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(Circulation Research. 2000;86:319.)
© 2000 American Heart Association, Inc.

Cellular Biology

Hypoxic Regulation of Inducible Nitric Oxide Synthase via Hypoxia Inducible Factor-1 in Cardiac Myocytes

Frank Jung, Lisa A. Palmer, Nan Zhou, Roger A. Johns

From the Departments of Internal Medicine, Division of Cardiology (F.J.), and Anesthesiology (L.A.P., N.Z., R.A.J.) University of Virginia Health Science Center, Charlottesville, Va; Department of Internal Medicine, Division of Cardiology (F.J.), University of Frankfurt, Germany; and Department of Anesthesiology and Critical Care (R.A.J.), The Johns Hopkins University School of Medicine, Baltimore, Md.

Correspondence to Roger A. Johns, Professor and Chairman, Department of Anesthesiology and Critical Care, Blalock 1415, The Johns Hopkins University School of Medicine, 600 North Wolfe St, Baltimore, MD 21287-4965. E-mail rajohns{at}jhmi.edu

	Abstract

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Abstract—The relationship between hypoxia and regulation of nitric oxide synthase (NOS) in myocardial tissue is not well understood. We investigated the role of hypoxia inducible factor-1 (HIF-1) on expression of the inducible NOS (iNOS) in myocardial cells in vivo and in vitro. In situ hybridization in myocardial tissue from rats exposed to hypoxia for 3 weeks demonstrated increased iNOS mRNA expression. Northern analysis of RNA from hearts of those animals and from cells exposed to hypoxia for 12 hours in vitro demonstrated an increase of HIF-1 RNA expression. Electrophoretic mobility shift assays using oligonucleotides containing the iNOS HIF-1 DNA binding site and nuclear extracts from cardiac myocytes showed induction of specific DNA binding in cells subjected to hypoxia. Transient transfection of cardiac myocytes using the murine iNOS promoter resulted in a 3.43-fold increase in promoter activity under hypoxia compared with normoxia. Mutation or deletion of the HIF-1 site eliminated the hypoxic response. As cytokines have been shown to regulate iNOS expression in myocardial cells, cultured neonatal cardiac myocytes were stimulated with interleukin-1ß causing a dramatic induction of iNOS protein expression under normoxia, with further augmentation under hypoxia. Transient transfection of cells stimulated with interleukin-1ß showed an increased iNOS promoter activity under normoxic conditions compared with unstimulated cells, with a further increase in response to hypoxia, which was dependent on HIF-1. These results demonstrate that hypoxia causes an increase in iNOS expression in cardiac myocytes and that HIF-1 is essential for the hypoxic regulation of iNOS gene expression.

Key Words: inducible nitric oxide synthase • hypoxia inducible factor-1 • heart • hypoxia

	Introduction

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Nitric oxide (NO) plays an important role as a regulator in the nervous, immune, and cardiovascular systems.¹ In cardiac tissue, basal production of NO is maintained by constitutive expression of endothelial NO synthase and contributes to the regulation of coronary circulation, heart rate, and myocardial contractility.² However, NO generated by the inducible form of NO synthase (iNOS) has been implicated in many pathophysiological states leading to myocardial dysfunction.^{3 4 5 6 7 8 9} It has also been reported that in ischemia/reperfusion injury, NO production can exhibit either a positive effect (attributed to a decrease in neutrophil and platelet adhesion or vasodilation) or a negative effect (related to the production of free radicals and inactivation of mitochondrial enzymes).^{10 11 12 13} Furthermore, there is recent evidence that iNOS plays a role as a mediator in the reduction of infarct size via late preconditioning.^{14 15}

The relationship between acute or chronic hypoxia and NO synthase regulation in cardiac myocytes is still not well established.¹⁶ Also, other factors, such as cytokines,^{1 3 17 18 19 20} may influence hypoxia-mediated iNOS regulation, although the exact mechanisms are not known.^{21 22 23 24 25}

