317 Abstract Frailty in the elderly is largely caused by loss of muscle mass and strength, increased susceptibility to injury, and impaired recovery following damage, particularly contraction-induced damage. The mechanisms responsible for the age-related loss of muscle mass and function are unclear although modified generation of Reactive Oxygen and Nitrogen Species (RONS) have been implicated in age-related tissue dysfunction. Many studies have provided evidence for the pivotal role of ROS in signal transduction and recognized these molecules as second messengers. Aberrant generation of RONS in the mitochondria and cyto- sol of cells and tissues of old mammals leads to an altered activation of crucial redox-responsive transcription factors at rest, following acute stress or during the regenerative process. Data suggest that targeted interventions to suppress altered mitochondrial ROS generation in muscle of old individuals are necessary to restore the signal for adaptive responses to contractions. Interventions based on antioxidant supplementation will suppress ROS signals in both mitochondrial and cytosolic compartments and hence be ineffective at prevention of age-related loss of muscle mass and function. Keywords Skeletal muscle • Ageing • ROS • RONS • HSPs • Adaptive responses • Mitochondria • Cytosol A. McArdle (*) and M.J. Jackson School of Clinical Sciences, University of Liverpool, UK e-mail: mdcr02@liverpool.ac.uk Reactive Oxygen Species Generation and Skeletal Muscle Wasting – Implications for Sarcopenia Anne McArdle and Malcolm J. Jackson G.S. Lynch (ed.), Sarcopenia – Age-Related Muscle Wasting and Weakness, DOI 10.1007/978-90-481-9713-2_14, © Springer Science+Business Media B.V. 2011 318 A. McArdle and M.J. Jackson 1 Skeletal Muscle Atrophy and Weakness Contribute to Physical Frailty in the Elderly Frailty in the elderly (Hadley et al. 1993) is largely caused by loss of muscle mass and strength, increased susceptibility to injury, and impaired recovery fol- lowing damage, particularly contraction-induced damage (Faulkner et al. 2007; Marcell 2003). By the age of 70, the cross-sectional area (CSA) of muscle is reduced by 25–30% (Porter et al. 1995) associated with a loss in absolute force generation (Grimby and Saltin 1983) and a decrease in specific force (per unit CSA) generation (Morse et al. 2005). After 70, strength continues to fall and power in the lower leg declines at ~3.5% per year (Skelton et al. 1994). These deficits profoundly impact on the quality of life of even healthy older people, as many are at, or near thresholds that limit the ability to carry out everyday tasks (Young and Skelton 1994). This age-related muscle weakness signifi- cantly increases the risk for elderly falling. Approximately 20% of community- dwelling elderly fall each year (Prudham and Evans 1981). Many elderly who fall suffer loss of independence and some never re-enter the community. One half of the accidental deaths in those over 65 are related to falls. While regular exercise can modify the rate of muscle deficits, even active elderly people show significant age-related declines in muscle mass and function (Wiswell et al. 2001). The mechanisms responsible for the age-related loss of muscle mass and function are unclear although modified generation of Reactive Oxygen and Nitrogen Species (RONS) have been implicated in age-related tissue dysfunction (Harman 2003). 2 The Source and Nature of the RONS Generated by Skeletal Muscle The source and nature of the RONS generated by muscle of young or adult mammals during contractions has been studied since the 1980s. Initial studies demonstrated increased generation of free radicals by contracting skeletal mus- cles (Davies et al. 1982; Jackson et al. 1983). The main reactive oxygen species (ROS) produced in the cell are free radical species, such as the superoxide anion and hydroxyl radical, and non-radical species, such as hydrogen peroxide (H2O2) (Palomero and Jackson 2010). The generation of specific RONS, including superoxide, nitric oxide and hydroxyl radicals by muscle were then described (Reid et al. 1992a,b; O’Neill et al. 1996; Balon and Nadler 1994; Kobzik et al 1994). Cells are required to preserve a delicate balance between ROS generation and elimination to maintain the correct redox status necessary to carry out vital functions. In excess, ROS can attack cellular structures, such as lipids, proteins, and DNA, thereby inducing irreversible changes that can lead to the disruption of 319Reactive Oxygen Species Generation and Skeletal Muscle Wasting – Implications cellular functions and integrity. Under normal physiologic