Article Text
Abstract
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.
- Dermatomyositis
- Polymyositis
- Inflammation
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Background
The idiopathic inflammatory myopathies (IIMs) have traditionally comprised polymyositis dermatomyositis (including juvenile polymyositis dermatomyositis), myositis occurring as part of another connective tissue disease and inclusion body myositis.1 Recent research makes it clear that the IIMs represent a growing spectrum of rare and heterogenous autoimmune disorders, where the IIM phenotype subset is predictable by the presence of a growing number of myositis specific autoantibodies and myositis associated autoantibodies.2 Patients with IIM exhibit proximal muscle weakness, which can be severe and very disabling. Rarely, patients with IIM may also succumb, most often due to associated malignancies or pulmonary complications.3 IIM are characterised histologically by the finding of inflammatory cell infiltrations in skeletal muscle in combination with upregulation of major histocompatibility complex I (MHC-I) within muscle cells and upon their cell surfaces. These abnormalities occur in combination with elevations of skeletal muscle enzyme levels (creatine kinase) and the presence of circulating myositis specific autoantibodies and myositis associated autoantibodies.2 The muscle inflammatory changes can occur alone, or in combination with inflammation of skin and/or lung (interstitial lung disease) and/or joints.
The current treatment of choice for IIM is glucocorticoids, used alone or in combination with various immunosuppressive agents, but the muscles of patients with IIM often remain weak despite this treatment. Such therapeutic failure may represent drug-resistance or be due to damage, that is, irreversible loss of muscle fibres, fibre atrophy and/or fatty replacement. However, it is increasingly recognised that muscle weakness may persist where disease activity (presence of infiltrating immune cells within muscle) or overt muscle damage are not present. Although inflammatory cell infiltrates were previously thought to represent the primary pathological process causing myositis-induced muscle weakness, there is growing evidence that other, non-inflammatory factors may also be involved. Thus, several studies indicate that the severity of muscle weakness correlates only poorly with the degree of inflammatory cell infiltrations.4–6 Moreover, weakness may persist when treatment has successfully cleared inflammatory infiltrates.5 In addition, in an established murine model of myositis, muscle weakness occurs before the characteristic inflammatory cell infiltrations appear.7 The mechanisms underlying this ‘non-inflammatory’ component of muscle weakness in myositis are poorly understood. Data indicate that the endoplasmic reticulum (ER) stress pathways are chronically activated in IIM and may play a major aetiological role in these disorders, but how ER stress pathways contribute to muscle weakness induction is currently unknown.8 In skeletal muscle the ER is also known as the sarcoplasmic reticulum (SR), in recognition of its specialised adaptions to enable the rapid calcium fluxes required for muscle contractions. However, the SR and ER are functionally similar (see later), so the terms are used interchangeably hereafter. Data from non-muscle cell lines suggest that ER stress pathway activation can result in altered redox homeostasis capable of causing oxidative damage to DNA, lipids and proteins via the generation of reactive oxygen species (ROS).9 How altered redox homeostasis and ROS relate to weakness-induction in IIM remains an unaddressed question. Studies examining muscle weakness in situations other than IIM, such as in ageing and in disuse, clearly suggest a role for altered ROS signalling in mediating muscle weakness.10–12 Although playing an important physiological role as cellular signalling mediators, increased ROS activities are associated with mitochondrial dysfunction, depressed force generation and activation of muscle catabolic and autophagy pathways.13–15 Despite this evidence suggesting a key role for modified ROS signalling in skeletal muscle weakness in myopathologies other than IIM, little research has examined the role of modified ROS generation in causing muscle weakness in the IIM.13 ,16 This review discusses the current knowledge regarding mechanisms of non-immune cell-mediated muscle weakness in IIM and provides a background to the role of modified ROS generation in muscle dysfunction, and we hypothesise that ROS-mediated mechanisms likely play a major role in the development of non-immune mediated muscle weakness in IIM.
