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Reactive oxygen species are generated by multiple mechanisms during contractile activity in exercising skeletal muscle and are recognised to play a role in signaling adaptations to the contractions. The sources of the superoxide and hydrogen peroxide generated are now relatively well understood but how the resulting low concentrations of hydrogen peroxide induce activation of multiple signaling pathways remains obscure. Several theories are presented together with accumulating evidence that 2-Cys peroxiredoxins may play a role of "effector" proteins in mediating the signaling actions of hydrogen peroxide. Identification of the mechanisms underlying these pathways offers the potential in the longer term for development of novel interventions to maintain exercise responses in the elderly with the potential to maintain muscle mass and function and consequent quality of life., Competing Interests: Declaration of competing interest The author declares no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

As a widespread global issue, protein deficiency hinders development and optimal growth in offspring. Maternal low-protein diet influences the development of age-related diseases, including sarcopenia, by altering the epigenome and organ structure through potential increase in oxidative stress. However, the long-term effects of lactational protein restriction or postnatal lifelong protein restriction on the neuromuscular system have yet to be elucidated. Our results demonstrated that feeding a normal protein diet after lactational protein restriction did not have significant impacts on the neuromuscular system in later life. In contrast, a lifelong low-protein diet induced a denervation phenotype and led to demyelination in the sciatic nerve, along with an increase in the number of centralised nuclei and in the gene expression of atrogenes at 18 months of age, indicating an induced skeletal muscle atrophy. These changes were accompanied by an increase in proteasome activity in skeletal muscle, with no significant alterations in oxidative stress or mitochondrial dynamics markers in skeletal muscle later in life. Thus, lifelong protein restriction may induce skeletal muscle atrophy through changes in peripheral nerves and neuromuscular junctions, potentially contributing to the early onset or exaggeration of sarcopenia., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The early life environment significantly affects the development of age-related skeletal muscle disorders. However, the long-term effects of lactational protein restriction on skeletal muscle are still poorly defined. Our study revealed that male mice nursed by dams fed a low-protein diet during lactation exhibited skeletal muscle growth restriction. This was associated with a dysregulation in the expression levels of genes related to the ribosome, mitochondria and skeletal muscle development. We reported that lifelong protein restriction accelerated loss of type-IIa muscle fibres and reduced muscle fibre size by impairing mitochondrial homeostasis and proteostasis at 18 months of age. However, feeding a normal-protein diet following lactational protein restriction prevented accelerated fibre loss and fibre size reduction in later life. These findings provide novel insight into the mechanisms by which lactational protein restriction hinders skeletal muscle growth and includes evidence that lifelong dietary protein restriction accelerated skeletal muscle loss in later life., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: Protein-energy malnutrition and the subsequent muscle wasting (sarcopenia) are common ageing complications. It is knowing to be also associated with dementia. Our programme will test the cytoprotective functions of vitamin E combined with the cortisol-lowering effect of chocolate polyphenols (PP), in combination with muscle anabolic effect of adequate dietary protein intake and physical exercise to prevent the age-dependent decline of muscle mass and its key underpinning mechanisms including mitochondrial function, and nutrient metabolism in muscle in the elderly., Methods and Analysis: In 2020, a 6-month double-blind randomised controlled trial in 75 predementia older people was launched to prevent muscle mass loss, in respond to the 'Joint Programming Initiative A healthy diet for a healthy life'. In the run-in phase, participants will be stabilised on a protein-rich diet (0.9-1.0 g protein/kg ideal body weight/day) and physical exercise programme (high-intensity interval training specifically developed for these subjects). Subsequently, they will be randomised into three groups (1:1:1). The study arms will have a similar isocaloric diet and follow a similar physical exercise programme. Control group (n=25) will maintain the baseline diet; intervention groups will consume either 30 g/day of dark chocolate containing 500 mg total PP (corresponding to 60 mg epicatechin) and 100 mg vitamin E (as RRR-alpha-tocopherol) (n=25); or the high polyphenol chocolate without additional vitamin E (n=25). Muscle mass will be the primary endpoint. Other outcomes are neurocognitive status and previously identified biomolecular indices of frailty in predementia patients. Muscle biopsies will be collected to assess myocyte contraction and mitochondrial metabolism. Blood and plasma samples will be analysed for laboratory endpoints including nutrition metabolism and omics., Ethics and Dissemination: All the ethical and regulatory approvals have been obtained by the ethical committees of the Azienda Ospedaliera Universitaria Integrata of Verona with respect to scientific content and compliance with applicable research and human subjects' regulation. Given the broader interest of the society toward undernutrition in the elderly, we identify four main target audiences for our research activity: national and local health systems, both internal and external to the project; targeted population (the elderly); general public; and academia. These activities include scientific workshops, public health awareness campaigns, project dedicated website and publication is scientific peer-review journals., Trial Registration Number: NCT05343611., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Progressive muscle atrophy and loss of muscle strength associated with old age have been well documented. Although age-associated impairments in skeletal muscle regeneration following injury have been demonstrated, less is known about whether aging impacts the regenerative response of neuromuscular junctions (NMJ) following contraction-induced injury. Reduced ability of NMJs to regenerate could lead to increased numbers of denervated muscle fibers and therefore play a contributing role to age-related sarcopenia. To investigate the relationship between age and NMJ regeneration following injury, extensor digitorum longus (EDL) muscles of middle-aged (18-19 months) and old mice (27-28 months) were subjected to a protocol of lengthening contractions (LC) that resulted in an acute force deficit of ~55% as well as functional and histological evidence of a similar magnitude of injury 3 days post LCs that was not different between age groups. After 28 days, the architecture and innervation of the NMJs were evaluated. The numbers of fragmented endplates increased and of fully innervated NMJs decreased post-injury for the muscle of both middle-aged and old mice and for contralateral uninjured muscles of old compared with uninjured muscles of middle-aged controls. Thus, the diminished ability of the skeletal muscle of old mice to recover following injury may be due in part to an age-related decrease in the ability to regenerate NMJs in injured muscles. The impaired ability to regenerate NMJs may be a triggering factor for degenerative changes at the NMJ contributing to muscle fiber weakness and loss in old age., (© 2023. The Author(s), under exclusive licence to American Aging Association.)