conditions, the reactive nature of ROS allows their incorporation into the structure of macromolecules in a reversible fashion. Such reversible oxidative modifications play a critical role in different signalling pathways that regulate different cellular functions and the fate of the cell (Sies and Jones 2007). Many studies have provided evidence for the pivotal role of ROS in signal transduction and recognized these molecules as second messengers (Powers and Jackson 2008). Most authors have assumed that the ROS generated by contractions are pre- dominantly generated by mitochondria due to the increased demand for energy, but recent data argue against this possibility (for discussion see Jackson 2008). In order to evaluate the relative magnitude of the increase in ROS activity that occurs in skeletal muscle fibres in response to contractions, Palomero et al (2008a) applied a protocol of electrically stimulated, isometric contractions to single isolated fibres from the mouse Flexor digitorum brevis (FDB) muscle. Fibres were loaded with 5- (and 6-) chloromethyl-2¢,7¢-dichlorodihydrofluorescein diacetate (CM-DCFH DA) and measurements of 5- (and 6-) chloromethyl-2¢,7¢- dichlorofluorescin (CM-DCF) fluorescence from individual fibers were obtained by microscopy to study ROS in skeletal muscle. This technique has advantages because of the maturity of the fibres compared with muscle cells in culture and the analysis of single cells prevents contributions from non-muscle cells. The contraction protocol used has been shown to (1) to induce release of superoxide and nitric oxide from muscle cells in culture and muscles of mice in vivo (McArdle et al. 2001; Pattwell et al. 2004), (2) to lead to a fall in muscle glu- tathione and protein thiol content (Vasilaki et al. 2006c) and (3) to stimulate redox-regulated adaptive responses (Vasilaki et al. 2006b) when applied to intact muscles in vivo. The increase in intracellular DCF fluorescence induced by the contraction protocol was less than that following exposure of the fibres to 1 uM hydrogen peroxide. We (Palomero et al. 2008a) calculated that the likely change in intracellular hydrogen peroxide following addition of 1 uM to the extracellular medium is ~0.1 uM. Thus it can be inferred that the absolute level of cytosolic ROS activity in muscle fibres that was achieved following contractile activity was potentially equivalent to ~0.1 uM hydrogen peroxide. Such levels of hydrogen peroxide have traditionally been associated with a signalling role for the oxidant and our recent data indicate that the ROS gener- ated by contractions are reduced by inhibitors of NADPH oxidase enzymes. The increase in ROS activity with contractions is also observed where dihydro- ethidium (DHE) is used as a probe. This probe is predominantly located in the cytosol, but when DHE is modified to locate within mitochondria (as a probe called Mito-HE or MitoSox) no increase in mitochondrial fluorescence was seen during contractions. We conclude that the source of ROS that acts as a signal for adaptive responses to contractions is not mitochondria, but is associ- ated with the cytosol. Inhibitor studies indicate that this is likely to be a mem- brane-located NADPH oxidase that is activated during contractions to generate superoxide (which is converted to hydrogen peroxide) and these ROS activate adaptive responses to contractions. 320 A. McArdle and M.J. Jackson 3 Modified ROS Generation Activates Redox-Sensitive Transcription Factors in Contracting Muscle Skeletal muscles of adult mice and humans adapt rapidly to contractile activity. Numerous proteins show adaptive responses to contraction, including the antioxidant defence enzymes and Heat Shock Proteins (HSPs) that protect against subsequent cellular damage (Hollander et al. 2003; McArdle et al. 2004, 2005). ROS have become increasingly recognised to mediate some adaptive responses of skeletal muscle to contractile activity through activation of redox-sensitive transcription fac- tors (Jackson et al. 2002; McArdle et al. 2004; Jackson 2005; Ji et al. 2006; Gomez- Cabrera et al. 2008; Ristow et al. 2009). Nuclear factor kappa B (NFkB) is one such factor, along with Activator Protein-1 (AP-1) and Heat Shock Factor 1 (Cotto and Morimoto 1999). These transcription factors are involved in remodelling, production of other cytoprotective proteins and production of inflammatory cytokines. ROS are principal regulators of NFkB activation in many situations (Moran et al. 2001). NFkB family members expressed in skeletal muscle play critical roles in modulating the specificity of NFkB (Bar-Shai et al. 2005; Hayden and Ghosh 2008). In skeletal