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Am J Physiol Heart Circ Physiol 294: H901-H908, 2008. First published December 14, 2007; doi:10.1152/ajpheart.00715.2007
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Prevention of HIF-1 activation and iNOS gene targeting by low-dose cadmium results in loss of myocardial hypoxic preconditioning in the rat

Elise Belaidi,1,2 Pauline C. Beguin,1,2 Patrick Levy,1,2,3 Christophe Ribuot,1,2 and Diane Godin-Ribuot1,2

1Laboratoire HP2, Hypoxie et Physiopathologies Cardiovasculaire et Respiratoire, Institut National de la Santé et de la Recherche Médicale ERI17, Grenoble, France; 2Faculté de Médecine, Université Grenoble 1, Grenoble, France; and 3Laboratoire EFCR, Hôpital A. Michallon, CHU, Grenoble, France

Submitted 20 June 2007 ; accepted in final form 7 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study aimed to underline the interaction between hypoxia-inducible factor-1 (HIF-1) and the inducible nitric oxide synthase (iNOS) gene in vivo and their contribution to the delayed myocardial preconditioning induced by acute intermittent hypoxia (IH) in the rat using chromatin immunoprecipitation and pharmacological inhibition by low-dose cadmium. Langendorff-perfused hearts of Wistar rats exposed to normoxia or IH 24 h earlier were submitted to global ischemia and reperfusion. Effects of iNOS inhibition by aminoguanidine (100 µM) before ischemia or of low-dose injection of cadmium chloride (1 mg/kg) before normoxia or IH were tested. Myocardial HIF-1 and iNOS quantification and in vivo chromatin immunoprecipitation of HIF-1 bound to the iNOS gene promoter were performed. IH-induced delayed cardioprotection resulted in an improvement in coronary flow and functional recovery at reperfusion and a decrease in infarct size. Myocardial HIF-1 activity was increased with resulting targeting of the iNOS gene. Aminoguanidine abolished the cardioprotective effects of IH. Cadmium chloride treatment before IH prevented myocardial HIF-1 activation (72.3 ± 4.0 vs. 42.1 ± 9.7 arbitrary units after cadmium chloride; P < 0.05), targeting of the iNOS gene, iNOS expression, and preconditioning (infarct size: 15.9 ± 5.6 vs. 30.1 ± 5.4% after cadmium chloride; P < 0.05). This study is the first to demonstrate the interaction of HIF-1 with the myocardial iNOS gene in situ after hypoxic preconditioning. Prevention of HIF-1 activation and iNOS gene targeting by a single low dose of cadmium abolished the delayed cardioprotective effects, bringing insight into the cardiovascular consequences of cadmium exposure.

intermittent hypoxia; cardioprotection; hypoxia inducible factor 1; tissue chromatin immunoprecipitation; cadmium chloride; inducible nitric oxide synthase

THE REDOX-SENSITIVE TRANSCRIPTION factor hypoxia-inducible factor (HIF)-1, a key regulator of the adaptative response to hypoxia, has been suggested to be a critical mediator of late-phase myocardial preconditioning (27). It is a heterodimer consisting of HIF-1{alpha} and HIF-1β subunits. Under normoxia (N), the HIF-1{alpha} protein is degraded after hydroxylation of two proline residues by O2-dependent prolyl-4-hydroxylases (23). This targets the subunit for the von Hippel-Lindau tumor suppressor protein E3 ubiquitin ligase complex, resulting in polyubiquitination and proteasomal degradation (8). Under conditions of hypoxia, the reduced activity of prolyl-4-hydroxylases leads to HIF-1{alpha} stabilization, nuclear translocation, and dimerization to the β-subunit. HIF-1 binding to hypoxia response elements (HRE) at the DNA consensus sequence 5'-RCGTG-3' in promoter regions drives the transcription of various genes involved in the adaptation to hypoxic-ischemic stress (27). Some of these are genes that have protein products with cardioprotective properties, with the most documented being inducible nitric oxide synthase (iNOS) (19, 24).

Cardiomyocyte protection has indeed been achieved by enhancing HIF-1{alpha} stabilization through pharmacological agents (31), expression of constitutively stable HIF-1{alpha} (11), or prolyl-4-hydroxylase gene silencing (24). More importantly, work on knockout mice has shown that HIF-1 signaling is involved in the development of hypoxia-induced delayed preconditioning (9).

Although these results point to a role for HIF-1 and iNOS in hypoxic preconditioning, no study has specifically assessed whether the interaction of these two factors is necessary to confer delayed cardioprotection in vivo, thus taking into account the complex regulatory pathways controlling the expression of target genes during the adaptation to hypoxia.