Research over almost 40 years has established that reactive oxygen species are generated at different sites in skeletal muscle and that the generation of these species is increased by various forms of exercise. Initially, this was thought to be potentially deleterious to skeletal muscle and other tissues, but more recent data have identified key roles of these species in muscle adaptations to exercise. The aim of this review is to summarise our current understanding of these redox signalling roles of reactive oxygen species in mediating responses of muscle to contractile activity, with a particular focus on the effects of ageing on these processes. In addition, we provide evidence that disruption of the redox status of muscle mitochondria resulting from age-associated denervation of muscle fibres may be an important factor leading to an attenuation of some muscle responses to contractile activity, and we speculate on potential mechanisms involved.

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.)

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., (Copyright © 2020. Published by Elsevier Inc.)

Skeletal muscle generates superoxide during contractions which is rapidly converted to H 2 O 2 . This molecule has been proposed to activate signalling pathways and transcription factors that regulate key adaptive responses to exercise but the concentration of H 2 O 2 required to oxidise and activate key signalling proteins in vitro is much higher than the intracellular concentration in muscle fibers following exercise. We hypothesised that Peroxiredoxins (Prx), which reacts with H 2 O 2 at the low intracellular concentrations found in muscle, would be rapidly oxidised in contracting muscle and hence potentially transmit oxidising equivalents to downstream signalling proteins as a method for their oxidation and activation. The aim of this study was to characterise the effects of muscle contractile activity on the oxidation of Prx1, 2 and 3 and determine if these were affected by aging. Prx1, 2 and 3 were all rapidly and reversibly oxidised following treatment with low micromolar concentrations of H 2 O 2 in C2C12 myotubes and also in isolated mature flexor digitalis brevis fibers from adult mice following a protocol of repeated isometric contractions. Significant oxidation of Prx2 was seen within 1 min (i.e. after 12 contractions), whereas significant oxidation was seen after 2 min for Prx1 and 3. In muscle fibers from old mice, Prx2 oxidation was significantly attenuated following contractile activity. Thus we show for the first time that Prx are rapidly and reversibly oxidised in response to contractile activity in skeletal muscle and hypothesise that these proteins act as effectors of muscle redox signalling pathways which are key to adaptations to exercise that are attenuated during aging., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2020 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Hydrogen peroxide appears to be the key reactive oxygen species involved in redox signalling, but comparisons of the low concentrations of hydrogen peroxide that are calculated to exist within cells with those previously shown to activate common signalling events in vitro indicate that direct oxidation of key thiol groups on "redox-sensitive" signalling proteins is unlikely to occur. A number of potential mechanisms have been proposed to explain how cells overcome this block to hydrogen peroxide-stimulated redox signalling and these will be discussed in the context of the redox-stimulation of specific adaptations of skeletal muscle to contractile activity and exercise. It is argued that current data implicate a role for currently unidentified effector molecules (likely to be highly reactive peroxidases) in propagation of the redox signal from sites of hydrogen peroxide generation to common adaptive signalling pathways., Competing Interests: Declaration of competing interest None of the authors have any relevant conflict of interest pertaining to this publication. The research work described has been funded by a number of external public bodies over several years following rigorous independent peer-review., (Copyright © 2020. Published by Elsevier B.V.)

