Hypoxia inducible factor-1 activation by prolyl 4-hydroxylase-2 gene silencing attenuates myocardial ischemia reperfusion injury.
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.
Natarajan
Ramesh
R
Division of Pulmonary Disease and Critical Care Medicine, Department of Internal Medicine, Virginia Commonwealth University Medical Center, Richmond, VA, USA.
Salloum
Fadi N
FN
Fisher
Bernard J
BJ
Kukreja
Rakesh C
RC
Fowler
Alpha A
AA
D000818:Animals; D002478:Cells, Cultured; D020868:Gene Silencing; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000502:physiology*; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D015428:Myocardial Reperfusion Injury / Q000517:prevention & control*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000502:physiology*; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D034741:RNA, Small Interfering / Q000494:pharmacology*
C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011393:Procollagen-Proline Dioxygenase; D052247:Nitric Oxide Synthase Type II