Abstract
During aging loss of skeletal muscle mass and function has a significant effect of an individual’s quality of life and ability to maintain independence. Both loss of muscle fibres and atrophy of the remaining fibres play a role in the muscle decline and this is associated with loss of motor units and a reduction in the number of motor neurons. Increased oxidative damage has long been claimed to be associated with aging and many studies have reported increased amounts of oxidative damage markers are found in tissues from old organisms. Reactive oxygen species (ROS) are recognised to play a major role in cell signalling and in muscle ROS generated during contractile play an important role in signalling adaptations to contractile activity. These ’redox-regulated’ pathways are beneficial adaptations which are attenuated during aging. This review will briefly cover what is currently known about the mechanisms underlying these muscle adaptations to exercise, how they are affected by aging and assess the importance of these pathways in age-related loss of skeletal muscle mass and function.
Introduction
Our initial work in this area examined free radical species in skeletal muscle using electron paramagnetic resonance (EPR) techniques and demonstrated an increased intensity of a free radical signal (likely originating from quinone species) in muscle of anaesthetised rats that were repeatedly contracted by electrically stimulation (Jackson et al. 1985). This work built on previous studies from Davies and colleagues who reported the presence of the same EPR signal in muscle and liver of rats run to exhaustion (Davies et al. 1982). At that time, we were also studying lipid peroxidation during muscle contractile activity or whole-body exercise together with other groups (Dillard et al. 1978, Brady et al. 1979, Gee & Tappel 1981, Jackson et al. 1983a ) and were able to show an increase in lipid peroxidation occurred during exercise and concluded that this process was occurring in skeletal muscle (Jackson et al. 1983a ).
Thus, these initial studies suggested that increased generation of reactive species such as free radicals was occurring in muscle during exercise and that this may be causing peroxidation of lipid molecules. The general contemporary view was that these species were potentially deleterious and hence should be avoided since they could oxidise lipids, proteins, DNA and RNA (see Halliwell & Gutteridge 1984) and many of our initial studies included attempts to reduce the deleterious effects using supplementary antioxidants, particularly vitamin E, (Brady et al. 1979, Jackson et al. 1983b ). This situation prevailed for several years before Sen and Packer (Sen & Packer 1996) and other researchers realised that in normal physiology, reactive oxygen species (ROS) had roles as mediators of cell signalling pathways and many subsequent studies have focussed on these signalling effects.
In this article we will focus on ROS generated during contractile activity in skeletal muscle and the roles that these species potentially play in adaptations to contractile activity. Some of these beneficial adaptations of muscle to exercise or contractile activity are also attenuated during aging and this appears particularly relevant for redox-regulated pathways. The review will therefore also cover what is known about this attenuation of redox responses during aging and the importance of this in the loss of muscle mass and function that occurs during aging.
ROS as mediators of muscle adaptations to exercise
Various ROS may be found in tissues, including superoxide (O2•–), singlet oxygen, hydrogen peroxide (H2O2), lipid peroxides (LOOH) and peroxynitrite (ONOO–), it has been recognised that the only ROS directly formed in skeletal muscle is superoxide with the important signalling molecule, hydrogen peroxide, being rapidly formed from superoxide. Nitric oxide (NO) is also formed in skeletal muscle from nitric oxide synthases and NO can potentially combine with superoxide to generate peroxynitrite. Superoxide and NO are generated from various intracellular sources within muscle fibres, and superoxide (Reid et al. 1992, McArdle et al. 2001), hydrogen peroxide (Viña et al. 2000) and NO (Balon & Nadler 1994, Kobzik et al. 1994) are also released into the interstitial space from muscle fibres (or generated on the extracellular side of the muscle plasma membrane).
