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Progressive muscle atrophy and loss of muscle strength associated with old age have been well documented. Although age-associated impairments in skeletal muscle regeneration following injury have been demonstrated, less is known about whether aging impacts the regenerative response of neuromuscular junctions (NMJ) following contraction-induced injury. Reduced ability of NMJs to regenerate could lead to increased numbers of denervated muscle fibers and therefore play a contributing role to age-related sarcopenia. To investigate the relationship between age and NMJ regeneration following injury, extensor digitorum longus (EDL) muscles of middle-aged (18–19 months) and old mice (27–28 months) were subjected to a protocol of lengthening contractions (LC) that resulted in an acute force deficit of ~55% as well as functional and histological evidence of a similar magnitude of injury 3 days post LCs that was not different between age groups. After 28 days, the architecture and innervation of the NMJs were evaluated. The numbers of fragmented endplates increased and of fully innervated NMJs decreased post-injury for the muscle of both middle-aged and old mice and for contralateral uninjured muscles of old compared with uninjured muscles of middle-aged controls. Thus, the diminished ability of the skeletal muscle of old mice to recover following injury may be due in part to an age-related decrease in the ability to regenerate NMJs in injured muscles. The impaired ability to regenerate NMJs may be a triggering factor for degenerative changes at the NMJ contributing to muscle fiber weakness and loss in old age.

Background: Ultraviolet radiation (UVR) generates reactive oxygen species in skin that can play a role in skin damage, but reports about the photoprotective properties of oral antioxidant supplements are conflicting. Objective: We examined the ability of 2 lipid-soluble antioxidants, vitamin E and [beta]-carotene, to reduce markers of oxidative stress and erythema in human skin exposed to UVR. Design: Sixteen healthy subjects took either [alpha]-tocopherol (n = 8; 400 IU/d) or [beta]-carotene (n = 8; 15 mg/d) for 8 wk. Biopsy samples before and after supplementation were taken from unexposed skin and skin 6 h after 120 mJ/[cm.sup.2] UVR. The effects of supplements on markers of oxidative stress in skin and the minimal erythema dose to UVR were assessed. Results: Supplementary vitamin E was bioavailable, the plasma concentration increased from 14.0 [+ or -] 0.66 ([bar.x] [+ or -] SEM) to 18.2 [+ or -] 0.64 [micro]g/mL (P < 0.01), and the skin concentration increased from 0.55 [+ or -] 0.09 to 1.6 [+ or -] 0.19 ng/mg protein (P < 0.01). Supplementary [beta]-carotene increased plasma concentrations from 1 [+ or -] 0.3 to 2.25 [+ or -] 0.3 [micro]g/mL (P < 0.05), but skin concentrations were undetectable. Before vitamin E supplementation, UVR increased the skin malondialdehyde concentration from 0.42 [+ or -] 0.07 to 1.24 [+ or -] 0.16 nmol/mg protein (P < 0.01), whereas oxidized or total glutathione increased from 9.98 [+ or -] 0.4% to 12.0 [+ or -] 1.0% (P < 0.05). Vitamin E supplementation significantly decreased the skin malondialdehyde concentration, but neither vitamin E nor [beta]-carotene significantly influenced other measures of oxidation in basal or UVR-exposed skin. Conclusions: Vitamin E or [beta]-carotene supplementation had no effect on skin sensitivity to UVR. Although vitamin E supplements significantly reduced the skin malondialdehyde concentration, neither supplement affected other measures of UVR-induced oxidative stress in human skin, which suggested no photoprotection of supplementation. KEY WORDS Lipid-soluble vitamins, oxidative stress, skin, photoprotection, human study

Background: Dietary selenium intakes in many countries, including the United Kingdom, are lower than international recommendations. No functional consequences of these lower intakes have been recognized, although experimental studies suggest that they might contribute to reduced immune function, increased cancer incidence, and increased susceptibility to viral disease. Objective: The objective was to assess whether administration of small selenium supplements to otherwise healthy UK subjects leads to functional changes in immune status and the rates of clearance and mutation of a picornavirus: live attenuated polio vaccine. Design: Twenty-two adult UK subjects with relatively low plasma selenium concentrations (< 1.2 [micro]mol/L, [approximately equal to] 60% of those screened) received 50 or 100 [micro]g Se (as sodium selenite) or placebo daily for 15 wk in a double-blind study. All subjects received an oral live attenuated poliomyelitis vaccine after 6 wk and enriched stable [sup.74]Se intravenously 3 wk later. Results: Selenium supplementation increased plasma selenium concentrations, the body exchangeable selenium pool (measured by using [sup.74]Se), and lymphocyte phospholipid and cytosolic glutathione peroxidase activities. Selenium supplements augmented the cellular immune response through an increased production of interferon 7 and other cytokines, an earlier peak T cell proliferation, and an increase in T helper cells. Humoral immune responses were unaffected. Selenium-supplemented subjects also showed more rapid clearance of the poliovirus, and the poliovirus reverse transcriptase-polymerase chain reaction products recovered from the feces of the supplemented subjects contained a lower number of mutations. Conclusions: The data indicate that these subjects had a functional selenium deficit with suboptimal immune status and a deficit in viral handling. They also suggest that the additional 100 [micro]g Se/d may be insufficient to support optimal function. KEY WORDS Selenium supplementation, poliovirus vaccine, immune function, viral mutation

