Francis McArdle

Adaptive responses of mouse skeletal muscle to contractile activity : The effect of age

This study has characterised the time course of two major transcriptional adaptive responses to exercise (changes in antioxidant defence enzyme activity and heat shock protein (HSP) content) in muscles of adult and old male mice following isometric contractions and has examined the mechanisms involved in the age-related reduction in transcription factor activation. Muscles of B6XSJL mice were subjected to isometric contractions and analysed for antioxidant defence enzyme activities, heat shock protein content and transcription factor DNA binding activity. Data demonstrated a significant increase in superoxide dismutase (SOD) and catalase activity and HSP content of muscles of adult mice following contractile activity which was associated with increased activation of the transcription factors, nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1) and heat shock factor (HSF) following contractions. Significant increases in SOD and catalase activity and heat shock cognate (HSC70) content were seen in quiescent muscles of old mice. The increase in antioxidant defence enzyme activity following contractile activity seen in muscles of adult mice was not seen in muscles of old mice and this was associated with a failure to fully activate NF-kappaB and AP-1 following contractions. In contrast, although the production of HSPs was also reduced in muscles of old mice following contractile activity compared with muscles of adult mice following contractions, this was not due to a gross reduction in the DNA binding activity of HSF.

Preconditioning of skeletal muscle against contraction‐induced damage: the role of adaptations to oxidants in mice

Adaptations of skeletal muscle following exercise are accompanied by changes in gene expression, which can result in protection against subsequent potentially damaging exercise. One cellular signal activating these adaptations may be an increased production of reactive oxygen and nitrogen species (ROS). The aim of this study was to examine the effect of a short period of non‐damaging contractions on the subsequent susceptibility of muscle to contraction‐induced damage and to examine the changes in gene expression that occur following the initial contraction protocol. Comparisons with changes in gene expression in cultured myotubes following treatment with a non‐damaging concentration of hydrogen peroxide (H2O2) were used to identify redox‐sensitive genes whose expression may be modified by the increased ROS production during contractions. Hindlimb muscles of mice were subjected to a preconditioning, non‐damaging isometric contraction protocol in vivo. After 4 or 12 h, extensor digitorum longus (EDL) and soleus muscles were removed and subjected to a (normally) damaging contraction protocol in vitro. Muscles were also analysed for changes in gene expression induced by the preconditioning protocol using cDNA expression techniques. In a parallel study, C2C12 myotubes were treated with a non‐damaging concentration (100 μm) of H2O2 and, at 4 and 12 h following treatment, myotubes were treated with a damaging concentration of H2O2 (2 mm). Myotubes were analysed for changes in gene expression at 4 h following treatment with 100 μm H2O2 alone. Data demonstrate that a prior period of non‐damaging contractile activity resulted in significant protection of EDL and soleus muscles against a normally damaging contraction protocol 4 h later. This protection was associated with significant changes in gene expression. Prior treatment of myotubes with a non‐damaging concentration of H2O2 also resulted in significant protection against a damaging treatment, 4 and 12 h later. Comparison of changes in gene expression in both studies identified haem oxygenase‐1 as the sole gene showing increased expression during adaptation in both instances suggesting that activation of this gene results from the increased ROS production during contractile activity and that it may play a role in protection of muscle cells against subsequent exposure to damaging activity.

Is oxidative stress a physiological cost of reproduction? An experimental test in house mice

Investment in reproduction is costly and frequently decreases survival or future reproductive success. However, the proximate underlying causes for this are largely unknown. Oxidative stress has been suggested as a cost of reproduction and several studies have demonstrated changes in antioxidants with reproductive investment. Here, we test whether oxidative stress is a consequence of reproduction in female house mice (Mus musculus domesticus), which have extremely high energetic demands during reproduction, particularly through lactation. Assessing oxidative damage after a long period of reproductive investment, there was no evidence of increased oxidative stress, even when females were required to defend their breeding territory. Instead, in the liver, markers of oxidative damage (malonaldehyde, protein thiols and the proportion of glutathione in the oxidized form) indicated lower oxidative stress in reproducing females when compared with non-reproductive controls. Even during peak lactation, none of the markers of oxidative damage indicated higher oxidative stress than among non-reproductive females, although a positive correlation between protein oxidation and litter mass suggested that oxidative stress may increase with fecundity. Our results indicate that changes in redox status occur during reproduction in house mice, but suggest that females use mechanisms to cope with the consequences of increased energetic demands and limit oxidative stress.

