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Description:
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Aging and skeletal muscle ischemia /reperfusion (I /R ) injury both lead to skeletal
muscle dysfunction , evidenced by decreased contractile force generation , particularly in
glycolytic muscle . The deficits in I /R are more severe and persistent in aged animals .
Previous studies in our lab led us to hypothesize that the expression of the glycolytic
enzyme glyceraldehyde -3 -phosphate dehydrogenase may be altered following I /R . We
further hypothesized that aging would enhance the oxidative stress and oxidative damage
experienced by the muscle . GAPDH protein levels were measured by Western blotting .
We observed that the enzyme is significantly decreased at 3 and 5 days of reperfusion in
the young muscle , while the enzyme was significantly decreased in the aged muscle at 1 ,
3 , 5 , and 7 days . Using PCR , we compared GAPDH mRNA levels at 5 days reperfusion
and found that the I /R tissue from both young and old have significant increases in
GAPDH transcript at this time point compared to control , suggesting that the protein
deficit is not due to decreased transcription . Finally , we examined tyrosine nitration . A
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spot selected following 2D gel electrophoresis and nitrotyrosine western blotting of
young and old muscle lysate was identified as GAPDH by mass spectrometry . We
compared tyrosine nitration over the time course of reperfusion . While total tyrosine
nitration does not increase in the I /R tissue in the young , nitration of GAPDH is
significantly increased at 1 and 3 days reperfusion . In contrast to the young , total
tyrosine nitration in the aged muscle was significantly increased at 1 , 3 , and 5 days of
reperfusion , with significant increases in nitration of GAPDH at the same time points .
We conclude that GAPDH protein levels are decreased following I /R , which could
interfere with metabolism and ATP generation . Further , this decrease is not likely
transcriptionally mediated . Based on the increases in tyrosine nitration , we propose that
oxidative modification enhances the degradation of GAPDH following I /R , and that the
persistence of decreased GAPDH in the aged muscle is due to the prolonged increases in
oxidative modification seen in that age group . This suggests that the aged muscle
experiences greater oxidative stress , protein modification , and GAPDH degradation ,
possibly contributing to the decreased muscle function reported in the literature . |