It has been shown that low oxygen tension regulates a number of other genes.^{26 27 28} cis-acting sequences responsible for the induction of hypoxia-induced transcription of the erythropoietin gene have been identified. The trans-acting factor, hypoxia inducible factor-1 (HIF-1), binds to a conserved region in the enhancer located in the 3'-flanking region of the erythropoietin gene, which is required for hypoxic inducibility.^{29 30} This DNA binding protein is a heterodimer composed of 2 subunits, HIF-1 and HIF-1ß.^{31 32 33} Functionally important binding sites for HIF-1 (consensus 5'-RCGTG-3') have also been found in a number of other genes known to be regulated by hypoxia, such as vascular endothelial growth factor^{34 35} ; the glycolytic enzymes aldolase A, enolase 1, and lactate dehydrogenase A; and phosphoglycerate kinase-1.^{36 37 38 39} A putative HIF-1 site in the murine iNOS gene was also shown to be required for hypoxia-induced transcription in a macrophage cell line and in pulmonary endothelial cells.^{22 23} We undertook the present study to examine what role HIF-1 and interleukin (IL)-1ß play on the effect of hypoxia-modulated iNOS expression in myocardium and isolated myocytes.

	Materials and Methods

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Primary neonatal cardiac myocytes were isolated from 1- to 2-day-old Sprague Dawley rat pups, using a neonatal cardiac myocyte isolation procedure kit according to the manufacturer’s instructions (NCMIS, Worthington Enzyme). Cardiac myocytes were then plated at a density of 2 to 3x10⁶ cells per 60-mm culture dish and allowed to seed for 24 hours before transfection or cytokine stimulation. For studies involving hypoxic conditions, cells were purged with 95% N₂–5% CO₂ for 20 minutes and then placed in a 1% to 2% O₂-5% CO₂–balanced N₂ incubator for 6 to 36 hours.

Cells were transfected using pGL-3 constructs containing the full-length iNOS promoter (pGLiNOS) or the iNOS promoter with a mutation of the HIF-1 binding site (pGL209) or the iNOS promoter with a deletion of the HIF-1 binding site (pGL220). After transfection, cells were placed into hypoxic (1% to 2% O₂) or nonhypoxic incubators for up to 36 hours before harvesting for luciferase assays. Cytokines (10 ng/mL) or vehicle (PBS/BSA) was added before hypoxic exposure. Promoter activity was measured in luciferase light units as fold increase over promoterless activity. ß-Galactosidase and protein concentrations using a standard BSA curve were used to normalize for transfection efficiency and cell number.

Nuclear extracts were prepared from cardiac myocytes exposed to normoxia and hypoxia for 36 hours as previously described.³¹ Extract (3 µg) was incubated with radiolabeled oligonucleotide probe (1.5 fmol) and loaded on a 4% nondenaturing polyacrylamide gel. Electrophoresis was carried out at 4°C. When used, competitor oligonucleotides were added at the beginning of the 5-minute preincubation period.

Procedures followed in the care and euthanization of the animals were approved by the Animal Research Committee of the University of Virginia. The protocol for the exposure of rats to hypoxia has been previously described.²⁵ Animals were euthanized after 3 weeks of exposure to normoxic or hypoxic conditions.

Crude protein from normoxic and hypoxic heart samples and cultured cardiac myocytes were prepared for Western blot analysis. Electrophoresis was carried out on a 7.5% SDS gel according to the method of Laemmli,⁴⁰ and blots were incubated with primary IgG iNOS antibody for 1 hour. Protein detection was carried out using enhanced chemiluminescence.

Total RNA was isolated from hearts of animals exposed to 3 weeks of hypoxia and normoxia and from normoxic and hypoxic myocytes. RNA was fractionated by glyoxyl-agarose gel electrophoresis and transferred to Hybond-N+ nylon membrane. cDNA probes for HIF-1 were labeled with ³-dCTP and hybridizations performed using 25 ng of end-labeled cDNA probe. The cDNA for HIF-1 has been previously described.⁴¹ Blots were exposed to autoradiography at -70°C.

In situ hybridization was performed on serial sections of formalin-fixed, paraffin-embedded tissue from left ventricles of animals exposed to normoxia and hypoxia for 3 weeks. The conditions of target pretreatment, hybridization, and probe generation have been extensively characterized.^{42 43} Sense and antisense orientation probes specific for iNOS mRNA were used. The in situ hybridization data were analyzed using both brightfield morphology as well as darkfield optics to better visualize the full distribution of the silver grains generating the autoradiographic signal.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hypoxia Induces iNOS mRNA in the Myocardium of Hearts From Rats Exposed to Chronic Hypoxia
To examine the effect of chronic hypoxia on iNOS expression in myocardium, in situ hybridization was used to determine the presence and cellular location of iNOS mRNA expression in hearts from rats subjected to 3 weeks of hypoxia. Compared with tissue sections from hearts of rats subjected to 3 weeks of normoxia (Figure 1A), iNOS mRNA expression was increased predominantly in the nuclei of myocardial cells from rat hearts exposed to chronic hypoxia (Figure 1B). These findings were observed independently of the origin of the tissue, in the right or left ventricle.