There are multiple potential sites in skeletal muscle for generation of superoxide/hydrogen peroxide and some specific sites have been recognised as important sources during contractile activity. Evidence that Nox2 (NAD(P)H oxidase 2) is activated in muscle during contractile activity comes from studies of superoxide generation by muscle fibres with inhibition of the enzyme by pharmacological or genetic approaches. We used isolated intact fibres from the mouseflexor digitorum brevis to examine superoxide generation from different sub-cellular sites. In these studies we were unable to demonstrate any role for mitochondria in contraction-induced superoxide generation, but found good evidence that Nox2 may play a role in contraction-induced ROS formation (Sakellariou et al. 2013). Protein and mRNA expression of NAD(P)H oxidase sub-units were demonstrated in single muscle fibres. NOX2, and p22phox sub-units were localised to the sarcolemma and transverse tubules and the regulatory p40phox and p67phox proteins were found in the cytoplasm of resting fibres, but following contractions, p40phox appeared to translocate to the sarcolemma indicating a mechanism for activation of Nox2 in exercising muscle. A specific p47phox-roGFP probe (Pal et al. 2013) has subsequently been used to demonstrate activation of Nox2 in muscle during exercise (Pal et al. 2013, Henríquez-Olguin et al. 2019b). Espinosa et al. (Espinosa et al. 2006) also hypothesised that the T-tubule-localised NAD(P)H oxidase might be activated by depolarisation of the T-tubules, but this has not been confirmed.
Other studies have examined in detail the mechanisms by which Nox2 may be activated in skeletal muscle and two particular processes, phosphorylation of p47phox and Rac1 activation have been reported (Pal et al. 2014, Ahn et al. 2015, Bost et al. 2015). These processes can potentially lead to translocation and binding of cytosolic sub-units to the cell membrane and Nox2/p22phox, but translocation to the cell membrane of regulatory units appears to have only been described in one paper (Sakellariou et al. 2013). Hence, whether translocation of sub-units is always a pre-requisite for superoxide generation by Nox2 in skeletal muscle remains an open question (Ferreira & Laitano 2016).
The availability of different mouse models which lack Nox2 activity has dramatically increased understanding of the roles of Nox2 in this area. Mice lacking either of the Nox2 regulatory sub-units p47phox or Rac1 did not show an increase in cytosolic ROS during exercise (Henríquez-Olguin et al. 2019b) and this was associated with modified physiological responses to exercise (Henríquez-Olguin et al. 2020).
An alternative homolog of NAD(P)H oxidase, Nox4, is expressed in many tissues and in different sub-cellular locations within those tissues. This includes mitochondria and sarcoplasmic reticulum of skeletal muscle (Ferreira & Laitano 2016). Deletion of Nox4 in skeletal muscle has been shown to reduce the hydrogen peroxide content of muscle post exercise, to compromise exercise capacity and promote the development of insulin resistance (Xirouchaki et al. 2021). The Nox4 enzyme complex has traditionally been considered to be constituently active, but the activity appears to be stimulated by a series of agonists including insulin, tumor necrosis factor alpha and angiotensin II. Nox4 is also reported to be important in skeletal muscle oxygen sensing (Sun et al. 2011), and Ferreira and Laitano (Ferreira & Laitano 2016) have speculated that changes in NADH:NAD ratio which occur during intense contractile activity and hypoxia might increase Nox4 ROS production. Surprisingly endothelial Nox4 has also been implicated in mediating muscle metabolic responses to exercise. Specht et al (Specht et al. 2021) demonstrated that mice lacking endothelial Nox4 had blunted glucose and fatty acid oxidation following exercise and impaired adaptations to exercise.
Despite extensive investigations, it does not appear that muscle mitochondrial electron transport chain-derived superoxide contributes to the increased ROS detected in skeletal muscle during contractile activity (Jackson 2008, Michaelson et al. 2010, Sakellariou et al. 2013); furthermore, Henriques-Olquin have demonstrated a decrease in oxidation of a mitochondria-localised hydrogen peroxide probe during exercise (Henríquez-Olguin et al. 2019a).