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.)

Previous studies have shown a significant increase in the mitochondrial generation of hydrogen peroxide (H 2 O 2 ) and other peroxides in recently denervated muscle fibers. The mechanisms for generation of these peroxides and how the muscle responds to these peroxides are not fully established. The aim of this work was to determine the effect of denervation on the muscle content of proteins that may contribute to mitochondrial peroxide release and the muscle responses to this generation. Denervation of the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles in mice was achieved by surgical removal of a small section of the peroneal nerve prior to its entry into the muscle. An increase in mitochondrial peroxide generation has been observed from 7 days and sustained up to 21 days following denervation in the TA muscle fibers. This increased peroxide generation was reduced by incubation of skinned fibers with inhibitors of monoamine oxidases, NADPH oxidases or phospholipase A2 enzymes and the muscle content of these enzymes together with peroxiredoxin 6 were increased following denervation. Denervated muscle also showed significant adaptations in the content of several enzymes involved in the protection of cells against oxidative damage. Morphological analyses indicated a progressive significant loss of muscle mass in the TA muscle from 7 days up to 21 days following denervation due to fiber atrophy but without fiber loss. These results support the possibility that, at least initially, the increase in peroxide production may stimulate adaptations in an attempt to protect the muscle fibers, but that these processes are insufficient and the increased peroxide generation over the longer term may activate degenerative and atrophic processes in the denervated muscle fibers., (Copyright © 2019 The Authors. Published by Elsevier B.V. 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.)

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., (Copyright © 2018. Published by Elsevier Inc.)

The retinal pigment epithelium (RPE) plays a central role in neuroretinal homoeostasis throughout life. Altered proteolysis and inflammatory processes involving RPE contribute to the pathophysiology of age-related macular degeneration (AMD), but the link between these remains elusive. We report for the first time the effect of advanced glycation end products (AGE)-known to accumulate on the ageing RPE's underlying Bruch's membrane in situ-on both key lysosomal cathepsins and NF-κB signalling in RPE. Cathepsin L activity and NF-κB effector levels decreased significantly following 2-week AGE exposure. Chemical cathepsin L inhibition also decreased total p65 protein levels, indicating that AGE-related change of NF-κB effectors in RPE cells may be modulated by cathepsin L. However, upon TNFα stimulation, AGE-exposed cells had significantly higher ratio of phospho-p65(Ser536)/total p65 compared to non-AGEd controls, with an even higher fold increase than in the presence of cathepsin L inhibition alone. Increased proportion of active p65 indicates an AGE-related activation of NF-κB signalling in a higher proportion of cells and/or an enhanced response to TNFα. Thus, NF-κB signalling modulation in the AGEd environment, partially regulated via cathepsin L, is employed by RPE cells as a protective (para-inflammatory) mechanism but renders them more responsive to pro-inflammatory stimuli., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

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.)

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.

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.)

Thioredoxin reductase 1 (TrxR1) is a selenocysteine-containing protein involved in cellular redox homeostasis which is downregulated in skeletal muscle differentiation. Here we show that TrxR1 decrease occurring during myogenesis is functionally involved in the coordination of this cellular process. Indeed, TrxR1 depletion reduces myoblasts growth by inducing an early myogenesis -related gene expression pattern which includes myogenin and Myf5 up-regulation and Cyclin D1 decrease. On the contrary, the overexpression of TrxR1 during differentiation delays myogenic process, by negatively affecting the expression of Myogenin and MyHC. Moreover, we found that miR-23a and miR-23b - whose expression was increased in the early stage of C2C12 differentiation - are involved in the regulation of TrxR1 expression through their direct binding to the 3' UTR of TrxR1 mRNA. Interestingly, the forced inhibition of miR-23a and miR-23b during C2C12 differentiation partially rescues TrxR1 levels and delays the expression of myogenic markers, suggesting the involvement of miR-23 in myogenesis via TrxR1 repression. Taken together, our results depict for the first time a novel molecular axis, which functionally acts in skeletal muscle differentiation through the modulation of TrxR1 by miR-23.