muscle, NFkB modulates expression of genes associated with myogenesis (Bakkar et al. 2008; Dahlman et al. 2009), catabolism-related genes (Bar-Shai et al. 2005; Peterson and Guttridge 2008; Van Gammeren et al. 2009) and cytoprotective pro- teins during adaptation to contractile activity (Vasilaki et al. 2006b). Moreover, skeletal muscle has been identified as an endocrine organ producing cytokines via NFkB activation following stresses such as systemic inflammation or physical strain (Lee et al. 2007). The specificity of the responses of skeletal muscle cells to NFkB activation is likely to be largely due to subtle differences in NFkB activation such as B binding sequences and NFkB dimer formation that regulate expression of specific genes (Bakkar et al. 2008). Activation of NFkB by ROS involves oxidation of key cysteine residues in upstream activators of NFkB and the process can be inhibited by antioxidants or reducing agents (Hansen et al. 2006) and more recently by HSPs (Chen and Currie 2006). Evidence from our laboratory and others have demonstrated that the HSP content of skeletal muscles increases rapidly following a demanding but non- damaging period of isometric contractions and this is termed the stress response (McArdle et al. 2001; Vasilaki et al. 2006b) and this increased HSP content is part of a more widespread adaptive response in transcription of cellular proteins (McArdle F et al. 2004). Data also demonstrated that this was associated with sig- nificant protection against subsequent damage (McArdle F et al. 2004). Definitive data demonstrating a functional role of HSPs in protection against damage and rapid recovery from damage was provided by a study using HSP70 overexpressor mice whereby muscles of these mice were protected against the secondary deficit characteristic of lengthening contraction – induced damage in mice and resulted in a more rapid recovery of maximum force generation (McArdle et al. 2004). The signal for increased HSP production following exercise has been a topic of interest for some time and oxidative stress, hyperthermia and modified energy 321Reactive Oxygen Species Generation and Skeletal Muscle Wasting – Implications supplies have all been proposed to play a role. Data from our laboratory has provided evidence that the primary signal for activation of transcription of HSPs in skeletal muscle in both rodents and humans following isometric contractions is an increased production of reactive oxygen species (ROS). Studies in mice have dem- onstrated that increased HSP production is associated with increased detection of ROS in the muscle extracellular space (McArdle et al. 2001) and a transient fall in protein sulphydryl groups and this occurred in the absence of any significant change in muscle temperature. Supplementation of human subjects with nutritional antioxidants abolished the exercise-induced increase in muscle HSP content (Khassaf et al. 2001). Further studies in humans have demonstrated that although the production of HSPs is dependent upon the intensity of exercise, exercise condi- tions and the training status of the individuals (Morton et al. 2008; Palomero et al. 2008b), an equivalent rise in muscle temperature without exercise did not result in increased muscle content of HSPs (Morton et al. 2007) although the cumulative effect of heat and ROS production may result in a reduction in threshold for ROS- induced HSP production. Changes in HSP content of muscle can play a direct role in modification of ROS production and thus feedback to modify the activation of the stress response. Neuronal nitric oxide synthase (nNOS) produces nitric oxide but also produces superoxide at low levels of L-arginine (Heinzel et al. 1992; Pou et al. 1992). nNOS is localised to the plasma membrane of muscle cells, associated with the dystrophin glycoprotein complex (Vranić et al. 2002) and HSP90 is also associated with nNOS. HSP90 is thought to modify the action of nNOS since the presence of HSP90 dose-dependently inhibits the superoxide anion radical generation from nNOS. At lower levels of L-arginine where marked superoxide anion radical generation occurred, HSP90 caused a more dramatic enhancement of NO synthesis from nNOS as compared to that under normal L-arginine (Song et al. 2002). The balance of production of NO and/or superoxide anion radical by nNOS may also be linked to the cellular localisation of nNOS since it has also been proposed that, in certain pathological conditions including Duchenne muscular dystrophy, deloca- lised nNOS produces altered patterns of NO/superoxide although the role of HSP90 in this production is unknown. The interaction between HSPs and other ROS generating systems is yet to be determined. 