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Disruption of skeletal muscle homeostasis by substitution with fibrotic tissue constitutes the principal cause of death in Duchenne muscular dystrophy (DMD) patients, yet the implicated fibrogenic mechanisms remain poorly understood. This study identifies the extracellular PAI-1/urokinase-type plasminogen activator (uPA) balance as an important regulator of microribonucleic acid (miR)-21 biogenesis, controlling age-associated muscle fibrosis and dystrophy progression. Genetic loss of PAI-1 in mdx dystrophic mice anticipated muscle fibrosis through these sequential mechanisms: the alteration of collagen metabolism by uPA-mediated proteolytic processing of transforming growth factor (TGF)-β in muscle fibroblasts and the activation of miR-21 expression, which inhibited phosphatase and tensin homologue and enhanced AKT signaling, thus endowing TGF-β with a remarkable cell proliferation-promoting potential. Age-associated fibrogenesis and muscle deterioration in mdx mice, as well as exacerbated dystrophy in young PAI-1(-/-) mdx mice, could be reversed by miR-21 or uPA-selective interference, whereas forced miR-21 overexpression aggravated disease severity. The PAI-1-miR-21 fibrogenic axis also appeared dysregulated in muscle of DMD patients, providing a basis for effectively targeting fibrosis and muscular dystrophies in currently untreatable individuals.

The repair of an injured tissue is a complex biological process involving the coordinated activities of tissue-resident and infiltrating cells in response to local and systemic signals. Following acute tissue injury, inflammatory cell infiltration and activation/proliferation of resident stem cells is the first line of defense to restore tissue homeostasis. However, in the setting of chronic tissue damage, such as in Duchenne Muscular Dystrophy, inflammatory infiltrates persist, the ability of stem cells (satellite cells) is blocked and fibrogenic cells are continuously activated, eventually leading to the conversion of muscle into nonfunctional fibrotic tissue. This review explores our current understanding of the cellular and molecular mechanisms underlying efficient muscle repair that are dysregulated in muscular dystrophy-associated fibrosis and in aging-related muscle dysfunction., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Chronic obstructive pulmonary disease is associated with systemic effects including reduced body weight, oxidative stress and altered circulating TNFalpha levels. The present study was aimed to investigate whether chronic exposure to cigarette smoke induces these systemic changes in a guinea pig model. Seven animals/group were exposed to the smoke of seven cigarettes/day, 5 days/week, during 2, 4 and 6 months (chronic exposure). Three animals/group were sacrificed immediately, 3 h or 24 h after exposure to seven cigarettes (acute exposure). Chronically smoke-exposed animals exhibited lower body weight gain, starting at 5th week, and goblet cell metaplasia in small bronchioles. At 6 months there was a trend for increased plasma and lung tissue TNFalpha levels. No changes, neither in skeletal muscle glutathione (GSH) nor in plasma lipid peroxidation, were observed at any time point after chronic exposure. However, skeletal muscle GSH decreased and plasma lipid peroxidation increased immediately after acute smoke exposure, equaling control levels thereafter. We conclude that cigarette smoke exposure in the guinea pig induces a transient and repeated oxidative effect, which might result in impaired systemic metabolism and consequent failure of smoke-exposed animals to gain weight. The effects of cigarette smoke on body weight antecede and appear to be independent from the alterations produced in small airways.

Background: A physiological increase in muscle glutathione after training is not seen in patients with chronic obstructive pulmonary disease (COPD), indicating abnormal peripheral muscle adaptations to exercise., Objective: We hypothesized that oxidative stress is primarily associated with low body mass index (BMI)., Methods: Eleven patients with preserved BMI (BMI(N): 28.2 +/- 1.2 kg.m(-2)), 9 patients with low BMI (BMI(L): 19.7 +/- 0.60 kg.m(-2)) and 5 age-matched controls (26.5 +/- 0.9 kg.m(-2)) were studied before and after 8 weeks of high-intensity endurance training. Reduced glutathione (GSH) and gamma-glutamyl cysteine synthase heavy-subunit chain mRNA expression (gammaGCS-HS mRNA) were measured in the vastus lateralis., Results: After training, exercise capacity increased (DeltaVO(2)PEAK, 13 +/- 5.2%; 10 +/- 5.6% and 15 +/- 4.3% in BMI(L), BMI(N) and controls, respectively; p < 0.05 each). GSH levels decreased in BMI(L) (from 5.2 +/- 0.7 to 3.7 +/- 0.8 nmol/mg protein, DeltaGSH -1.5 +/- 0.7 nmol/mg protein, p < 0.05); no changes were seen in BMI(N) (from 5.4 +/- 0.7 to 6.7 +/- 0.9 nmol/mg protein, DeltaGSH 1.3 +/- 0.9 nmol/mg protein), whereas GSH markedly increased in controls (from 4.6 +/- 1 to 8.7 +/- 0.4 nmol/mg protein, DeltaGSH 4.1 +/- 1 nmol/mg protein, p < 0.01). DeltaGSH in BMI(L) was different from DeltaGSH in BMI(N) and controls (p < 0.05, each). Consistent changes were observed in gammaGCS-HS mRNA expression., Conclusions: GSH depletion after training in BMI(L) may suggest that oxidative stress plays a key role in muscle wasting in COPD patients.