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.)

Frailty is a geriatric syndrome that is an important public health problem for the older adults living in the USA. Although several methods have been developed to measure frailty in humans, we have very little understanding of its etiology. Because the molecular basis of frailty is poorly understood, mouse models would be of great value in determining which pathways contribute to the development of frailty. More importantly, mouse models would be critical in testing potential therapies to treat and possibly prevent frailty. In this article, we present data showing that Sod1KO mice, which lack the antioxidant enzyme, Cu/Zn superoxide dismutase, are an excellent model of frailty, and we compare the Sod1KO mice to the only other mouse model of frailty, mice with the deletion of the IL-10 gene. 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. The Sod1KO mice also show alterations in pathways that have been proposed to play a role in the etiology of frailty: oxidative stress, mitochondrial dysfunction, and cell senescence. Using Sod1KO mice, we show that dietary restriction can delay/prevent characteristics of frailty in mice.

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).)

Age-related loss of skeletal muscle mass and function is a major contributor to morbidity and has a profound effect on the quality of life of older people. The potential role of age-dependent mitochondrial dysfunction and cumulative oxidative stress as the underlying cause of muscle aging remains a controversial topic. Here we show that the pharmacological attenuation of age-related mitochondrial redox changes in muscle with SS31 is associated with some improvements in oxidative damage and mitophagy in muscles of old mice. However, this treatment failed to rescue the age-related muscle fiber atrophy associated with muscle atrophy and weakness. Collectively, these data imply that the muscle mitochondrial redox environment is not a key regulator of muscle fiber atrophy during sarcopenia but may play a key role in the decline of mitochondrial organelle integrity that occurs with muscle aging.

An increasingly sophisticated array of approaches are now available for the study of the activities of reactive oxygen species and oxidative modifications in skeletal muscle, but the most up-to-date techniques are not readily available to many researchers in this field due to their requirement for sophisticated mass spectrometry, imaging or other high cost technologies. Most papers published therefore rely on a number of established approaches although the choice of approach is also clearly dependent upon the experimental model and access to skeletal muscle that is available to the investigator, how much detail is required and the overall question to be addressed. Numerous reports have described the problems associated with some of the popular approaches that are widely followed, including measurement of thiobarbituric acid substances and the sole use of fluorescence-based probes such as dichlorodihydrofluorescein. This brief review reports the areas in which methods are improving to allow valid assessments to made in this area and indicates some of the more recent developments that provide alternative ways to assess the activity of individual species and endpoints in the various experimental models that may be examined., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