UVR-induced oxidative stress in human skin in vivo: effects of oral vitamin C supplementation

Previous studies of cultured skin cells and murine skin in vivo have indicated that UVR-induced damage involves the generation of reactive oxygen species and depletion of endogenous antioxidant systems. In order to explore the relevance of this to UVR-induced damage to human skin, we have undertaken a detailed examination of the time-course of changes in markers of oxidative stress in human skin following exposure to physiological amounts of UVR in vivo. In addition, we have examined the skin bioavailability of a common nutritional antioxidant, vitamin C, and have assessed the effects of supplementation on markers of oxidative stress. Our hypothesis was that acute exposure of human skin to UVR in vivo would lead to oxidation of cellular biomolecules that could be prevented by prior vitamin C treatment. A UVR-challenge of 120 mJ/cm2 of broadband UVB (peak 310 nm, range 270-400 nm) was applied to buttock skin of 8 healthy volunteers. This caused a rapid and significant rise in activity of skin catalase at 1 h and an increase in the oxidized/total glutathione ratio at 6 h post-UVR. AP-1 DNA binding also peaked at 1-6 h post-UVR, then declined rapidly to baseline levels. No significant changes were seen in skin malonaldehyde content. Oral vitamin C supplements (500 mg/day) were taken by 12 volunteers for 8 weeks resulting in significant rises in plasma and skin vitamin C content. Supplementation had no effect on the UVR-induced erythemal response. The skin malonaldehyde content was reduced by vitamin C supplementation, but surprisingly, reductions in the skin content of total glutathione and protein thiols were also seen. We speculate that this apparently paradoxical effect could be due to regulation of total reductant capacity by skin cells, such that vitamin C may have been replacing other reductants in these cells. No evidence was obtained for an effect of the supplementary vitamin C on the mild oxidative stress seen in human skin following UVR exposure.

Vitamin E and the Oxidative Stress of Exercise

Abstract: There is clear evidence that contracting skeletal muscle generates a complex set of reactive oxygen and nitrogen species and that the pattern and magnitude of this generation is influenced by the type and frequency of the muscle contraction protocol. The functions of these species in exercising organisms are still unclear although data have been presented indicating that they play a role in contraction‐induced muscle damage and/or in signaling adaptive responses to contractions. Vitamin E has been claimed to exert a regulatory effect on the actions of contraction‐induced oxidants for a considerable time, although evidence for any specific role in this area is lacking. A review of studies in this area suggests that vitamin E supplements are unlikely to reliably reduce the severity of contraction‐induced muscle damage but, in contrast, appear capable of modulating redox‐regulated adaptive responses to contractions. Full evaluation of the roles of oxidants and antioxidants such as vitamin E in responses of muscle to contractions should enable the manipulation of these processes with potential beneficial effects on maintenance of optimal muscle function.

Effect of antioxidant supplementation on the adaptive response of human skin fibroblasts to UV-induced oxidative stress

The effect of supplementation with substances having antioxidant properties on the adaptive responses of human skin fibroblasts to UV-induced oxidative stress was studied in vitro. UVR was found to induce a substantial oxidative stress in fibroblasts, resulting in an increased release of superoxide anions and an increase in lipid peroxidation (shown by an elevated malonaldehyde content). Sub-lethal doses of UVR were also found to induce adaptive responses in the fibroblast antioxidant defences, with a transient rise in catalase and superoxide dismutase activities followed by a slower, large increase in cellular glutathione content. Supplementation of the fibroblasts with the antioxidants, Trolox (a water soluble analogue of alpha-tocopherol), ascorbic acid or beta-carotene, had differential effects on these responses. Trolox supplementation reduced the UVR-induced cellular oxidative stress and adaptive response in a predictable concentration-dependent manner. This was in contrast to ascorbic acid which increased superoxide release from fibroblasts. At low doses, ascorbate supplements also reduced the magnitude of the adaptive increases in catalase and superoxide dismutase activities and increase in glutathione content. Beta-carotene had a similar effect to ascorbic acid, reducing the extent of the adaptations to UVR at lower doses while simultaneously increasing superoxide release and malonaldehyde content. These in vitro data indicate that only the vitamin E analogue suppressed UVR-induced oxidative stress in a predictable manner and suggest that common dietary antioxidants may not be equally effective in reducing the potential deleterious effects of UVR-induced oxidative stress in skin.