Background: Zinc homeostasis and normal plasma zinc concentrations are maintained over a wide range of intakes. Objective: The objective was to identify the homeostatic response to severe zinc depletion by using compartmental analysis. Design: Stable zinc isotope tracers were administered intravenously to 5 men at baseline (12.2 mg dietary Zn/d) and after 5 wk of acute zinc depletion (0.23 mg/d). Compartmental modeling of zinc metabolism was performed by using tracer and mass data in plasma, urine, and feces collected over 6-14 d. Results: The plasma zinc concentration fell 65% on average after 5 wk of zinc depletion. The model predicted that fractional zinc absorption increased from 26% to essentially 100%. The rate constants for zinc excretion in the urine and gastrointestinal tract decreased 96% and 74%, respectively. The rate constants describing the distribution kinetics of plasma zinc did not change significantly. When zinc depletion was simulated by using an average mass model of zinc metabolism at baseline, the only change that accounted for the observed fall in plasma zinc concentration was a 60% reduction in the rate constant for zinc release from the most slowly turning over zinc pool. The large changes in zinc intake, excretion, and absorption--even when considered together--only explained modest reductions in plasma zinc mass. Conclusion: The kinetic analysis with a compartmental model suggests that the profound decrease in plasma zinc concentrations after 5 wk of severe zinc depletion was mainly due to a decrease in the rate of zinc release from the most slowly turning over body zinc pool. Am J Clin Nutr 2001;74:116-24. KEY WORDS Zinc depletion, compartmental model, kinetic analysis, rate constants, plasma zinc, zinc homeostasis, men

The loss of muscle mass and weakness that accompanies ageing is a major contributor to physical frailty and loss of independence in older people. A failure of muscle to adapt to physiological stresses such as exercise is seen with ageing and disruption of redox regulated processes and stress responses are recognized to play important roles in theses deficits. The role of redox regulation in control of specific stress responses, including the generation of heat shock proteins (HSPs) by muscle appears to be particularly important and affected by ageing. Transgenic and knockout studies in experimental models in which redox and HSP responses were modified have demonstrated the importance of these processes in maintenance of muscle mass and function during ageing. New data also indicate the potential of these processes to interact with and influence ageing in other tissues. In particular the roles of redox signalling and HSPs in regulation of inflammatory pathways appears important in their impact on organismal ageing. This review will briefly indicate the importance of this area and demonstrate how an understanding of the manner in which redox and stress responses interact and how they may be controlled offers considerable promise as an approach to ameliorate the major functional consequences of ageing of skeletal muscle (and potentially other tissues) in man.

The restoration of oxygenated blood to ischaemic myocardium --reperfusion--halts the process leading to infarction. Early reperfusion is the only way to prevent progression to myocardial necrosis and thus to limit [...]

The molecular mechanisms underlying skeletal muscle aging and associated sarcopenia have been linked to an altered oxidative status of redox-sensitive proteins. Reactive oxygen and reactive nitrogen species (ROS/RNS) generated by contracting skeletal muscle are necessary for optimal protein function, signaling, and adaptation. To investigate the redox proteome of aging gastrocnemius muscles from adult and old male mice, we developed a label-free quantitative proteomic approach that includes a differential cysteine labeling step. The approach allows simultaneous identification of up- and downregulated proteins between samples in addition to the identification and relative quantification of the reversible oxidation state of susceptible redox cysteine residues. Results from muscles of adult and old mice indicate significant changes in the content of chaperone, glucose metabolism, and cytoskeletal regulatory proteins, including Protein DJ-1, cAMP-dependent protein kinase type II, 78 kDa glucose regulated protein, and a reduction in the number of redox-responsive proteins identified in muscle of old mice. Results demonstrate skeletal muscle aging causes a reduction in redox-sensitive proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response. Data is available via ProteomeXchange with identifier PXD001054.

