Anne McArdle

Effect of Vitamin C Supplements on Antioxidant Defence and Stress Proteins in Human Lymphocytes and Skeletal Muscle

Oxidative stress induces adaptations in the expression of protective enzymes and heat shock proteins (HSPs) in a variety of tissues. We have examined the possibility that supplementation of subjects with the nutritional antioxidant, vitamin C, influences the ability of lymphocytes to express protective enzymes and HSPs following exposure to an exogenous oxidant and the response of skeletal muscle to the physiological oxidative stress that occurs during exercise in vivo. Our hypothesis was that an elevation of tissue vitamin C content would reduce oxidant‐induced expression of protective enzymes and HSP content. Lymphocytes from non‐supplemented subjects responded to hydrogen peroxide with increased activity of superoxide dismutase (SOD) and catalase, and HSP60 and HSP70 content over 48 h. Vitamin C supplementation at a dose of 500 mg day−1 for 8 weeks was found to increase the serum vitamin C concentration by ∼50 %. Lymphocytes from vitamin C‐supplemented subjects had increased baseline SOD and catalase activities and an elevated HSP60 content. The SOD and catalase activities and the HSP60 and HSP70 content of lymphocytes from supplemented subjects did not increase significantly in response to hydrogen peroxide. In non‐supplemented subjects, a single period of cycle ergometry was found to significantly increase the HSP70 content of the vastus lateralis. Following vitamin C supplementation, the HSP70 content of the muscle was increased at baseline with no further increase following exercise. We conclude that, in vitamin C‐supplemented subjects, adaptive responses to oxidants are attenuated, but that this may reflect an increased baseline expression of potential protective systems against oxidative stress (SOD, catalase and HSPs).

Overexpression of HSP70 in mouse skeletal muscle protects against muscle damage and age-related muscle dysfunction.

Ageing is associated with skeletal muscle atrophy, a deficit in force generation, an increased susceptibility to contraction‐induced injury, and a permanent force deficit following severe injury. Muscles of young mice adapt rapidly following exercise by an increase in the production of heat shock proteins (HSPs), whereas muscles of old mice show a severely diminished response. We hypothesized that overexpression of HSP70 in muscle throughout life would reduce age‐related changes in function. The maximum tetanic force of extensor digitorum longus (EDL) muscles of adult and old wild‐type (WT) and HSP70 overexpressor transgenic mice was determined. EDL muscles were subjected to damaging lengthening contractions and the ability to generate force was assessed for up to 28 days following the contractions. Overexpression of HSP70 in muscles of old transgenic mice prevented the specific force deficit observed in muscles of old WT mice. The complete recovery of muscles of old HSP70 transgenic mice by 14 days following the contraction protocol was in contrast to the 44% force deficit, which remained in muscles of old WT mice at 28 days following the protocol. These data indicate that a diminished production of HSP70 in muscles of old mammals has a major effect on age‐related functional deficits.

The exercise-induced stress response of skeletal muscle, with specific emphasis on humans.

Skeletal muscle adapts to the stress of contractile activity via changes in gene expression to yield an increased content of a family of highly conserved cytoprotective proteins known as heat shock proteins (HSPs). These proteins function to maintain homeostasis, facilitate repair from injury and provide protection against future insults. The study of the exercise-induced production of HSPs in skeletal muscle is important for the exercise scientist as it may provide a valuable insight into the molecular mechanisms by which regular exercise can provide increased protection against related and non-related stressors. As molecular chaperones, HSPs are also fundamental in facilitating the cellular remodelling processes inherent to the training response.Whilst the exercise-induced stress response of rodent skeletal muscle is relatively well characterized, data from humans are more infrequent and less insightful. Data indicate that acute endurance- and resistance-type exercise protocols increase the muscle content of ubiquitin, aB-crystallin, HSP27, HSP60, HSC70 and HSP70. Although increased HSP transcription occurs during exercise, immediately post-exercise or several hours following exercise, time-course studies using western blotting techniques have typically demonstrated a significant increase in protein content is only detectable within 1–2 days following the exercise stress. However, comparison amongst studies is complicated by variations in exercise protocol (mode, intensity, duration, damaging, non-damaging), muscle group examined, predominant HSP measured and, perhaps most importantly, differences in subject characteristics both within and between studies (training status, recent activity levels, nutritional status, age, sex, etc.). Following ‘non-damaging’ endurancetype activities (exercise that induces no overt structural and functional damage to the muscle), the stress response is thought to be mediated by redox signalling (transient and reversible oxidation of muscle proteins) as opposed to increases in contracting muscle temperature per se. Following ‘damaging’ forms of exercise (exercise that induces overt structural and functional damage to the muscle), the stress response is likely initiated by mechanical damage to protein structure and further augmented by the secondary damage associated with inflammatory processes occurring several days following the initial insult. Exercise training induces an increase in baseline HSP levels, which is dependent on a sustained and currently unknown dose of training and also on the individual’s initial training status. Furthermore, trained subjects display an attenuated or abolished stress response to customary exercise challenges, likely due to adaptations of baseline HSP levels and the antioxidant system.Whilst further fundamental work is needed to accurately characterize the exercise-induced stress response in specific populations following varying exercise protocols, exercise scientists should also focus their efforts on elucidating the precise biological significance of the exercise-induced induction of HSPs. In addition to their potential cytoprotective properties, the role of HSPs in modulating cell signalling pathways related to both exercise adaptation and health and disease also needs further investigation. As a nonpharmacological intervention, exercise and the associated up-regulation of HSPs and the possible correction of maladapted pathways may therefore prove effective in providing protection against protein misfolding diseases and in preserving muscle function during aging.

