide anion alone can lead to molecular damage without the conversion to a hydroxyl radical. In this respect, superoxide dismutase-deficient mice have been shown to develop higher levels of amyloid-β plaque, a major contributor to Alzheimer’s disease (Massaad et al. 2009). Furthermore, superoxide dismutase deficiency can cause severe mitochondrial damage leading to reduced ATP regeneration (Aquilano et al. 2006), a state which could negatively impact performance and overall cellular function. Additional studies indicate that exercise-trained superoxide dismutase knockdown mice develop a form of dilated cardiomyopathy, a maladaptive response to aerobic exercise (Richters et al. 2011). In summary, the elimination of superoxide dismutase leads to excess levels of due to the cell’s inability to convert the ROS into H₂O₂ and O₂.

In summary, the electron transport chain is the site for mitochondrial respiration resulting in oxygen consumption leading to ATP synthesis. In many eukaryotic cells (but certainly not all), and particularly in basal situations, the electron transport chain is a major site for ROS production, an event that is mostly viewed as physiologically expected. In fact and on a percentage basis, free radical generation in resting conditions is higher than in active or exercising situations. However, when oxygen consumption increases (such as during exercise), an excess of ROS is generated, which may overload the antioxidant enzymes (SOD, GSX, catalase) leading to high levels of oxygen species. An imbalance in the redox state between oxidant production and removal can lead to an enhanced release of superoxides, ultimately resulting in damage to the mitochondria as well as other important cellular structures. The extent of this damage will go on to hinder the ability of the cells to adapt to homeostatic demands, particularly in the face of physical exercise (Riksen et al. 2006, Richters et al. 2011). However, it is important to realise that a balance of ROS production and removal must be struck as evidence exists that too much is detrimental to vibrant cellular function, while a calibrated amount of radical production is needed for optimal cellular function (Powers et al. 2011).

1.2.2. Xanthine Oxidase Pathway

The xanthine oxidase pathway leads to production and may also contribute to the production of more potent free radicals such as H₂O₂ and the hydroxyl radical (•OH-). The pathway is triggered under conditions of heavy skeletal muscle contraction, hypoxia or ischaemia. Xanthine oxidase is an enzyme that catalyses the reaction of hypoxanthine to xanthine, uric acid and a superoxide anion. In healthy tissue, xanthine oxidase exists as xanthine dehydrogenase, and is involved in purine metabolism. Under conditions of hypoxia, ischaemia or large bouts of muscular contractions ATP is depleted to ADP and AMP. AMP is further deaminated to IMP by AMP deaminase with additional conversion of AMP into adenosine by 5´ nucleotidase leading to inosine production (Riksen et al. 2006). Purine nucleoside phosphorylase, a key enzymatic step in purine metabolism, converts inosine into hypoxanthine (Canduri et al. 2004) and the large calcium release from muscle contraction triggers a calcium-activated protease, which further changes xanthine dehydrogenase to xanthine oxidase. Activation of xanthine oxidase catalyses the conversion of hypoxanthine into xanthine, which ultimately yields uric acid; superoxide is produced as part of the process as well and is diagrammed in Figure 1.3 (Askew 2002, Sasaki and Joh 2007).

FIGURE 1.3

Xanthine oxidase pathway and production of superoxide and hydroxyl radicals.

Once the xanthine oxidase pathway is triggered, excess production occurs requiring removal through pathways involving MnSOD, glutathione and catalase. As previously reviewed, is converted into H₂O₂ with the potential for conversion into the hydroxyl radical. Under hypoxic or ischaemic reperfusion scenarios, xanthine oxidase conversion increases and under these conditions is thought to be a primary contributor to ROS production. In cardiac tissue, pathophysiology of ischaemia–reperfusion leads to tissue damage and eventual heart failure where the xanthine oxidase pathway is involved (Minhas et al. 2006, Burgoyne et al. 2012). In this respect, Minhas et al. (2006) inhibited xanthine oxidase, which ultimately led to a restoration of cardiac function in a rat heart failure model. Furthermore, ROS production from the xanthine oxidase pathway may impair skeletal muscle function, where inhibition results in greater maximal isometric force generation (Ryan et al. 2011).

Superoxide production from the xanthine oxidase pathway, however, might be species dependent and the overall impact of this pathway in human muscle remains to be fully appreciated. Although it is clear that superoxide is produced through this pathway in rat muscle (Gomez-Cabrera et al. 2005), other studies have documented that human skeletal muscle contains much lower levels of xanthine oxidase (Linder et al. 1999, Gomez-Cabrera et al. 2003). As a result, much debate exists as to how much impact the xanthine oxidase pathway contributes to superoxide production in human skeletal muscle.

