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Reactive oxygen species (ROS) are recognized as important signaling molecules in healthy skeletal muscle. Redox sensitive proteins can respond to intracellular changes in ROS by oxidation of reactive thiol groups on cysteine (Cys) residues. Exercise is known to induce the generation of superoxide and nitric oxide, resulting in the activation of several adaptive signaling pathways; however, it has been suggested that aging attenuates these redox-regulated adaptations to acute exercise. In the present study, we used redox proteomics to study the vastus lateralis muscles of Adult (n = 6 male, 6 female; 18-30 yrs) and Old (n = 6 male, 6 female; 64-79 yrs) adults. Participants completed a bout of high intensity cycling exercise consisting of five sets of 2-min intervals performed at 80% maximal aerobic power output (PPO), with 2 min recovery cycling at 40% PPO between sets. Muscle biopsies were collected prior to exercise, and immediately following the first, second, and fifth high intensity interval. Global proteomic analysis indicated differences in abundance of a number of individual proteins between skeletal muscles of Adult and Old subjects at rest with a significant exacerbation of these differences induced by the acute exercise. In particular, we observed an exercise-induced decrease in abundance of mitochondrial proteins in muscles from older subjects only. Redox proteome analysis revealed cysteines from five cytosolic proteins in older subjects with lower oxidation (i.e. greater reduction) than was seen in muscle from the young adults at rest. Redox homeostasis was well maintained in Adult subjects following exercise, but there was significant increase in oxidation of multiple mitochondrial and cytosolic protein cysteines in Old subjects. We also observed that oxidation of peroxiredoxin 3 occurred following exercise in both Adult and Old groups, supporting the possibility that this is a key effector protein for mitochondrial redox signaling. Thus, we show, for the first time that exercise reveals a lack of resilience in muscle of older human participants, that is apparent as a loss of mitochondrial proteins and oxidation of multiple protein cysteines that are not seen in younger subjects. The precise consequences of this redox disruption are unclear, but this likely play a role in the attenuation of multiple adaptations to exercise that are classically seen with aging. Such changes were only seen following the acute stress of exercise., highlighting the need to consider not only basal differences seen during aging but also the difference following physiological challenge., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Age is the greatest risk factor for the major chronic musculoskeletal disorders, osteoarthritis, osteoporosis and age-related loss of skeletal muscle mass and function (sarcopenia). Dramatic advances in understanding of the fundamental mechanisms underlying the ageing process are being exploited to understand the causes of these age-related disorders and identify approaches to prevent or treat these disorders. This review will focus on one of these fundamental mechanisms, redox regulation, and the role of redox changes in age-related loss of skeletal muscle mass and function (sarcopenia). Key to understanding the role of such pathways has been the development and study of experimental models of musculoskeletal ageing that are designed to examine the effect of modification of ROS regulatory enzymes. These have primarily involved genetic deletion of regulatory enzymes for ROS in mice. Many of the models studied show increased oxidative damage in tissues, but no clear relationship with skeletal muscle aging has been seen The exception to this has been mice with disruption of the superoxide dismutases and, in particular, deletion of Cu,ZnSOD (SOD1) localised in the cytosol and mitochondrial intermembrane space. Studies of tissue specific models lacking SOD1 have highlighted the potential role that disrupted redox pathways can play in muscle loss and weakness and have demonstrated the need to study both motor neurons and muscle to understand age-related loss of skeletal muscle. The complex interplay that has been identified between changes in redox homeostasis in the motor neuron and skeletal muscle and their role in premature loss of muscle mass and function illustrates the utility of modifiable models to establish key pathways that may contribute to age-related changes and identify potential logical approaches to intervention., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mice lacking Cu/Zn-superoxide dismutase (Sod1 -/- or Sod1KO mice) show high levels of oxidative stress/damage and a 30% decrease in lifespan. The Sod1KO mice also show many phenotypes of accelerated aging with the loss of muscle mass and function being one of the most prominent aging phenotypes. Using various genetic models targeting the expression of Cu/Zn-superoxide dismutase to specific tissues, we evaluated the role of motor neurons and skeletal muscle in the accelerated loss of muscle mass and function in Sod1KO mice. Our data are consistent with the sarcopenia in Sod1KO mice arising through a two-hit mechanism involving both motor neurons and skeletal muscle. Sarcopenia is initiated in motor neurons leading to a disruption of neuromuscular junctions that results in mitochondrial dysfunction and increased generation of reactive oxygen species (ROS) in skeletal muscle. The mitochondrial ROS generated in muscle feedback on the neuromuscular junctions propagating more disruption of neuromuscular junctions and more ROS production by muscle resulting in a vicious cycle that eventually leads to disaggregation of neuromuscular junctions, denervation, and loss of muscle fibers., (Published by Elsevier Inc.)

The complexities and heterogeneity of the ageing process have slowed the development of consensus on appropriate biomarkers of healthy ageing. The MRC-Arthritis Research UK Centre for Integrated research into Musculoskeletal Ageing (CIMA) is a collaboration between researchers and clinicians at the Universities of Liverpool, Sheffield and Newcastle. One of CIMA's objectives is to 'Identify and share optimal techniques and approaches to monitor age-related changes in all musculoskeletal tissues, and to provide an integrated assessment of musculoskeletal function', i.e. to develop a toolkit for assessing musculoskeletal ageing. This toolkit is envisaged as an instrument that can be used to characterise and quantify musculoskeletal function during 'normal' ageing, lend itself to use in large-scale, internationally important cohorts, and provide a set of biomarker outcome measures for epidemiological and intervention studies designed to enhance healthy musculoskeletal ageing. Such potential biomarkers include: biochemical measurements in biofluids or tissue samples, in vivo measurements of body composition, imaging of structural and physical properties, and functional tests. The CIMA Toolkit Working Group assessed candidate biomarkers of musculoskeletal ageing under these four headings, detailed their biological bases, strengths and limitations, and made practical recommendations for their use. In addition, the CIMA Toolkit Working Group identified gaps in the evidence base and suggested priorities for further research on biomarkers of musculoskeletal ageing.

