DIFFERENTIAL GENE EXPRESSION IN CULTURED SKELETAL MUSCLE CELLS FOLLOWING OXIDATIVE STRESS





S. SPIERS*, F. McARDLE, M. J. JACKSON

UNIVERSITY OF LIVERPOOL
DEPARTMENT OF MEDICINE
UCD5
DUNCAN BUILDING
LIVERPOOL
L69 3GA
UK




The molecular mechanisms involved in the deterioration of muscle function that is seen with age are poorly defined although an increased inability to adapt to oxidative stress has been implicated. We aim to elucidate those genes involved in adaptation to non-damaging oxidative stress using mRNA differential display and cDNA microarray technology and to investigate any attenuation in the adaptation process in aged and adult mice following exercise-induced oxidative stress.
Total RNA has been collected from 5-day-old C2C12 myotubes 4 and 12hrs following treatment with a single bolus dose of 0.1mM hydrogen peroxide (control cells are untreated). Using mRNA differential display approximately 120 differentially expressed products have been isolated and these are now being verified by Northern blotting and RT-PCR. Sequence data has shown the early, transient down-regulation of components of the mitochondrial complex I and up-regulation of a number of transcription factor activators.
Analysis using stress specific cDNA microarrays has given a more global picture of the adaptive response. Expressed genes fell broadly into two categories:- those genes down-regulated at 4hrs post adaptation have been classified as stress response effectors/regulators whilst those genes specifically up-regulated at 12hrs post treatment have been shown to be involved in nucleotide excision repair/homologous recombination or drug/xenobiotic metabolism. Four hours post treatment of C2C12 myotubes with 0.1mM hydrogen peroxide 90.2% of genes expressed in control cells were down-regulated. This response was transient and at 12hrs post treatment expression levels were seen to return to within control values. In striking contrast to the overall down-regulation seen at 4hrs was the highly significant increase in mRNA expression for the molecular chaperone heme oxygenase.
Measurements of total glutathione and protein thiol levels in 5-day-old C2C12 myotubes at 4 and 12hrs following exposure to 0.1mM hydrogen peroxide showed no significant changes with hydrogen peroxide treatment. Both glutathione peroxidase and catalase activities were not detectable prior to or following oxidative stress. Western blot analysis of C2C12 myotubes at 4 and 12hrs post treatment with mild oxidative stress(0.1mM hydrogen peroxide)showed no significant changes in the expression or phosphorylation of HSP25 or levels of HSP70, HSC70 or HSP60. However a marked increase was detected in the expression of heme oxygenase at both 4 and 12hrs which is in agreement with our mRNA results.
The authors would like to thank the Biotechnology and Biological Sciences Research Council (BBSRC) for their financial support.




Key words: oxidative stress, skeletal muscle, differential display,







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