Proteinuria is pathogenic to proximal tubular cells (PTC) and linked with progression to renal failure. The aim of this study was to determine the effects of human serum albumin (HSA) overload on the changes in gene and protein expression stimulated by oxidative stress in PTC and any interaction with ANG II that is pivotal in disease pathogenesis. Markers of oxidative stress, antioxidant defences, transcription factor activation, and the expression of stress-related genes were measured in human PTC (HK-2 cells) overloaded with either globulin-free fatty acid free (GF/FAF) HSA or globulin-free (GF) HSA. The effects of ANG II were also determined. HSA overload in HK-2 cells caused PTC hyperfunction, increased oxidative stress, and an upregulation of adaptive responses and stress-related genes. Some responses were common to both HSAs but others were unique to either HSA and unaffected by addition of ANG II or candesartan (a specific ANG II type 1 receptor blocker). ANG II also independently induced oxidative stress and upregulated other stress-related genes. HSA overload in HK-2 cells stimulated increased oxidative stress and upregulated changes in stress-related gene expression indicating new pathways of PTC injury that are not mediated via ANG II type 1 receptors.

Skeletal muscle has been shown to generate a complex set of reactive oxygen and nitrogen species (ROS) both at rest and during contractile activity. The primary ROS generated are superoxide and nitric oxide and the pattern and magnitude of their generation is influenced by the nature of the contractile activity. It is increasingly clear that the ROS generated by skeletal muscle play an important role in influencing redox-regulated processes that control, at least some of, the adaptive responses to contractile activity. These processes are also recognized to be modified during ageing and in some disease states, providing the potential that interventions affecting ROS activity may influence muscle function or viability in these situations.

PATTWELL, D. M., and M. J. JACKSON. Contraction-induced oxidants as mediators of adaptation and damage in skeletal muscle. Exerc. Sport Sci. Rev., Vol. 32, No. 1, pp. 14–18, 2004. Contracting skeletal muscle generates reactive oxygen and nitrogen species (ROS) that can induce changes in gene expression or cell damage depending upon the pattern of production and the endogenous protective systems. The hypothesis is presented that skeletal muscle uses contraction-induced ROS as signals to induce adaptive responses including maintenance of oxidant homeostasis and prevention of oxidative damage.

The aim of this study was to investigate the production of HSP70 in gastrocnemius muscles from adult (6‐month‐old) and aged (28‐month‐old) rats following contractile activity. At 24 h following a period of repeated isometric contractions, muscles from adult rats contained significantly elevated levels of HSP70 compared with nonexercised muscle. This was not evident in muscles from aged rats. This attenuated response may play a major role in development of the age‐related functional deficit that occurs in skeletal muscle. © 2002 Wiley Periodicals, Inc. Muscle Nerve 25: 000–000, 2002

Previous studies in animals have demonstrated that a single period of aerobic exercise induces a rise in the skeletal muscle activity of the antioxidant enzymes superoxide dismutase and catalase and an increase in the muscle content of heat shock proteins (HSPs). The purpose of this study was to examine the time course of response of human skeletal muscle superoxide dismutase and catalase activities and the content of HSP60 and HSP70 after a period of exhaustive, nondamaging aerobic exercise. Seven volunteers undertook one-legged cycle ergometry at 70% maximal oxygen uptake for 45 min. Biopsies were obtained from the vastus lateralis muscle 7 days before and at 1, 2, 3, and 6 days after exercise. Muscle superoxide dismutase activity increased to a peak at 3 days postexercise, muscle catalase activities were unchanged, and muscle content of HSP60 and the inducible HSP70 increased by variable amounts to reach means of 190% and 3,100% of preexercise values, respectively, by 6 days postexercise. These data indicate that human skeletal muscle responds to a single bout of nondamaging exercise by increasing superoxide dismutase activity and provide the first evidence of an increase in HSP content of human skeletal muscle after a submaximal exercise bout.