Xanthine oxidase has also been claimed to be an important source for generation of superoxide in exercising muscle and use of the xanthine oxidase inhibitors, allopurinol, or its active metabolite oxypurinol, has been associated with a decrease in the levels of indicators of oxidative damage and markers of muscle damage after exhaustive exercise protocols in humans and rats (Gómez-Cabrera et al. 2003, Gomez-Cabrera et al. 2005). Despite lack of evidence the xanthine oxidase is present in muscle fibres, we demonstrated that xanthine oxidase in muscle contributed to increased superoxide activity in the extracellular fluid of skeletal muscles and affected skeletal muscle contractile function during an isometric contraction protocol (Gomez-Cabrera et al. 2010). The mechanism of activation of the xanthine oxidase pathway is unclear, but most studies argue that in relatively hypoxic tissues, anaerobic metabolism leads to proteolytic modification of xanthine dehydrogenase to form xanthine oxidase (Nishino et al. 2008) and to the increased availability of the xanthine oxidase substrates, hypoxanthine and xanthine (Pacher et al. 2006). This is consistent with the argument that superoxide generation by contracting muscle during exercise is greatest with fatigue or exhaustion of energy supplies (Viña et al. 2000).
A summary of the main sites that have been proposed for superoxide and NO generation in skeletal muscle fibres is presented in Fig. 1A.
Left panel: Updated schematic diagram illustrating the potential sub-cellular sites for generation of superoxide and NO is skeletal muscle fibres. Modified from (Jackson 2011). Major potential reactions of superoxide and NO are shown leading to secondary reactive species. Recently recognised sites for aquaporin location in skeletal muscle are shown where this protein may regulate H2O2 transport across membranes. Right panel: Examples of pathways involved in adaptations of skeletal muscle that have been reported to be inhibited by antioxidants and contain redox sensitive components that may facilitate their activation by oxidants.
Citation: Redox Experimental Medicine 2023, 1; 10.1530/REM-23-0014
Target pathways for redox control during exercise
It has proven difficult to identify specific redox-sensitive targets that are activated during exercise in skeletal muscle, but studies in humans and animals using very high levels of antioxidants have provided evidence that the these treatments inhibited cytoprotective responses (e.g. exercise-induced increase in heat shock and other stress proteins) (Venditti et al. 2014), reduced mitochondrial biogenesis (Gomez-Cabrera et al. 2008, Ristow et al. 2009, Paulsen et al. 2014), prevented an increase in muscle insulin sensitivity (Ristow et al. 2009), and inhibit the release of cytokines and inflammatory mediators (Wuyts et al. 2003). These studies have been controversial (Higashida et al. 2011, Gomez-Cabrera et al. 2012), but other adaptations potentially activated by ROS have been identified in genetic knockout mouse models, designed to delete ROS-generating enzymes. For instance, Nox2 knockout mice show reductions in post-exercise glucose uptake via impaired GLUT4 translocation (Henríquez-Olguin et al. 2019a,b ), a Nox4 knockout in mice was found to lead to development of insulin resistance (Xirouchaki et al. 2021), and a specific endothelial Nox4 knockout leads to impaired metabolic adaptations to chronic exercise (Specht et al. 2021). Thus, together, these studies indicate that the range of adaptive pathways activated during exercise and regulated by redox pathways is likely to be extensive.
Key processes involved in muscle adaptations to exercise have been intensively studied for a number of years and multiple pathways have been identified where redox regulation appears important including the pathways leading to the adaptations described above (Jackson 2020) (Fig. 1B).
Mechanisms of activation of signalling pathways by hydrogen peroxide
We have argued that the nanomolar levels of H2O2 found in muscle cytosol or mitochondria are not sufficiently high to directly oxidise redox-sensitive proteins in the muscle adaptive signalling pathways discussed previously (Jackson et al. 2020). Other researchers have proposed potential ways by which these physiological (nM) cytosolic or mitochondrial levels of H2O2 might oxidise cysteine thiols in redox-sensitive proteins (Stocker et al. 2018). The first is by direct oxidation and some authors have argued that this is facilitated by proximity of the target protein to the source of generation of H2O2 where local concentrations may be increased (Ushio-Fukai 2009), although computational modelling has indicated that even near sites of H2O2 generation the concentrations are too low to oxidise typical redox targets (Travasso et al. 2017). The ‘floodgate’ hypothesis modifies this initial proposal and suggests that local scavengers of H2O2 become rapidly oxidised and inactivated subsequently permitting a local increase in H2O2 concentration to micromolar levels (Wood et al. 2003).
