Oxidative Damage in Muscular Dystrophy Correlates with the Severity of the Pathology: Role of Glutathione Metabolism

Muscular dystrophies (MDs) such as Duchenne muscular dystrophy (DMD), sarcoglycanopathy (Sgpy) and dysferlinopathy (Dysfy) are recessive genetic neuromuscular diseases that display muscle degeneration. Although these MDs have comparable endpoints of muscle pathology, the onset, severity and the course of these diseases are diverse. Different mechanisms downstream of genetic mutations might underlie the disparity in these pathologies. We surmised that oxidative damage and altered antioxidant function might contribute to these differences. The oxidant and antioxidant markers in the muscle biopsies from patients with DMD (n = 15), Sgpy (n = 15) and Dysfy (n = 15) were compared to controls (n = 10). Protein oxidation and lipid peroxidation was evident in all MDs and correlated with the severity of pathology, with DMD, the most severe dystrophic condition showing maximum damage, followed by Sgpy and Dysfy. Oxidative damage in DMD and Sgpy was attributed to the depletion of glutathione (GSH) and lowered antioxidant activities while loss of GSH peroxidase and GSH-S-transferase activities was observed in Dysfy. Lower GSH level in DMD was due to lowered activity of gamma-glutamyl cysteine ligase, the rate limiting enzyme in GSH synthesis. Similar analysis in cardiotoxin (CTX) mouse model of MD showed that the dystrophic muscle pathology correlated with GSH depletion and lipid peroxidation. Depletion of GSH prior to CTX exposure in C2C12 myoblasts exacerbated oxidative damage and myotoxicity. We deduce that the pro and anti-oxidant mechanisms could be correlated to the severity of MD and might influence the dystrophic pathology to a different extent in various MDs. On a therapeutic note, this could help in evolving novel therapies that offer myoprotection in MD.     

Halofuginone and muscular dystrophy

Muscular dystrophies (MDs) include different inherited diseases that all result in progressive muscle degeneration, impaired locomotion and often premature death. The major focus of MD research has been on alleviating the primary genetic deficit - using gene therapy and myoblast-transfer approaches to promote expression of the deficient or mutated genes in the muscle fibers. Although promising, these approaches have not yet entered into clinical practice and unfortunately for MD patients, there is currently no cure. Thus, the development of complementary and supportive therapies that slow disease progression and improve patients' quality of life is critically important. The main features of MDs are sarcolemmal instability and increased myofiber vulnerability to mechanical stress, resulting in myofiber degeneration. Fibrosis, with progressive replacement of muscle tissue, is a prominent feature in some MDs, preventing complete regeneration and hampering muscle functions. TGFβ is the leading candidate for activating fibroblasts and eliciting overproduction of extracellular matrix (ECM) proteins. Halofuginone, an inhibitor of Smad3 phosphorylation downstream of TGFβ signaling, inhibits the activation of fibroblasts and their ability to synthesize ECM, regardless of their origin or location. In animal models of MDs with prominent muscle fibrosis, halofuginone treatment has resulted in both prevention of collagen production in young animals and resolution of established fibrosis in older ones: the reduction in muscle collagen content was associated with improved muscle histopathology and major improvements in muscle function. Recently, these halofuginone-dependent improvements were also observed in MD with minor fibrosis involvement, probably due to a direct effect of halofuginone on muscle cells, resulting in myotube fusion that is dependent on Akt and MAPK pathway activation. In summary, halofuginone improves muscle histopathology and muscle functions in various MDs, via inhibition of muscle fibrosis on the one hand, and increased myotube fusion on the other.     