The potential role of ER stress in IIM
The biology of ER stress and the unfolded protein response
The ER has a key responsibility for correctly folding and assembling newly synthesised proteins. It is a highly specialised cellular compartment, allowing formation of disulphide bonds.17 The ER is able to respond to changes in folding capacity requirements, so as to ensure precision in protein assembly. The ER responds to protein load changes via the unfolded protein response (UPR), achieving increased folding capacity by elevating the expression of the chaperones Grp78/BiP and Grp94, and by suppression of translation by eiF2α de-phosphorylation via protein kinase-like endoplasmic reticulum kinase (PERK).17 Accumulation of misfolded or aggregated proteins within the ER results in activation of the UPR. The latter is divided into three pathways, each characterised by different transmembrane signalling proteins (figure 1): inositol requiring enzyme 1 (IRE1), eukaryotic translation initiation factor 2-α kinase 3 (PERK) and activating transcription factor 6 (ATF6). Chronic overactivation of the UPR typically results in cell death, an evolutionarily favourable characteristic of the UPR, allowing organisms to remove cells which are inaccurately assembling proteins.18 ER stress has been described in the pathogenesis of numerous diseases, from metabolic dysfunction and neurodegenerative disorders to certain muscular dystrophies.19–21
ER stress in IIM
ER stress has recently been shown to be directly associated with muscle fibre degeneration and dysfunction in IIM.8 Muscle tissue from the murine H-2kb myositis model and from patients with myositis, displays ER stress-related markers such as increased Grp78 and Caspase12.8 ,22 Additional examination of the H-2kb murine model, which was generated to be immunodeficient, demonstrated profound muscle weakness, which further supports the hypothesis that non-immune mechanisms (eg, ER stress) could play a key role in muscle dysfunction in IIM.23 A recent study of muscle biopsies from patients with myositis confirmed colocalisation of the ER stress-related proteins Grp75 and Grp94, within fibres which show upregulated levels of MHC-I.8 ,24 Studies of patients diagnosed with another IIM, sporadic inclusion body myositis, have similarly demonstrated activation of the ER stress pathway, as evidenced by upregulation of homocysteine-induced ER protein.25
Overall, these data suggest that ER stress may play an important role in non-immune cell mediated muscle weakness in IIM but the processes mediating these responses are poorly understood.26 Recent work has reported a potential mechanistic link between ER stress induction and the generation of ROS via altered redox homeostasis, augmented by calcium transfer through the mitochondrial associated membranes (MAMs).27 The MAMs are a collection of proteins enriched between the ER and mitochondria, which form a transient complex, allowing bidirectional communication between the two compartments.28 Based on this and the involvement of modified ROS signalling in myopathologies other than IIM, it appears feasible that modified ROS may play a role in mediating muscle weakness in IIM.
ROS as mediators of skeletal muscle weakness
Background
ROS is the term used to describe a family of highly reactive ‘species’, which are either oxygen- (eg, superoxide, O2−,) or derivatives of oxygen (eg, hydrogen peroxide, H2O2).29 ROS are generated in several subcellular sites, as respiration products within the mitochondrial electron transport chain (ETC) or as integral components of the protein folding machinery within the ER.30 Mitochondria are a significant source of ROS, together with the plasma membrane and endothelium.31 ,32 Superoxide is the primary radical species generated within mitochondria, generated by complex III of the ETC, and is typically converted to H2O2 by the antioxidant defence enzyme manganese superoxide dismutase, located within the mitochondrial matrix. H2O2 can readily cross membranes into the cytosol, where it can be converted to H2O and O2 by catalase.32 Superoxide can form the highly reactive peroxynitrite species (ONOO−) by reacting with nitric oxide (NO), generated by the NO synthase enzyme family. Cytosolic superoxide can also be converted by copper zinc superoxide dismutase, to generate H2O2.33 Superoxide generated from the enzyme xanthine oxidase in the endothelium can cross into, and exert effects, in the extracellular environment. Alternative sources of superoxide generating enzymes are the lipoxygenases, cyclo-oxygenases and NAD(P)H oxidases.31 A schematic representation of ROS generation in the skeletal muscle appears in figure 2.
ROS were previously considered only in the context of toxicity, potentially causing oxidative damage to DNA, proteins and lipids.30 However, ROS are now considered as key signalling molecules, playing important roles in cellular homeostasis, for example, mediating adaptive responses via activation of redox-sensitive transcription.34 However, during cellular stress, for example, from infection and inflammation, this tightly regulated network is perturbed, causing aberrant ROS generation capable of causing dysregulation of the adaptive responses.10
ER stress induces oxidative damage to cellular components
Maintenance of the redox status of a cell and its compartments is vital for cellular homeostasis.30 The ER in particular requires a highly oxidised environment to enable the formation of disulphide bonds, via the thiol-disulphide exchange mechanism, during protein assembly.35 As part of the protein folding machinery the glutathione/glutathione disulphide redox couple helps maintain ER redox status.29 During protein folding and assembly, the transfer of electrons from protein disulphide isomerase to O2, necessary for disulphide bond formation, generates H2O2.36 Under physiological conditions networks of antioxidant defence enzymes regulate ROS actions.29 However, during significant ER stress, accumulation of misfolded proteins perpetuates H2O2 production, as a result of persistent protein refolding attempts.37 The H2O2 produced crosses the ER membrane into other cellular compartments and either directly oxidises proteins and lipids, etc, or reacts with additional species to generate even more highly reactive radicals such as the hydroxyl anion.38 Studies using antioxidants, such as butylated hydroxyanisole, show that these agents protect against ER stress-induced protein misfolding and subsequent cell death.9 Thus, the ER itself may be considered a site of ROS production during ER stress, with implications for whole cell redox homeostasis.