An alternative proposal involves the utilisation of highly oxidisable effectors of redox signalling in a ‘redox relay’ that allows transmission of the oxidising equivalents from H2O2 to less oxidisable target signalling molecules. Proteins including peroxiredoxins, thioredoxins, glutathione peroxidases and catalase are present in skeletal muscle and can react with hydrogen peroxide at the low concentrations found intracellularly and Winterbourn has undertaken calculations of the selectivity of hydrogen peroxide for peroxiredoxins in comparison with redox-sensitive signalling proteins such as PTPs and GSH at estimated cellular concentrations. She concluded that Prx reacts with almost all of the peroxide (Winterbourn 2008), further concluding that ‘the oxidation (of PTPs and related enzymes) observed in cells is likely to be an indirect effect of peroxide reacting with a primary sensor’. Some studies have also recently indicated that Prxs can function as a signal peroxidase to activate specific pathways. Prx1 activates the transcription factor ASK1 (apoptosis signal-regulating kinase 1) (Jarvis et al. 2012) and Prx2 forms a ‘redox relay’ with the transcription factor STAT3 such that oxidative equivalents flow from Prx2 to STAT3 (signal transducer and activator of transcription 3) by generating disulphide-linked STAT3 oligomers with modified transcriptional activity (Sobotta et al. 2015).
Our studies examined Prx oxidation in muscle during contractions and concluded that muscle Prx1, 2 and 3 oxidation occurred rapidly during muscle contractions and that this increased Prx oxidation was rapidly reversed following cessation of contractions (Stretton et al. 2020) (Fig. 2). No contraction-induced formation of hyperoxidised Prx was seen. Thus contractile activity leads to the rapid and specific oxidation of Prx isoforms and we argue that this is due to oxidation by the physiological concentrations of H2O2 generated in skeletal muscle during contractile activity. The apparent essentiality of this process in mediating some adaptations to contractile activity has been recently examined using a C. Elegans model and gene knockout strategy in which it has been shown that peroxiredoxin 2 is required for redox-mediated adaptations to exercise in this model. These data therefore provide the first direct support for a role for Prx in mediating redox signalling of adaptations to exercise (Xia et al. 2023). The precise mechanism by which oxidation of Prx can lead to activation of specific signalling pathways involved in muscle adaptations to contractile activity or exercise is the subject of current research (Jackson 2020).
Oxidation and recovery of peroxiredoxin (Prx) 1-3 in isolated flexor digitorum brevis (FDB) fibres during and following 15 min of electrically stimulated contractions. Adapted from Stretton et al. (Stretton et al. 2020). * P < 0.05.
Citation: Redox Experimental Medicine 2023, 1; 10.1530/REM-23-0014
Effects of aging on skeletal muscle
In older people, declining muscle mass and function causes instability and increased risk of falls with a loss of independence (Young & Skelton 1994). By age 70, the cross-sectional area of skeletal muscle is reduced by 25–30% and muscle strength by 30–40% (Porter et al. 1995). Both a decrease in the number of muscle fibres, and atrophy and weakening of those fibres remaining (Lexell et al. 1986, Brooks & Faulkner 1988, Lexell et al. 1988) appear to contribute to the reduction in muscle mass and function with age in humans and rodents. This is termed sarcopenia and the intrinsic and extrinsic changes regulating muscle aging in humans also occur in rodents, indicating that mice and rats are relevant models of human sarcopenia (Demontis et al. 2013, Cobley et al. 2014). The loss of muscle that occurs with aging occurs in parallel with loss of motor units in both humans and rodents (Campbell et al. 1973, Sheth et al. 2018). A 25–50% reduction in the number of motor neurons occurs in both man and rodents with aging (Tomlinson & Irving 1977, Rowan et al. 2012). Loss of innervation of individual fibres occurs in muscles of aged humans and animals and our study which indicated that ∼15% of muscle fibres from old mice are completely denervated and ∼80 % of NMJs showed some disruption (Vasilaki et al. 2016).