The complexities and heterogeneity of the ageing process have slowed the development of consensus on appropriate biomarkers of healthy ageing. The Medical Research Council-Arthritis Research UK Centre for Integrated research into Musculoskeletal Ageing (CIMA) is a collaboration between researchers and clinicians at the Universities of Liverpool, Sheffield and Newcastle. One of CIMA's objectives is to 'Identify and share optimal techniques and approaches to monitor age-related changes in all musculoskeletal tissues, and to provide an integrated assessment of musculoskeletal function'-in other words to develop a toolkit for assessing musculoskeletal ageing. This toolkit is envisaged as an instrument that can be used to characterise and quantify musculoskeletal function during 'normal' ageing, lend itself to use in large-scale, internationally important cohorts, and provide a set of biomarker outcome measures for epidemiological and intervention studies designed to enhance healthy musculoskeletal ageing. Such potential biomarkers include: biochemical measurements in biofluids or tissue samples, in vivo measurements of body composition, imaging of structural and physical properties, and functional tests. This review assesses candidate biomarkers of musculoskeletal ageing under these four headings, details their biological bases, strengths and limitations, and makes practical recommendations for their use. In addition, we identify gaps in the evidence base and priorities for further research on biomarkers of musculoskeletal ageing.

Age-related loss of muscle mass and function is associated with increased frailty and loss of independence. The mechanisms underlying the susceptibility of different muscle types to age-related atrophy are not fully understood. Reactive oxygen species (ROS) are recognised as important signalling molecules in healthy muscle and redox sensitive proteins can respond to intracellular changes in ROS concentrations modifying reactive thiol groups on Cysteine (Cys) residues. Conserved Cys residues tend to occur in functionally important locations and can have a direct impact on protein function through modifications at the active site or determining protein conformation. The aim of this work was to determine age-related changes in the redox proteome of two metabolically distinct murine skeletal muscles, the quadriceps a predominantly glycolytic muscle and the soleus which contains a higher proportion of mitochondria. To examine the effects of aging on the global proteome and the oxidation state of individual redox sensitive Cys residues, we employed a label free proteomics approach including a differential labelling of reduced and reversibly oxidised Cys residues. Our results indicate the proteomic response to aging is dependent on muscle type but redox changes that occur primarily in metabolic and cytoskeletal proteins are generally preserved between metabolically distinct tissues., Biological Significance: Skeletal muscle containing fast twitch glycolytic fibres are more susceptible to age related atrophy compared to muscles with higher proportions of oxidative slow twitch fibres. Contracting skeletal muscle generates reactive oxygen species that are required for correct signalling and adaptation to exercise and it is also known that the intracellular redox environment changes with age. To identify potential mechanisms for the distinct response to age, this article combines a global proteomic approach and a differential labelling of reduced and reversibly oxidised Cysteine residues in two metabolically distinct skeletal muscles, quadriceps and soleus, from adult and old mice. Our results indicate that the global proteomic changes with age in skeletal muscles are dependent on fibre type. However, redox specific changes are preserved across muscle types and accompanied with a reduction in the number of redox sensitive Cysteine residues., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Disruption of neuromuscular junctions and denervation of some muscle fibers occurs in ageing skeletal muscle and contribute to loss of muscle mass and function. Aging is associated with mitochondrial dysfunction and loss of redox homeostasis potentially occurs through increased mitochondrial generation of reactive oxygen species (ROS). No specific link between increased mitochondrial ROS generation and denervation has been defined in muscle ageing. To address this, we have examined the effect of experimental denervation of all fibers, or only a proportion of the fibers, in the mouse tibialis anterior (TA) muscle on muscle mitochondrial peroxide generation. Transection of the peroneal nerve of mice caused loss of pre-synaptic axons within 1-3 days with no significant morphological changes in post-synaptic structures up to 10 days post-surgery when decreased TA mass and fiber size were apparent. Mitochondria in the denervated muscle showed increased peroxide generation by 3 days post-transection. Use of electron transport chain (ETC) substrates and inhibitors of specific pathways indicated that the ETC was unlikely to contribute to increased ROS generation, but monoamine oxidase B, NADPH oxidase and phospholipase enzymes were implicated. Transection of one of the 3 branches of the peroneal nerve caused denervation of some TA muscle fibers while others retained innervation, but increased mitochondrial peroxide generation occurred in both denervated and innervated fibers. Thus the presence of recently denervated fibers leads to increased ROS generation by mitochondria in neighboring innervated fibers providing a novel explanation for the increased mitochondrial oxidative stress and damage seen with aging in skeletal muscles., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Skeletal muscle ageing is characterised by atrophy, a deficit in specific force generation, increased susceptibility to injury, and incomplete recovery after severe damage. The hypothesis that increased generation of reactive oxygen species (ROS) in vivo plays a key role in the ageing process has been extensively studied, but remains controversial. Skeletal muscle generates ROS at rest and during exercise. ROS can cause oxidative damage particularly to proteins. Indeed, products of oxidative damage accumulate in skeletal muscle during ageing and the ability of muscle cells to respond to increased ROS becomes defective. The aim of this review is to examine the evidence that ROS manipulation in peripheral nerves and/or muscle modifies mechanisms of proteostasis in skeletal muscle and plays a key role in initiating sarcopenia., (© 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