The mitochondrial DNA polymerase as a target of oxidative damage

The mitochondrial respiratory chain is a source of reactive oxygen species (ROS) that are responsible for oxidative modification of biomolecules, including proteins. Due to its association with mitochondrial DNA, DNA polymerase γ (pol γ) is in an environment to be oxidized by hydrogen peroxide and hydroxyl radicals that may be generated in the presence of iron ions associated with DNA. We tested whether human pol γ was a possible target of ROS with H₂O₂ and investigated the effect on the polymerase activities and DNA binding efficiency. A 1 h treatment with 250 µM H₂O₂ significantly inhibited DNA polymerase activity of the p140 subunit and lowered its DNA binding efficiency. Addition of p55 to the p140 catalytic subunit prior to H₂O₂ treatment offered protection from oxidative inactivation. Oxidatively modified amino acid residues in pol γ  resulting from H₂O₂ treatment were observed in vitro as well as in vivo, in SV40-transfected human fibroblasts. Pol γ was detected as one of the major oxidized mitochondrial matrix proteins, with a detectable decline in polymerase activity. These results suggest pol γ as a target of oxidative damage, which may result in a reduction in mitochondrial DNA replication and repair capacities.     

Loss of Fis1 impairs proteostasis during skeletal muscle aging in Drosophila

Increased levels of dysfunctional mitochondria within skeletal muscle are correlated with numerous age‐related physiopathological conditions. Improving our understanding of the links between mitochondrial function and muscle proteostasis, and the role played by individual genes and regulatory networks, is essential to develop treatments for these conditions. One potential player is the mitochondrial outer membrane protein Fis1, a crucial fission factor heavily involved in mitochondrial dynamics in yeast but with an unknown role in higher‐order organisms. By using Drosophila melanogaster as a model, we explored the effect of Fis1 mutations generated by transposon Minos‐mediated integration. Mutants exhibited a higher ratio of damaged mitochondria with age as well as elevated reactive oxygen species levels compared with controls. This caused an increase in oxidative stress, resulting in large accumulations of ubiquitinated proteins, accelerated muscle function decline, and mitochondrial myopathies in young mutant flies. Ectopic expression of Fis1 isoforms was sufficient to suppress this phenotype. Loss of Fis1 led to unbalanced mitochondrial proteostasis within fly muscle, decreasing both flight capabilities and lifespan. Fis1 thus clearly plays a role in fly mitochondrial dynamics. Further investigations into the detailed function of Fis1 are necessary for exploring how mitochondrial function correlates with muscle health during aging.     

Proteins modulation in human skeletal muscle in the early phase of adaptation to hypobaric hypoxia

High altitude hypoxia is a paraphysiological condition triggering redox status disturbances of cell organization leading, via oxidative stress, to proteins, lipids, and DNA damage. In man, skeletal muscle, after prolonged exposure to hypoxia, undergoes mass reduction and alterations at the cellular level featuring a reduction of mitochondrial volume density, accumulation of lipofuscin, a product of lipid peroxidation, and dysregulation of enzymes whose time course is unknown. The effects of 7-9 days exposure to 4559 m (Margherita Hut, Monte Rosa, Italy) on the muscle proteins pattern were investigated, pre- and post-exposure, in ten young subjects, by 2-D DIGE and MS. Ten milligram biopsies were obtained from the mid part of the vastus lateralis muscle at sea level (control) and at altitude, after 7-9 days hypoxia. Differential analysis indicates that proteins involved in iron transport, tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and oxidative stress responses were significantly (p<0.05) decreased in hypoxia. Parenthetically, hypoxia markers such as hypoxia inducible factor 1 alpha (HIF-1alpha) and pyruvate dehydrogenase kinase 1 (PDK1) were still at the pre-hypoxia levels, whereas the mammalian target of rapamycin (mTOR), a marker of protein synthesis, was reduced.     

In inclusion-body myositis muscle fibers Parkinson-associated DJ-1 is increased and oxidized