The accurate measurement of the chemical activators of pain in skeletal muscle has proved to be a major challenge. This study examined the applicability of microdialysis to the measurement of pain‐producing substances in skeletal muscle using a defined model of ischemia and reperfusion in the rat. Microdialysis probes were placed into muscle of anesthetized rats. Ischemia was induced for 4 h, followed by reperfusion for 1 h. Perfusates were analyzed for hypoxanthine, potassium, prostaglandin (PG) E2and histamine. A 20‐fold increase in perfusate hypoxanthine concentration was seen prior to reperfusion (70.1 ± 27.1 μM for ischemic versus 3.7 ± 1.9 μM for control; P< 0.05). An initial increase in PGE2concentration was seen during ischemia (7.4 ± 2.0 nM versus 3.4 ± 1.4 nM; P< 0.05) and immediately post‐reperfusion (17.9 ± 5.2 nM versus 4.0 ± 1.1 nM; P< 0.05). Potassium concentration was significantly increased following occlusion and reperfusion. This indicates the applicability of microdialysis to the measurement of pain‐producing substances in muscle during ischemia and reperfusion. Further use will provide novel information on muscle pain both in defined model systems and in clinical situations in humans. © 1999 John Wiley & Sons, Inc. Muscle Nerve 22: 1047–1052, 1999

The accurate measurement of the chemical activators of pain in skeletal muscle has proved to be a major challenge. This study examined the applicability of microdialysis to the measurement of pain-producing substances in skeletal muscle using a defined model of ischemia and reperfusion in the rat. Microdialysis probes were placed into muscle of anesthetized rats. Ischemia was induced for 4 h, followed by reperfusion for 1 h. Perfusates were analyzed for hypoxanthine, potassium, prostaglandin (PG) E2 and histamine. A 20-fold increase in perfusate hypoxanthine concentration was seen prior to reperfusion (70.1 ± 27.1 μM for ischemic versus 3.7 ± 1.9 μM for control; P &lt; 0.05). An initial increase in PGE2 concentration was seen during ischemia (7.4 ± 2.0 nM versus 3.4 ± 1.4 nM; P &lt; 0.05) and immediately post-reperfusion (17.9 ± 5.2 nM versus 4.0 ± 1.1 nM; P &lt; 0.05). Potassium concentration was significantly increased following occlusion and reperfusion. This indicates the applicability of microdialysis to the measurement of pain-producing substances in muscle during ischemia and reperfusion. Further use will provide novel information on muscle pain both in defined model systems and in clinical situations in humans. © 1999 John Wiley & Sons, Inc. Muscle Nerve 22: 1047–1052, 1999

1.Duchenne and Becker muscular dystrophies are X-linked disorders caused by defects in muscle dystrophin. The mdx mouse is an animal model for Duchenne muscular dystrophy which has a point mutation in the dystrophin gene, resulting in little (< 3%) or no expression of dystrophin in muscle. Mdx mice show a characteristic pattern of muscle necrosis and regeneration. Muscles are normal until the third postnatal week when widespread necrosis commences. This is followed by muscle regeneration, with the persistence of centrally nucleated fibres. 2.This work has examined the hypothesis that the onset of this muscle necrosis is associated with postnatal maturation of the thyroid endocrine system and that pharmacological inhibition of thyroid hormone synthesis delays the onset of muscle necrosis. 3.Serum T4 and T3 concentrations of mice were found to rise immediately before the onset of muscle necrosis in the mdx mouse, and induction of hypothyroidism by treatment of animals with propylthiouracil was found to delay the onset of muscle necrosis. 4.The results provide the first demonstration of experimental delay of muscle necrosis by manipulation of the endocrine system in muscle lacking dystrophin, and provide a novel insight into the way in which a lack of dystrophin interacts with postnatal development to precipitate muscle necrosis in the mdx mouse.

Van der Meulen, Jack H., Anne McArdle, Malcolm J. Jackson, and John A. Faulkner. Contraction-induced injury to the extensor digitorum longus muscles of rats: the role of vitamin E.J. Appl. Physiol.83(3): 817–823, 1997.—Three days after a protocol of 225 pliometric (lengthening) contractions was administered to in situ extensor digitorum longus muscles of rats, the force deficit was 64 ± 7% and the percentage of damaged muscle fibers was 38 ± 5% of the control values. We then tested the hypothesis that at 3 h and 3 days after the protocol an elevation in the muscle vitamin E content would decrease the force deficit, the percentage of damaged muscle fibers, and the serum activities of creatine kinase and pyruvate kinase. The 5–8 days of intravenous injections of α-tocopherol increased muscle vitamin E content threefold compared with vehicle (ethanol)-treated rats. Despite the difference in vitamin E content, the force deficit and number of damaged fibers were not different. After the contraction protocol, the serum creatine kinase and pyruvate kinase activities of the vehicle-treated rats increased fourfold at 3 h and twofold at 3 days, whereas the vitamin E-treated rats showed no change. We conclude that vitamin E treatment did not ameliorate either the induction of the injury or the more severe secondary injury at 3 days. Despite the absence of evidence for an antioxidant function, the lack of any increase in serum enzyme activities for vitamin E-treated rats at 3 h and 3 days supported a role for vitamin E in the prevention of enzyme loss after muscle damage.

