Abstract 74: Nitrative Inactivation of Thioredoxin-1 and Its Role in Post-Ischemic Myocardial Apoptosis
Recent studies have demonstrated that intracellular proteins involved in oxidative stress and apoptosis are nitrated in diseased but not in normal tissues. However, definitive evidence that supports a causative link between a specific protein that is nitratively modified with tissue injury in a specific disease is currently lacking. The aims of the present study were to determine whether thioredoxin (Trx), a novel cytoprotective molecule, is susceptible to nitrative inactivation in vitro and in vivo and to establish a causative link between Trx nitration and post-ischemic myocardial apoptosis. In vitro exposure of human Trx-1 to SIN-1 (a peroxynitrite donor, 100 μM) resulted in significant Trx-1 nitration and almost abolished Trx-1 activity (>85% inhibition). SIN-1 induced Trx-1 nitration and inactivation were completely blocked by MnTE-2-PyP5+ (a SOD mimic, 500 μM) and markedly attenuated by PTIO (an NO scavenger, 500 μM). Administration of either reduced or oxidized Trx-1 in vivo (2 mg/kg) markedly attenuated myocardial ischemia/reperfusion (MI/R) injury as determined by apoptosis and infarct size (P<0.01). However, administration of nitrated Trx-1 failed to exert any cardioprotective effect. To determine whether nitrative Trx inactivation may play a causative role in MI/R-induced cardiomyocyte apoptosis, two additional experiments were performed. Significant Trx-1 nitration was detected in ischemic/reperfused cardiac tissue, and Trx activity was markedly inhibited (P<0.01). Most interestingly, Trx-1/ASK1 complex formation, a mechanism by which Trx inhibits apoptosis, was abolished and ASK1 activity was increased in I/R cardiac tissue. Treatment with either a novel peroxynitrite decomposition catalyst (FP15) or a SOD mimic (MnTE-2-PyP5+) 10 min before reperfusion blocked nitrative Trx inactivation, attenuated ASK1 activation and reduced post-ischemic myocardial apoptosis. These results strongly suggest that nitrative inactivation of Trx plays a pro-apoptotic role under those pathologic conditions where production of reactive nitrogen species is increased, and that anti-nitrating treatment may have therapeutic value in those diseases, such as myocardial ischemia/ reperfusion, where pathologic apoptosis is increased.