Liposomal tetrahydrobiopterin preserves eNOS coupling in the post-ischemic heart conferring in vivo cardioprotection

Tetrahydrobiopterin (BH₄) is an essential cofactor of nitric oxide synthase (NOS), and reduced BH₄ availability leads to endothelial NOS (eNOS) uncoupling and increased reactive oxygen species (ROS) generation. Questions remain regarding the functional state of eNOS and role of BH₄ availability in the process of in vivo myocardial ischemia-reperfusion (I/R) injury. Rats were subjected to 60min of in vivo left coronary artery occlusion and varying periods of reperfusion with or without pre-ischemic liposomal BH₄ supplementation (1mg/kg, iv). Myocardial infarction was correlated with cardiac BH₄ content, eNOS protein level, NOS enzyme activity, and ROS generation. In the vehicle group, 60-min ischemia drastically reduced myocardial BH₄ content in the area at risk (AAR) compared to non-ischemic (NI) area and the level remained lower during early reperfusion followed by recovery after 24-h reperfusion. Total eNOS, activated eNOS protein level (eNOS Ser1177 phosphorylation) and NOS activity were also significantly reduced during ischemia and/or early reperfusion, but recovered after 24-h reperfusion. With liposomal BH₄ treatment, BH₄ levels were identical in the AAR and NI area during ischemia and/or early reperfusion, and were significantly higher than with vehicle. BH₄ pre-treatment preserved eNOS Ser1177 phosphorylation and NOS activity in the AAR, and significantly reduced myocardial ROS generation and infarction compared to vehicle. These findings provide direct evidence that in vivo I/R induces eNOS dysfunction secondary to BH₄ depletion, and that pre-ischemic liposomal BH₄ administration preserves eNOS function conferring cardioprotection with reduced oxidative stress.