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., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Age-related skeletal muscle dysfunction is the underlying cause of morbidity that affects up to half the population aged 80 and over. Considerable evidence indicates that oxidative damage and mitochondrial dysfunction contribute to the sarcopenic phenotype that occurs with aging. To examine this, we administered the mitochondria-targeted antioxidant mitoquinone mesylate {[10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)decyl] triphenylphosphonium; 100 μM} to wild-type C57BL/6 mice for 15 wk (from 24 to 28 mo of age) and investigated the effects on age-related loss of muscle mass and function, changes in redox homeostasis, and mitochondrial organelle integrity and function. We found that mitoquinone mesylate treatment failed to prevent age-dependent loss of skeletal muscle mass associated with myofiber atrophy or alter a variety of in situ and ex vivo muscle function analyses, including maximum isometric tetanic force, decline in force after a tetanic fatiguing protocol, and single-fiber-specific force. We also found evidence that long-term mitoquinone mesylate administration did not reduce mitochondrial reactive oxygen species or induce significant changes in muscle redox homeostasis, as assessed by changes in 4-hydroxynonenal protein adducts, protein carbonyl content, protein nitration, and DNA damage determined by the content of 8-hydroxydeoxyguanosine. Mitochondrial membrane potential, abundance, and respiration assessed in permeabilized myofibers were not significantly altered in response to mitoquinone mesylate treatment. Collectively, these findings demonstrate that long-term mitochondria-targeted mitoquinone mesylate administration failed to attenuate age-related oxidative damage in skeletal muscle of old mice or provide any protective effect in the context of muscle aging.-Sakellariou, G. K., Pearson, T., Lightfoot, A. P., Nye, G. A., Wells, N., Giakoumaki, I. I., Griffiths, R. D., McArdle, A., Jackson, M. J. Long-term administration of the mitochondria-targeted antioxidant mitoquinone mesylate fails to attenuate age-related oxidative damage or rescue the loss of muscle mass and function associated with aging of skeletal muscle., (© The Author(s).)

Ageing is associated with loss of skeletal muscle fibres, atrophy of the remaining fibres and weakness. These changes in muscle are accompanied by disruption of motor neurons and neuromuscular junctions although the direct relationship between the nerve and muscle degeneration is not understood. Oxidative changes have been implicated in the mechanisms leading to age-related loss of muscle mass and in degeneration of the central nervous system, but little is known about age-related changes in oxidation in specific peripheral nerves that supply muscles that are affected by ageing. We have therefore examined the sciatic nerve of old mice at an age when loss of tibialis anterior muscle mass and function is apparent. Sciatic nerve from old mice did not show a gross increase in oxidative damage, but electron paramagnetic resonance (EPR) studies indicated an increase in the activity of superoxide and/or peroxynitrite in the nerves of old mice at rest that was further exacerbated by electrical stimulation of the nerve to activate muscle contractions. Proteomic analyses indicated that specific redox-sensitive proteins are increased in content in the nerves of old mice that may reflect an adaptation to regulate the increased superoxide/peroxynitrite and maintain redox homoeostasis. Analysis of redox active cysteines showed some increase in reversible oxidation in specific proteins in nerves of old mice, but this was not universally seen across all redox-active cysteines. Detailed analysis of the redox-active cysteine in one protein in the nerve of old mice that is key to redox signalling (Peroxiredoxin 6, Cys 47) showed a minor increase in reversible oxidation that would be compatible with a change in its redox signalling function. In conclusion, the data presented indicate that sciatic nerve from old mice does not show a gross increase in oxidative damage similar to that seen in the TA and other muscles that it innervates. Our results indicate an adaptation to increased oxidation with minor changes in the oxidation of key cysteines that may contribute to defective redox signalling in the nerve., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Superoxide and nitric oxide are generated by skeletal muscle, and these species are increased by contractile activity. Mitochondria have long been assumed to play the primary role in generation of superoxide in muscle, but recent studies indicate that, during contractile activity, membrane-localized NADPH oxidase(s) rapidly generate(s) superoxide that plays a role in redox signaling. This process is important in upregulation of rapid and specific cytoprotective responses that aid maintenance of cell viability following contractile activity, but the overall extent to which redox signaling contributes to regulation of muscle metabolism and homeostasis following contractile activity is currently unclear, as is identification of key redox-sensitive protein targets involved in these processes. Reactive oxygen and nitrogen species have also been implicated in the loss of muscle mass and function that occurs with aging, although recent work has questioned whether oxidative damage plays a key role in these processes. A failure of redox signaling occurs in muscle during aging and may contribute to the age-related loss of muscle fibers. Whether such changes in redox signaling reflect primary age-related changes or are secondary to the fundamental mechanisms is unclear. For instance, denervated muscle fibers within muscles from aged rodents or humans appear to generate large amounts of mitochondrial hydrogen peroxide that could influence adjacent innervated fibers. Thus, in this instance, a "secondary" source of reactive oxygen species may be potentially generated as a result of a primary age-related pathology (loss of neurons), but, nevertheless, may contribute to loss of muscle mass and function during aging., (Copyright © 2015 the American Physiological Society.)