1.3. ROS IN CARDIAC PHYSIOLOGY AND PATHOPHYSIOLOGY

The role as well as the involvement of ROS production in cardiac pathophysiology has been well documented and shown to contribute to contractile dysfunction, cardiomyopathy, arrhythmia, ischaemic reperfusion injury and mitochondrial DNA damage (Burgoyne et al. 2012). In many pathological states, there is a shift in the redox state resulting in an excess production of ROS relative to antioxidant defences (Belch et al. 1991, Hill and Singal 1996). The primary source of excess ROS production is indeed cardiac myocyte mitochondria (ETC), xanthine oxidase and excess NADPH oxidase, along with contribution from neutrophils via respiratory burst actions (Tsutsui et al. 2011). Inside failing hearts when compared with healthy hearts, the electron transport chain produces greater O₂^-, and evidence suggests that complex I is a major site for superoxide production (Sawyer and Colucci 2000, Tsutsui et al. 2011). Furthermore, elevated levels of inflammatory cytokines (e.g. tumour necrosis factor-α) and angiotensin II are common in patients with heart failure, which are linked to increased activity of NADPH oxidase (Tsutsui et al. 2011). In addition, these cellular stimuli also activate p47^phox, which accelerates a cascade of events that ultimately leads to increased production of superoxide via NADPH oxidase (Li et al. 2006). In addition, xanthine oxidase activity is greater in heart failure patients due to the ischaemic/reperfusion cycle, ATP depletion and production of H₂O₂ as well as •OH- (Cappola et al. 2001). Inhibition of this increased xanthine oxidase activity in a murine heart failure model via allopurinol administration has been shown to improve contractile function (secondary to reduced free radical production) (Cappola et al. 2001).

Recent literature indicates that the mechanism of how excess ROS production causes cardiac dysfunction most likely involves abnormalities in calcium homeostasis (Burgoyne et al. 2012). As indicated previously, ROS production facilitates normal cardiac function through the activation of ryanodine receptor 1 (RyR1) receptors inducing Ca²⁺ release. However, ROS production at high levels can lead to RyR1 malfunction and aberrant calcium release leading to contractile dysfunction, arrhythmia and myopathy (Sag et al. 2011) with further upstream molecular targets being protein kinase A and calcium calmodulin kinase (Sag et al. 2011). Paradoxically, mitochondria are a source of ROS production in the diseased heart, but are also the targets of oxidative damage mainly because much of the ROS produced in the mitochondria do not cross the inner mitochondrial membrane, leaving them trapped (and reactive) in the inner membrane space (Sag et al. 2011). Additionally, mitochondria have their own genomic system for the transcription of mitochondrial DNA, which is disrupted by ROS production and can decrease rates of mitochondrial protein synthesis (Ballinger et al. 2000).

From an exercise perspective, acute fatiguing exercise or several days of exhaustive exercise has been shown to produce ROS in contracting muscle tissue (Fisher-Wellman and Bloomer 2009). During exercise and as expected, both myocardial oxygen consumption and mitochondrial ATP production increase. These acute responses lead to a large increase in electron transport chain activity which opens the door to excessive reduction of molecular oxygen to a superoxide. Indeed, Bo and colleagues have reported an increase in superoxide levels approximately 90 and 120 min after exercise (Bo et al. 2008); the major site of mitochondrial O₂ production is complexes I and II of the ETC (Ji and Mitchell 1994). ROS production causes mitochondrial membrane damage and also disrupts the proton gradient inside this organelle ultimately leading to a reduction in ATP generation (Ji and Mitchell 1994). Training status appears to be a key consideration in this respect as Leichtweis demonstrated a significant reduction in mitochondrial ROS production from aerobically trained rats when compared with lesser trained rats (Leichtweis et al. 1997). In conjunction, trained rats have been shown to have a greater antioxidant response during as well as post exercise (Bo et al. 2008). Whether chronic exercise induced myocardial ROS production leads to cardiomyopathy or dysfunction of a healthy heart is not known. All current research has utilised animal models, but long-term studies have yet to be conducted, particularly in the human species. In summary, balanced ROS production is critical to heart development and also facilitates optimal calcium handling and contractility, but overproduction is unfavourable and in the long run can contribute to the development of heart failure.