Skeletal muscle differentation is a complex process regulated at multiple levels. This study addressed the effect of glutathione (GSH) depletion on the transition of murine skeletal muscle C2C12 myoblasts into myocytes induced by growth factor inactivation. Cellular GSH levels increased within 24 hours on myogenic stimulation of myoblasts due to enhanced GSH synthetic rate accounted for by stimulated glutamate-L-cysteine ligase (also known as gamma-glutamylcysteine synthetase) activity. In contrast, the synthesis rate of GSH using gamma-glutamylcysteine and glutamate as precursors, which reflects the activity of the GSH synthetase, did not change during differentiation. The stimulation of GSH stores preceded the myogenic differentiation of C2C12 myoblasts monitored by expression of muscle-specific genes, creatine kinase (CK), myosin heavy chain (MyHC), and MyoD. The pattern of DNA binding activity of NF-kappaB and AP-1 in differentiating cells was similar both displaying an activation peak at 24 hours after myogenic stimulation. Depletion of cellular GSH levels 24 hours after stimulation of differentiation abrogated myogenesis as reflected by lower CK activity, MyHC levels, MyoD expression, and myotubes formation, effects that were reversible on GSH replenishment by GSH ethyl ester (GHSEE). Moreover, GSH depletion led to sustained activation of NF-kappaB, while GSHEE prevented it. Furthermore, inhibition of NF-kappaB activation restored myogenesis despite GSH depletion. Thus, GSH contributes to the formation of myotubes from satellite myoblasts by ensuring inactivation of NF-kappaB, and hence maintaining optimal GSH levels may be beneficial in restoring muscle mass in chronic inflammatory disorders.

Post-training downregulation of muscle tumour necrosis factor (TNF)-alpha messenger ribonucleic acid (mRNA) expression and decrease in cellular TNF-alpha levels have been reported in the elderly. It is hypothesised that chronic obstructive pulmonary disease (COPD) patients may not show these adaptations due to their reduced ability to increase muscle antioxidant capacity with training. Eleven COPD patients (forced expiratory volume in one second 40 +/- 4.4% of the predicted value) and six age-matched controls were studied. Pre- and post-training levels of TNF-alpha, soluble TNF receptors (sTNFRs: sTNFR55 and sTNFR75) and interleukin (IL)-6 in plasma at rest and during exercise and vastus lateralis TNF-alpha mRNA were examined. Moderate-intensity constant-work-rate exercise (11 min at 40% of pretraining peak work-rate) increased pretraining plasma TNF-alpha levels in COPD patients (from 17 +/- 3.2 to 23 +/- 2.7 pg x mL(-1); p<0.005) but not in controls (from 19 +/- 4.6 to 19 +/- 3.2 pg x mL(-1)). No changes were observed in sTNFRs or IL-6 levels. After 8 weeks' endurance training, moderate-intensity exercise increased plasma TNF-alpha levels similarly to pretraining (from 16 +/- 3 to 21 +/- 4 pg x mL(-1); p<0.01). Pretraining muscle TNF-alpha mRNA expression was significantly higher in COPD patients than in controls (29.3 +/- 13.9 versus 5.0 +/- 1.5 TNF-alpha/18S ribonucleic acid, respectively), but no changes were observed after exercise or training. It is concluded that moderate-intensity exercise abnormally increases plasma tumour necrosis factor-alpha levels in chronic obstructive pulmonary disease patients without exercise-induced upregulation of the tumour necrosis factor-alpha gene in skeletal muscle.