The molecular mechanisms underlying skeletal muscle aging and associated sarcopenia have been linked to an altered oxidative status of redox-sensitive proteins. Reactive oxygen and reactive nitrogen species (ROS/RNS) generated by contracting skeletal muscle are necessary for optimal protein function, signaling, and adaptation. To investigate the redox proteome of aging gastrocnemius muscles from adult and old male mice, we developed a label-free quantitative proteomic approach that includes a differential cysteine labeling step. The approach allows simultaneous identification of up- and downregulated proteins between samples in addition to the identification and relative quantification of the reversible oxidation state of susceptible redox cysteine residues. Results from muscles of adult and old mice indicate significant changes in the content of chaperone, glucose metabolism, and cytoskeletal regulatory proteins, including Protein DJ-1, cAMP-dependent protein kinase type II, 78 kDa glucose regulated protein, and a reduction in the number of redox-responsive proteins identified in muscle of old mice. Results demonstrate skeletal muscle aging causes a reduction in redox-sensitive proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response. Data is available via ProteomeXchange with identifier PXD001054.

Skeletal muscle represents a physiologically relevant model for the application of redox proteomic techniques to dissect its response to exercise and aging. Contracting skeletal muscles generate ROS (reactive oxygen species) and RNS (reactive nitrogen species) necessary for the regulation of many proteins involved in excitation-contraction coupling. The magnitude and species of ROS/RNS generated by contracting muscles will have downstream effects on specific protein targets and cellular redox signalling. Redox modifications on specific proteins are essential for the adaptive response to exercise and skeletal muscle can develop a dysregulated redox response during aging. In the present article, we discuss how redox proteomics can be applied to identify and quantify the reversible modifications on susceptible cysteine residues within those redox-sensitive proteins, and the integration of oxidative and non-oxidative protein modifications in relation to the functional proteome.

The ability of muscles to regenerate successfully following damage diminishes with age and this appears to be a major contributor to the development of muscle weakness and physical frailty. Successful muscle regeneration is dependent on appropriate reinnervation of regenerating muscle. Age-related changes in the interactions between nerve and muscle are poorly understood but may play a major role in the defective regeneration. During aging there is defective redox homeostasis and an accumulation of oxidative damage in nerve and muscle that may contribute to defective regeneration. The aim of this review is to summarise the evidence that abnormal reactive oxygen species (ROS) generation in nerve and/or muscle may be responsible for the defective regeneration that contributes to the degeneration of skeletal muscle observed during aging. Identifying the importance of ROS generation in skeletal muscle during aging could have fundamental implications for interventions to prevent muscle degeneration and treatments to reverse the age-related decline in muscle mass and function., (© 2013 Published by Elsevier Inc.)

Significance: Aging leads to a loss of skeletal muscle mass and function that causes instability, increased risk of falls, and need for residential care. This is due to a reduction in the muscle mass and strength that is primarily due caused by a decrease in the number of muscle fibers, particularly, type II fibers, and atrophy and weakening of those remaining., Recent Advances: Although increased oxidative damage was originally thought to be the key to the aging process, data now indicate that reactive oxygen species (ROS) may be one of the several components of the degenerative processes in aging. The skeletal muscle shows important rapid adaptations to the ROS generated by contractions that are attenuated in aged organisms and transgenic studies have indicated that overcoming these attenuated responses can prevent the age-related loss of muscle mass and function., Critical Issues: Elucidation of the mechanisms by which the skeletal muscle adapts to the ROS generated to contractions and the way in which these processes are attenuated by aging is critical to the development of logical approaches to prevent age-related loss of muscle mass and function., Future Directions: Future studies are likely to focus on the redox regulation of adaptive pathways and their maintenance during aging as an approach to maintain and improve muscle function.

An increase in the activity of reactive oxygen species (ROS) has been implicated in the mechanisms of loss of skeletal muscle that occurs during aging, but few studies have attempted to directly assess activities in intact muscle fibers. The current project used the nonspecific fluorescent probe for ROS and reactive nitrogen species, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (CM-DCFH), in single, isolated, mature skeletal muscle fibers from adult and old mice in addition to biochemical measurements of key regulatory proteins for ROS in muscles of these animals. Data confirmed the changes in key regulatory processes for ROS (increased glutathione peroxidase 1 and catalase activities and reduced total glutathione content) previously reported in muscle from old mice and showed increased CM-DCFH oxidation in muscle fibers from old mice at rest and indicate that these changes are likely due to an increase in generation of oxidants rather than a lack of scavenging capacity. The increased CM-DCFH oxidation persisted even when cellular defenses against oxidants were increased by loading fibers from young and old mice with glutathione. During contractile activity, and in contrast to the increase observed in fibers from young mice, there was no further increase in CM-DCFH oxidation in muscle fibers from old mice. These data also suggest that the defect in short-term adaptations to contractions that occurs in old mice may be related to a diminished, or absent, increase in the muscle generation of ROS and/or reactive nitrogen species that normally accompanies contractile activity in young mice.