Sporadic inclusion-body myositis (s-IBM) is the most common muscle disease of older persons. The muscle-fiber molecular phenotype exhibits similarities to both Alzheimer-disease (AD) and Parkinson-disease (PD) brains, including accumulations of amyloid-beta, phosphorylated tau, alpha-synuclein, and parkin, as well as evidence of oxidative stress and mitochondrial abnormalities. Early-onset autosomal-recessive PD can be caused by mutations in the DJ-1 gene, leading to its inactivation. DJ-1 has antioxidative and mitochondrial-protective properties. In AD and PD brains, DJ-1 is increased and oxidized. We studied DJ-1 in 17 s-IBM and 18 disease-control and normal muscle biopsies by: (1) immunoblots of muscle homogenates and mitochondrial fractions; (2) real-time PCR; (3) oxyblots evaluating DJ-1 oxidation; (4) light- and electron-microscopic immunocytochemistry. Compared to controls, in s-IBM muscle fibers DJ-1 was: (a) increased in the soluble fraction, monomer 2-fold (P = 0.01), and dimer 2.8-fold (P = 0.004); (b) increased in the mitochondrial fraction; (c) highly oxidized; and (d) aggregated in about 15% of the abnormal muscle fibers. DJ-1 mRNA was increased 3.5-fold (P = 0.034). Accordingly, DJ-1 might play a role in human muscle disease, and thus not be limited to human CNS degenerations. In s-IBM muscle fibers, DJ-1 could be protecting these fibers against oxidative stress, including protection of mitochondria.     

Myofiber damage accompanying intramuscular parasitism by Sarcocystis muris

Myofiber degeneration which results from Sarcocystis infection exhibits a number of fine structural features suggestive of other myopathies and several well-defined fine structural features not characteristic of other myopathies. Some of these fine structural features are similar to those observed in intramuscular infections of Trichinella spiralis, another muscle parasite. Major alterations of the myofibrillar contractile apparatus occur at the periphery of the membrane bound parasitophorous vacuole which include splitting and fragmentation of the myofibrils at the longitudinal ends of the parasitophorous vacuole and Z line dissolution at the radial periphery. Membranous structural elements including mitochondria, sarcoplasmic reticulum and T system components become disarrayed as the myofibrils degenerate. Some minor hypertrophy of the sarcoplasmic reticulum occurs in conjunction with initial fragmentation of the myofibrils bu no major dilation or hypertrophy has been observed. There is a distinctive membranous organization of the interface of the parasitophorous vacuole. The presence of pycnotic and fragmenting nuclei, sarcolemmal invaginations with accompanying fibrous connective tissue invasion and large areas of undifferentiated cytoplasm suggest the ultimate necrosis and destruction of infected myofibers. The similarity between morphological features of myofibrillar degeneration accompanying intramuscular Sarcocystis muris infections and those associated with a variety of myopathies resulting from other causes suggests that a common mechanism of muscle response to damage might result in the observed structural degeneration.     

Muscle regeneration in mitochondrial myopathies

Mitochondrial myopathies cover a diverse group of disorders in which ragged red and COX-negative fibers are common findings on muscle morphology. In contrast, muscle degeneration and regeneration, typically found in muscular dystrophies, are not considered characteristic features of mitochondrial myopathies. We investigated regeneration in muscle biopsies from 61 genetically well-defined patients affected by mitochondrial myopathy. Our results show that the perturbed energy metabolism in mitochondrial myopathies causes ongoing muscle regeneration in a majority of patients, and some were even affected by a dystrophic morphology. The results add to the complexity of the pathogenesis underlying mitochondrial myopathies, and expand the knowledge about the impact of energy deficiency on another aspect of muscle structure and function.     

Mitochondrial intermembrane inclusion bodies: The common denominator between human mitochondrial myopathies and creatine depletion due to impairment of cellular energetics

Mitochondrial inclusion bodies are often described in skeletal muscle of patients suffering diseases termed mitochondrial myopathies. A major component of these structures was discovered as being creatine kinase. Similar creatine kinase enriched inclusion bodies in the mitochondria of creatine depleted adult rat cardiomyocytes have been demonstrated. Structurally similar inclusion bodies are observed in mitochondria of ischemic and creatine depleted rat skeletal muscle. This paper describes the various methods for inducing mitochondrial inclusion bodies in rodent skeletal muscle, and compares their effects on muscle metabolism to the metabolic defects of mitochondrial myopathy muscle. We fed rats with a creatine analogue guanidino propionic acid and checked their soled for mitochondrial inclusion bodies, with the electron microscope. The activity of creatine kinase was analysed by measuring creatine stimulated oxidative phosphorylation in soleus skinned fibres using an oxygen electrode . The guanidino propionic acid-rat soleus mitochondria displayed no creatine stimulation, whereas control soleus did, even though the GPA soled had a five fold increase in creatine kinase protein per mitochondrial protein. The significance of these results in light of their relevance to human mitochondrial myopathies and the importance of altered muscle metabolism in the formation of these crystalline structures are discussed. (Mol Cell Biochem 174: 283–289, 1997)     