View larger version (86K): [in this window] [in a new window]   Figure 1. Hypoxia increases iNOS mRNA expression in myocardial cells in hearts of rats exposed to chronic hypoxia. In situ hybridization analysis was performed on hearts of rats exposed to 10% O2 (hypoxia) (A) or room air (normoxia) (B) for 3 weeks. Tissues from left ventricles were probed with the antisense cRNA for iNOS to demonstrate iNOS RNA expression. Tissues from hypoxic hearts were also probed with the control sense cRNA for iNOS (C).

Hypoxia Induces HIF-1 mRNA Expression in Hearts From Rats Subjected to Chronic Hypoxia
It has not been shown whether HIF-1 is influenced by conditions of chronic hypoxia in cardiac cells in vivo or in vitro. To determine whether expression of RNA encoding HIF-1 increased in response to chronic hypoxia in myocardial cells and tissue, Northern blot analysis was performed using total RNA from hearts of rats subjected to normoxia and hypoxia for 3 weeks and from neonatal cardiac myocytes subjected to normoxia or hypoxia for up to 36 hours. In normoxic rat hearts and neonatal cardiac myocytes cultured under nonhypoxic conditions, low-level RNA expression of HIF-1 was observed, whereas HIF-1 RNA levels were increased in hearts from rats exposed to hypoxia (Figure 2A) and in isolated neonatal cardiac myocytes exposed to prolonged hypoxia (Figure 2B).

View larger version (23K): [in this window] [in a new window]   Figure 2. Analysis of HIF-1 RNA expression in hypoxic rat hearts and hypoxic neonatal cardiac myocytes. Northern blot analysis was performed on total RNA from the hearts of rats exposed to room air (normoxia, n=4) or 10% O2 (hypoxia, n=4) for 3 weeks (A) and from isolated neonatal cardiac myocytes exposed to 20% O2 (normoxia) or 1% O2 (hypoxia) up to 12 hours. Blots were hybridized to probes for HIF-1 (B). Data are mean±SD of 3 experiments performed. Differences of photometric activity between normoxia and hypoxia were statistically significant using a 2-way variance analysis (*P<0.001).

Hypoxia and IL-1ß Have an Additive Effect on the Induction of iNOS Protein Expression in Cardiac Myocytes
Cytokines, such as IL-1ß, are involved in the induction of iNOS expression not only in cardiac myocytes but also in other cell types.^{44 45} However, the effect of hypoxia on modulation of IL-1ß–induced iNOS expression is controversial. Therefore, we examined the effect of IL-1ß stimulation of cardiac myocytes on iNOS expression under normoxic and hypoxic conditions. In control cells stimulated with vehicle (PBS/BSA), no iNOS protein expression was observed under normoxia, whereas there was only weak induction under hypoxia. In contrast, stimulation of cells with IL-1ß caused a significant induction of iNOS protein expression under normoxia, which was further increased under hypoxic conditions (Figure 3).

View larger version (25K): [in this window] [in a new window]   Figure 3. IL-1ß induces iNOS protein expression in cardiac myocytes, which is potentiated by hypoxia. Western blot analysis was performed using total protein from cardiac myocytes exposed to normoxia or hypoxia. The cytokine IL-1ß (0.1 or 10 ng/mL) or vehicle (PBS/BSA) was added before cells were subjected to normoxia or hypoxia. Cellular protein was prepared after a 36-hour exposure. Blots were hybridized with primary IgG iNOS antibody (Transduction Laboratories).