Thioredoxins (Trx's) regulate redox signaling and are localized to various cellular compartments. Specific redox-regulated pathways for adaptation of skeletal muscle to contractions are attenuated during aging, but little is known about the roles of Trx's in regulating these pathways. This study investigated the susceptibility of Trx1 and Trx2 in skeletal muscle to oxidation and reduction in vitro and the effects of aging and contractions on Trx1, Trx2, and thioredoxin reductase (TrxR) 1 and 2 contents and nuclear and cytosolic Trx1 and mitochondrial Trx2 redox potentials in vivo. The proportions of cytosolic and nuclear Trx1 and mitochondrial Trx2 in the oxidized or reduced forms were analyzed using redox Western blotting. In myotubes, the mean redox potentials were nuclear Trx1, -251 mV; cytosolic Trx1, -242mV; mitochondrial Trx2, -346mV, data supporting the occurrence of differing redox potentials between cell compartments. Exogenous treatment of myoblasts and myotubes with hydrogen peroxide or dithiothreitol modified glutathione redox status and nuclear and cytosolic Trx1, but mitochondrial Trx2 was unchanged. Tibialis anterior muscles from young and old mice were exposed to isometric muscle contractions in vivo. Aging increased muscle contents of Trx1, Trx2, and TrxR2, but neither aging nor endogenous ROS generated during contractions modified Trx redox potentials, although oxidation of glutathione and other thiols occurred. We conclude that glutathione redox couples in skeletal muscle are more susceptible to oxidation than Trx and that Trx proteins are upregulated during aging, but do not appear to modulate redox-regulated adaptations to contractions that fail during aging., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Skeletal muscle is repeatedly exposed to passive stretches due to the activation of antagonist muscles and to external forces. Stretch has multiple effects on muscle mass and function, but the initiating mechanisms and intracellular signals that modulate those processes are not well understood. Mechanical stretch applied to some cell types induces production of reactive oxygen species (ROS) and nitric oxide that modulate various cellular signalling pathways. The aim of this study was to assess whether intracellular activities of ROS and nitric oxide were modulated by passive stretches applied to single mature muscle fibres isolated from young and old mice. We developed a novel approach to apply passive stretch to single mature fibres from the flexor digitorum brevis muscle in culture and to monitor the activities of ROS and nitric oxide in situ by fluorescence microscopy. Passive stretch applied to single skeletal muscle fibres from young mice induced an increase in dihydroethidium oxidation (reflecting intracellular superoxide) with no increase in intracellular DAF-FM oxidation (reflecting nitric oxide activity) or CM-DCFH oxidation. In contrast, in fibres isolated from muscles of old mice passive stretch was found to induce an increase in intracellular nitric oxide activities with no change in DHE oxidation., (© 2012 Informa UK, Ltd.)

Skeletal muscle generates superoxide and nitric oxide at rest and this generation is increased by contractile activity. In young and adult animals and man, an increase in activities of these species and the secondary products derived from them (reactive oxygen species, ROS) stimulate redox-sensitive signalling pathways to modify the cellular content of cytoprotective regulatory proteins such as the superoxide dismutases, catalase and heat shock proteins that prevent oxidative damage to tissues. The mechanisms underlying these adaptive responses to contraction include activation of redox-sensitive transcription factors such as nuclear factor B (NFB), activator protein-1 (AP1) and heat shock factor 1 (HSF1). During ageing all tissues, including skeletal muscle, demonstrate an accumulation of oxidative damage that may contribute to loss of tissue homeostasis. The causes of this increased oxidative damage are uncertain, but substantial data now indicate that the ability of skeletal muscle from aged organisms to respond to an increase in ROS generation by increased expression of cytoprotective proteins through activation of redox-sensitive transcription factors is severely attenuated. This age-related lack of physiological adaptations to the ROS induced by contractile activity appears to contribute to a loss of ROS homeostasis and increased oxidative damage in skeletal muscle.

In muscle, aging is associated with a failure of adaptive responses to contractile activity, and this is hypothesized to play an important role in age-related loss of muscle mass and function. Mice lacking the Cu,Zn superoxide dismutase (Cu,ZnSOD, SOD1) show an accelerated, age-related loss of muscle mass and function. This work determined whether adult mice lacking Cu,ZnSOD (Sod1(-/-) mice) show a premature failure of adaptive responses to contractions in a similar manner to old wild-type (WT) mice. Adult Sod1(-/-) mice (6-8 months of age) had a ∼30% reduction in gastrocnemius muscle mass compared with age-matched WT mice. This lower muscle mass was associated with an activation of DNA binding by NFκB and AP-1 at rest. Measurements of the activity of reactive oxygen species (ROS) in single fibres from the muscles of Sod1(-/-) mice at rest indicated an elevation in activity compared with fibres from WT mice. Following 15 min of isometric contractions, muscle fibres from WT mice showed an increase in the intracellular ROS activities and activation of NFκB and AP-1, but no changes in either ROS activity or NFκB and AP-1 activation were seen in the muscles of Sod1(-/-) mice following contractions. This pattern of changes mimics that seen in the muscles of old WT mice, suggesting that the attenuated responses to contractile activity seen in old mice result from chronic exposure to increased oxidant activity. Data support the use of the Sod1(-/-) mouse model to evaluate potential mechanisms that contribute to the loss of muscle mass and function in the elderly., (© 2010 Crown in the right of Canada. Aging Cell © 2010 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

Skeletal muscle atrophy and weakness are major contributors to frailty and impact significantly on quality of life of older people. Muscle aging is characterized by a loss of maximum tetanic force (P(o)) generation, primarily due to muscle atrophy, to which mitochondrial dysfunction is hypothesized to contribute. We hypothesized that lifelong overexpression of the mitochondrial heat shock protein (HSP) HSP10 in muscle of mice would protect against development of these deficits. P(o) generation by extensor digitorum longus muscles of adult and old wild-type and HSP10-overexpressing mice was determined in situ. Muscles were subjected to damaging lengthening contractions, and force generation was remeasured at 3 h or 28 days to examine susceptibility to, and recovery from, damage, respectively. Muscles of old wild-type mice had a 23% deficit in P(o) generation and a 10% deficit in muscle cross-sectional area compared with muscles of adult wild-type mice. Overexpression of HSP10 prevented this age-related fall in P(o) generation and reduction in cross-sectional area observed in muscles of old wild-type mice. Additionally, overexpression of HSP10 protected against contraction-induced damage independent of age but did not improve recovery if damage occurred. Preservation of muscle force generation and CSA by HSP10 overexpression was associated with protection against the age-related accumulation of protein carbonyls. Data demonstrate that development of age-related muscle weakness may not be inevitable and show, for the first time, that lifelong overexpression of an HSP prevents the age-related loss of P(o) generation. These findings support the hypothesis that mitochondrial dysfunction is involved in the development of age-related muscle deficits.