Flux control of cytochrome c oxidase in human skeletal muscle

In the present work, by titrating cytochrome c oxidase (COX) with the specific inhibitor KCN, the flux control coefficient and the metabolic reserve capacity of COX have been determined in human saponin-permeabilized muscle fibers. In the presence of the substrates glutamate and malate, a 2.3 +/- 0.2-fold excess capacity of COX was observed in ADP-stimulated human skeletal muscle fibers. This value was found to be dependent on the mitochondrial substrate supply. In the combined presence of glutamate, malate, and succinate, which supported an approximately 1.4-fold higher rate of respiration, only a 1.4 +/- 0.2-fold excess capacity of COX was determined. In agreement with these findings, the flux control of COX increased, in the presence of the three substrates, from 0.27 +/- 0.03 to 0.36 +/- 0.08. These results indicate a tight in vivo control of respiration by COX in human skeletal muscle. This tight control may have significant implications for mitochondrial myopathies. In support of this conclusion, the analysis of skeletal muscle fibers from two patients with chronic progressive external ophthalmoplegia, which carried deletions in 11 and 49% of their mitochondrial DNA, revealed a substantially lowered reserve capacity and increased flux control coefficient of COX, indicating severe rate limitations of oxidative phosphorylation by this enzyme.     

Modeling Cardiac Action Potential Shortening Driven by Oxidative Stress-Induced Mitochondrial Oscillations in Guinea Pig Cardiomyocytes

Ischemia-induced shortening of the cardiac action potential and its heterogeneous recovery upon reperfusion are thought to set the stage for reentrant arrhythmias and sudden cardiac death. We have recently reported that the collapse of mitochondrial membrane potential (ΔΨ_m) through a mechanism triggered by reactive oxygen species (ROS), coupled to the opening of sarcolemmal ATP-sensitive potassium (K_ATP) channels, contributes to electrical dysfunction during ischemia-reperfusion. Here we present a computational model of excitation-contraction coupling linked to mitochondrial bioenergetics that incorporates mitochondrial ROS-induced ROS release with coupling between the mitochondrial energy state and electrical excitability mediated by the sarcolemmal K_ATP current (I_K,ATP). Whole-cell model simulations demonstrate that increasing the fraction of oxygen diverted from the respiratory chain to ROS production triggers limit-cycle oscillations of ΔΨ_m, redox potential, and mitochondrial respiration through the activation of a ROS-sensitive inner membrane anion channel. The periods of transient mitochondrial uncoupling decrease the cytosolic ATP/ADP ratio and activate I_K,ATP, consequently shortening the cellular action potential duration and ultimately suppressing electrical excitability. The model simulates emergent behavior observed in cardiomyocytes subjected to metabolic stress and provides a new tool for examining how alterations in mitochondrial oxidative phosphorylation will impact the electrophysiological, contractile, and Ca²⁺ handling properties of the cardiac cell. Moreover, the model is an important step toward building multiscale models that will permit investigation of the role of spatiotemporal heterogeneity of mitochondrial metabolism in the mechanisms of arrhythmogenesis and contractile dysfunction in cardiac muscle.     