A relative increase in oxidation of lipids, proteins and DNA has been recognised to occur in the circulation and tissues of exercising humans and animals since the late 1970s and throughout the ensuing 40 years a great deal of work has been undertaken to elucidate the potential source(s) of this exercise-induced "oxidative stress". Specific aspects of physical exercise (e.g. contractile activity, relative hypoxia, hyperaemia) may theoretically induce increased generation of reactive oxygen species in a number of potential tissues, but data strongly indicate that contractile activity of skeletal muscle predominates as the source of oxidants and contributes to local oxidation and that of extracellular biomaterials. Taken together with the relatively large mass of muscle compared with other tissues and cells it appears that muscle fibres are the major contributor to the relative increase in whole body "oxidative stress" during some forms of exercise. The sub-cellular sources of this increased oxidation have also been the subject of considerable research with early studies predominantly indicating that muscle mitochondria were the likely increased source of oxidants, such as hydrogen peroxide, but assessments of the relative concentrations of hydrogen peroxide in skeletal muscle fibres at rest and during contractile activity do not support this possibility. In contrast, several recent studies have identified NADPH oxidase enzymes in skeletal muscle that appear to play a signalling role in physiological responses exercise and together with xanthine oxidase enzymes may contribute to the relative increase in whole body oxidation. A fuller understanding of the relative roles of these sources and the function(s) of the species generated appears increasingly important in attempts to harness the beneficial effects of exercise for maintenance of health in aging and a variety of chronic conditions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Skeletal muscles of old mice demonstrate a profound inability to regenerate fully following damage. Such a failure could be catastrophic to older individuals where muscle loss is already evident. Degeneration and regeneration of muscle fibres following contraction-induced injury in adult and old mice are well characterised, but little is known about the accompanying changes in motor neurons and neuromuscular junctions (NMJs) following this form of injury although defective re-innervation of muscle following contraction-induced damage has been proposed to play a role in sarcopenia. This study visualised and quantified structural changes to motor neurons and NMJs in Extensor digitorum longus (EDL) muscles of adult and old Thy1-YFP transgenic mice during regeneration following contraction-induced muscle damage. Data demonstrated that the damaging contraction protocol resulted in substantial initial disruption to NMJs in muscles of adult mice, which was reversed entirely within 28 days following damage. In contrast, in quiescent muscles of old mice, ∼15 % of muscle fibres were denervated and ∼80 % of NMJs showed disruption. This proportion of denervated and partially denervated fibres remained unchanged following recovery from contraction-induced damage in muscles of old mice although ∼25 % of muscle fibres were completely lost by 28 days post-contractions. Thus, in old mice, the failure to restore full muscle force generation that occurs following damage does not appear to be due to any further deficit in the percentage of disrupted NMJs, but appears to be due, at least in part, to the complete loss of muscle fibres following damage., Competing Interests: Compliance with ethical standards Conflict of interests The authors declare no conflict of interest.

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., (Copyright © 2016. Published by Elsevier Ltd.)

Diets rich in fruits and vegetables (FV), which contain (poly)phenols, protect against age-related inflammation and chronic diseases. T-lymphocytes contribute to systemic cytokine production and are modulated by FV intake. Little is known about the relative potency of different (poly)phenols in modulating cytokine release by lymphocytes. We compared thirty-one (poly)phenols and six (poly)phenol mixtures for effects on pro-inflammatory cytokine release by Jurkat T-lymphocytes. Test compounds were incubated with Jurkat cells for 48 h at 1 and 30 µm, with or without phorbol ester treatment at 24 h to induce cytokine release. Three test compounds that reduced cytokine release were further incubated with primary lymphocytes at 0·2 and 1 µm for 24 h, with lipopolysaccharide added at 5 h. Cytokine release was measured, and generation of H2O2 by test compounds was determined to assess any potential correlations with cytokine release. A number of (poly)phenols significantly altered cytokine release from Jurkat cells (P<0·05), but H2O2 generation did not correlate with cytokine release. Resveratrol, isorhamnetin, curcumin, vanillic acid and specific (poly)phenol mixtures reduced pro-inflammatory cytokine release from T-lymphocytes, and there was evidence for interaction between (poly)phenols to further modulate cytokine release. The release of interferon-γ induced protein 10 by primary lymphocytes was significantly reduced following treatment with 1 µm isorhamnetin (P<0·05). These results suggest that (poly)phenols derived from onions, turmeric, red grapes, green tea and açai berries may help reduce the release of pro-inflammatory mediators in people at risk of chronic inflammation.

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.)

Although it is now clear that reactive oxygen species (ROS) are not the key determinants of longevity, a number of studies have highlighted the key role that these species play in age-related diseases and more generally in determining individual health span. Age-related loss of skeletal muscle mass and function is a key contributor to physical frailty in older individuals and our current understanding of the key areas in which ROS contribute to age-related deficits in muscle is through defective redox signalling and key roles in maintenance of neuromuscular integrity. This topical review will describe how ROS stimulate adaptations to contractile activity in muscle that include up-regulation of short-term stress responses, an increase in mitochondrial biogenesis and an increase in some catabolic processes. These adaptations occur through stimulation of redox-regulated processes that lead to the activation of transcription factors such as NF-κB, AP-1 and HSF1 which mediate changes in gene expression. They are attenuated during ageing and this appears to occur through an age-related increase in mitochondrial hydrogen peroxide production. The potential for redox-mediated cross-talk between motor neurons and muscle is also described to illustrate how ROS released from muscle fibres during exercise may help maintain the integrity of axons and how the degenerative changes in neuromuscular structure that occur with ageing may contribute to mitochondrial ROS generation in skeletal muscle fibres., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