It is recognised that reactive oxygen species (ROS) are both key regulators of cellular signalling and initiators of oxidative damage to DNA, lipids and proteins under different circumstances. Thus in skeletal muscle from animals and humans, studies indicate that ROS can potentially contribute to the pathophysiology of the age-related loss of skeletal muscle mass and function, while additionally acting as a key signal for contraction-induced adaptations in the tissue. The specific nature and sources of generation of the ROS contributing to these actions remain unknown, but the combination of physiological and pathological roles of ROS imply that interventions based on a simple suppression of ROS activities through use on non-specific antioxidants are unlikely to retard or improve the age-related declines in muscle mass and function. This review will briefly describe the background to this area and describe alternative strategies aimed at correcting specific redox-related changes in ageing muscle, such as the decline in oxidative signalling pathways, that may indicate rational interventions to help maintain muscle mass and function in the elderly.

During aging, a significant loss of skeletal muscle mass and function occurs that can have a dramatic impact on the quality of life of older individuals. The processes underlying this loss of mass and function are unknown, but a chronic increase in cellular superoxide has been implicated in the contributory mechanisms. Mitochondria are a major cellular site for superoxide generation at complexes I or III of the electron transport chain. Within the mitochondrial matrix, superoxide is converted to hydrogen peroxide though activity of Mn-superoxide dismutase. A portion of the superoxide generated at complex III is also released into the mitochondrial intermembrane space, which contains a recently identified copper, zinc superoxide dismutase (Cu,ZnSOD). Deletion of Cu,ZnSOD has been shown to lead to a phenotype of accelerated age-related loss of skeletal muscle mass and function, although it is unclear whether loss of the enzyme in the intermembrane space or cytosol is important in this respect. It is hypothesized that the processes underlying loss of muscle mass and function in the Cu,ZnSOD knockout mice provide a model that can inform identification of the processes that occur during normal aging.

Skeletal muscle atrophy and weakness are major causes of frailty in the elderly. Functional deficits in muscles of old humans and rodents are associated with attenuated production of heat shock proteins (HSPs) after exercise, and transgenic overexpression of HSP70 reverses this functional decline. We hypothesized that training would increase HSP70 content of muscle in adult and old wild-type mice and that this would protect against the development of age-related functional deficits. A 10-wk treadmill training protocol at 15 m/min, for 15 min, 3 days/wk resulted in a significant increase in HSP70 content of muscles of adult mice. Muscles of old untrained mice demonstrated a significant increase in HSP70 protein content and a reduction in HSP70 mRNA content compared with adult untrained mice. Training for 12 mo starting at age 12-14 mo old or for 10 wk starting from age 24 mo old resulted in modification of HSP70 protein and mRNA content to levels of adult mice. Training did not change force generation of extensor digitorum longus muscles of old mice or improve recovery after damaging contractions. The twofold increase in HSP70 content in muscles of adult mice after training may have not been sufficient to provide protection in this instance.

Skeletal muscle aging is characterized by atrophy, a deficit in specific force generation, increased susceptibility to injury, and incomplete recovery after severe injury. The ability of muscles of old mice to produce heat shock proteins (HSPs) in response to stress is severely diminished. Studies in our laboratory using HSP70 overexpressor mice demonstrated that lifelong overexpression of HSP70 in skeletal muscle provided protection against damage and facilitated successful recovery after damage in muscles of old mice. The mechanisms by which HSP70 provides this protection are unclear. Aging is associated with the accumulation of oxidation products, and it has been proposed that this may play a major role in age-related muscle dysfunction. Muscles of old wild-type (WT) mice demonstrated increased lipid peroxidation, decreased glutathione content, increased catalase and superoxide dismutase (SOD) activities, and an inability to activate nuclear factor (NF)-kappaB after contractions in comparison with adult WT mice. In contrast, levels of lipid peroxidation, glutathione content, and the activities of catalase and SOD in muscles of old HSP70 overexpressor mice were similar to adult mice and these muscles also maintained the ability to activate NF-kappaB after contractions. These data provide an explanation for the preservation of muscle function in old HSP70 overexpressor mice.

The ability of muscles of old mice to recover force generation following substantial damage is severely impaired, particularly during the late phase of regeneration. This inability to recover successfully may be associated with the attenuated ability of muscles of old mice to produce heat shock proteins (HSPs) in response to stress since muscles of old mice overexpressing HSP70 recover successfully following damage. The capacity of mature mammalian skeletal muscle to regenerate following damage is due to the presence of undifferentiated mononuclear myogenic precursor cells (satellite cells) at the periphery of mature skeletal muscle fibres. HSP expression is under the primary transcriptional control of heat shock factors 1 and 2 (HSF1 and HSF2). The aim of this study was to examine the expression of heat shock factors 1 and 2 by western blotting in mouse-derived C2C12 myoblasts as an experimental model system for investigating skeletal muscle regeneration. Data demonstrated that the HSF2 content of myotubes was significantly increased during the early stages of regeneration. In contrast, the HSF1 content of myotubes remained relatively low until late during regeneration. Thus, abnormal activation of HSF1 may play a role in the defective regeneration seen in muscles of old mice.

