Abstract 17410: Pre-Treatment with Liposomal 6R-BH4 but not 6S-BH4 Induces in vivo Cardioprotection Through Maintenance of Nitric Oxide Synthase Coupling

Abstract

Tetrahydrobiopterin (BH4) is an essential co-factor for nitric oxide synthase (NOS) activity and BH4 depletion leads to endothelial NOS (eNOS) uncoupling with increased superoxide (O 2 -. ) generation. Myocardial BH4 levels are decreased with ischemia/reperfusion (I/R) and have been hypothesized to play a critical role in modulating eNOS activity and myocardial salvage. BH4 treatment exerts myocardial protection; however, questions remain regarding the related mechanism and role of NOS coupling versus nonspecific antioxidative effects of BH4. To address this issue, rats were subjected to 60-min in vivo coronary artery occlusion and varying periods (10-min, 20-min or 24-hr) of reperfusion with or without liposomal 6R-BH4 or 6S-BH4 supplementation (1 mg/kg, iv, 5-min before ischemia). Myocardial NO and O 2 -. production were measured in frozen heart sections using the fluorescence probes (CuFL and DHE, respectively) in the absence or presence of NOS inhibitor or SOD mimetic. Myocardial infarct size was determined 24-hr after reperfusion and compared with NOS inhibitor (L-NAME) or 6R-BH4 or 6S-BH4 (that lacks NOS co-factor activity). Compared to vehicle group, 6R-BH4 treatment caused an increase in NO with decrease in O 2 -. in I/R hearts. Postischemic NO or O 2 -. -derived fluorescence was largely abolished by added NOS inhibitor L-NAME (1 mM) or the SOD-mimetic MnTBAP (50 μM), respectively. 6R-BH4 reduced myocardial infarct size by ∼50% compared to vehicle group (31±2% vs. 59±2%, n=8). L-NAME pre-treatment (10 mg/kg, iv, 15-min before vehicle or 6R-BH4) had no effect on vehicle group, but it abolished the infarct-limiting effect of 6R-BH4 treatment. Compared to vehicle, 6S-BH4 had minimal effect on myocardial infarction (59±2% vs. 50±3%, n=6-8). Thus, these findings demonstrate that the protective effect of BH4 is primarily due to restoration of NOS coupling with suppression of O 2 -. and enhancement of NO production from the enzyme.