1.4. ROS IN SKELETAL MUSCLE

Arguably, the balance of free radicals inside the skeletal muscle is more important, particularly in the context of exercise and sport, than any other tissue. Section 1.3 attempted to highlight some of the key considerations of another important tissue relative to exercise, cardiac muscle, but in vivo human work is lacking in this tissue. Fortunately, the available literature in skeletal muscle is more prevalent and recent reviews have highlighted the changes relative to oxidative stress in this tissue. It is now commonly accepted that contracting skeletal muscle is a distinct source of ROS and nitrogen species (Pattwell et al. 2004). Focusing this discussion towards exercise and as mentioned previously, initial reports that exercise increases radical generation led to much controversy and speculation relative to the importance and potential danger of these circumstances (Powers et al. 2010, 2011). Thus, much of the dogma surrounding oxidative stress and exercise 20–30 years ago suggested that oxidative stress was unhealthy and would result in circumstances that negatively impacted on health and performance.

Currently, it is no longer debated that exercise increases oxidative stress throughout the body as this outcome has been illustrated in a number of exercise modes including swimming (Gougoura et al. 2007), prolonged cycling (Michailidis et al. 2007) and eccentric exercise (Nikolaidis et al. 2007, 2008) to name a few. Skeletal muscle readily produces the two parent molecules of both radical species, superoxide () and NO. As illustrated in Figure 1.4, superoxide production inside the skeletal muscle occurs in a number of locations: the mitochondria, sarcoplasmic reticulum, transverse tubules, sarcolemma and the cytosol (Powers et al. 2011). As highlighted in previous sections, superoxide production inside the mitochondria of the skeletal muscle is focused upon complexes I and III of the electron transport chain (Barja 1999), but recent reports dispute the notion that this location is responsible for an appreciable amount of superoxide production in this tissue. For years, reports that as much as 2–5% of the total oxygen consumed by mitochondria was reduced to superoxide (Boveris and Chance 1973, Loschen et al. 1974) were made and it was thus thought that the more oxygen that was consumed the more radical species were produced. More recent studies using modern-day technology with greater specificity report that only 0.15% of all oxygen consumed by the mitochondria is used to form superoxide (St-Pierre et al. 2002). Moreover, multiple studies also report that more superoxide production may occur during basal respiration than during any form of active respiration (Di Meo and Venditti 2001). In addition to the mitochondria, superoxide is also produced via NADPH oxidases located within the sarcoplasmic reticulum, transverse tubules and sarcolemma (Powers and Jackson 2008), but limited evidence into these mechanisms precludes any further discussion. In addition, xanthine oxidase activity in the skeletal muscle of rat muscle indicates that this mechanism can also be responsible for appreciable superoxide production (Gomez-Cabrera et al. 2005); however, human muscle contains much less of this enzyme making its contribution to superoxide production in human muscle to be negligible (Linder et al. 1999, Gomez-Cabrera et al. 2005). Finally, other sources of superoxide production may be evident at the level of the plasma membrane (e.g. phospholipase A2-dependent), but more research is needed to more fully discuss the impact of these processes (Jackson 2009).

FIGURE 1.4

Illustration of the potential cellular sites for the production of superoxide in muscle fibres. Note that primary sites for cellular superoxide production include mitochondria, NADPH oxidases (located within the sarcoplasmic reticulum, transverse tubules (more...)

To effectively balance the production of ROS that occur, muscles contain a vast network of endogenous antioxidant enzymes that quench ROS (Ferreira and Reid 2008). In this respect, the sarcoplasm contains CuZn-superoxide dismutase (aka SOD1), catalase and GPX. Moreover, the mitochondrial matrix contains MnSOD (aka SOD2), in addition to GPX. In addition, other less popular thiol-based antioxidant systems exist and include thioredoxin, thioredoxin reductase and peroxiredoxins, although little is known about their regulation and function in skeletal muscle. Multiple non-enzymatic antioxidants exist including lipid-soluble components found nearly exclusively in cellular membranes such as vitamin E, carotenes and ubiquinol. Water-soluble components exist and are ubiquitously distributed across the muscle cell; these include ascorbate (vitamin C), lipoate (lipoic acid), urate and glutathione. In addition, several herbal and botanical species contain various components (e.g. flavonoids, polyphenols, catechins) that are also known to exert antioxidant properties. Of these, glutathione is the most abundant non-protein thiol and also exerts the greatest controls in skeletal muscle over the redox balance of the cell. In this respect, a common marker used in research to represent redox balance is the ratio of oxidised glutathione (GSSG) to reduced glutathione (GSH). For these reasons, glutathione is considered by many to be the most important non-enzymatic antioxidants (Ferreira and Reid 2008).

