Tyrosine nitration and altered protein pool levels of glyceraldehyde-3-phosphate dehydrogenase following ischemia/reperfusion: The influence of aging in glycolytic skeletal muscle
Aging and skeletal muscle ischemia/reperfusion (I/R) injury both lead to skeletal\r\nmuscle dysfunction, evidenced by decreased contractile force generation, particularly in\r\nglycolytic muscle. The deficits in I/R are more severe and persistent in aged animals.\r\nPrevious studies in our lab led us to hypothesize that the expression of the glycolytic\r\nenzyme glyceraldehyde-3-phosphate dehydrogenase may be altered following I/R. We\r\nfurther hypothesized that aging would enhance the oxidative stress and oxidative damage\r\nexperienced by the muscle. GAPDH protein levels were measured by Western blotting.\r\nWe observed that the enzyme is significantly decreased at 3 and 5 days of reperfusion in\r\nthe young muscle, while the enzyme was significantly decreased in the aged muscle at 1,\r\n3, 5, and 7 days. Using PCR, we compared GAPDH mRNA levels at 5 days reperfusion\r\nand found that the I/R tissue from both young and old have significant increases in\r\nGAPDH transcript at this time point compared to control, suggesting that the protein\r\ndeficit is not due to decreased transcription. Finally, we examined tyrosine nitration. A\r\nvii\r\nspot selected following 2D gel electrophoresis and nitrotyrosine western blotting of\r\nyoung and old muscle lysate was identified as GAPDH by mass spectrometry. We\r\ncompared tyrosine nitration over the time course of reperfusion. While total tyrosine\r\nnitration does not increase in the I/R tissue in the young, nitration of GAPDH is\r\nsignificantly increased at 1 and 3 days reperfusion. In contrast to the young, total\r\ntyrosine nitration in the aged muscle was significantly increased at 1, 3, and 5 days of\r\nreperfusion, with significant increases in nitration of GAPDH at the same time points.\r\nWe conclude that GAPDH protein levels are decreased following I/R, which could\r\ninterfere with metabolism and ATP generation. Further, this decrease is not likely\r\ntranscriptionally mediated. Based on the increases in tyrosine nitration, we propose that\r\noxidative modification enhances the degradation of GAPDH following I/R, and that the\r\npersistence of decreased GAPDH in the aged muscle is due to the prolonged increases in\r\noxidative modification seen in that age group. This suggests that the aged muscle\r\nexperiences greater oxidative stress, protein modification, and GAPDH degradation,\r\npossibly contributing to the decreased muscle function reported in the literature.