The present study was undertaken to test whether endurance training in patients with COPD, along with enhancement of muscle bioenergetics, decreases muscle redox capacity as a result of recurrent episodes of cell hypoxia induced by high intensity exercise sessions. Seventeen patients with COPD (FEV(1), 38 +/- 4% pred; PaO2), 69 +/- 2.7 mm Hg; PaCO2, 42 +/- 1.7 mm Hg) and five age-matched control subjects (C) were studied pretraining and post-training. Reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, and gamma-glutamyl cysteine synthase heavy subunit chain mRNA expression (gammaGCS-HS mRNA) were measured in the vastus lateralis. Pretraining redox status at rest and after moderate (40% Wpeak) constant-work rate exercise were similar between groups. After training (DeltaWpeak, 27 +/- 7% and 37 +/- 18%, COPD and C, respectively) (p < 0.05 each), GSSG levels increased only in patients with COPD (from 0.7 +/- 0.08 to 1.0 +/- 0.15 nmol/ mg protein, p < 0.05) with maintenance of GSH levels, whereas GSH markedly increased in C (from 4.6 +/- 1.03 to 8.7 +/- 0.41 nmol/ mg protein, p < 0.01). Post-training gammaGCS-HS mRNA levels increased after submaximal exercise in patients with COPD. No evidence of lipid peroxidation was observed. We conclude that although endurance training increased muscle redox potential in healthy subjects, patients with COPD showed a reduced ability to adapt to endurance training reflected in lower capacity to synthesize GSH.

Ceramide has been identified as a putative lipid messenger that mediates diverse cellular processes including cell death. Since glutathione (GSH) depletion is known to sensitize cells to many cytotoxic agents and as a result of the reported regulation of neutral sphyngomyelinase (NSMase) by GSH, the present study compared the role of individual SMases in the induction of oxidative stress, regulation of cellular GSH, and apoptosis of rat hepatocytes. Exposure of cultured rat hepatocytes to exogenous Bacillus cereus sphingomyelinase (bSMase), a neutral SMase, or human placenta sphingomyelinase (hSMase), an acidic SMase (ASMase), generated similar ceramide levels in a dose-dependent manner. However, whereas bSMase increased hepatocellular GSH levels, hSMase depleted GSH stores, an effect that was prevented by monensin and mannose 6-phosphate (M-6-P), suggesting that exogenous hSMase enters hepatocytes by endocytosis and is delivered to an endosomal/lysosomal acidic compartment. Interestingly, despite the differential effect of either SMases on cell GSH levels, both bSMase and hSMase increased gamma-glutamylcysteine synthetase heavy-subunit chain (gamma-GCS-HS) mRNA levels. Consistent with these findings on GSH regulation, hSMase, but not bSMase, generated reactive oxygen species (ROS), being accompanied by mitochondrial depolarization, suggesting that hSMase targeted mitochondria, leading to oxidative stress. Accordingly, hepatocytes displayed a selective sensitivity to hSMase in contrast to bSMase exposure, and depletion of GSH stores enhanced susceptibility to hSMase as a result of potentiation of ROS formation and caspase 3 activation. Thus, these findings reveal the ability of ASMase to induce oxidative stress as a result of the targeting of mitochondria, and that GSH depletion sensitizes hepatocytes to the ASMase-induced apoptosis.

Because reactive oxygen species (ROS) have been implicated as mediators of inflammatory bowel disease (IBD), the purpose of the present work was to determine the functional role of mucosal GSH in the trinitrobenzenesulfonic acid in 50% ethanol (TNBS+ethanol)-induced colitis in rats. Mucosal samples were taken to evaluate the temporal relationship between the extent of injury, the levels of glutathione (GSH) during acute colitis induced by TNBS+ethanol, and the effect of N-acetylcysteine (NAC) administration. In vitro assays revealed the interaction of TNBS with GSH leading to the almost instantaneous disappearance of GSH, while the reductive metabolism of TNBS by GSSG reductase generated ROS. Mucosal samples from TNBS+ethanol-treated rats indicated a direct correlation between GSH depletion and injury detected as soon as 30 minutes after TNBS+ethanol administration that persisted 24 hours post treatment. Although, short term depletion of mucosal GSH per se by diethylmaleate did not result in mucosal injury, the oral administration of NAC (40 mM) 4 hours after TNBS+ethanol treatment increased GSH stores (2-fold), decreasing the extent of mucosal injury (60-70%) examined at 24 hours post treatment. However, an equimolar dose of dithiothreitol failed to increase GSH levels and protect mucosa from TNBS+ethanol-induced injury. Interestingly, GSH levels in TNBS+ethanol-treated rats recovered by 1-2 weeks, an effect that was accounted for by an increase of gamma-glutamylcysteine synthetase (gamma-GCS) activity due to an induction of gamma-GCS-heavy subunit chain mRNA. Thus, TNBS promotes two independent mechanisms of injury, GSH depletion and ROS generation, both being required for the manifestation of mucosal injury as GSH limitation renders intestine susceptible to the TNBS-induced ROS overgeneration. Accordingly, in vivo administration of NAC attenuates the acute colitis through increased mucosal GSH levels, suggesting that GSH precursors may be of relevance in the acute relapse of IBD.