Aging is also associated with a failure of adaptations to stress (Pomatto & Davies 2017). In skeletal muscle this is seen as an attenuation of important adaptations to exercise including acute stress responses (Vasilaki et al. 2006a), mitochondrial biogenesis (Viña et al. 2009) and anabolic responses (Cuthbertson et al. 2005).
Linkages between ROS and muscle aging
It has been recognised for some time that tissues of aged organisms contain greater oxidative damage to lipids, DNA and proteins than is found in young organisms (Drew et al. 2003, Sastre et al. 2003, Vasilaki et al. 2006b). Some initial interventions to reduce ROS activities in non-mammalian models were reported to extend lifespan (Orr & Sohal 1993, 1994, 2003, Melov et al. 2000), but these effects were not confirmed (Gems & Doonan 2009, Pérez et al. 2009) and it now seems clear that the level of ROS generation and oxidative damage is not a fundamental determinant of lifespan. Some authors have argued that the age-related changes in ROS activities and oxidative damage are important mediators of age-related disorders (Hamilton et al. 2012). Several of the ROS-stimulated responses to exercise are also attenuated in old mice including increased stress responses (Vasilaki et al. 2006a) and mitochondrial biogenesis (Viña et al. 2009, Cobley et al. 2015) and mitochondrial peroxide generation has been repeatedly reported to be increased in skeletal muscle during aging (Vasilaki et al. 2006b, Jang & Van Remmen 2009).
Manipulation of ROS to attempt to modify skeletal muscle aging
In an attempt to experimentally examine the role of ROS/oxidative damage in aging in mammalian systems, Arlan Richardson and colleagues examined 18 different experimental mouse models in which multiple regulatory proteins for ROS were transgenically deleted or overexpressed. These manipulations caused major changes in the level of oxidative damage in tissues (Pérez et al. 2009) but did not have any effect on lifespan. Their conclusion was that their ‘data calls into serious question the hypothesis that alterations in oxidative damage/stress play a role in the longevity of mice’. In subsequent studies these same authors re-examined the data from these large cohorts of mice and refined their conclusion to indicate that under chronic stress, including pathological phenotypes that diminish optimal health, oxidative stress/damage plays a major role in aging. Under these conditions, enhanced antioxidant defences were found to exert an ‘antiaging’ action, leading to changes in lifespan, age-related pathology, and physiological function (Salmon et al. 2010).
The exception to the lack of a major effect of the many genetic manipulations on lifespan and muscle aging was mice lacking Sod1 (Cu, Zn superoxide dismutase) which showed a ~20% decrease in lifespan (Pérez et al. 2009) and an overt accelerated skeletal muscle aging phenotype (Muller et al. 2006).