As highlighted throughout, contracting skeletal muscle contributes radical species from a number of locations, but the notion that these radicals may be needed or may be in some ways beneficial to contracting muscle is an exciting perspective. In this respect, more and more evidence is available to highlight that the balance between the production and quenching of radicals is critically important (Jackson 2009, Powers et al. 2011). On one end of the spectrum, disuse animal models clearly illustrate that this situation is deleterious and results in excessive production of free radicals inside the muscle (Powers et al. 2005, 2007). In a somewhat alarming fashion, other studies show that production of ROS may be a required signal for normal healthy (and desired) remodelling to occur in response to various exercise stimuli (Droge 2002, Jackson 2008). How exactly the muscle tissue and its systemic response identifies and regulates whether the presence of ROS is harmful or helpful is still unknown. While evidence mounts to reveal that factors such as the site of where ROS are produced may differ between an inactive or active state (Powers and Jackson 2008), no clear answer remains. What is known, however, is that multiple cellular signalling pathways throughout the skeletal muscle are impacted and, to some extent, regulation by ROS. Towards this aim, Jackson highlighted and reviewed several cellular signalling pathways that to some extent are impacted by ROS production. As highlighted in Figure 1.5, these pathways represent a wide array of cellular functions including inflammation, proteolysis, hypertrophy, oxidative phenotype and mitochondrial biogenesis (Jackson 2009). More recently, Powers and colleagues elegantly reviewed the evidence surrounding the contribution of two signalling pathways in skeletal muscle known to be heavily influenced by ROS: NF-κB and PGC-1α (Powers et al. 2010, 2011). Briefly, NF-κB is implicated in a large number of cellular processes including inflammation, cell growth, stress responses and apoptosis (Kramer and Goodyear 2007). Evidence in this pathway reveals that ROS and nitrogen species are used by this pathway to transfer signals from inside the cytoplasm to the nucleus ultimately impacting gene expression and eventual phenotypic expression (Droge 2002, Ji et al. 2006). Similarly, another pathway in which the presence and balance of reactive oxygen and nitrogen species must be adequately managed involves PGC-1α; a pathway known to control mitochondrial biogenesis and be a primary regulator of an oxidative phenotype inside contracting muscle. As examples, these pathways nicely highlight the key understanding which must be acquired regarding the production and balance of radical species. Additionally, it remains very likely that as more research is completed in this area, the appropriate balance between a pro-oxidant and antioxidant cellular state will also influence the activity and regulation of several other cellular mechanisms inside the contracting muscle.

FIGURE 1.5

Candidate signalling pathways in skeletal muscle in which reactive oxygen and/or nitrogen species exert some level of influence over its activity and regulation. (From Jackson, M.J. 2009. Free Radic Biol Med. 47(9):1267–1275. With permission.) (more...)

1.5. CONCLUSION

Exercising a skeletal muscle deals with a complex array of positive and negative signals that must be appropriately balanced and coordinated to achieve optimal health and performance as well as to optimally promote desired adaptations from exercise training. The impact of free radical species (ROS and reactive nitrogen) in working cells has undergone a vast overhaul in understanding. Gone should be the initial fears of the 1970s where the mere production of radical species was deemed harmful and replacing it should be the need to appreciate and better understand how these factors combine to favourably impact a myriad of cell functions. A number of pathways exist where the parent molecules of both ROS (superoxide) and reactive nitrogen (NO) species are produced inside cells, but most evidence points towards the majority of them being produced throughout the mitochondria, as part of NADPH oxidase activity, and xanthine oxidase involvement. To counteract any excessive production of these radical species and to maintain a favourable state of redox balance, a number of enzymatic and non-enzymatic molecules exist inside cells, in particular muscle cells. Current evidence clearly points towards acquiring a better understanding of what factors result in a cellular environment that results in damage and destruction or regulation and favourable adaptations. In this respect, sound evidence is available to highlight the importance of redox control over pathways involving NF-κB and PGC-1α, and as more interest and research is completed, our understanding of the impact of radical species on other key signalling pathways will also develop. In closing, radical production is expected and should not be viewed as a ‘necessary evil’, but rather as a potential partner in allowing your body to most favourably adapt and respond to the physical challenges brought upon it.

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