OBJECTIVES: We assessed the hypothesis that overexpression of the antioxidant enzyme heme oxygenase (HO)-1 may protect against chronic recurrent ischemia/reperfusion injury. BACKGROUND: Multiple and recurring episodes of myocardial ischemia can result in significant myocardial damage, including myocyte death, fibrosis, and wall thinning, leading to impaired ventricular function and cardiac failure. METHODS: In this study we used a closed-chest rodent model of chronic recurring myocardial ischemia and reperfusion to investigate the efficacy of pre-emptive gene therapy in overexpressing the antioxidant enzyme HO-1, using adeno-associated virus (AAV)-2 as the delivery vector. RESULTS: We show that constitutive overexpression of HO-1 can prevent myocardial wall thinning, inflammation, fibrosis, and deterioration of cardiac function (as measured by echocardiography, histology, and immunohistochemistry) induced by repeated transient myocardial ischemia and reperfusion injury. With HO-1 therapy, there was a significant reduction in apoptosis as determined by levels of markers of survival proteins and terminal deoxynucleotidyltransferase dUTP nick end-labeling staining. This prevention of tissue damage was also associated with reduction in superoxide generation. CONCLUSIONS: Taken together we provide the first evidence of the therapeutic efficacy of pre-emptive AAV-HO-1 delivery for prevention against multiple ischemic injury. This approach protects myocytes by simultaneously activating protective response and inhibiting pathological left ventricular remodeling and, therefore, may be a useful cardio-protective strategy for patients with coronary artery disease at a high risk for recurrent myocardial ischemia.