We have described a delayed form of myocardial preconditioning induced by acute intermittent hypoxia (IH) in the rat (3, 4). The aim of the present study was thus to investigate the interaction of HIF-1 with the myocardial iNOS gene in this setting using myocardial chromatin immunoprecipitation, a powerful technique that allows the direct evaluation of transcription factor binding on gene promoters in vivo (26). Furthermore, we also wanted to confirm the pivotal role of HIF-1 by evaluating whether prevention of its activation in vivo could abolish the delayed cardioprotection. For this, we have chosen a pharmacological approach and have pretreated the animals before hypoxic preconditioning with a single low dose of cadmium, a metal known to enhance HIF-1{alpha} degradation in vitro (10).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
IH model. The recommendations of the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, National Research Council, Washington, DC: National Academy Press, 1996) were followed in these experiments, which were approved by the local ethics committee and the Direction des Services Vétérinaires de l'Isère, France.

Experiments were conducted on adult male Wistar rats (weight range 330–350 g) from Elevage Janvier (Le Genest st. Isles, France) housed in controlled conditions and provided with standard rat chow ad libitum. For exposure to IH or N, animals were housed in identical custom-made cylindrical Plexiglas chambers (length = 28 cm, diameter = 10 cm, volume = 2.2 liters) with tightly fitted lids. During 4 h, rats received repeated 1-min cycles of IH composed of 40 s of hypoxia and 20 s of N via software-driven timed solenoid valves. Hypoxia was provided by mixing pure nitrogen and compressed air in latex balloons to obtain a 10% inspired O2 fraction (FIO2). N consisted of administration of compressed air to allow a return to 21% FIO2. Animals exposed to N only received similar cycles (with the same disturbances and noise as IH) of compressed air. The FIO2 level in the chambers was controlled throughout the exposure with an O2 analyzer (model ML206; AD Instrument, Oxfordshire, UK). Figure 1 provides an illustration of the FIO2 profile produced by the IH protocol.


Figure 1
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  		Fig. 1. Representative tracing of the changes in inspired O2 fraction (FIO2) in the chambers during exposure to intermittent hypoxia (IH). FIO2 was determined by continuous sampling from the chamber using an O2 analyzer. Hypoxia was achieved by flushing nitrogen into the chamber until the O2 concentration reached 10%. Hypoxia was maintained for 40 s, after which room air was flushed into the chamber for 20 s to reestablish normoxia (21% FIO2).

 
Ischemia-reperfusion protocol. Twenty-four hours after IH or N, rats were anesthetized with pentobarbital sodium (60 mg/kg ip) and treated with heparin (500 IU/kg iv). Hearts were excised, immersed in frozen Krebs-Henseleit buffer (in mM: 118.0 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25.2 NaHCO3, 11.0 glucose, and 0.5 EDTA, pH 7.4), and perfused by the aorta, in a retrograde manner at a constant pressure of 75 mmHg, according to the Langendorff method. The perfusion medium was gassed with 95% O2-5% CO2. Myocardial temperature was controlled with a thermoprobe inserted into the left ventricle (LV) and maintained at 37°C. A water-filled latex balloon (no. 4; Hugo Sachs, Germany) connected to a pressure transducer was inserted into the LV and inflated to obtain a LV end-diastolic pressure (LVEDP) between 5 and 15 mmHg. The ischemia-reperfusion (I/R) protocol consisted of 20 min of stabilization, 30 min of no-flow global ischemia, and 120 min of reperfusion (Fig. 2). Coronary flow (CF) was measured at timed intervals by collecting the coronary venous effluent during 1 min and was expressed relative to heart weight (ml·min–1·g–1). LV developed pressure (in mmHg), its maximal and minimal rates of rise (in mmHg/s), LVEDP, and heart rate (beats/min) were recorded throughout the protocol and measured at the end of stabilization, at the end of ischemia, and at various times during reperfusion.


Figure 2
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  		Fig. 2. Twenty-four hours after IH or normoxia (N), animals were subjected to an ischemia-reperfusion (I/R) protocol composed of stabilization (20 min), no-flow global ischemia (30 min), and reperfusion (120 min) periods. Some rats were pretreated with a low dose of cadmium chloride (CdCl2; 1 mg/kg ip), 1 h before exposure to IH or N. Some hearts were perfused during the last 10 min of stabilization with aminoguanidine (Ag; 100 µM) before I/R.

 
After the I/R protocol, hearts were removed, weighed, frozen, and cut from apex to base into 2-mm-thick transverse slices (6 or 7 per heart). The slices were incubated at 37°C in 1% triphenyltetrazolium chloride in phosphate buffer (pH 7.4) for 10 min, fixed in 10% formaldehyde, and photographed. Areas of infarcted tissue were measured by a computerized planimetric technique (Image Tool software) and expressed as a percentage of total ventricular area minus cavities.