4 HSPs Interact with and Mediate Activation of Transcription Factors The dependence of a stress response in muscles following non-damaging exercise on the initial level of HSPs in the quiescent muscle seems to be due to a feedback mechanism by which increased cellular HSPs deactivate Heat Shock Factor 1 (HSF1), the main transcription factor thought to be responsible for the acute stress response (Pirkkala et al. 2001). It is also possible that other adaptations to exercise 322 A. McArdle and M.J. Jackson may play a role in this lack of response, such as an increase in ROS defences (McArdle et al. 2001) which would also reduce the ROS signal. Thus, the threshold for activation of the stress response changes in muscles with altered HSP content or altered oxidant/antioxidant status (termed redox status). Data have demonstrated interactions between cellular HSPs and the activation of other transcription factors, particularly NFkB and AP-1, which are involved in remodelling, production of other cytoprotective proteins and production of inflam- matory cytokines. These studies have concentrated on the protective role of HSPs in ameliorating the activation of the pro-inflammatory pathways of NFkB whereby high levels of HSP70 and HSP27 have been shown to suppress the pro-inflammatory pathway of NFkB (Chen and Currie 2006). Heat shock treatment suppresses NFkB activation in mucosal cells of endotoxin treated mice by inhibiting the phosphoryla- tion and degradation of the NFkB inhibitor, IkB-a and prior heat shock treatment also inhibits IkB kinase (IKK) activation and results in a decreased cytoplasmic level of IKK-a and IKK complex insolublisation (Pritts et al. 2000; Yoo et al. 2000; Chen et al. 2004). In non-muscle cells, HSP70 and HSP27 have been found to interact directly with NFkB, IkB-a, IKK-a, and IKK-b in suppress, resulting in the suppres- sion of NFkB (Shimizu et al. 2002; Guzhova et al. 1997; Park et al. 2003). It is entirely feasible that a similar interaction is present in skeletal muscle cells and that this interaction not only modulates cytokine production by skel- etal muscle, but other pathways in which NFkB and AP-1 may be involved. The pattern and time course of HSP production in skeletal muscles to different forms of exercise and other stresses differs and our data have shown that differ- ent HSPs provide specific protection to various aspects of damage and regen- eration. It is likely that there is some specificity in these interactions with specific HSPs modulating different aspects of transcription factor activation or inhibitor degradation and the induction of the stress response in skeletal muscle may act as a shut-down mechanism of NFkB - mediated cytokine production by muscle cells. The interaction of HSPs with AP-1 and NFkB is further complicated since sev- eral HSPs are known to contain promoters which can be regulated by both NFkB and AP-1. For example, HSP90 contains a promoter which is regulated by NFkB and downregulation of the p65 component of NFkB resulted in reduced constitutive expression of HSP90 (Ammirante et al. 2008). HSPs can also contain an AP-1 promoter (e.g. Hosokawa et al. 1993). 5 Changes in and HSP Content and Redox Status of Muscles Facilitate Successful Myogenesis and Rapid Regeneration Following Damage Controlled changes in transcription factor activation and deactivation are crucial to successful myogenesis and regeneration. During myoblast proliferation and fusion, the HSP content of cells is relatively high and this is primarily due to the expression 323Reactive Oxygen Species Generation and Skeletal Muscle Wasting – Implications and activation of the developmental Heat Shock Factor 2 (HSF2). HSP content then falls gradually with maturation of the cells, along with expression of HSF2 (McArdle et al. 2006). Expression of HSF1 increases at the later stages of myogen- esis once myotubes have been formed (McArdle et al. 2006) such that these cells are now stress responsive. Data from our laboratory examining NFkB activation in vivo following muscle damage have demonstrated a secondary and relatively late phase of NFkB activation at 14 and 28 days post-damage, a time associated with a secondary phase of remodelling, maturation and reinnervation of skeletal muscle fibres. Myogenesis and regeneration are dependent on changes in ROS generation since muscle cells with altered ROS production demonstrate a failure of successful myo- genesis in culture. This may be associated with aberrant activation of redox-respon- sive transcription factors. For example, primary myoblasts from glutathione peroxidase 1 null mice do not fuse to form multinuclear myotubes in culture (Lee et al. 2006). Thus, it is clear that RONS generation plays a major role in determining tran- scriptional activation in skeletal muscle during contraction-induced adaptive responses and alteration of such generation results in adaptive and functional deficits. 