Background & Aims: The molecular mechanisms responsible for leukocyte recruitment in experimental colitis are poorly understood. The aims of this study were to measure expression of endothelial intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) and to determine their role in leukocyte recruitment in experimental colitis., Methods: Rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis and control rats were studied 1, 7, or 21 days after treatment. ICAM-1 and VCAM-1 expressions were measured by the double radiolabeled antibody technique. Leukocyte-endothelial cell interactions were determined in colonic venules by fluorescence intravital microscopy. Therapeutic effects of treatment with anti-VCAM-1 antibodies were also assessed., Results: Colonic endothelial ICAM-1 was constitutively expressed and did not increase in colitic animals. In contrast, constitutive expression of VCAM-1 was low but markedly increased (6-fold) 1 and 7 days after induction of colitis. Increased colonic expression of VCAM-1 paralleled macroscopic damage score, myeloperoxidase activity, and increased leukocyte adhesion in colonic venules. The latter was significantly decreased by immunoneutralization of ICAM-1 and completely abrogated by immunoneutralization of VCAM-1. Long-term administration of anti-VCAM-1 antibody resulted in significant attenuation of colitis., Conclusions: Induction of colitis in rats by TNBS is followed by up-regulation of endothelial VCAM-1. VCAM-1 and constitutive ICAM-1 are major determinants of leukocyte recruitment to the inflamed intestine.

Background & Aims: Tumor necrosis factor (TNF)-alpha induces cell injury by generating oxidative stress from mitochondria. The purpose of this study was to determine the effect of ethanol on the sensitization of hepatocytes to TNF-alpha., Methods: Cultured hepatocytes from ethanol-fed (ethanol hepatocytes) or pair-fed (control hepatocytes) rats were exposed to TNF-alpha, and the extent of oxidative stress, gene expression, and viability were evaluated., Results: Ethanol hepatocytes, which develop a selective deficiency of mitochondrial glutathione (mGSH), showed marked susceptibility to TNF-alpha. The susceptibility to TNF-alpha, manifested as necrosis rather than apoptosis, was accompanied by a progressive increase in hydrogen peroxide that correlated inversely with cell survival. Nuclear factor kappaB activation by TNF-alpha was significantly greater in ethanol hepatocytes than in control hepatocytes, an effect paralleled by the expression of cytokine-induced neutrophil chemoattractant. Similar sensitization of normal hepatocytes to TNF-alpha was obtained by depleting the mitochondrial pool of GSH with 3-hydroxyl-4-pentenoate. Restoration of mGSH by S-adenosyl-L-methionine or by GSH-ethyl ester prevented the increased susceptibility of ethanol hepatocytes to TNF-alpha., Conclusions: These results indicate that mGSH controls the fate of hepatocytes in response to TNF-alpha. Its depletion caused by alcohol consumption amplifies the power of TNF-alpha to generate reactive oxygen species, compromising mitochondrial and cellular functions that culminate in cell death.

It has been widely postulated that age-dependent changes in the mitochondrial genetic system may contribute to the human aging process. We recently reported unchanged specific activities of mitochondrial respiratory chain enzymes and a decrease in oxidation capacity of different substrates with aging, due, in part, to some confounding variables such as physical activity or tobacco consumption. The present study deals with age-related changes in muscle mtDNA structure and its biogenesis in humans. We found a low prevalence of mtDNA rearrangements with aging, only detected by PCR. The mtDNA content increased significantly with age (b = 0.0115, P < 0.0001). Also, an unchanged steady-state level of mitochondrial transcripts, a reduced transcription rate (P < 0.0001), and an increase in mitochondrial membrane lipid peroxidation (P < 0.0001) were observed in aging. These data demonstrate that minor structural mtDNA changes appear during the human aging process. By contrast, alterations in mitochondrial homeostasis ultimately producing modifications in mitochondrial biogenesis rates could play a role in the process of human senescence.