Ageing is associated with skeletal muscle atrophy, a deficit in force generation, an increased susceptibility to contraction-induced injury, and a permanent force deficit following severe injury. Muscles of young mice adapt rapidly following exercise by an increase in the production of heat shock proteins (HSPs), whereas muscles of old mice show a severely diminished response. We hypothesized that overexpression of HSP70 in muscle throughout life would reduce age-related changes in function. The maximum tetanic force of extensor digitorum longus (EDL) muscles of adult and old wild-type (WT) and HSP70 overexpressor transgenic mice was determined. EDL muscles were subjected to damaging lengthening contractions and the ability to generate force was assessed for up to 28 days following the contractions. Overexpression of HSP70 in muscles of old transgenic mice prevented the specific force deficit observed in muscles of old WT mice. The complete recovery of muscles of old HSP70 transgenic mice by 14 days following the contraction protocol was in contrast to the 44% force deficit, which remained in muscles of old WT mice at 28 days following the protocol. These data indicate that a diminished production of HSP70 in muscles of old mammals has a major effect on age-related functional deficits.

The aim of this study was to investigate the production of HSP70 in gastrocnemius muscles from adult (6-month-old) and aged (28-month-old) rats following contractile activity. At 24 h following a period of repeated isometric contractions, muscles from adult rats contained significantly elevated levels of HSP70 compared with nonexercised muscle. This was not evident in muscles from aged rats. This attenuated response may play a major role in development of the age-related functional deficit that occurs in skeletal muscle., (Copyright 2002 Wiley Periodicals, Inc.)

Responding appropriately to exercise is essential to maintenance of skeletal muscle mass and function at all ages and particularly during aging. Here, a hypothesis is presented that a key component of the inability of skeletal muscle to respond effectively to exercise in aging is a denervation-induced failure of muscle redox signalling. This novel hypothesis proposes that an initial increase in oxidation in muscle mitochondria leads to a paradoxical increase in the reductive state of specific cysteines of signalling proteins in the muscle cytosol that suppresses their ability to respond to normal oxidising redox signals during exercise. The following are presented for consideration:Transient loss of integrity of peripheral motor neurons occurs repeatedly throughout life and is normally rapidly repaired by reinnervation, but this repair process becomes less efficient with aging. Each transient loss of neuromuscular integrity leads to a rapid, large increase in mitochondrial peroxide production in the denervated muscle fibers and in neighbouring muscle fibers. This peroxide may initially act to stimulate axonal sprouting and regeneration, but also stimulates retrograde mitonuclear communication to increase expression of a range of cytoprotective proteins in an attempt to protect the fiber and neighbouring tissues against oxidative damage. The increased peroxide within mitochondria does not lead to an increased cytosolic peroxide, but the increases in adaptive cytoprotective proteins include some located to the muscle cytosol which modify the local cytosol redox environment to induce a more reductive state in key cysteines of specific signalling proteins. Key adaptations of skeletal muscle to exercise involve transient peroxiredoxin oxidation as effectors of redox signalling in the cytosol. This requires sensitive oxidation of key cysteine residues. In aging, the chronic change to a more reductive cytosolic environment prevents the transient oxidation of peroxiredoxin 2 and hence prevents essential adaptations to exercise, thus contributing to loss of muscle mass and function. Experimental approaches suitable for testing the hypothesis are also outlined. Image 1 • It is hypothesised that denervation leads to failed redox signalling and attenuated muscle exercise responses in aging. • Loss of peripheral motor neurons leads to increased mitochondrial peroxide in denervated and neighbouring muscle fibers. • This peroxide stimulates increased expression of cytoprotective proteins to protect the fiber against oxidative damage. • Increased mitochondrial peroxide does not increase cytosolic peroxide, but adaptations reduce the cytosol redox state. • Key adaptations of skeletal muscle to exercise involve transient peroxiredoxin oxidation in the cytosol. • Peroxiredoxin oxidation is suppressed during aging by a chronic change to a more reductive local cytosolic environment. [ABSTRACT FROM AUTHOR]

Age‐associated loss of muscle mass and function (sarcopenia) has a profound effect on the quality of life in the elderly. Our previous studies show that CuZnSOD deletion in mice (Sod1−/− mice) recapitulates sarcopenia phenotypes, including elevated oxidative stress and accelerated muscle atrophy, weakness, and disruption of neuromuscular junctions (NMJs). To determine whether deletion of Sod1 initiated in neurons in adult mice is sufficient to induce muscle atrophy, we treated young (2‐ to 4‐month‐old) Sod1flox/SlickHCre mice with tamoxifen to generate i‐mn‐Sod1KO mice. CuZnSOD protein was 40‐50% lower in neuronal tissue in i‐mn‐Sod1KO mice. Motor neuron number in ventral spinal cord was reduced 28% at 10 months and more than 50% in 18‐ to 22‐month‐old i‐mn‐Sod1KO mice. By 24 months, 22% of NMJs in i‐mn‐Sod1KO mice displayed a complete lack of innervation and deficits in specific force that are partially reversed by direct muscle stimulation, supporting the loss of NMJ structure and function. Muscle mass was significantly reduced by 16 months of age and further decreased at 24 months of age. Overall, our findings show that neuronal‐specific deletion of CuZnSOD is sufficient to cause motor neuron loss in young mice, but that NMJ disruption, muscle atrophy, and weakness are not evident until past middle age. These results suggest that loss of innervation is critical but may not be sufficient until the muscle reaches a threshold beyond which it cannot compensate for neuronal loss or rescue additional fibers past the maximum size of the motor unit. [ABSTRACT FROM AUTHOR]

