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Circulation Research. 2005
Published online before print November 23, 2005, doi: 10.1161/01.RES.0000197816.63513.27
A more recent version of this article appeared on January 6, 2006
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Submitted on August 12, 2005
Revised on November 3, 2005
Accepted on November 11, 2005

Hypoxia Inducible Factor-1 Activation by Prolyl 4-Hydroxylase-2 Gene Silencing Attenuates Myocardial Ischemia Reperfusion Injury

Ramesh Natarajan ; Fadi N. Salloum ; Bernard J. Fisher ; Rakesh C. Kukreja ; and Alpha A. Fowler III *

From the Divisions of Pulmonary Disease and Critical Care Medicine (R.N., B.J.F., A.A.F.) and Cardiology (F.N.S., R.C.K.), Department of Internal Medicine, Virginia Commonwealth University Medical Center, Richmond, Va.

* To whom correspondence should be addressed. E-mail: aafowler{at}vcu.edu.

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-1{alpha}-prolyl 4-hydroxylases, which target HIF-1{alpha} 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-1{alpha}-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-1{alpha} protein levels while significantly increasing HIF-1 transcriptional activity and iNOS mRNA expression. Wild-type mice infused with PHD2 siRNA (1.5 µg/g body weight) showed a 61±2.4% (P<0.05) reduction in cardiac PHD2 mRNA within 24 hours. In addition HIF-1{alpha} 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.
Key words: small interfering RNA • hypoxia inducible factor-1{alpha}-prolyl 4-hydroxylase-2 • cardiac ischemia/reperfusion • inducible NO synthase


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