doi: 10.1161/01.RES.0000200398.52220.cc
© 2006 American Heart Association, Inc.
Editorials |
Induction of HIF-1
and iNOS With siRNA
A Novel Mechanism for Myocardial Protection
From the Department of Medicine, Division of Cardiology, Albert Einstein College of Medicine, Bronx, NY.
Correspondence to David J. Lefer, PhD, Department of Medicine, Division of Cardiology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail dlefer{at}aecom.yu.edu
See related article, pages 133–140
Key Words: siRNA • prolyl 4-hydroxylase-2 • gene silencing • myocardial infarction • cardiac function
Hypoxia-inducible factor-1 (HIF-1) is a dimeric transcriptional complex composed of HIF-1
and HIF-1ß subunits that has been recognized primarily for its role in the maintenance of oxygen and energy homoeostasis.1–3 Although the HIF-1ß subunit is constitutively expressed, the expression and function of HIF-1 are precisely regulated by cellular oxygen concentrations.1–3 Under conditions of normoxia HIF-1 is rapidly degraded by the 26S proteasome and nonfunctional whereas HIF-1 accumulates instantaneously under hypoxic conditions.3–5 HIF-1 represents a highly important redox-sensitive transcription factor that regulates the expression of numerous genes under pathological conditions including: heme oxygenase-1 (HO-1), inducible nitric oxide synthase (iNOS), VEGF, glycolytic enzymes, glucose transporters, erythropoietin, and insulin-like growth factor 2 (IGF-2).4,5 All of these genes encode proteins that are prosurvival in nature and can result in cytoprotection. Because the HIF-1 subunit is highly sensitive to oxygen and is degraded by the proteasome after prolyl-hydroxylation and ubiquitination in normoxic cells, attempts have been made to enhance the expression of HIF-1 as a means to protect cells against ischemic injury. Previous studies have demonstrated that stable expression or activation of HIF-1 can protect isolated cardiac myocytes6 and isolated hearts7 against ischemia-reperfusion (I/R) injury. Additionally, it has been previously reported that inducible nitric oxide synthase (iNOS) is regulated under conditions of hypoxia by HIF-1 in cardiac myocytes.8
In the present issue of Circulation Research Natarajan and colleagues9 have reported that inhibition of HIF-1-prolyl-4-hydroxylase-2 (PDH2) with small interfering RNA (siRNA) protects the ischemic-reperfused murine myocardium. In these carefully performed experiments these investigators treated mice with PHD2 siRNA 24 hours before initiation of global cardiac ischemia-reperfusion (I/R) injury and observed a 70% reduction in myocardial infarct size and significantly preserved left ventricular postischemic myocardial contractile function. Treatment with PHD2 siRNA significantly increased HIF-1 in both isolated endothelial cells and in the murine heart with concomitant elevations in endothelial and myocardial iNOS protein levels. The authors hypothesized that increases in iNOS protein were in part responsible for the cardioprotective effects of PDH2 siRNA (Figure) and clearly demonstrated a lack of myocardial protection with PDH2 siRNA in iNOS deficient (–/–) mice and mice treated with an iNOS inhibitor (ie, 1400W). The authors failed to observe increases in endothelial nitric oxide synthase (eNOS) in mice receiving the PDH2 siRNA which further supports iNOS-dependent signaling and nitric oxide-mediated cardioprotection. Interestingly, previous reports indicate that NO donors can induce HIF-1 expression under normoxic conditions.10 Thus, it is plausible that increased iNOS expression and NO generation after HIF-1 activation with PDH2 siRNA might trigger further HIF-1 activity and NO generation via a positive feedback mechanism. The authors have previously reported that a nonspecific prolyl hydroxylase inhibitor, dimethyloxallyl glycine (DMOG), reduced myocardial infarct size in rabbits in an HO-1–dependent manner.7 The results of the present study using siRNA to inhibit PHD2 are in agreement with this previous study of pharmacological inhibition of prolyl hydroxylases. The authors focused the present study on the effects of PHD2 inhibition on iNOS upregulation and myocardial protection in the setting of I/R. However, it is likely that the protective effects of PHD2 might be mediated by a host of cardioprotective gene products under the regulation of HIF-1 such as VEGF, IGF-2, erythropoietin, and HO-1. Future studies might investigate the effects of HIF-1a activation cardioprotection with respect to other protective proteins. The present study serves to confirm and extend the concept of NO-mediated cardioprotection in the setting of myocardial I/R injury. Although the present study is truly novel and very exciting, future studies of PDH2 siRNA in in vivo animal models of myocardial I/R will certainly provide additional support for the present study and add to the likelihood that the results of the current study could be translated to the clinic. Furthermore, it would also be of interest to evaluate the time course of HIF-1 activation before ischemia and the extent of cardioprotection to further understand the critical time necessary for pretreatment with HIF-1 activators.
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Numerous studies have defined a role for NO-mediated signaling in the protection of the myocardium against myocardial injury including: acute therapy for I/R injury,11 congestive heart failure,12 and myocardial preconditioning.13 In recent years nitric oxide has emerged as a very proximal and potentially important signaling molecule whose precise molecular mechanisms of action remain unknown. Nitric oxide has been shown to modulate coronary blood flow,11,14,15 inhibit platelet function,15 attenuate leukocyte-mediated inflammation,11,15 inhibit cardiac apoptosis,15 modulate mitochondrial function and respiration,16 and influence heart rate and cardiac contractility.12,15 Any or all of these physiological actions of nitric oxide could be responsible for the protective effects observed in the study of Natarajan et al.9
One additional and very exciting aspect of the present study is the application of siRNA to animals in vivo as a means to modify protein expression and ultimately modulate cardiovascular physiology. In the present study, siRNA was administered to mice via intraperitoneal injection at 24 hours before cardiac ischemia at which time hearts were excised and subjected to global ischemia and reperfusion. It was previously thought that siRNA experiments were limited to transfection of cells in vitro and that these reagents and would not be effective under in vivo conditions. The authors have clearly demonstrated that PHD2 siRNA does upregulate HIF-1 protein expression in the myocardium after systemic injection, thereby setting the stage for future in vivo animal studies of siRNA therapy.
The authors should be congratulated for a very novel and highly exciting study that presents additional insights into the mechanisms of protection of the ischemic myocardium. The present study confirms the protective actions of both HIF-1 and iNOS but also significantly extends our current understanding of myocardial reperfusion injury. The authors clearly demonstrate for the first time the efficacy of gene silencing with siRNA in the intact animal and sets the stage for numerous future studies of gene silencing in vivo as a therapeutic approach for the treatment of various cardiovascular diseases.
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Acknowledgments |
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Dr Lefer’s research laboratory is supported by research grants from the Heart, Lung, and Blood Institute of the National Institutes of Health (2RO1 HL-60849 to D.J.L.) and from the American Diabetes Association (7-04-RA-59 to D.J.L.).
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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Related Article:
Circ. Res. 2006 98: 133-140.
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