Hypoxia Induces Nuclear Proteins That Bind to the HIF-1 Sequence in the iNOS Promoter
The HIF-1 binding site in the 5'-flanking region of the murine iNOS promoter has been shown to be involved in the regulation of iNOS gene expression in macrophages and endothelial cells.^{22 23} To determine whether hypoxic exposure of neonatal cardiac myocytes induces nuclear proteins that bind to the HIF-1 binding site, electrophoretic mobility shift assays (EMSAs) were performed using nuclear extracts prepared from cells cultured under normoxic or hypoxic conditions and a 30-bp wild-type (WT) oligonucleotide containing the consensus HIF-1 binding site (Figure 4A). Two constitutively expressed DNA binding activities were present in extracts from cardiac cells cultured under normoxic or hypoxic conditions. However, another DNA binding activity was specifically induced by hypoxia (Figure 4B, arrow). This DNA binding activity could be detected in cells as early as 6 hours after exposure to hypoxia. Competition experiments with excess unlabeled WT or mutated oligonucleotides (Figure 4A) demonstrated that binding of this hypoxia-induced factor was specific (Figure 4B).

View larger version (47K): [in this window] [in a new window]   Figure 4. Hypoxia induces a factor that binds to iNOS gene promoter sequences. A, Sequences of the oligonucleotides used in the EMSA. The WT oligonucleotide contains the WT 9-bp sequence (underlined), which is identical to the HIF-1 binding site in the erythropoietin gene enhancer, in the context of the murine iNOS 5'-flanking region. MUT contains a 3-bp mutation previously shown to eliminate HIF-1 binding to the erythropoietin gene enhancer.48 B, EMSAs were performed on nuclear extracts prepared from neonatal cardiac myocytes exposed to 20% O2 (normoxia, N) or 1% O2 (hypoxia, H) for 12 hours, using an oligonucleotide probe (WT) consisting of a WT 30-bp sequence from the iNOS promoter. Arrow denotes the location of the hypoxia-induced complex. Competition was performed using increasing molar excess (50x, 100x, and 200x) of unlabeled WT or mutated (Mut) oligonucleotides.

Hypoxia Increases Transcriptional Activity of the iNOS Promoter in Cardiac Myocytes via HIF-1
To determine whether iNOS promoter activity was affected by hypoxia in cardiac myocytes, transient transfection experiments were performed using pGLiNOS (Figure 5A), which contains 1588 bp of the 5'-flanking promoter region of the murine iNOS gene linked to a luciferase reporter gene. iNOS promoter activity was increased 3.43-fold in cells subjected to hypoxia compared with cells exposed to normoxia (Figure 5B).

View larger version (26K): [in this window] [in a new window]   Figure 5. Hypoxia increases promoter activity of the iNOS gene via HIF-1. Augmentation by IL-1ß of the iNOS promoter activity under hypoxic conditions is dependent on HIF-1 binding to the iNOS promoter. A, Schematic diagram illustrating the location of the HIF-1 binding site (-227 to -219 bp) in the 5'-flanking region of the murine iNOS gene (pGLiNOS), the HIF-1 binding site deletion construct (pGL220), and the construct containing the HIF-1 mutation (pGL209). Other cis-acting sequences are putative binding sites for activator protein-1 (rectangle), nuclear factor B (square), and p53 (oval). LUC indicates luciferase. B and C, Neonatal cardiac myocytes were transiently transfected with the WT pGLiNOS, the HIF-1 deletion (pGL220), and HIF-1 mutation constructs (pGL209) or with backbone vector (pGLbasic) alone (not shown). After transfection, cells were cultured under nonhypoxic (open bars) or hypoxic (filled bars) conditions for 36 hours. Before exposure, cells were either incubated with vehicle (PBS/BSA) (B) or stimulated with IL-1ß cytokine (10 ng/mL) (C). Cells were harvested and luciferase activity was determined. Promoter activity was expressed in luciferase light units as fold increase over promoterless activity (not shown). Protein concentrations using a standard BSA curve were used to normalize for cell number in the luciferase and ß-galactosidase assay. Induction of iNOS promoter activity under hypoxia is shown relative to the activity of the construct under normoxic conditions. Data are mean±SD of triplicate samples. Results are shown for 3 representative experiments. Differences of luciferase activity between normoxia and hypoxia were statistically significant using a 2-way variance analysis (*P<0.05).

To determine whether the HIF-1 binding site is functionally required for hypoxic induction of the murine iNOS promoter in cardiac myocytes, transient transfection experiments were performed using constructs with a deletion (pGL220) or a mutation (pGL209) of the HIF-1 binding site (Figure 5A). Both deletion and mutation of the HIF-1 binding site eliminated the increase in iNOS promoter activity seen in response to hypoxia when compared with cells exposed to normoxia (Figure 5B). This confirms that the HIF-1 binding site is required for transcriptional activation of the iNOS gene in cardiac myocytes under hypoxic conditions.

