N-Acetylcysteine and Allopurinol Confer Synergy in Attenuating Myocardial Ischemia Injury via Restoring HIF-1 alpha/HO-1 Signaling in Diabetic Rats

Objectives: To determine whether or not the antioxidants N-acetylcysteine (NAC) and allopurinol (ALP) confer synergistic cardioprotection against myocardial ischemia/reperfusion (MI/R) injury by stabilizing hypoxia inducible factor 1 alpha (HIF-1 alpha)/heme oxygenase 1 (HO-1) signaling in diabetic myocardium. Methods: Control or diabetic [streptozotocin (STZ)-induced] Sprague Dawley rats received vehicle or NAC, ALP or their combination for four weeks starting one week after STZ injection. The animals were then subjected to thirty minutes of coronary artery occlusion followed by two hours reperfusion in the absence or presence of the selective HO-1 inhibitor, tin protoporphyrin-IX (SnPP-IX) or the HIF-1 alpha inhibitor 2-Methoxyestradiol (2ME2). Cardiomyocytes exposed to high glucose were subjected to hypoxia/re-oxygenation in the presence or absence of HIF-1 alpha and HO-1 achieved by gene knock-down with related siRNAs. Results: Myocardial and plasma levels of 15-F2t-isoprostane, an index of oxidative stress, were significantly increased in diabetic rats while cardiac HO-1 protein and activity were reduced; this was accompanied with reduced cardiac protein levels of HIF-1 alpha, and increased post-ischemic myocardial infarct size and cellular injury. NAC and ALP given alone and in particular their combination normalized cardiac levels of HO-1 and HIF-1 alpha protein expression and prevented the increase in 15-F2t-isoprostane, resulting in significantly attenuated post-ischemic myocardial infarction. NAC and ALP also attenuated high glucose-induced post-hypoxic cardiomyocyte death in vitro. However, all the above protective effects of NAC and ALP were cancelled either by inhibition of HO-1 or HIF-1 alpha with SnPP-IX and 2ME2 in vivo or by HO-1 or HIF-1 alpha gene knock-down in vitro. Conclusion: NAC and ALP confer synergistic cardioprotection in diabetes via restoration of cardiac HIF-1 alpha and HO-1 signaling.