Smooth muscle cell sarcolemma. Purification and properties of plasma membranes from the rat uterus

A membrane fraction with sarcolemmal properties was purified from the smooth muscle layers (myometrium) of rat uterus by successive differential and equilibrium centrifugation in sucrose. The putative sarcolemmal fraction was identified by iodination with [^125I]iodosulfanilic acid, had an equilibrium density of 1.15, and was enriched in enzyme activities usually associated with the plasma membrane including 5′-nucleotidase (EC 3.1.3.5) and (Na⁺ + K⁺) ATPase (EC 3.6.1.3). These membranes were free of mitochondrial or nuclear membrane contamination, suggesting the relative enrichment of sarcolemmal membranes in the fraction. Proteins of the membranes were heterogeneous with respect to molecular weight, but only a few were labelled when intact muscle was radioiodinated. Uniform resistance of sarcolemmal proteins to trypsin digestion and salt extraction suggested many are tightly bound or intrinsic membrane proteins and was a further indication of the homogeneity of membranes in this fraction.     

Methionine sulfoxide reductase 2 reversibly regulates Mge1, a cochaperone of mitochondrial Hsp70, during oxidative stress

Peptide methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in protein(s). Although these reductases have been implicated in several human diseases, there is a dearth of information on the identity of their physiological substrates. By using Saccharomyces cerevisiae as a model, we show that of the two methionine sulfoxide reductases (MXR1, MXR2), deletion of mitochondrial MXR2 renders yeast cells more sensitive to oxidative stress than the cytosolic MXR1. Our earlier studies showed that Mge1, an evolutionarily conserved nucleotide exchange factor of Hsp70, acts as an oxidative sensor to regulate mitochondrial Hsp70. In the present study, we show that Mxr2 regulates Mge1 by selectively reducing MetO at position 155 and restores the activity of Mge1 both in vitro and in vivo. Mge1 M155L mutant rescues the slow-growth phenotype and aggregation of proteins of mxr2Δ strain during oxidative stress. By identifying the first mitochondrial substrate for Mxrs, we add a new paradigm to the regulation of the oxidative stress response pathway.     

Proteomic signatures of in vivo muscle oxidative capacity in healthy adults

Adequate support of energy for biological activities and during fluctuation of energetic demand is crucial for healthy aging; however, mechanisms for energy decline as well as compensatory mechanisms that counteract such decline remain unclear. We conducted a discovery proteomic study of skeletal muscle in 57 healthy adults (22 women and 35 men; aged 23–87 years) to identify proteins overrepresented and underrepresented with better muscle oxidative capacity, a robust measure of in vivo mitochondrial function, independent of age, sex, and physical activity. Muscle oxidative capacity was assessed by ³¹P magnetic resonance spectroscopy postexercise phosphocreatine (PCr) recovery time (τ_PCr) in the vastus lateralis muscle, with smaller τ_PCr values reflecting better oxidative capacity. Of the 4,300 proteins quantified by LC‐MS in muscle biopsies, 253 were significantly overrepresented with better muscle oxidative capacity. Enrichment analysis revealed three major protein clusters: (a) proteins involved in key energetic mitochondrial functions especially complex I of the electron transport chain, tricarboxylic acid (TCA) cycle, fatty acid oxidation, and mitochondrial ABC transporters; (b) spliceosome proteins that regulate mRNA alternative splicing machinery, and (c) proteins involved in translation within mitochondria. Our findings suggest that alternative splicing and mechanisms that modulate mitochondrial protein synthesis are central features of the molecular mechanisms aimed at maintaining mitochondrial function in the face of impairment. Whether these mechanisms are compensatory attempt to counteract the effect of aging on mitochondrial function should be further tested in longitudinal studies.     

Impaired Mitochondrial Energy Production Causes Light-Induced Photoreceptor Degeneration Independent of Oxidative Stress