TNF-α is a key inflammatory mediator and is proposed to induce transcriptional responses via the mitochondrial generation of Reactive Oxygen Species (ROS). The aim of this study was to determine the effect of TNF-α on the production of myokines by skeletal muscle. Significant increases were seen in the release of IL-6, MCP-1/CCL2, RANTES/CCL5 and KC/CXCL1 and this release was inhibited by treatment with Brefeldin A, suggesting a golgi-mediated release of cytokines by muscle cells. An increase was also seen in superoxide in response to treatment with TNF-α, which was localised to the mitochondria and this was also associated with activation of NF-κB. The changes in superoxide, activation of NF-kB and release of myokines were attenuated following pre-treatment with SS-31 peptide indicating that the ability of TNF-α to induce myokine release may be mediated through mitochondrial superoxide, which is, at least in part, associated with activation of the redox sensitive transcription factor NF-kB., (Copyright © 2015. Published by Elsevier B.V.)

Background: Household air pollution in low income countries is an important cause of mortality from respiratory infection. We hypothesised that chronic smoke exposure is detrimental to alveolar macrophage function, causing failure of innate immunity. We report the relationship between macrophage function and prior smoke exposure in healthy Malawians., Methods: Healthy subjects exposed daily to cooking smoke at home volunteered for bronchoalveolar lavage. Alveolar macrophage particulate content was measured as a known correlate of smoke exposure. Phagocytosis and intraphagosomal function (oxidative burst and proteolysis) were measured by a flow cytometric assay. Cytokine responses in macrophages were compared following re-exposure in vitro to wood smoke, before and after glutathione depletion., Results: Volunteers had a range of alveolar macrophage particulate loading. The macrophage capacity for phagosomal oxidative burst was negatively associated with alveolar macrophage particulate content (n = 29, r2 = 0.16, p = 0.033), but phagocytosis per se and proteolytic function were unaffected. High particulate content was associated with lower baseline CXCL8 release (ratio 0.51, CI 0.29-0.89) and lower final concentrations on re-exposure to smoke in vitro (ratio 0.58, CI 0.34-0.97). Glutathione depletion augmented CXCL8 responses by 1.49x (CI 1.02-2.17) compared with wood smoke alone. This response was specific to smoke as macrophages response to LPS were not modulated by glutathione., Conclusion: Chronic smoke exposure is associated with reduced human macrophage oxidative burst, and dampened inflammatory cytokine responses. These are critical processes in lung defence against infection and likely to underpin the relationship between air pollution and pneumonia.

The small heat shock protein α-B-crystallin (CRYAB) is critically involved in stress-related cellular processes such as differentiation, apoptosis, and redox homeostasis. The up-regulation of CRYAB plays a key role in the cytoprotective and antioxidant response, but the molecular pathway driving its expression in muscle cells during oxidative stress still remains unknown. Here we show that noncytotoxic exposure to sodium meta-arsenite (NaAsO2) inducing redox imbalance is able to increase the CRYAB content of C2C12 myoblasts in a transcription-dependent manner. Our in silico analysis revealed a genomic region upstream of the Cryab promoter containing two putative antioxidant-responsive elements motifs and one AP-1-like binding site. The redox-sensitive transcription factors Nrf2 and the AP-1 component c-Jun were found to be up-regulated in NaAsO2-treated cells, and we demonstrated a specific NaAsO2-mediated increase of c-Jun and Nrf2 binding activity to the genomic region identified, supporting their putative involvement in CRYAB regulation following a shift in redox balance. These changes also correlated with a specific phosphorylation of JNK and p38 MAPK kinases, the well-known molecular mediators of signaling pathways leading to the activation of these transcription factors. Pretreatment of C2C12 cells with the JNK inhibitor SP600125 induced a decrease in c-Jun and Nrf2 content and was able to counteract the NaAsO2-mediated increase in CRYAB expression. Thus these data show a direct role of JNK in CRYAB regulation under redox imbalance and also point to a previously unrecognized link between c-Jun and Nrf2 transcription factors and redox-induced CRYAB expression in muscle cells., (Copyright © 2015 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 data provides information in support of the research article, "Differential Cysteine Labeling and Global Label-Free Proteomics Reveals an Altered Metabolic State in Skeletal Muscle Aging", Journal of Proteome Research, 2014, 13 (11), 2008-21 [1]. Raw data is available from ProteomeXchange [2] with identifier PDX001054. The proteome of gastrocnemius muscle from adult and old mice was analyzed by global label-free proteomics and the relative quantification of specific reduced and reversibly oxidized Cysteine (Cys) residues was performed using Skyline [3]. Briefly, reduced Cysteine (Cys) containing peptides was alkylated using N-ethylmalemide (d0-NEM). Samples were desalted and reversibly oxidized Cys residues were reduced using tris(2-carboxyethyl)phosphine (TCEP) and the newly formed reduced Cys residues were labeled with heavy NEM( d5-NEM). Label-free analysis of the global proteome of adult (n=5) and old (n=4) gastrocnemius muscles was performed using Peaks7™ mass spectrometry data analysis software [4]. Relative quantification of Cys containing peptides that were identified as reduced (d(0) NEM labeled) and reversibly oxidized d(5)-NEM labeled was performed using the intensity of their precursor ions in Skyline. Results indicate that muscles from old mice show reduced redox flexibility particularly in proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response.