Exercise and skeletal muscle ageing: cellular and molecular mechanisms

As we age, our skeletal muscle becomes smaller and weaker. In addition, the remaining muscle is more susceptible to damage, particularly following exercise, recovery from damage is severely impaired and muscle is unable to adapt rapidly following sequential periods of exercise. The mechanisms by which skeletal muscle damage occurs are poorly understood and the role that an increased production of free radical species plays in this damage is controversial. However, evidence is emerging which suggests that an increased production of free radicals may act as an activator of the adaptive response in skeletal muscle, resulting in the increased production of antioxidant enzymes and heat shock proteins (HSPs). The increased content of these proteins facilitates rapid remodelling of muscle and provides considerable protection against subsequent periods of damaging exercise. There is considerable evidence that the production of free radicals is modified during the ageing process. The aim of this review is to examine the possible effects of this modification on the ability of muscle cells to respond to stress and the functional effect that this may have on our muscles as we age.

Free radical generation by skeletal muscle of adult and old mice: effect of contractile activity

Oxidative modification of cellular components may contribute to tissue dysfunction during aging. In skeletal muscle, contractile activity increases the generation of reactive oxygen and nitrogen species (ROS). The question of whether contraction‐induced ROS generation is further increased in skeletal muscle of the elderly is important since this influences recommendations on their exercise participation. Three different approaches were used to examine whether aging influences contraction‐induced ROS generation. Hind limb muscles of adult and old mice underwent a 15‐min period of isometric contractions and we examined ROS generation by isolated skeletal muscle mitochondria, ROS release into the muscle extracellular fluid using microdialysis techniques, and the muscle glutathione and protein thiol contents. Resting skeletal muscle of old mice compared with adult mice showed increased ROS release from isolated mitochondria, but no changes in the extracellular levels of superoxide, nitric oxide, hydrogen peroxide, hydroxyl radical activity or muscle glutathione and protein thiol contents. Skeletal muscle mitochondria isolated from both adult and old mice after contractile activity showed significant increases in hydrogen peroxide release compared with pre‐contraction values. Contractions increased extracellular hydroxyl radical activity in adult and old mice, but had no significant effect on extracellular hydrogen peroxide or nitric oxide in either group. In adult mice only, contractile activity increased the skeletal muscle release of superoxide. A similar decrease in muscle glutathione and protein thiol contents was seen in adult and old mice following contractions. Thus, contractile activity increased skeletal muscle ROS generation in both adult and old mice with no evidence for an age‐related exacerbation of ROS generation.

In vivo model of muscle pain: Quantification of intramuscular chemical, electrical, and pressure changes associated with saline-induced muscle pain in humans

Abstract Intramuscular injection of hypertonic saline is a good model to study human muscle pain (Kellgren 1938). The present study concerns the intramuscular (i.m.) pain mediators in saline‐induced muscle pain. In experiment 1, the diffusion of infused hypertonic and isotonic saline (0.5 ml) in m. tibialis anterior was illustrated by magnetic resonance imaging (MRI) in one subject. In experiment 2, six volunteers received four sequential infusions (0.5 ml given at 5 min intervals) of isotonic saline and thereafter four sequential infusions (0.5 ml given at 5 min intervals) of hypertonic saline into m. tibialis anterior. The isotonic and hypertonic saline infusions were computer‐controlled and separated by 20 min. The muscle pain intensity was assessed by continuous recordings on a visual analogue scale (VAS). One microdialysis probe was inserted 1 cm from the infusion needle in m. tibialis anterior and another probe in the other m. tibialis anterior. Concentrations of the i.m. sodium, potassium, magnesium, and prostaglandin E2 (PGE2) were assessed from the dialysates. Intramuscular electromyography (EMG) and pressure were assessed in the area of the infused saline. In experiment 1, the infusion of hypertonic and isotonic saline created a visible saline‐pool on the MRI scans. These saline‐pool volumes were stable and not correlated to the pain scores. In experiment 2, infusion of isotonic saline produced little pain compared to infusion of hypertonic saline. Maximal pain was reported after the first infusion of hypertonic saline and thereafter the pain gradually decreased with subsequent infusions of hypertonic saline. During infusion of hypertonic saline the i.m. sodium and potassium concentrations increased significantly, i.m. magnesium concentration tended to be increased, and the i.m. PGE2 concentration tended to be decreased although these changes were not significant. The i.m. EMG was smaller during and after infusions of hypertonic saline compared with isotonic saline. The i.m. pressure was not different during the infusions of hypertonic and isotonic saline but was increased between the infusions of hypertonic saline. This study has shown that i.m. infusion of hypertonic saline produced a saline‐pool, causing the i.m. pressure to increase. Possibly, pain activation and cessation are related to increased intramuscular sodium and potassium content respectively.