Two insults often underlie a variety of eye diseases including glaucoma, optic atrophy, and retinal degeneration—defects in mitochondrial function and aberrant Rhodopsin trafficking. Although mitochondrial defects are often associated with oxidative stress, they have not been linked to Rhodopsin trafficking. In an unbiased forward genetic screen designed to isolate mutations that cause photoreceptor degeneration, we identified mutations in a nuclear-encoded mitochondrial gene, ppr, a homolog of human LRPPRC. We found that ppr is required for protection against light-induced degeneration. Its function is essential to maintain membrane depolarization of the photoreceptors upon repetitive light exposure, and an impaired phototransduction cascade in ppr mutants results in excessive Rhodopsin1 endocytosis. Moreover, loss of ppr results in a reduction in mitochondrial RNAs, reduced electron transport chain activity, and reduced ATP levels. Oxidative stress, however, is not induced. We propose that the reduced ATP level in ppr mutants underlies the phototransduction defect, leading to increased Rhodopsin1 endocytosis during light exposure, causing photoreceptor degeneration independent of oxidative stress. This hypothesis is bolstered by characterization of two other genes isolated in the screen, pyruvate dehydrogenase and citrate synthase. Their loss also causes a light-induced degeneration, excessive Rhodopsin1 endocytosis and reduced ATP without concurrent oxidative stress, unlike many other mutations in mitochondrial genes that are associated with elevated oxidative stress and light-independent photoreceptor demise.     

Curcumin ameliorates CKD-induced mitochondrial dysfunction and oxidative stress through inhibiting GSK-3β activity

Curcumin has been reported to attenuate muscle atrophy. However, the underling mechanism remains unclear. The aim of this study was to investigate whether curcumin could improve chronic kidney disease (CKD)-induced muscle atrophy and mitochondrial dysfunction by inhibiting glycogen synthase kinase-3β (GSK-3β) activity. The sham and CKD mice were fed either a control diet or an identical diet containing 0.04% curcumin for 12 weeks. The C2C12 myotubes were treated with H₂O₂ in the presence or absence of curcumin. In addition, wild-type and muscle-specific GSK-3β knockout (KO) CKD model mice were made by 5/6 nephrectomy, and the sham was regarded as control. Curcumin could exert beneficial effects, including weight maintenance and improved muscle function, increased mitochondrial biogenesis, alleviated mitochondrial dysfunction by increasing adenosine triphosphate levels, activities of mitochondrial electron transport chain complexes and basal mitochondrial respiration and suppressing mitochondrial membrane potential. In addition, curcumin modulated redox homeostasis by increasing antioxidant activity and suppressed mitochondrial oxidative stress. Moreover, the protective effects of curcumin had been found to be mediated via inhibiting GSK-3β activity in vitro and in vivo. Importantly, GSK-3β KO contributed to improved mitochondrial function, attenuated mitochondrial oxidative damage and augmented mitochondrial biogenesis in muscle of CKD. Overall, this study suggested that curcumin alleviated CKD-induced mitochondrial oxidative damage and mitochondrial dysfunction via inhibiting GSK-3β activity in skeletal muscle.     

Low intensity training of mdx mice reduces carbonylation and increases expression levels of proteins involved in energy metabolism and muscle contraction

High intensity training induces muscle damage in dystrophin-deficient mdx mice, an animal model for Duchenne muscular dystrophy. However, low intensity training (LIT) rescues the mdx phenotype and even reduces the level of protein carbonylation, a marker of oxidative damage. Until now, beneficial effects of LIT were mainly assessed at the physiological level. We investigated the effects of LIT at the molecular level on 8-week-old wild-type and mdx muscle using 2D Western blot and protein–protein interaction analysis. We found that the fast isoforms of troponin T and myosin binding protein C as well as glycogen phosphorylase were overcarbonylated and downregulated in mdx muscle. Some of the mitochondrial enzymes of the citric acid cycle were overcarbonylated, whereas some proteins of the respiratory chain were downregulated. Of functional importance, ATP synthase was only partially assembled, as revealed by Blue Native PAGE analysis. LIT decreased the carbonylation level and increased the expression of fast isoforms of troponin T and of myosin binding protein C, and glycogen phosphorylase. In addition, it increased the expression of aconitate hydratase and NADH dehydrogenase, and fully restored the ATP synthase complex. Our study demonstrates that the benefits of LIT are associated with lowered oxidative damage as revealed by carbonylation and higher expression of proteins involved in energy metabolism and muscle contraction. Potentially, these results will help to design therapies for DMD based on exercise mimicking drugs.