Hypoxic Augmentation of IL-1ß–Induced iNOS Promoter Activity Is Dependent on HIF-1 Binding to the iNOS Promoter
To determine whether the inducibility of iNOS expression by IL-1ß is also occurring at the transcriptional level and whether the increase of promoter activity under hypoxic conditions is dependent on interaction of IL-1ß with HIF-1, we performed transient transfection experiments in cardiac myocytes with and without IL-1ß stimulation (Figure 5B and 5C). Overall, stimulation with IL-1ß caused a significant increase in iNOS promoter activity under normoxic conditions, which was further potentiated when cells were exposed to hypoxia. Using constructs containing a deletion (pGL220) or a mutation (pGL209) of the HIF-1 binding site (Figure 5A) still eliminated the increase in promoter activity seen in response to hypoxia in cells stimulated with IL-1ß (Figure 5C). This confirms that hypoxic augmentation of IL-1ß–induced iNOS promoter activity in cardiac myocytes is dependent on HIF-1 binding to the iNOS promoter.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our studies were designed to investigate the mechanism of hypoxia-induced regulation of iNOS in cardiac myocytes in vitro and in vivo. We have demonstrated that iNOS mRNA expression is increased in hearts from rats exposed to 3 weeks of hypoxia. iNOS mRNA expression was equally present in the right and left ventricles. There are now several other studies demonstrating increased iNOS expression under hypoxic conditions in cell types other than cardiac myocytes. Melillo et al²² found that interferon-–induced iNOS transcription is increased by hypoxia in a macrophage cell line. Additional evidence was provided earlier from our laboratory showing that iNOS gene expression was induced by hypoxia in pulmonary endothelial cells.²³ Kitakaze et al¹⁶ have shown that short-term exposure of cardiac myocytes to hypoxia resulted in increased NO production, although they did not specify whether this effect was mediated by the constitutive or the inducible form of NO synthase.

However, it is not entirely clear what mechanisms regulate iNOS gene expression under hypoxic conditions in cardiac myocytes. HIF-1 is one transcription factor known to play a role in oxygen regulation of several genes, such as erythropoietin, vascular endothelial growth factor, and recently the iNOS gene in pulmonary endothelial cells.^{23 29 34} In our study, we have uncovered for the first time a specific mechanism of hypoxia-induced iNOS gene regulation in cardiac myocytes that is mediated via the transcription factor HIF-1. HIF-1 DNA binding activity was only detected in nuclear extracts of cells grown under hypoxic conditions. Also, transient transfection experiments revealed that for the murine iNOS gene, HIF-1 is essential for the increased promoter activity in cardiac myocytes exposed to hypoxia, as mutation or deletion of the HIF-1 binding site abolished hypoxic induction of the iNOS promoter activity. It is known that both HIF-1 mRNA and protein are ubiquitously expressed in all organs of human and rodents⁴⁶ and that they are rapidly induced by hypoxia and rapidly decay upon return to nonhypoxic conditions.^{30 31 32 33 47} Our in vivo data demonstrated that induction of HIF-1 RNA expression was observed in hearts of rats subjected to 3 weeks of hypoxia.

The role HIF-1 plays in the transcriptional regulation of gene expression in response to hypoxia may be both cell type specific and gene specific. For example, Semenza et al⁴⁸ have shown that in the human hepatoblastoma cell line Hep3B, which is transcriptional activation mediated by HIF-1, requires the binding of a second unidentified factor at site 2 of the erythropoietin gene enhancer. Comparison of sequences around the HIF-1 site present in the 5'-flanking region of the iNOS gene and the 3'-enhancer of the erythropoietin gene shows a region of similarity 10 bp downstream of the HIF-1 site. This 5-bp 5'-CACTG-3' sequence eliminated the ability of the erythropoietin enhancer to activate transcription in response to hypoxia. Thus, it is possible that the 5'-CACTG-3' sequence in the iNOS gene may also be involved in the hypoxia-induced increase of iNOS expression.