In animal models, oxygen-derived free radicals have been found to be important mediators of reperfusion injury to ischemic but viable myocardium. However, in humans, there is no direct evidence of free radical production after the restoration of coronary artery patency in acute myocardial infarction. The purpose of this study was to quantitate and assess the time course of free radical production in coronary venous outflow in patients with acute myocardial infarction undergoing successful recanalization of the infarct-related artery by primary percutaneous transluminal coronary angioplasty (PTCA). Primary PTCA was performed in 17 patients with acute myocardial infarction of <6 hours duration. Direct free radical production was assessed by coronary venous effluent blood sampling before PTCA and at timed intervals up to 24 hours (or 48 hours in 6 patients) after recanalization. All samples were added to the spin trapping agent α-phenyl N-tert butyl nitrone and analyzed by electron paramagnetic resonance spectroscopy. Vessel patency resulted in a sharp increase in free radical signal. Relative to the level before PTCA, the changes reached statistical significance after only 15 minutes (p <0.05). Peak signals were observed between 112 and 312 hours (p < 0.001), then declined up to 5 hours. A second increase in signal level was detected between 18 and 24 hours despite no angiographic evidence of reocclusion. A gradual decline was observed after 24 hours. These findings provide the first direct and quantitative evidence of free radical production in the immediate postrecanalization phase after thrombotic occlusion of a major coronary artery in humans.

1. Electron paramagnetic resonance spectroscopy was used to study free-radical signals in freeze-clamped frozen liver tissue from rats after a 1 year period of dietary supplementation with alcohol, iron, or alcohol and iron. In alcohol-fed, iron-fed and alcohol- and iron-fed animals, mild histological damage was seen on light microscopy and evidence of mitochondrial and nuclear injury was identified by electron microscopy. 2. Subcellular fractionation studies showed an increase in the activity of the peroxisomal marker catalase (P <0.01) in alcohol-fed rats compared with controls, but a fall of 82% (P <0.001) in alcohol- and iron-fed animals. The activity of the mitochondrial marker succinate dehydrogenase rose by 7% (not significant) in alcohol-fed animals and by 17% (not significant) in iron-fed animals, but fell by 94% (P <0.001) in alcohol- and iron-fed animals, suggesting serious impairment of mitochondrial function. 3. Iron overload was substantial in the iron-fed animals and there was an excellent correlation between liver iron concentration and iron-derived signals by electron paramagnetic resonance spectroscopy (P <0.001). A clear free-radical signal of g = 2.003–2.005 was detected in all liver samples, but there was no significant difference in the magnitude of this signal in any study group. 4. The absence of any increase in the stable free-radical signal, even in the presence of considerable hepatic damage, does not support the hypothesis that free radicals mediate alcoholic liver disease in this animal model, although the results cannot be taken as proof against this hypothesis.

Skeletal muscle has the unique ability to increase the rate of oxygen usage during contraction. This has led several workers to suggest that by‐products of this increased oxygen consumption, oxygen‐derived free radicals, may be primarily responsible for exercise‐induced damage to skeletal muscle. However, because of this rapidly changing redox state, skeletal muscle has developed a number of different endogenous mechanisms which adapt rapidly following a period of exercise. These include numerous structural and biochemical changes such as increased muscle activity of antioxidant enzymes and content of stress or heat shock proteins (HSPs). This adaptation is associated with protection against the potentially damaging effects of a second period of exercise. In addition, we have recently demonstrated a significant increase in free radical production during a period of nondamaging exercise, which is rapidly followed by a significant increase in the expression of antioxidant enzymes and HSPs, suggesting that a change in redox state of the muscle may act as signal for adaptation.

Oxygen-derived free radicals (FR) have been found to be important mediators of myocardial reperfusion injury in animal studies. In man, most studies of FR measurement after reperfusion have relied on indirect markers alone. However, their accuracy and relationship to FR's has been controversial. We have therefore used primary PTCA for AMI as a model of acute reperfusion to compare directFR measurement using electron paramagnetic resonance (EPR) spectroscopy and the spin trap agent α-phenyl N-tert butyl nitrone, with two of the most commonly used indirectmarkers. These were: [1] the percentage molar ratio (PMR) of the diene conjugate 9,11-octa-decadieneoic acid to the naturally-occurring isomer 9,12-linoleic acid, and [2] serum malonaldehyde (MDA).