6 Modified Generation of Reactive Oxygen Species (ROS) Have Been Implicated in Age-Related Skeletal Muscle Dysfunction The mechanisms responsible for the age-related loss of muscle mass and function are unclear. Initial studies implicated an increase in oxidative damage in all tissues, including skeletal muscle, in the functional decline of those tissues (Sastre et al. 2003; Drew et al. 2003; Vasilaki et al. 2006b,c). Detrimental roles of ROS in tissues have been widely studied and a chronic increase in the production of ROS has been implicated in a number of pathological conditions such as cancer and ageing (Jackson et al. 2002). In contrast, it is now accepted that acute changes in ROS generation are essential for physiological sig- nalling processes. These include ROS acting as short-lived messengers in signal transduction pathways such as those involved in cellular differentiation, prolifera- tion, maturation and programmed cell death via activation of redox-responsive transcription factors (Jackson et al. 2002). However, these processes are still poorly defined and in particular there is a lack of information on the magnitude, time course and localisation of such redox changes in tissues. A chronic accumulation of oxidative damage has been postulated as a major component of the ageing process for over 50 years (Harman 1956). Mitochondria have been claimed to be the major site of reactive oxygen species (ROS) generation that contributes to increased oxidative damage during ageing (see Sanz et al. 2006 for a review) and isolated skeletal muscle mitochondria from old organisms release a greater amount 324 A. McArdle and M.J. Jackson of hydrogen peroxide that is attributable to increased superoxide generation by electron transport chain complexes (Lass et al. 1998; Mansouri et al. 2006; Vasilaki et al. 2006c). Studies of the mutations in mitochondrial DNA in a number of cell types have shown that these accumulate with age (Shah et al. 2009; Taylor et al. 2003). Mutations in mitochondrial DNA can theoretically disrupt the function of the respiratory chain thereby compromising the production of ATP from oxidative phosphorylation. Although much of the current data has concentrated on mitochon- dria as a predominant site for ROS generation during ageing, alternative cellular sites for ROS generation are receiving increasing attention. For example, copper, zinc superoxide dismutase (SOD1) is normally located in the cytosol and mitochon- drial intermembrane space and mice lacking SOD1 show a shortened lifespan and an acceleration of the normal age-related changes in structure and function of several tissues (Muller et al. 2007). It must be noted however that, although the oxidative stress theory of ageing is by far the most popular theory on ageing, data in support of this theory in mammalian systems is sparce (Pérez et al. 2009). We have undertaken a number of studies to define the site of the defect in adap- tive responses following contractile activity in muscle from aged mice. We exam- ined the effect of contractile activity on various indicators of ROS activity in muscle from old compared with adult mice. A protocol of contractile activity caused a significant fall in the total glutathione content of contracting muscles from adult mice, but less of a fall in muscles from old animals and this was associated with a diminished release of extracellular superoxide from the muscles of old mice (Vasilaki et al. 2006c). Vasilaki et al. (2007) also reported a contraction-induced increase in the 3-nitrotyrosine content of muscle from adult mice that was not seen in the muscle from old mice. These data all suggest that the contraction-induced increase in ROS activities is reduced in muscle from old mice compared with that from muscle of adult mice. The chronic increase in the activities of regulatory enzymes for ROS (SOD1 and SOD2 and catalase) and HSP content seen in muscle from old mice (Kayani et al. 2008b) appears to reflect an attempt to adapt to a chronic increase in ROS activities. Despite this attempted adaptation, increased muscle oxidation remains evident in the muscle from old mice (Broome et al. 2006). The effects of these changes on the ROS signals that normally stimulate adaptations to contractions are unknown. 