Abstract

Hypoxia inducible factor-1 (HIF-1) regulates changes in transcription of key genes such as inducible NO synthase (iNOS) in hypoxic/ischemic environments. In normoxia, HIF-1 activation is controlled by HIF-1alpha-prolyl 4-hydroxylases, which target HIF-1alpha for ubiquitination and proteasomal degradation. We hypothesized that normoxic HIF-1 preservation could attenuate cardiac ischemia/reperfusion injury via a preconditioning effect. HIF-1 preservation was achieved by using small interfering RNA (siRNA) to silence murine HIF-1alpha-prolyl-4 hydroxylase-2 (PHD2). PHD2 siRNA reduced PHD2 mRNA expression 89+/-1.5% (P<0.001) in a time- and concentration-dependent manner in normoxic murine microvascular endothelial cells (EC). PHD2 silencing in normoxic EC stabilized HIF-1alpha protein levels while significantly increasing HIF-1 transcriptional activity and iNOS mRNA expression. Wild-type mice infused with PHD2 siRNA (1.5 microg/g body weight) showed a 61+/-2.4% (P<0.05) reduction in cardiac PHD2 mRNA within 24 hours. In addition HIF-1alpha protein levels and HIF-1-dependent iNOS mRNA levels were increased. PHD2 siRNA-transfected hearts from wild-type mice (n=6) subjected to 30 minutes ischemia followed by 60 minutes reperfusion exhibited reduced infarct size when compared with saline-treated controls (9.7+/-1.9% versus 31.6+/-1.8%, respectively, P<0.0001, n=6) and to control mice transfected with a nontargeting siRNA control (28.4+/-3.0%, P<0.0001, n=6). Hearts from iNOS knockout mice receiving PHD2 siRNA by identical injection protocol (n=6) exhibited infarct size indistinguishable from saline controls (28.7+/-1.3%). These results show that in vitro and in vivo, PHD2 silencing using a siRNA strategy produces transcriptionally active HIF-1. Normoxic activation of HIF-1 in hearts following in vivo PHD2 siRNA administration attenuates reperfusion injury via an iNOS-dependent pathway.