Compared with the murine sequence, there is 85% homology in the 5'-flanking region of the rat iNOS gene, which also contains an intact HIF-1 consensus site.⁴⁹ It is therefore likely that the rat iNOS gene is regulated in a similar manner under hypoxic conditions. Conversely, in the human iNOS gene, there is no known HIF-1 binding site contained within the published sequence. It is possible that a HIF-1 site is present upstream of the known published sequence and may still be involved in the hypoxic regulation of the human iNOS gene. Alternatively, other factors may be responsible for the regulation of the human iNOS gene under hypoxic conditions. Putative binding sites of factors such as activator protein-1 and nuclear factor B, which have previously been implicated in the regulation of other genes by low oxygen tension, are also present in the human iNOS gene and may participate in its regulation by hypoxia.

It is not known whether additional factors are required for the activation of the iNOS gene via HIF-1 in cardiac myocytes under hypoxic conditions. For example, regulation of the lactate dehydrogenase A gene by hypoxia in the human cervical carcinoma cell line HeLa is augmented by forskolin and is dependent on the HIF-1 binding site and a cAMP response element.³⁹ In the murine macrophage line ANA-1, the effects of hypoxia on iNOS transcription, which requires the HIF-1 binding site, are augmented by interferon- treatment.²¹ Similarly, our transient transfection experiments with cardiac myocytes demonstrated that stimulation with IL-1ß further increased promoter activity not only under normoxic but also under hypoxic conditions. In contrast, mutation and deletion of the HIF-1 site still resulted in abolishment of the hypoxia-dependent response. Thus, our data suggest that IL-1ß is able to induce iNOS expression and that the increased iNOS gene expression under hypoxic conditions is still dependent on HIF-1.

Also on the translational level, stimulation of cells with IL-1ß resulted in a significant induction of iNOS protein expression in cells subjected to normoxia, which was further augmented under hypoxic conditions, which is in contrast to another study in which exposure of cardiac myocytes to prolonged hypoxia (48 hours) did not cause an increase of iNOS expression in cardiac myocytes. iNOS mRNA and protein expression as well as NO release in cardiac myocytes was only induced after stimulation of cells with IL-1ß. Interestingly, exposure of cells to prolonged hypoxia then led to a significant decrease in IL-1ß–mediated NO release and iNOS induction.²¹ This discrepancy with our study could be explained by differences in the stimulation protocol with IL-1ß. Although previous data from our laboratory suggested that the hypoxic response of iNOS expression was dependent on the concentration of the IL-1ß stimulus in cell types other than cardiac myocytes, we did not find a difference in hypoxia-induced iNOS expression in cardiac myocytes when they were stimulated with 2 different concentrations of IL-1ß (0.1 or 10 ng/mL). Also, the time of stimulation with IL-1ß may play a critical role in the response pattern of gene regulation in certain cell types. For example, Friedlander et al⁵⁰ demonstrated that IL-1ß exhibited pro- or antiapoptotic effects on hepatocytes, depending on the time of stimulation of cells exposed to hypoxia. This raises new, interesting thoughts regarding different roles of cytokines in specific cell types.

In summary, the mechanisms by which iNOS gene expression is increased in cardiac myocytes under pathophysiological conditions in which oxygen availability is compromised are not known. For the first time, we have shown that increased expression of HIF-1 results in the transcriptional activation of iNOS gene expression under hypoxic conditions in myocardial cells, thus demonstrating a specific mechanism of hypoxia-induced iNOS expression in this cell type. Our studies have also demonstrated that IL-1ß induced iNOS gene and protein expression in cardiac myocytes, which is further augmented under hypoxic conditions via the transcription factor HIF-1. The functional role of NO production under conditions of low oxygen tension in cardiac myocytes is not established. One could speculate that the increase of NO production and iNOS gene expression in cardiac myocytes observed under different pathophysiological conditions such as myocardial ischemia/reperfusion or myocardial infarction, stunning, or hibernation, may potentially contribute to the impairment in myocardial contraction. However, there is increasing evidence that increased NO production and expression of iNOS may have a protective role as a mediator in late cardiac preconditioning.^{14 15} Therefore, identification and elucidation of regulatory pathways, such as the HIF-1 pathway, on the regulation of iNOS gene expression may have important therapeutic consequences and remains a subject for further investigation.

	Acknowledgments

 
This work was supported by NIH Grants GM49111 and HL39706 (both to R.A.J.) and by a Scientist Development Grant (to L.A.P.) from the American Heart Association.

	Footnotes

 
This manuscript was sent to Stephen F. Vatner, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received August 9, 1999; accepted November 22, 1999.

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