The loss of muscle mass and weakness that accompanies ageing is a major contributor to physical frailty and loss of independence in older people. A failure of muscle to adapt to physiological stresses such as exercise is seen with ageing and disruption of redox regulated processes and stress responses are recognized to play important roles in theses deficits. The role of redox regulation in control of specific stress responses, including the generation of heat shock proteins (HSPs) by muscle appears to be particularly important and affected by ageing. Transgenic and knockout studies in experimental models in which redox and HSP responses were modified have demonstrated the importance of these processes in maintenance of muscle mass and function during ageing. New data also indicate the potential of these processes to interact with and influence ageing in other tissues. In particular the roles of redox signalling and HSPs in regulation of inflammatory pathways appears important in their impact on organismal ageing. This review will briefly indicate the importance of this area and demonstrate how an understanding of the manner in which redox and stress responses interact and how they may be controlled offers considerable promise as an approach to ameliorate the major functional consequences of ageing of skeletal muscle (and potentially other tissues) in man.
Age-related changes in skeletal muscle
In older people, declining muscle mass and function lead to instability and increased risk of falls . By age 70, the cross-sectional area of skeletal muscle is reduced by 25–30% and muscle strength is reduced by 30–40% . The age-related reduction in muscle mass and function in humans and rodents appears primarily due to a decrease in the number of muscle fibres, and atrophy and weakening of those remaining [3–5], although the relative contribution of atrophy of fibres versus loss of fibres has been questioned . Most of the intrinsic and extrinsic changes regulating muscle ageing in humans have been observed in rodents and ageing mice and rats are models of human sarcopenia [7,8]. Advancing age is associated with other functional changes in the remaining muscle fibres including a slowing of the phenotype  and in man an ∼25% reduction in the number of motor neurons occurs with ageing. The causes of this loss are unknown, but small motor neurons (which tend to innervate type I fibres) appear to be preserved relative to large motor neurons. Over time, the loss of large motor neurons is thought to be partially compensated by a sprouting phenomenon through which small motor neurons re-innervate those type II fibres that have become temporarily denervated and hence these fibres acquire a slower phenotype . This process appears to be incomplete and eventually the new ‘giant’ motor units are lost. Atrophy and loss of axons have been reported in older individuals , together with abnormalities in peripheral nerves, including segmental demyelination [11,12], swollen demyelinated and remyelinated axons and denervated Schwann cell columns . Some recent data from rodents indicate that despite the loss of peripheral axons with ageing, the number of motor neuron cell bodies in the lumbar spinal cord are unchanged suggesting that the initial changes may predominantly occur in peripheral regions of motor units . Human and rodent studies both indicate that substantial net loss of motor units occurs with increasing age [15–17] and motor neuron loss occurs in parallel with muscle fibre loss and loss of muscle function [4,5,18]. It is unclear which of these is the primary event [17,19].
Reactive oxygen species play a crucial role in muscle physiology through redox-signalling pathways and are mediators of adaptations to contractile activity
Contractile activity increases the generation of superoxide and nitric oxide (NO) by skeletal muscle fibres with the formation of secondary reactive oxygen species (ROS) and reactive nitrogen species [20–22]. NO generation is regulated by the nitric oxide synthases and recent studies indicate that NAD(P)H oxidases (located with the muscle plasma membrane. T-tubules or triads) are the major source of superoxide in contracting muscle [23,24]. Although ROS can be deleterious to cells causing oxidative damage to lipids, DNA and proteins , in normal physiology ROS mediate some adaptive processes to physiological stresses through changes in gene expression [26–28]. Signalling by these reactive molecules appears to be mainly achieved by targeted modifications of specific residues in proteins . In skeletal muscle the increased ROS generated during contractile activity initiate some adaptive responses. Hydrogen peroxide (H2O2) appears particularly important and activates a number of redox-regulated transcription factors, including NF-κB, AP-1, HSF-1 and nrf2 [28,30–32] with a subsequent robust increase in expression of heat shock proteins (HSPs), regulatory enzymes for ROS and cytoprotective proteins [33–35]. The overall extent to which redox-regulated processes mediate other adaptations to exercise is unclear, but data also support roles in stimulating the expression of genes associated with catabolism [36–38] and mitochondrial biogenesis [39,40].