Physical frailty in the elderly is driven by loss of muscle mass and function and hence preventing this is the key to reduction in age-related physical frailty. Our current understanding of the key areas in which ROS contribute to age-related deficits in muscle is through increased oxidative damage to cell constituents and/or through induction of defective redox signalling. Recent data have argued against a primary role for ROS as a regulator of longevity, but studies have persistently indicated that aspects of the aging phenotype and age-related disorders may be mediated by ROS. There is increasing interest in the effects of defective redox signalling in aging and some studies now indicate that this process may be important in reducing the integrity of the aging neuromuscular system. Understanding how redox-signalling pathways are altered by aging and the causes of the defective redox homeostasis seen in aging muscle provides opportunities to identify targeted interventions with the potential to slow or prevent age-related neuromuscular decline with a consequent improvement in quality of life for older people. [ABSTRACT FROM AUTHOR]

Increased extracellular generation of reactive oxygen species (ROS) as a result of increasing reliance on glycolytic metabolism by old mitochondria-rich tissues has been claimed to contribute to the propagation of oxidative damage during aging (the reductive hotspot hypothesis), but the process has not been examined experimentally in old animals. Superoxide activity in the extracellular fluid of gastrocnemius muscle and markers of oxidation in blood and the liver were examined in adult and old mice at rest and following a period of demanding isometric contractions. The activity of superoxide in muscle microdialysates did not differ between adult and old mice at rest, but during contractile activity, there was a significant increase in the superoxide activity in microdialysates from adult muscle but no increase in microdialysates from old muscle. At rest, the liver of old mice contained an increased malonaldehyde content and a decreased protein thiol content in comparison with adult mice, but following the contraction protocol, only the adult mice showed significant, transient increases in the serum and liver malonaldehyde content and a decrease in liver glutathione and protein thiol content. Further studies revealed that the lack of superoxide release from contracting muscle of old mice was not due to reduced force generation by these muscles. These data provide no evidence for an increased extracellular superoxide in resting or contracting skeletal muscle of old mice, or that release of superoxide from muscle contributes to oxidation of blood components in the liver in old mice as is predicted from the reductive hotspot hypothesis. [ABSTRACT FROM AUTHOR]

Abstract Age-associated frailty is predominantly due to loss of muscle mass and function. The loss of muscle mass is also associated with a greater loss of muscle strength, suggesting that the remaining muscle fibres are weaker than those of adults. The mechanisms by which muscle is lost with age are unclear, but in this review we aim to pull together various strands of evidence to explain how muscle contractions support proteostasis in non-muscle tissues, particularly focussed on the production and potential transfer of Heat Shock Proteins (HSPs) and how this may fail during ageing, Furthermore we will identify logical approaches, based on this hypothesis, by which muscle loss in ageing may be reduced. Skeletal muscle generates superoxide and nitric oxide at rest and this generation is increased by contractile activity. In adults, this increased generation of reactive oxygen and nitrogen species (RONS) activate redox-sensitive transcription factors such as nuclear factor κB (NFκB), activator protein-1 (AP1) and heat shock factor 1 (HSF1), resulting in increases in cytoprotective proteins such as the superoxide dismutases, catalase and heat shock proteins that prevent oxidative damage to tissues and facilitate remodelling and proteostasis in both an intra- and inter-cellular manner. During ageing, the ability of skeletal muscle from aged organisms to respond to an increase in ROS generation by increased expression of cytoprotective proteins through activation of redox-sensitive transcription factors is severely attenuated. This age-related lack of physiological adaptations to the ROS induced by contractile activity appears to contribute to a loss of ROS homeostasis, increased oxidative damage and age-related dysfunction in skeletal muscle and potentially other tissues. Graphical abstract fx1 Highlights • Skeletal muscle generates increased ROS following contractile activity. • This increase in ROS activates transcription of cytoprotective proteins. • The cytoprotective proteins promote intra- and inter-cellular proteostasis. • Generation of ROS and expression of protective proteins is altered during ageing. • This contributes to age-related dysfunction in muscle and potentially other tissues. [ABSTRACT FROM AUTHOR]

Aims: Lack of Cu,Zn-superoxide dismutase (CuZnSOD) in homozygous knockout mice ( Sod1−/−) leads to accelerated age-related muscle loss and weakness, but specific deletion of CuZnSOD in skeletal muscle ( mSod1KO mice) or neurons ( nSod1KO mice) resulted in only mild muscle functional deficits and failed to recapitulate the loss of mass and function observed in Sod1−/− mice. To dissect any underlying cross-talk between motor neurons and skeletal muscle in the degeneration in Sod1−/− mice, we characterized neuromuscular changes in the Sod1−/− model compared with mSod1KO mice and examined degenerative molecular mechanisms and pathways in peripheral nerve and skeletal muscle. Results: In contrast to mSod1KO mice, myofiber atrophy in Sod1−/− mice was associated with increased muscle oxidative damage, neuromuscular junction degeneration, denervation, nerve demyelination, and upregulation of proteins involved in maintenance of myelin sheaths. Proteomic analyses confirmed increased proteasomal activity and adaptive stress responses in muscle of Sod1−/− mice that were absent in mSod1KO mice. Peripheral nerve from neither Sod1−/− nor mSod1KO mice showed increased oxidative damage or molecular responses to increased oxidation compared with wild type mice. Differential cysteine (Cys) labeling revealed a specific redox shift in the catalytic Cys residue of peroxiredoxin 6 (Cys47) in the peripheral nerve from Sod1−/− mice. Innovation and Conclusion: These findings demonstrate that neuromuscular integrity, redox mechanisms, and pathways are differentially altered in nerve and muscle of Sod1−/− and mSod1KO mice. Results support the concept that impaired redox signaling, rather than oxidative damage, in peripheral nerve plays a key role in muscle loss in Sod1−/− mice and potentially sarcopenia during aging. Antioxid. Redox Signal. 28, 275-295. [ABSTRACT FROM AUTHOR]