The association between ER stress and modified redox homeostasis can be explained by an intrinsic link between the ER and mitochondria. In skeletal muscle the ER has a physical link with mitochondria, via the calcium release unit (CRU).39 Mitochondria are precisely positioned at the junction of the A and I bands along the length of the fibre, resulting in a close proximity to the CRU.40 This structural link provides a mechanism of rapid and efficient delivery of ATP and Ca2+ to the sarcomere during contraction.41 Ca2+ released from muscle ER can readily be taken up by mitochondria in myofibres.42 This is associated with alterations in mitochondrial membrane potential (ΔΨm), increased cellular respiration and elevated ROS production.43 We hypothesise that enhanced ROS generation downstream of ER-mitochondria signalling, results in oxidative damage to key cellular components. Skeletal muscle SR, and the ER of other cells, share marked similarities in composition and function, muscle SR being highly enriched in ER stress proteins (eg, Grp78, Calnexin), and ER and SR play an important role in Ca2+ homeostasis, although muscle SR has evolved a very specialised role for the latter.44 Based on this we reason that activation of ER stress proteins (eg, Grp78) in IIM reflect perturbations in SR function, which may have a direct impact on mitochondria.8 Studies in non-muscle systems have also reported direct intraorganelle contact via the MAM, that is, analogous to the CRU in muscle and again providing a regulated mechanism for bidirectional Ca2+ transport.45 During ER stress Ca2+ transport between the ER and mitochondria increases, resulting in increased respiration, causing enhanced ROS production.46 Ca2+ released from the ER stimulates the NO synthase enzymes, enhancing NO production, which inhibits ETC complexes and results in elevated ROS generation.47 Furthermore, Ca2+ stimulates release of cytochrome c from the mitochondrial matrix, which inhibits ETC complexes, again resulting in elevated ROS production.48 Lastly, cytochrome c changes may result in additional Ca2+ release from SR/ER and uptake by mitochondria, further fuelling ER stress-induced ROS generation.49 Interestingly, examination of mitochondrial enzyme citrate synthase, an important component of the tricarboxylic acid cycle, in the H-2kb murine model of IIM reported no changes in enzyme activity in response to MHC-I upregulation and activation of the ER stress pathway.50 However, another recent study has reported that the impact of ER stress on mitochondrial activity is via direct inhibition of the ETC downstream of tricarboxylic acid cycle enzymes.51 Therefore, further examination of all components of the respiratory pathway warrants significant investigation to clarify on this issue. There is thus a clear link between SR/ER stress and modified redox homeostasis, either as a result of direct mechanisms derived from protein folding, or crosstalk between ER and mitochondria. To what extent these mechanisms contribute to muscle weakness in IIM remains to be elucidated, although clearly ROS plays an important generic role in mediating muscle function and dysfunction in myopathologies other than IIM.10 ,52 Alternate non-immune mechanisms of weakness such as hypoxia have been described in the context of IIM, with patients with myositis having a lower muscle capillary density.53 ,54 Moreover, the relationship between hypoxia and modified ROS generation has received significant attention. Many studies indicate that hypoxia induces changes in ROS generation, whereby severe hypoxia in skeletal muscle results in elevated H2O2 generation.55 However, there is a large element of controversy surrounding the application of these findings in the context of pathology.56
Impact of modified ROS generation on skeletal muscle function
Isolated mitochondria taken from quiescent skeletal muscle of old mice produce elevated H2O2 levels compared with mitochondria from younger mice.57 Increased mitochondrial superoxide generation in old rodents is the likely source of H2O2.14 ,58 Oxidative damage localised to the mitochondria is observed in skeletal muscle of old mice.59 Supplementation of mice with antioxidants precludes disuse-induced muscle atrophy and dysfunction, clearly suggesting that ROS are important mediators in muscle mass and functional losses in this situation.60 Muscle dysfunction associated with altered redox homeostasis has been demonstrated in transgenic murine studies. Knock out of a key endogenous antioxidant enzyme, copper zinc superoxide dismutase, results in skeletal muscle weakness and disturbed neuromuscular signalling.61 This transgenic model also displays accelerated loss of muscle mass and mitochondrial dysfunction.62 Ablation of manganese superoxide dismutase, a key mitochondrial antioxidant enzyme, results in increased oxidative damage in muscle alongside impaired mitochondrial function.63 ,64 Rodent models of dysferlinopathies and Duchenne muscular dystrophy demonstrate elevated levels of protein oxidative damage, suggesting that perturbed redox homeostasis also plays an aetiopathological role in these pathologies.65 These findings suggest that regulation of ROS generation by endogenous antioxidants is of crucial importance, while perturbations may clearly be associated with muscle dysfunction.