The idiopathic inflammatory myopathies (IIMs) are a group of rare autoimmune disorders, collectively known as myositis. Affected patients present with proximal muscle weakness, which usually improves following treatment with immunosuppressants, but often incompletely so, thus many patients remain weak. IIMs are characterised histologically by inflammatory cell infiltrates into skeletal muscle and overexpression of major histocompatibility complex I on muscle cell surfaces. Although inflammatory cell infiltrates represent a major feature of myositis there is growing evidence that muscle weakness correlates only poorly with the degree of cellular infiltration, while weakness may in fact precede such infiltrations. The mechanisms underpinning such non-immune cell mediated weakness in IIM are poorly understood. Activation of the endoplasmic reticulum stress pathways appears to be a potential contributor. Data from non-muscle cells indicate that endoplasmic reticulum stress results in altered redox homeostasis capable of causing oxidative damage. In myopathological situations other than IIM, as seen in ageing and sepsis, evidence supports an important role for reactive oxygen species (ROS). Modified ROS generation is associated with mitochondrial dysfunction, depressed force generation and activation of muscle catabolic and autophagy pathways. Despite the growing evidence demonstrating a key role for ROS in skeletal muscle dysfunction in myopathologies other than IIM, no research has yet investigated the role of modified generation of ROS in inducing the weakness characteristic of IIM. This article reviews current knowledge regarding muscle weakness in the absence of immune cells in IIM, and provides a background to the potential role of modified ROS generation as a mechanism of muscle dysfunction. The authors suggest that ROS-mediated mechanisms are potentially involved in non-immune cell mediated weakness seen in IIM and outline how these mechanisms might be investigated in this context. This appears a timely strategy, given recent developments in targeted therapies which specifically modify ROS generation., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Compromised mitochondrial respiratory function is associated with advancing age. Damage due to an increase in reactive oxygen species (ROS) with age is thought to contribute to the mitochondrial deficits. The coenzyme nicotinamide adenine dinucleotide in its reduced (NADH) and oxidized (NAD(+)) forms plays an essential role in the cyclic sequence of reactions that result in the regeneration of ATP by oxidative phosphorylation in mitochondria. Monitoring mitochondrial NADH/NAD(+) redox status during recovery from an episode of high energy demand thus allows assessment of mitochondrial function. NADH fluoresces when excited with ultraviolet light in the UV-A band and NAD(+) does not, allowing NADH/NAD(+) to be monitored in real time using fluorescence microscopy. Our goal was to assess mitochondrial function by monitoring the NADH fluorescence response following a brief period of high energy demand in muscle from adult and old wild-type mice. This was accomplished by isolating whole lumbrical muscles from the hind paws of 7- and 28-month-old mice and making simultaneous measurements of force and NADH fluorescence responses during and after a 5 s maximum isometric contraction. All muscles exhibited fluorescence oscillations that were qualitatively similar and consisted of a brief transient increase followed by a longer transient period of reduced fluorescence and, finally, an increase that included an overshoot before recovering to resting level. Compared with the adult mice, muscles from the 28 mo mice exhibited a delayed peak during the first fluorescence transient and an attenuated recovery following the second transient. These findings indicate an impaired mitochondrial capacity to maintain NADH/NAD(+) redox homeostasis during contractile activity in skeletal muscles of old mice.