Attenuated responses of skeletal muscle to stresses, including contractile activity are a characteristic of ageing
During ageing a number of important adaptations to exercise are attenuated or missing and this appears to contribute to reduced muscle mass and function. The attenuated responses appear to particularly affect ROS-stimulated responses that include stress responses and mitochondrial biogenesis [32,39–41]. These changes are apparent in mouse models and studies in man indicate that the age-related attenuation in responses are generally replicated, although some specific responses may be restored by exercise training . Figure 1 shows examples of the attenuated responses of muscle HSPs, SOD and catalase to exercise in mice and humans [32,41].
Attenuated adaptations to contractile activity in mouse and human muscle
In young or adult organisms the HSP response occurs in a robust manner in many cell types, including muscle, but this response is not ubiquitously demonstrated by all cells and those cells with a weaker response also demonstrate an increased susceptibility to cell damage and death [42,43]. It has been proposed that such weaker cellular stress responses are supported by exosome transfer of HSPs from those tissues with a robust response, such as skeletal muscle and that this communication is critical to ensure that proteostasis is maintained across the different tissues of the whole organism . Muscle has been shown to release HSPs  and preliminary data from our laboratory indicate that, whereas muscle fibres from adult mice release HSPs in response to treatment with a non-damaging concentration of tumour necrosis factor alpha (TNF-α), fibres from old mice do not show any HSP release. Thus, the age-related loss of a robust increase in production of HSPs by skeletal muscle of older mammals may have an impact beyond that on muscle tissue alone, affecting also proteostasis in distant tissues.
The potential causes of the attenuated responses in muscle from old rodents and man have been examined in a number of studies. Palomero et al.  examined the increase in intracellular ROS generation following contractile activity in isolated muscle fibres from old and young mice and reported that fibres from old mice showed a chronic increase in ROS activity at rest. In contrast with the robust increase in ROS activity observed following contractions in fibres from young/adult mice, those from old mice showed no further increase following contractions. Taken together with data that show a chronic elevation of mitochondrial hydrogen peroxide generation in muscle fibres from old mice , these data may indicate that the attenuated redox responses in muscle from old mice are associated with a chronic increase in oxidation in the fibres from old mice that arises from increased mitochondrial generation of hydrogen peroxide (see Figure 2 and Jackson and McArdle ). It is noteworthy that an age-related increase in mitochondrial generation of hydrogen peroxide by skeletal muscle has not been universally observed in studies of rodents  or man .
Schematic illustration of effect of ageing on responses to contractile activity
Effects of modification of stress and redox-regulated responses
A number of studies have examined the effect of modification of HSPs and redox processes in muscle during ageing in order to determine the importance of these pathways in age-related loss of muscle.
Overexpression and pharmacological modification of heat shock proteins
Data from transgenic studies indicate that maintenance of the ability to activate adaptations to increased muscle ROS generation during contractions is important for the maintenance of muscle mass and function in ageing since, in a transgenic mouse model, lifelong overexpression of the stress chaperone HSP70 protected against the age-related deficit in muscle-specific force generation and preserved regenerative capacity of muscle following lengthening contractions . Transgenic overexpression of the mitochondrial chaperone HSP10 resulted in preservation of muscle tetanic force generation and fibre cross-sectional area in old mice and this was associated with evidence of reduced oxidative damage in muscle mitochondria of old mice . Thus, there is evidence that HSPs play a fundamental role in skeletal muscle function, and inability to induce individual HSPs during ageing may have deleterious effects on muscle structure and function.