7 The Altered Generation of ROS in Muscles of Old Mice is Associated with an Inability of Muscles of Old Individuals to Adapt to Stress Activation of redox-responsive transcription factors in response to an acute stress such as exercise is aberrant in muscles of old humans and mice. These muscles demonstrate both chronic constitutive activation of redox-sensitive transcription factors (Vasilaki et al. 2006b; Cuthbertson et al. 2005) and an inability to further activate these transcription factors following an acute non-damaging contraction 325Reactive Oxygen Species Generation and Skeletal Muscle Wasting – Implications protocol (Vasilaki et al. 2006b). The chronic activation of transcription factors such as NFkB in muscles of old mice is associated with chronic increases in the expres- sion of a number of genes. For example, increased content and activities of antioxi- dant defence enzymes such as the superoxide dismutases and catalase (Broome et al. 2006), increased content of HSPs (Vasilaki et al. 2006b; Kayani et al. 2008b) and increased production of cytokines and chemokines by muscle cells (Febbraio and Pedersen 2005). The inability to further activate NFkB in response to an acute contraction protocol is associated with severe attenuation of normal changes in expression of cytoprotective genes (Demirel et al. 2003; Heydari et al. 2000; Locke and Tanguay 1996; Muramatsu et al. 1996; Rao et al. 1999; Vasilaki et al. 2006b). We have shown that the increases in HSP content and antioxidant enzyme activities stimu- lated by isometric contractions in muscles of adult rodents were abolished in muscles of old rodents (Vasilaki et al. 2002, 2006b). These severely blunted adaptive responses to acute contractions in muscles from old rodents contribute to age-related muscle dysfunction (McArdle et al. 2004a; Broome et al. 2006) and can be overcome by activation of the transcription factor through alternative, pharma- cological routes (Kayani et al. 2008a). Transgenic overexpression of HSP70 in skeletal muscle throughout life partially preserved muscle function in old mice and prevented the age-related chronic activation of transcription factors and changes in muscle content of cytoprotective proteins at rest (McArdle et al. 2004b; Broome et al. 2006). The mechanisms by which an increased muscle content of HSP70 exerts these effects on NFkB are unclear although overexpression of HSP70 throughout life also prevented the accumulation of markers of oxidative damage in muscle from old mice (Broome et al. 2006). A diminished ability to respond to the stress of contractions plays an important role in other age-related defects in muscle function and adaptation. Ljubicic and Hood (2008) reported a severe attenuation of the signalling pathways involved in mitochondrial biogenesis in type II muscle fibres of old rats following contractions compared with that seen in fibres from young rats. ROS play an important role in the activation of these signalling cascades (Irrcher et al. 2009). These authors sug- gest that ROS affect mitochondrial biogenesis via the upregulation of transcrip- tional regulators as peroxisome proliferator-activated receptor-gamma coactivator-1 protein-alpha (PGC-1alpha), suggesting that an aberrant activation of ROS genera- tion following contractions may be responsible for the diminished mitochondrial biogenesis in muscles of old rats. This blunted or absent adaptation to stress in muscle of old humans and mice is not limited to the exercise response. Skeletal muscle of healthy elderly humans demonstrates a reduction in anabolic sensitivity and responsiveness of muscle protein synthesis pathways. Cuthbertson et al. (2005) demonstrated a reduction in the phosphorylation of mTOR and downstream translational regulators in response to essential amino acid (EAA) ingestion when compared with the young despite a greater plasma EAA availability in elderly subjects. The authors concluded that the nutrient signal was not transduced as well by old as by young muscle, resulting in a lower protein synthesis response to the same stimulus. . Skeletal Muscle Wasting – Implications for Sarcopenia Anne McArdle and Malcolm J. Jackson G.S. Lynch (ed.), Sarcopenia – Age-Related Muscle Wasting and Weakness, DOI 10.1007/978-90-481-9713-2_14,. people show significant age-related declines in muscle mass and function (Wiswell et al. 2001). The mechanisms responsible for the age-related loss of muscle mass and function are unclear. signals in both mitochondrial and cytosolic compartments and hence be ineffective at prevention of age-related loss of muscle mass and function. Keywords Skeletal muscle • Ageing • ROS • RONS