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.

Skeletal muscle generation of reactive oxygen species (ROS) is increased following contractile activity and these species interact with multiple signaling pathways to mediate adaptations to contractions. The sources and time course of the increase in ROS during contractions remain undefined. Confocal microscopy with specific fluorescent probes was used to compare the activities of superoxide in mitochondria and cytosol and the hydrogen peroxide content of the cytosol in isolated single mature skeletal muscle (flexor digitorum brevis) fibers prior to, during, and after electrically stimulated contractions. Superoxide in mitochondria and cytoplasm were assessed using MitoSox red and dihydroethidium (DHE) respectively. The product of superoxide with DHE, 2-hydroxyethidium (2-HE) was acutely increased in the fiber cytosol by contractions, whereas hydroxy-MitoSox showed a slow cumulative increase. Inhibition of nitric oxide synthases increased the contraction-induced formation of hydroxy-MitoSox only with no effect on 2-HE formation. These data indicate that the acute increases in cytosolic superoxide induced by contractions are not derived from mitochondria. Data also indicate that, in muscle mitochondria, nitric oxide (NO) reduces the availability of superoxide, but no effect of NO on cytosolic superoxide availability was detected. To determine the relationship of changes in superoxide to hydrogen peroxide, an alternative specific approach was used where fibers were transduced using an adeno-associated viral vector to express the hydrogen peroxide probe, HyPer within the cytoplasmic compartment. HyPer fluorescence was significantly increased in fibers following contractions, but surprisingly followed a relatively slow time course that did not appear directly related to cytosolic superoxide. These data demonstrate for the first time temporal and site specific differences in specific ROS that occur in skeletal muscle fibers during and after contractile activity.

Deletion of copper-zinc superoxide dismutase (CuZnSOD) in Sod1(-/-) mice leads to accelerated loss of muscle mass and force during aging, but the losses do not occur with muscle-specific deletion of CuZnSOD. To determine the role of motor neurons in the muscle decline, we generated transgenic Sod1(-/-) mice in which CuZnSOD was expressed under control of the synapsin 1 promoter (SynTgSod1(-/-) mice). SynTgSod1(-/-) mice expressed CuZnSOD in brain, spinal cord, and peripheral nerve, but not in other tissues. Sciatic nerve CuZnSOD content in SynTgSod1(-/-) mice was ~20% that of control mice, but no reduction in muscle mass or isometric force was observed in SynTgSod1(-/-) mice compared with control animals, whereas muscles of age-matched Sod1(-/-) mice displayed 30-40% reductions in mass and force. In addition, increased oxidative damage and adaptations in stress responses observed in muscles of Sod1(-/-) mice were absent in SynTgSod1(-/-) mice, and degeneration of neuromuscular junction (NMJ) structure and function occurred in Sod1(-/-) mice but not in SynTgSod1(-/-) mice. Our data demonstrate that specific CuZnSOD expression in neurons is sufficient to preserve NMJ and skeletal muscle structure and function in Sod1(-/-) mice and suggest that redox homeostasis in motor neurons plays a key role in initiating sarcopenia during aging.

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.

We have previously shown that deletion of CuZnSOD in mice (Sod1(-/-) mice) leads to accelerated loss of muscle mass and contractile force during aging. To dissect the relative roles of skeletal muscle and motor neurons in this process, we used a Cre-Lox targeted approach to establish a skeletal muscle-specific Sod1-knockout (mKO) mouse to determine whether muscle-specific CuZnSOD deletion is sufficient to cause muscle atrophy. Surprisingly, mKO mice maintain muscle masses at or above those of wild-type control mice up to 18 mo of age. In contrast, maximum isometric specific force measured in gastrocnemius muscle is significantly reduced in the mKO mice. We found no detectable increases in global measures of oxidative stress or ROS production, no reduction in mitochondrial ATP production, and no induction of adaptive stress responses in muscle from mKO mice. However, Akt-mTOR signaling is elevated and the number of muscle fibers with centrally located nuclei is increased in skeletal muscle from mKO mice, which suggests elevated regenerative pathways. Our data demonstrate that lack of CuZnSOD restricted to skeletal muscle does not lead to muscle atrophy but does cause muscle weakness in adult mice and suggest loss of CuZnSOD may potentiate muscle regenerative pathways.