The Sod1KO mouse
Discovery of the accelerated skeletal muscle aging phenotype in whole body Sod1KO mice (Muller et al. 2006) prompted detailed investigations of the changes in this model in comparison with aging of muscle in wild-type control mice. Sod1 is found in the cell cytosol and mitochondrial inter-membrane space (IMS) and skeletal muscles of Sod1KO mice were found to exhibit mitochondrial abnormalities, degeneration of neuromuscular junctions (NMJs) and loss of innervation, and loss of contractile force during adulthood that is similar to the hind limb muscle phenotype seen in old wild-type mice (Brooks & Faulkner 1988, 1994, Jang et al. 2010, Larkin et al. 2011, Bhaskaran et al. 2020). In order to try and determine the tissue sites most crucial to the effects of lack of Sod1 in causing early skeletal muscle loss, mice with a genetic deletion of Sod1 specifically targeted to skeletal muscle were examined. These studies surprisingly found that CuZnSOD deletion targeted to skeletal muscle does not induce loss of muscle mass even in older adult mice (up to 16–17 months) and contractile function was only marginally reduced (Zhang et al. 2013). Conversely, to determine whether the muscle decline associated in Sod1KO mice was initiated by redox changes in motor neurons, a mouse model in which the human CuZnSOD gene was specifically targeted to neurons on a whole body Sod1KO background (SynTgSodKO mice) was examined (Sakellariou et al. 2014). In these ‘nerve rescue’ mice, the neuron-specific expression of Sod1 prevented the muscle atrophy, NMJ degeneration and muscle weakness phenotypes that occur in the Sod1KO mice, suggesting that the redox balance in motor neurons was a critical mediator of muscle innervation and atrophy (Sakellariou et al. 2014).
To address the potential role of a motor neuron lack of Sod1 in the muscle phenotype, mice with a constitutive embryonic neuron-specific deletion of Sod1 were generated (Sataranatarajan et al. 2015), but these mice did not show significant atrophy of muscles even at 20 months of age, although mild contractile dysfunction and signs of denervation were evident. These data appeared to contradict the conclusions from studies of the nerve rescue (SynTgSodKO) mice (Sakellariou et al. 2014), and it was hypothesised that the specific approach used to delete neuronal Sod1, using the nestin-cre, may not have been sufficient to induce a phenotype, or that the deletion during embryonic development may induce compensatory effects that altered the effect of motor neuron deletion in the mice at later ages (Bhaskaran et al. 2020). A mouse model with inducible deletion of Sod1 (i-mn-Sod1KO) targeted by a neuron-specific cre recombinase driven by the Thy1 promoter was generated. The findings obtained showed that neuronal deletion of Sod1 in adult mice resulted in accelerated atrophy and contractile dysfunction of skeletal muscle as well as a disruption of NMJ morphology in older mice (Bhaskaran et al. 2020).
Lessons from genetic manipulation of ROS control pathways for muscle aging
Importance of motor neurons
Studies of muscle mitochondrial H2O2 production in aging animals and other models have consistently shown an increase in H2O2 production with advancing age (Vasilaki et al. 2006b, Jang & Van Remmen 2009) and in the Sod1KO mice data indicate that disruption of neuromuscular integrity regulates muscle mitochondrial ROS generation (Muller et al. 2007, Zhang et al. 2013, Sakellariou et al. 2014, Sakellariou et al. 2018, Bhaskaran et al. 2020). Further evidence for a role for motor neurons in regulation of muscle mitochondria peroxide generation comes from experiments where transection of nerves innervating muscles caused a large increase in muscle mitochondrial peroxide generation (Muller et al. 2007). Furthermore, muscles of old mice show considerable evidence of localised fibre denervation (Vasilaki et al. 2016), which is associated with an increased mitochondrial peroxide generation (Staunton et al. 2019). We also examined the effect of partial denervation of the mouse TA muscle and found a substantial increase in mitochondrial peroxide generation in the denervated fibres and also in neighbouring innervated fibres (Pollock et al. 2017). These data strongly suggest that loss of innervation in muscle fibres contributes to increased muscle mitochondrial ROS generation (Pollock et al. 2017) and associated muscle mitochondrial degeneration (Scalabrin et al. 2019) in aging. Furthermore, by analogy, we speculate that the increased number of denervated fibres seen in muscles of old mice and humans suggests that denervation may play a role in the increased mitochondrial peroxide generation seen during aging (Pollock et al. 2017).