Role of superoxide-nitric oxide interactions in the accelerated age-related loss of muscle mass in mice lacking Cu, Zn superoxide dismutase

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.

Effects of dietary carbohydrate on delayed onset muscle soreness and reactive oxygen species after contraction induced muscle damage

Background: Delayed onset muscle soreness (DOMS) occurs after unaccustomed exercise and has been suggested to be attributable to reactive oxygen species (ROS). Previous studies have shown increased ROS after lengthening contractions, attributable to invading phagocytes. Plasma glucose is a vital fuel for phagocytes, therefore carbohydrate (CHO) status before exercise may influence ROS production and DOMS Objective: To examine the effect of pre-exercise CHO status on DOMS, ROS production, and muscle function after contraction induced muscle damage. Method: Twelve subjects performed two downhill runs, one after a high CHO diet and one after a low CHO diet. Blood samples were drawn for analysis of malondialdehyde, total glutathione, creatine kinase, non-esterified fatty acids, lactate, glucose, and leucocytes. DOMS and muscle function were assessed daily. Results: The high CHO diet resulted in higher respiratory exchange ratio and lactate concentrations than the low CHO diet before exercise. The low CHO diet resulted in higher non-esterified fatty acid concentrations before exercise. DOMS developed after exercise and remained for up to 96 hours, after both diets. A biphasic response in creatine kinase occurred after both diets at 24 and 96 hours after exercise. Malondialdehyde had increased 72 hours after exercise after both diets, and muscle function was attenuated up to this time. Conclusions: Downhill running resulted in increased ROS production and ratings of DOMS and secondary increases in muscle damage. CHO status before exercise had no effect.

Formation of 3-nitrotyrosines in carbonic anhydrase III is a sensitive marker of oxidative stress in skeletal muscle

Oxidation of skeletal muscle proteins has been reported to occur following contractions, with ageing, and with a variety of disease states, but the nature of the oxidised proteins has not been identified. A proteomics approach was utilised to identify major proteins that contain carbonyls and/or 3‐nitrotyrosine (3‐NT) groups in the gastrocnemius (GTN) muscles of adult (5–11 months of age) and old (26–28 months of age) wild type (WT) mice and adult mice lacking copper, zinc superoxide dismutase (Sod1−/− mice), manganese superoxide dismutase (Sod2+/− mice) or glutathione peroxidase 1 (GPx1−/− mice). In quiescent GTN muscles of adult and old WT mice, protein carbonylation and/or formation of 3‐NT occurred in several proteins involved in glycolysis, as well as creatine kinase and carbonic anhydrase III. Following contractions, the 3‐NT intensity was increased in specific protein bands from GTN muscles of both adult and old WT mice. In quiescent GTN muscles from adult Sod1−/−, Sod2+/− or GPx1−/− mice compared with age‐matched WT mice only carbonic anhydrase III showed a greater 3‐NT content. We conclude that formation of 3‐NT occurs readily in response to oxidative stress in carbonic anhydrase III and this may provide a sensitive measure of oxidative damage to muscle proteins.

Are there functional consequences of a reduction in selenium intake in UK subjects

Dietary Se levels in the UK have fallen over the last 20 years and recent surveys indicate that average Se intakes are 30-40 microg/d, which is well below the current UK reference nutrient intake for adult men (75 microg/d) or women (60 microg/d). Functional consequences of this decline have not been recognised, although epidemiological data suggest it may contribute to increased risk of infections and incidence of some cancers. Previous data have indicated that biochemical changes in Se-dependent proteins occur in otherwise healthy UK subjects given small Se supplements. The current studies have focused on the effect of small Se supplements on the immune response since there is evidence of specific interactions between Se intake and viral replication, and since the potential anti-cancer effects of Se may be mediated by non-antioxidant effects of Se such as changes in immune function. Data indicate that subjects given small Se supplements (50 or 100 microg Se/d) have changes in the activity of Se-dependent enzymes and evidence of improved immune function and clearance of an administered live attenuated virus in the form of poliovirus vaccine. Responses of individual subjects to Se supplements are variable, and current work is evaluating potential explanations for this variability, including genetic variability and pre-existing Se status.