| 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 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. |