Disruption of site-specific regulation of superoxide rather than generalised oxidative stress is key to the muscle phenotype of Sod1KO mice
Detailed proteomic studies of the changes that occur in muscle and nerve of Sod1KO mice compared with muscle-specific Sod1KO mice (mSod1KO mice) examined degenerative molecular mechanisms and pathways in peripheral nerve and skeletal muscle (Sakellariou et al. 2018). The data obtained suggested that neuromuscular integrity, redox mechanisms, and pathways are differentially altered in nerve and muscle of Sod1KO and mSod1KO mice and concluded that impaired redox signalling, rather than oxidative damage, in peripheral nerve was likely to play a key role in muscle loss in Sod1−/− mice and hence potentially sarcopenia during aging. This conclusion is generally in accord with previous studies which indicated that the level of oxidative damage was not a prime determinant of longevity in mammalian species (Pérez et al. 2009).
Other studies of Sod1KO mice have examined the specific ROS and reactive nitrogen species that are modified by the deficiency since lack of Sod1 may theoretically lead to increased superoxide, reduced nitric oxide (NO), and/or increased peroxynitrite, each of which could initiate muscle fibre loss (Sakellariou et al. 2011). Using a combination of fluorescent intracellular probes for superoxide and mice with modified NO generation, we concluded that that formation of peroxynitrite in muscle fibres is a major effect of lack of Sod1 in Sod1KO mice and may contribute to fibre loss in this model, and that NO regulates superoxide availability and peroxynitrite formation in muscle (Sakellariou et al. 2011). Again this work indicates that the effect of lack of Sod1 are relatively specific in leading to an increase in peroxynitrite activity in tissues, particularly the motor neuron. Such changes would lead to specific and deleterious modifications of key molecules in the motor neurons (e.g. nitration of proteins such as nerve growth factors (Pehar et al. 2006) which would not specifically occur in the presence of a generalised oxidative stress. A further useful comparison has been made between the phenotypic effects of Sod1 deletion and muscle specific loss of Sod2 (MnSOD) (Lustgarten et al. 2009). Muscle Sod2 is localised to the mitochondrial matrix, while Sod1 localises to the cytosol and mitochondrial IMS, but Sod2 loss severely affected muscle aerobic metabolism but did not induce a premature aging phenotype in comparison to the effects of lack of Sod1.
Value of Sod1KO mice as a model of accelerated skeletal muscle aging
As previously mentioned the Sod1KO mouse shows many of the muscle phenotypes of old wild-type mice at a much earlier age and the major effect of the Sod1KO appears to be through effects at the level of the motor neuron. This model has been proposed as a useful experimental model of frailty since Sod1KO mice exhibit four characteristics that have been used to define human frailty: weight loss, weakness, low physical activity, and exhaustion. In addition, Sod1KO mice show increased inflammation and sarcopenia, which are strongly associated with human frailty (Deepa et al. 2017).
In recent studies we have examined in detail the changes in motor neurons and the NMJ which occur in inducible neuron-specific Sod1KO mice (i-mnSod1KO mice) which present with an early onset of muscle loss (Pollock et al. 2023). Surprisingly no specific effect of a lack of neuronal Sod1 was seen, but rather all of the changes seen in aging were accelerated. Thus the i-mnSod1KO mice showed an increase in denervated NMJ, reduced numbers of large axons and increased number of small axons compared with old WT mice. A large proportion of the innervated NMJs in old i-mnSod1KO mice also displayed a simpler structure than that seen in adult or old WT mice (see Fig. 3). We concluded that although previous work had showed that neuronal deletion of Sod1 induced exaggerated loss of muscle in old mice, this deletion leads to a reduced axonal area, increased proportion of denervated NMJ, and reduced acetyl choline receptor complexity and other changes in nerve and NMJ structure that are also seen in WT mice at a more advanced age (Pollock et al. 2023). Thus, the Sod1KO mouse model and its tissue-specific derivatives have provided a great deal of valuable information on the tissue interactions and mechanisms that lead to muscle loss in aging. Clearly a simple lack of Sod1 does not occur during aging in WT animals or humans but the detailed analogies in phenotype and mechanisms seen in aging wild-type mice and Sod1KO mice provide confidence in the relevance and utility of this model.