In parallel with these transgenic experiments, pharmacological intervention to increase HSP expression using a known HSP inducer, 17-(allylamino)-17-demethoxygeldanamycin (17AAG), was found to significantly increase the HSP70 content of muscles of adult and old mice and to improve the ability for muscles of old mice to regenerate following lengthening contractions . More recently, an alternate HSP70 inducing drug, arimoclomol, has been shown to attenuate development of pathology in a model of amytrophic lateral sclerosis  suggesting that treatment targeted to increase HSP70 content of muscle may provide a potential therapeutic intervention in treatment of age-related muscle dysfunction.
Overexpression and knockout of regulatory enzymes for reactive oxygen species
A small number of studies have reported that specific manipulation of mitochondrial ROS activities can reduce mitochondrial oxidation  and preserve muscle function during ageing . Our group has collaborated with colleagues in the U.S.A. to undertake studies to examine the effects of deletion of regulatory enzymes for ROS on neuromuscular ageing in mice. Despite frequent observation of increased oxidative damage in mouse models lacking important regulatory enzymes for ROS, no clear relationship with neuromuscular ageing was generally seen. The exception to this pattern was in mice with a whole body deletion of Cu,Zn superoxide dismutase (Sod1) which show neuromuscular changes with ageing that have been claimed to reflect an accelerated skeletal muscle ageing process . Adult Sod1 knockout (KO) mice show a decline in skeletal muscle mass, loss of muscle fibres and a decline in the number of motor units, loss of motor function and contractility, partial denervation and mitochondrial dysfunction by 8 months of age . These are all changes that are also seen in old wild-type (WT) mice, but not until after 22–24 months of age .
Sod1 is present in both the cytosol of cells and within the mitochondrial inter-membrane space  and hence lack of Sod1 may influence redox homoeostasis in the mitochondria and cytosol. Jang et al.  showed that this model was associated with a large increase in mitochondrial H2O2 production and in our studies we concluded that increased peroxynitrite in muscle may play an important role in the phenotype of Sod1KO mice . We also showed that, in common with old WT mice, muscles of Sod1KO mice demonstrated a constitutive activation of NF-κB with increased production of pro-inflammatory cytokines and a constitutive increase in the content of a number of HSPs in muscle at rest and also failed to further activate cytoprotective adaptive responses to contractile activity. This results in diminished acute additional expression of HSPs and other cytoprotective proteins following contractile activity. Thus, a further effect of the lack of Sod1 that mimics that seen in old WT mice is a failure of redox-mediated signalling of adaptive responses to contractile activity [60,61].
In subsequent work, our group of investigators has examined whether the muscle atrophy in this model is initiated by changes within muscle fibres or motor neurons. Surprisingly, mice with skeletal muscle-specific deletion of Sod1 (mSod1KO mice) show no evidence of neuromuscular junction degeneration or loss of muscle fibres and indeed showed some muscle hypertrophy . Other changes seen in Sod1KO mice were not observed in the muscles of mSod1KO mice, including the increases in 3-NT, catalase and peroxiredoxin V previously reported in muscles of Sod1KO mice . To determine the role of motor neurons in the loss of muscle mass and function in Sod1KO mice, we subsequently established a transgenic Sod1KO mouse in which human SOD1 is expressed in neurons under the control of a synapsin 1 promoter (nSOD1-Tg-Sod1KO mice). These ‘nerve rescue’ mice expressed SOD1 in central and peripheral neurons but not other tissues. Sciatic nerve CuZnSOD content in nSOD1-Tg-Sod1KO mice was ∼20% of WT control mice, but they showed no loss of muscle mass or maximum isometric-specific force production at 8–12 months of age, when significant reductions were seen in whole body Sod1KO mice . Thus, these data implicate a lack of Sod1 specifically in motor neurons in the pathogenesis of the accelerated muscle ageing phenotype seen in the Sod1KO mice. We have also recently examined the effect of neuron-specific Sod1 KO in nSod1KO mice, but this model also does not recapitulate the full sarcopenia phenotype seen in Sod1KO mice and shows only minor changes in muscle mass and function . The implication of this work appears to be that both neurons and muscle contribute to maintenance of neuromuscular function in this model and that deletion of Sod1 in both tissues is necessary to generate the full sarcopenic phenotype.
