Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, C78

Oral Communications

Skeletal muscles from mice lacking Cu,Zn superoxide dismutase show accelerated loss of muscle mass and function and attenuated responses to contractile activity

L. Zibrik1, A. Vasilaki1, H. Van Remmen3, A. G. Richardson3, J. A. Faulkner2, M. J. Jackson1, A. McArdle1

1. School of Clinical Sciences, The University of Liverpool, Liverpool, United Kingdom. 2. Institute of Gerontology, University of Michigan, Michigan, Michigan, USA. 3. Health Science Centre, Department of Physiology, University of Texas, San Antonio, Texas, USA.

Reactive oxygen species (ROS) are increased in skeletal muscles by isometric contractions and muscle adapts to this by increasing transcription of cytoprotective proteins though activation of redox-sensitive transcription factors such as Nuclear Factor kB (NF-kB) and Activator protein-1 (AP-1). ROS may play a crucial role in ageing and increased mitochondrial superoxide generation is implicated in this process. We previously showed aberrant DNA binding activity of NF-kB and AP-1 in muscles of old mice compared with adult both at rest and following contractile activity. Mice lacking Cu, Zn superoxide dismutase (SOD1; Sod1-/- mice) show accelerated loss of skeletal muscle mass and function (1). The aim of this study was to examine ROS in isolated skeletal muscle fibres from adult Sod1-/- mice and the effect of a lack of SOD1 on the adaptive responses in muscle following contractile activity. Adult C57/BL6 wild type (WT) mice and Sod1-/- mice were anaesthetised (65mg/100g pentobarbitone sodium) and the hind limbs subjected to an isometric contraction protocol (2). Immediately following the contractions mice were killed according to UK legislation. Single muscle fibres were isolated from the flexor digitorum brevis (FDB) and intracellular superoxide activity monitored using dihydroethidium (DHE) at rest and after electrically stimulated contractions (0.5 sec every 5 sec at 50 Hz for 15 min). Apocynin (an inhibitor of NAD(P)H oxidases) was used to determine the potential source of ROS. Gastrocnemius muscles were analysed for NF-kB and AP-1 DNA binding activity by Electrophoresis Mobility Shift Assay and for components of the NF-kB activation pathway by western blotting. Data indicated aberrant DNA binding activity of AP-1 and NF-kB in muscles of Sod1-/- mice, similar to that seen in muscles of old WT mice (2) and identified p50 and p65 as components of the NF-kB complex. Phosphorylated IkBa was increased by ~60% in muscles of Sod1-/- mice. Electrical stimulated contractions of FDB fibres from WT mice induced a significant increase in ethidium fluorescence that was abolished by apocynin treatment. There were no differences in ethidium fluorescence in quiescent muscle fibres from 6 month Sod1-/- mice compared with fibres from WT mice. These findings indicate that a lack of SOD1 causes attenuation of adaptive responses of skeletal muscle to contractile activity analogous to that seen in muscle from aged mice, but this is not associated with increased cytosolic superoxide activity measured by oxidation of DHE.

Where applicable, experiments conform with Society ethical requirements