Impact of enhanced ROS generation on muscle weakness in IIM
Chronic activation of ER stress pathways is clearly documented in IIM, while the association between ER stress and alterations in redox homeostasis is well characterised in non-muscle systems.8 ,27 Based on evidence demonstrating that modified ROS generation is associated with muscle dysfunction in myopathologies other than IIM, an obvious question arising is whether altered redox homeostasis in response to ER stress is also a key contributor to muscle weakness in IIM.10 In the acute phase, that is, immediately following activation of the ER pathway, elevated H2O2 levels within the ER could readily pass into other cellular compartments, react with other free radicals to form other highly reactive species, and so cause oxidative damage to cytosolic components, such as the contractile proteins, which are rich in readily oxidisable thiol residues. Sustained or chronic activation of the ER stress response, as likely occurs in the IIM, could result in persistent but futile attempts at protein folding, and so sustain H2O2 generation within the ER lumen. Additionally, persistent Ca2+ transfer from the ER to the mitochondria would result in further uncontrolled ROS generation. Cumulative oxidative damage to key muscle components, including contractile apparatus, could then impair force generation.66 Moreover, based on previous findings, chronically elevated ROS production by the mitochondria would result in uncoupling of the ETC, decreasing the rate of respiration and attenuating ATP production, potentially creating an energy deficit and so further impairing contractile performance.67 ,68 We propose that immediate and sustained phases of ER stress may play critical aetiological roles in causing muscle weakness and dysfunction in the IIM (figure 3). Recent studies have reported an association between dysregulated ROS generation and a role in autoimmunity. Specifically, mutations in the ncf1 gene, which encodes the cytosolic subunit of the ROS generating enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2, result in an overall depression of ROS generation. This mutation and altered ROS response have been demonstrated to be associated with increased severity of arthritis in a rodent model, and attributed to an attenuated respiratory burst response.69 Furthermore the ncf1 mutation and modified ROS response have been recently identified as causing activation of the Interferon (IFN) signalling pathway.70 In the context of IIM this is an important observation as the IFN pathway has been implicated in the pathogenesis of myositis.71
Interventions to target ROS-mediated muscle dysfunction
The use of ‘broad spectrum’ antioxidant compounds has received significant attention, but several studies have reported deleterious effects, suggesting that this approach may not be feasible.72–74 Research into the development of novel compounds targeting specific cellular sites of ROS generation may provide a more elegant approach. For example, supplementation with a small permeable peptide termed Szeto-Schiller 31 (SS-31), which localises to the mitochondrial intermembrane space, has shown significant beneficial effects in other models of skeletal muscle dysfunction. SS-31 associates with mitochondrial cardiolipin, improves ATP production, reduces mitochondrial ROS production, and lowers oxidative damage and in a rodent model, disuse muscle atrophy was attenuated by SS-31; a process demonstrated to be mediated by mitochondria-derived ROS.75 In another rodent model of disuse atrophy, induced by using mechanical ventilation, SS-31 peptide protected against oxidative damage to reduce mitochondrial and contractile dysfunction.11 In aging rodent studies, SS-31 improved several measures of muscle performance.76 Another specific targeted antioxidant (MitoQ) has, in mice, attenuated mitochondrial dysfunction in quadriceps and in a rodent model of amyotrophic lateral sclerosis improved muscle strength.77 ,78 Targeting processes upstream of alterations in redox homeostasis, for instance focusing on muscle ER/SR stress pathways, may represent another potential therapeutic avenue. Studies using salubrinal, an inhibitor of eIF2α de-phosphorylation, appear to show protection against ER stress and cell death.79 ,80 It is possible that upstream targeting to prevent/reduce activation of the ER stress pathway could ameliorate downstream ROS production and so mitochondrial and contractile dysfunction. Thus, SS-31, MitoQ and salubrinal, or similar products, are therapeutic options worthy of investigation in IIM.
Summary
Persistent muscle weakness in the absence of inflammatory cell infiltrates in the IIM remains a poorly understood phenomenon. Sustained activation of ER stress pathways is a well characterised hallmark of IIM, so a strong mechanistic link between ER stress and perturbed redox homeostasis is thus hypothesised (figure 4). These processes are likely intrinsically linked in IIM, and perturbations in redox homeostasis likely play a role in causing non-immune cell mediated muscle weakness. In view of the ongoing development of novel compounds to target these pathways, it appears timely to now investigate the role of modified ROS in inducing muscle weakness in IIM.
Acknowledgments
The authors thank Dr Giorgos Sakellariou and Dr Caroline Cotton for their insightful comments and critique in preparation of this manuscript.
References
Footnotes
Handling editor Tore K Kvien
Contributors The four authors contributed to the writing and preparation of this article.
Competing Interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.