Thus, studies of the Sod1KO model have demonstrated the importance of nerve–muscle interactions in the maintenance of neuromuscular function where ROS homoeostasis is compromised during ageing. Since, adult mice lacking Sod1 replicate many of the features seen in old WT mice they may indicate key mechanisms that lead to loss of muscle fibres and function that are relevant to the ageing of WT mice.
Interactions between attenuated stress and redox responses and other ageing mechanisms
Heat shock proteins and inflammation
During ageing, mammals develop an elevated level of low-grade chronic systemic inflammation . This is characterized by increased circulating levels of several pro-inflammatory cytokines such as interleukin (IL)-6, TNF-α and C-reactive protein  in association with a reduction in anti-inflammatory factors such as IL-10 . More recently, skeletal muscle has been demonstrated to be a potential source of a diverse range of cytokines, termed myokines . The role and impact of myokines in skeletal muscle ageing has not been extensively explored, however, it raises the possibility that myokines may influence the local muscle environment and that muscle could be a significant source of cytokines during ageing.
Inflammation has been proposed as a key driver of skeletal muscle ageing, and the impact of inflammatory cytokines on skeletal muscle has been widely studied. For example, exposure of skeletal muscle to TNF-α results in muscle weakness associated with a loss of total muscle protein, evidenced by increased ubiquitin conjugating activity, with increased NF-κB activation and mediated, at least in-part, by ROS [69,70]. Thus, the development of therapeutic interventions to target inflammatory pathways may provide a potential treatment for at least some aspects of age-related muscle dysfunction.
Increased intracellular HSP expression attenuates plasma concentrations of the proinflammatory cytokines IL-1β and tumor necrosis factor (TNF)-α in both in vitro and in vivo models [71–73]. Adenoviral transvection of HSP70 protects pulmonary epithelium against lung injury . The mechanism by which HSP70 provides this protection is thought to be through stabilization of I-κB kinase . In patients with severe trauma, a correlation was shown between survival and the ability to mount a greater HSP response .
Recent research has identified actions of HSPs located in the extracellular environment. The mechanism by which HSPs are released into the extracellular environment seems crucial to the functioning of these HSPs and both active and passive pathways have been proposed . Passive release is defined as non-specific release, often as a result of cell death and necrosis. The active (specific) release of HSPs by exocytosis provides an attractive explanation for the protective effect of HSP overexpression against age-related loss of muscle. Interaction of muscle-derived HSPs with immune cells indicates the ability of cross-talk to occur between skeletal muscle and the immune system in a paracrine or endocrine manner (Figure 3).
Schematic illustration of the potential role of muscle-derived extracellular HSPs (eHSPs) in regulation of inflammation in non-muscle tissues.
eHSPs can function in a cytokine-like manner through immunostimulation and immunomodulation . eHSPs have been widely characterized as “danger signals” for the immune system during the early stages of trauma/infection and studies have implicated the immunomodulatory capacity of eHSP60 and eHSP70. eHSP72 has been shown to have a direct inflammatory effect on airway epithelium, resulting in up-regulation in the production of several inflammatory cytokines in the airway, such as IL-8 and TNF-α . This up-regulation of inflammatory cytokines occurred exclusively via Toll-like receptor (TLR) 4 and was mediated by NF-κB.
