Skeletal muscle generates a number of free radical species during contraction. Muscles have developed adaptive mechanisms to protect against this increased production of free radicals by the up-regulation of antioxidant enzymes (including catalase and superoxide dismutase (SOD)) and heat shock proteins (HSPs) (McArdle et al. 2002). We have demonstrated that the ability of muscles of aged rodents to adapt following contractile activity is severely attenuated (Vasilaki et al. 2002), although the mechanisms responsible for this attenuation have not been established.
The aim of this study was to characterise the extent and time course of production of antioxidant enzymes and HSPs in muscles of adult and aged mice following a severe contraction protocol and to examine the mechanisms responsible for attenuation of the stress response in skeletal muscle of aged mice following exercise.
Male adult (14Ð16 months old) and aged (29Ð32 months old) B6XSJL mice were anaesthetised with an I.P. injection of sodium pentobarbitone (65 mg (100 g body wt)-1). Hindlimbs were subjected to a 15 min period of isometric contractions via surface electrodes. Mice were killed humanely by an overdose of anaesthetic at different time points following the contraction protocol and anterior tibialis muscles were analysed for activity of catalase and superoxide dismutase and HSP content at various time points (McArdle et al. 2001). Muscles were analysed for heat shock factor 1 (HSF1) and nuclear factor κB (NF-κB) DNA binding activity immediately following the contraction protocol (Mosser et al. 1988). Data were analysed with analysis of variance and modified Bonferonni’s t test, n = 5Ð6. Data are presented as means ± S.E.M.
The period of exercise resulted in a significant rise in catalase activity (non-exercised: 0.6 ± 0.2 U (mg protein)-1, 12 h post-exercise: 2.4 ± 0.6 U (mg protein)-1, P < 0.05) and SOD activity (non-exercised: 32.3 ± 5.4 U (mg protein)-1, 12 h post-exercise: 49.7 ± 3.7 U (mg protein)-1, P < 0.05) in muscles of adult mice. In addition, the HSC70 content of muscles from adult mice was significantly increased following the exercise protocol (e.g. 4 h post-exercise: adult mice: 144.4 ± 8.4 % of non-exercised control, P < 0.05). This response was not evident in muscles of aged mice. In muscles of aged mice, binding of HSF1 to the heat shock element (HSE) binding domain was not grossly altered. However, the binding of NF-κB from muscles of old mice to the NF-κB binding domain was severely attenuated.
These data suggest that the inability of muscles of aged mice to produce HSPs following contractile activity may not be due to an altered binding of HSF1 to the HSE. In contrast, this may be the mechanism by which production of antioxidant enzymes is altered in aged mice following exercise.
The authors would like to thank the University of Liverpool and Research into Ageing for financial support.
All procedures accord with current UK legislation.