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 κB (NFκB), 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.
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.
Schematic illustration of the process of redox signalling of responses to contractile activity and their modification during ageing. In muscle from young and adult subjects, contractile activity leads to activation of muscle NADPH oxidase(s) with generation of superoxide that is rapidly converted to hydrogen peroxide with local oxidation of redox‐active thiols and activation of specific redox‐sensitive transcriptional pathways. This mediates multiple adaptations to the contractile activity including stress responses and mitochondrial biogenesis. In old subjects, this process is attenuated by over‐production of hydrogen peroxide by mitochondria which at the level of individual fibres may be related to the partially or full fibre denervation.
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.
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.
Summary
A common characteristic of aging is defective regeneration of skeletal muscle. The molecular pathways underlying age‐related decline in muscle regenerative potential remain elusive. microRNAs ...are novel gene regulators controlling development and homeostasis and the regeneration of most tissues, including skeletal muscle. Here, we use satellite cells and primary myoblasts from mice and humans and an in vitro regeneration model, to show that disrupted expression of microRNA‐143‐3p and its target gene, Igfbp5, plays an important role in muscle regeneration in vitro. We identified miR‐143 as a regulator of the insulin growth factor‐binding protein 5 (Igfbp5) in primary myoblasts and show that the expression of miR‐143 and its target gene is disrupted in satellite cells from old mice. Moreover, we show that downregulation of miR‐143 during aging may act as a compensatory mechanism aiming at improving myogenesis efficiency; however, concomitant upregulation of miR‐143 target gene, Igfbp5, is associated with increased cell senescence, thus affecting myogenesis. Our data demonstrate that dysregulation of miR‐143‐3p:Igfbp5 interactions in satellite cells with age may be responsible for age‐related changes in satellite cell function.
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.
Using deletion of CuZnSOD in motor neurons to induce increased oxidative stress and mimic loss of motor neurons in spinal cord, we show that neuronal loss induces NMJ disruption that progresses over time to cause muscle atrophy and weakness in i‐mn‐Sod1KO mice. The delay between loss of motor neuron number and significant atrophy and weakness suggests there are compensatory mechanisms at play to mitigate the impact of reduced innervation (possibly including increased sprouting) that eventually fail over time.
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.
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.
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.
Summary
Mice lacking Cu,Zn superoxide dismutase (SOD1) show accelerated, age‐related loss of muscle mass. Lack of SOD1 may lead to increased superoxide, reduced nitric oxide (NO), and increased ...peroxynitrite, each of which could initiate muscle fiber loss. Single muscle fibers from flexor digitorum brevis of wild‐type (WT) and Sod1−/− mice were loaded with NO‐sensitive (4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate, DAF‐FM) and superoxide‐sensitive (dihydroethidium, DHE) probes. Gastrocnemius muscles were analyzed for SOD enzymes, nitric oxide synthases (NOS), and 3‐nitrotyrosine (3‐NT) content. A lack of SOD1 did not increase superoxide availability at rest because no increase in ethidium or 2‐hydroxyethidium (2‐HE) formation from DHE was seen in fibers from Sod1−/− mice compared with those from WT mice. Fibers from Sod1−/− mice had decreased NO availability (decreased DAF‐FM fluorescence), increased 3‐NT in muscle proteins indicating increased peroxynitrite formation and increased content of peroxiredoxin V (a peroxynitrite reductase), compared with WT mice. Muscle fibers from Sod1−/− mice showed substantially reduced generation of superoxide in response to contractions compared with fibers from WT mice. Inhibition of NOS did not affect DHE oxidation in fibers from WT or Sod1−/− mice at rest or during contractions, but transgenic mice overexpressing nNOS showed increased DAF‐FM fluorescence and reduced DHE oxidation in resting muscle fibers. It is concluded that formation of peroxynitrite in muscle fibers is a major effect of lack of SOD1 in Sod1−/− mice and may contribute to fiber loss in this model, and that NO regulates superoxide availability and peroxynitrite formation in muscle.
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