In patients with acute lung injury, progression of the trauma has been correlated with serum levels of HSP60 . This link to pathogenesis has been associated with the ability of HSP60 to interact with T cells via TLR2, resulting in down-regulation of chemokine receptor expression on the cell surface, and the prevention of T-cell chemotaxis . In addition to direct cell interaction studies, HSP72 has been found to have chemotactic properties to neutrophils, a function that seemed to be abrogated upon antagonism of TLR2 . Furthermore, this indicates the importance and specificity of eHSPs for surface TLRs, and the CD14 dependency of this interaction [83,84].
An attractive hypothesis that may help explain the protective effect of HSPs against age-related decline in skeletal muscle is that in adult animals and man, these proteins are generated by muscle as part of a stress response and are released into the extracellular space in exosomes where they can play an immunomodulatory role. In contrast, in old organisms there is an inability to generate HSPs in muscle following physiological stress and hence lack of any immunomodulatory effects. Reversal of the age-related inability to generate HSPs through transgenic overexpression will therefore restore the release of eHSPs, modifying the chronic age-related pro-inflammatory state with beneficial effects on muscle and other tissues.
Redox control in muscle and denervation
The studies cited above for the nerve rescue Sod1KO mice  provide an example of how restoration of neuronal ROS homoeostasis can restore defective function in muscle mitochondria that is associated with increased ROS generation. An analogous situation appears to occur in experimental denervation or nerve crush which has been found to lead to activation of a number of degenerative pathways in the denervated muscle, including an increased mitochondrial generation of ROS species  and increased generation of pro-inflammatory cytokines . Muller et al.  reported a remarkably large increase in muscle mitochondrial H2O2 generation following denervation and subsequent unpublished studies in our laboratory have shown that this increased mitochondrial peroxide release is already apparent within 3 days of nerve transection. The reason for this rapid activation of specific degradatory pathways is unclear. It is feasible that this response initially reflects an attempt to restore innervation, since products such as cytokines are released from the muscle fibre and have been claimed to stimulate axonal sprouting, but if prolonged will inevitably lead to degradation of the denervated muscle fibres. Further studies also showed that other peroxides in addition to H2O2 were released from mitochondria from denervated muscle and that inhibition of 12/15 lipoxygenase could ameliorate some of the muscle atrophy induced by denervation . Thus, together these data suggest that muscle mitochondrial ROS generation plays a role in the muscle degeneration seen following denervation. We have hypothesized that denervation-induced generation of ROS by muscle mitochondria may be key to understanding the increase in mitochondrial ROS generation reported in skeletal muscle from old mice . The extent of the increase in muscle mitochondrial hydrogen peroxide generation following denervation is very substantial (up to 100-fold),  and hence the presence of a small proportion of denervated fibres at any time point might explain the relative increase in ROS generation observed in isolated mitochondria from aged animals (e.g. ).
Redox regulation of metabolism has become increasingly recognized as an important component of muscle responses to stresses, including exercise. The role of redox regulation in control of specific stress responses including the generation of HSPs appears to be particularly important and affected by ageing. It is clear from transgenic and KO studies in experimental models that manipulation of redox and HSP responses can modify the severe functional consequences of ageing on skeletal muscle and new data are also indicating the potential of these processes to interact with and influence ageing in other tissues. Understanding how these processes interact and how they can be controlled offers considerable promise as an approach to ameliorate major functional consequences of ageing in man.
Loss of muscle mass and function with ageing is a major contributor to frailty in the elderly.
Redox-mediated pathways play a key role in skeletal muscle adaptations to to exercise and include increased generation of stress-related proteins including HSPs.
These redox-related responses are attenuated in the elderly leading to to increased suceptibility to damage and inability to regulate inflammatory responses.
Interventions to restore responses of skeletal muscle to contractile activity offer a route to help preserve skeletal muscle mass and function in the elderly.
The authors would like to acknowledge the major contributions of their multiple collaborators and co-workers in this research together with generous continued financial support from major U.K. (Medical Research Council, Biotechnology and Biological Sciences Research Council, Arthritis Research UK, Research into Ageing) and U.S. (National Institute on Aging) funders.
The authors declare no competing interests.