Changes in NMJ innervation and fragmentation in differing ages of wild-type and i-mnSod1KO mice and adult Sod1KO mice. Representative images of NMJ and axonal structure are shown following bungarotoxin staining (magenta) and Thy1-YFP/CFP (green) visualisation in whole EDL muscles from adult, mid-age and old WT, mid-age and old i-mnSod1KO and Sod1KO-Thy1-CFP mice; scale bar = 50 μm. For full details, see Pollock et al. (2023).
Citation: Redox Experimental Medicine 2023, 1; 10.1530/REM-23-0014
Role of denervation in age-related changes in skeletal muscle and attenuation of redox-regulated responses to contractile activity
Studies by our group have demonstrated that genetic interventions designed to overcome some of the attenuated responses to exercise that occur during aging can mitigate some of the deleterious effects of aging in mouse models (McArdle et al. 2004, Kayani et al. 2010). These data support the hypothesis that attenuation of responses to exercise may play an important role in the loss of muscle mass and function that accompanies aging and early studies of the Sod1KO mice indicated that adult mice with this genetic defect showed an analogous attenuation of responses to contractile activity to that seen in old wild-type mice (Vasilaki et al. 2010).
We have linked the attenuation of responses to contractile activity seen in both aging and the Sod1KO mice to an increase in muscle mitochondrial hydrogen peroxide production in both situations. We speculated that this increase would lead to an increased expression of regulatory enzymes for ROS (Prx, GPx, TrX, etc.) which would suppress the likelihood of oxidation of critical cysteines in signalling proteins during contractions generation (Jackson 2020). Furthermore, since cycles of localised denervation and re-innervation appear to occur throughout life and may contribute to disrupted mitochondrial peroxide generation (Jackson 2020) and mitochondrial structure and function (Scalabrin et al. 2019), we have speculated that the focal denervation seen in both adult Sod1KO and old wild-type mice leads to the increased mitochondrial peroxide production in both the denervated and neighbouring innervated muscle fibres which would drive the attenuation of redox-regulated adaptive mechanisms (Jackson 2020) (Fig. 4). This provides a testable mechanism by which focal and intermittent denervation during aging have a deleterious effect in suppressing key responses of muscle to exercise.
Schematic representation of the proposed changes which occur at the NMJ to affect muscle viability and lead to attenuation of redox-regulated muscle responses in adult wild-type mice, Sod1KO mice and denervated muscle fibres of old wild-type mice. In adult wild-type mice (A), neural stimulation of contraction in the muscle leads to activation of Nox2 generation of superoxide and hydrogen peroxide leading to stimulation of signalling responses. In Sod1KO mice (B), the lack of Sod1 leads to increased generation of peroxynitrite in motor neurons which accelerates the effect of aging on the NMJ (C). In B and C, denervation of fibres leads to increased production of mitochondrial hydrogen peroxide and other peroxides which propagates to affect mitochondrial peroxide production in local innervated fibres and suppresses redox-regulated adaptations to contractions.
Citation: Redox Experimental Medicine 2023, 1; 10.1530/REM-23-0014
Conclusions and future perspectives
It is clear from the preceding sections that ROS play an important role in muscle adaptations to exercise and details of some of the pathways involved are becoming clearer. How these pathways are influenced by aging and what potential there is for manipulation of the processes to benefit older people remains much less clear. Current data suggest that simple supplementation with ROS-scavenging antioxidants is unlikely to have any beneficial effect on the aberrant muscle redox signalling in older individuals and animals, but targeted, site-specific interventions to correct defects at a sub-cellular level (e.g. increased mitochondrial peroxide generation) may hold some promise in this area. Further elucidation of pathways are likely to provide an increased opportunity for uncovering interventions to preserve muscle function and mass in older individuals.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Acknowledgements
The author would like to acknowledge the multiple colleagues, collaborators and students who have contributed to this work over many years and to thank the funding agencies, including the UKRI Medical Research Council (MRC) and Biotechnology and Biological Sciences Research Council (BBSRC) and US National Institutes of Health (NIH) for generous and repeated funding.
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