Effect of lifelong overexpression of HSP70 in skeletal muscle on age-related oxidative stress and adaptation after nondamaging contractile activity

Skeletal muscle aging is characterized by atrophy, a deficit in specific force generation, increased susceptibility to injury, and incomplete recovery after severe injury. The ability of muscles of old mice to produce heat shock proteins (HSPs) in response to stress is severely diminished. Studies in our laboratory using HSP70 overexpressor mice demonstrated that lifelong overexpression of HSP70 in skeletal muscle provided protection against damage and facilitated successful recovery after damage in muscles of old mice. The mechanisms by which HSP70 provides this protection are unclear. Aging is associated with the accumulation of oxidation products, and it has been proposed that this may play a major role in age‐related muscle dysfunction. Muscles of old wild‐type (WT) mice demonstrated increased lipid peroxidation, decreased glutathione content, increased catalase and superoxide dismutase (SOD) activities, and an inability to activate nuclear factor (NF)‐κB after contractions in comparison with adult WT mice. In contrast, levels of lipid peroxidation, glutathione content, and the activities of catalase and SOD in muscles of old HSP70 overexpressor mice were similar to adult mice and these muscles also maintained the ability to activate NF‐κB after contractions. These data provide an explanation for the preservation of muscle function in old HSP70 overexpressor mice.—Broome, C. S., Kayani, A. C., Palomero, J., Dillmann, W. H., Mestril, R., Jackson, M. J., McArdle, A. Effect of lifelong overexpression of HSP70 in skeletal muscle on age‐related oxidative stress and adaptation after nondamaging contractile activity. FASEB J. 20, E855–E860 (2006)

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.

Reduced carbohydrate availability does not modulate training-induced heat shock protein adaptations but does upregulate oxidative enzyme activity in human skeletal muscle

The primary aim of the present study was to test the hypothesis that training with reduced carbohydrate availability from both endogenous and exogenous sources provides an enhanced stimulus for training-induced heat shock protein (HSP) adaptations of skeletal muscle. A secondary aim was to investigate the influence of reduced carbohydrate availability on oxidative adaptations and exercise performance. Three groups of recreationally active men performed 6 wk of high-intensity intermittent running occurring four times per week. Group 1 (n = 8; Low + Glu) and 2 (n = 7; Low + Pla) trained twice per day, 2 days/wk, and consumed a 6.4% glucose or placebo solution, respectively, immediately before every second training session and at regular intervals throughout exercise. Group 3 (n = 8; Norm) trained once per day, 4 days/wk, and consumed no beverage throughout training. Training induced significant improvements in maximal oxygen uptake (Vo(2max)) (P = 0.001) and distance covered on Yo-Yo Intermittent Recovery Test 2 (P = 0.001) in all groups, with no difference between conditions. Similarly, training resulted in significant increases in HSP70, HSP60, and alphaB-crystallin in the gastrocnemius (P = 0.03, 0.02, and 0.01, respectively) and vastus lateralis (P = 0.01, 0.02, and 0.003, respectively) muscles in all groups, with no difference between conditions. In contrast, training resulted in significant increases in succinate dehydrogenase (SDH) activity of the gastrocnemeius (Low + Glu, Low + Pla, and Norm: 27, 76, and 53% increases, respectively; P = 0.001) and vastus lateralis muscles (Low + Glu, Low + Pla, and Norm: 17, 70, and 19% increases, respectively; P = 0.001) where the magnitude of increase in SDH activity was significantly larger for both muscles (P = 0.03 and 0.04 for gastrocnemius and vastus lateralis, respectively) for subjects training in the Low + Pla condition. Data provide the first evidence that in whole body exercise conditions, carbohydrate availability appears to have no modulating effect on training-induced increases of the HSP content of skeletal muscle. In contrast, training under conditions of reduced carbohydrate availability from both endogenous and exogenous sources provides an enhanced stimulus for inducing oxidative enzyme adaptations of skeletal muscle although this does not translate to improved performance during high-intensity exercise.