Drug pretreatment and experimental groups. Forty-six rats were divided into six groups (Fig. 2). Two groups were exposed to IH (n = 8) or N (n = 8) for 4 h. Two groups, CdIH (n = 9) and CdN (n = 7), were pretreated 1 h before IH or N exposure, by a single intraperitoneal injection of cadmium chloride (Sigma-Aldrich) at a dose of 1 mg/kg. This dose was chosen because it has been shown to be the minimal single dose necessary to induce significant cellular responses in the rat without cytotoxic effects (15). In two additional groups, AgIH (n = 7) and AgN (n = 7), hearts were perfused during the last 10 min of stabilization with the iNOS inhibitor aminoguanidine (Ag; Sigma-Aldrich) at a dose (100 µM) shown to effectively inhibit iNOS activity and cell death under ischemic conditions (17) and to significantly decrease myocardial nitrate/nitrite content during I/R in the isolated rat heart (12).

ELISA-based measurement of myocardial HIF-1 activation. In additional animals (n = 4 per group) exposed to IH or N with or without prior cadmium chloride treatment, myocardial samples were collected immediately at the end of the 4-h exposure. Nuclear extracts were obtained with a commercial kit (Nuclear Extract Kit; Active Motif Europe, Rixensart, Belgium). Activation of HIF-1 was quantified by a DNA-binding ELISA kit (TransAM; Active Motif Europe) based on the binding of activated HIF-1 to an oligonucleotide containing an HRE (5'-TACGTGCT-3') from the erythropoietin gene.

In vivo chromatin immunoprecipitation assay of myocardial HIF-1{alpha} binding to the iNOS gene. The chromatin immunoprecipitation (ChIP) assay was performed according to the procedure described by Orlando et al. (26), with minor modifications. Myocardial samples (~300 mg) from IH and N rats were minced and treated for 10 min with formaldehyde (1% final concentration in PBS) at room temperature. Cross-linking was stopped by the addition of glycine to a final concentration of 125 mM. Cross-linked extracts were fragmented enzymatically (Enzymatic Chromatin Shearing kit; Active Motif Europe) (input sample). Then, 40 µg of chromatin extract were precleared with protein A Sepharose beads (Amersham Biosciences, Orsay, France) for 1 h. After centrifugation, supernatants were incubated overnight at 4°C with a ChIP-grade anti-HIF-1{alpha} antibody (ab2185; Abcam, Cambridge, UK) or without antibody (mock condition sample). Immunoprecipitated DNA-protein complexes were bound to protein A Sepharose beads after 3 h of incubation at 4°C and washed in low-salt buffer, high-salt buffer, LiCl buffer, and Tris-EDTA buffer successively, as described by Duong et al. (13). After protein elimination with proteinase K (200 µg; Promega) and phenol extraction, the DNA was precipitated, suspended in water, and used as a template for PCR. PCR primers were selected to amplify a 132-bp DNA sequence containing the HRE of the iNOS gene (partial nucleotide sequence accession code AJ230461) according to Keinänen et al. (21). The forward primer sequence used was 5'-CGGGTAAGTTCCTTAACCTGC-3', and the reverse primer sequence was 5'-GTTACAAATCAAGTGGCTGGG-3'. PCR amplification products were analyzed on ethidium bromide-stained 3% agarose gels during the exponential phase of the PCR (33 cycles). The quantitative aspect of the PCR at 33 cycles was verified by serial dilutions of the input.

Western blot analysis of myocardial iNOS expression. In a last series of animals (n = 4/group) exposed to IH or N with or without prior cadmium chloride treatment, myocardial samples were collected 24 h after exposure. Hearts were homogenized in lysis buffer and centrifuged at 13,000 rpm (20 min). The supernatants were ultracentrifuged at 100,000 g (1 h, 4°C) to collect the cytosolic fraction. Cytosolic proteins (50 µg) were separated on 8% SDS-polyacrylamide gels and transferred to nitrocellulose membranes, which were incubated with anti-iNOS antibody (1/1,000; Santa Cruz Biotechnology) overnight at 4°C. Bound antibody was visualized by use of horseradish peroxidase-conjugated goat anti-rabbit antibody (1/20,000; Jackson Immunoresearch). Equal loading was confirmed by GAPDH immunoblotting.

Statistical analysis. Experimental data are presented as means ± SE. Infarct sizes and myocardial HIF-1 contents were compared by one-way ANOVA. Hemodynamic data were analyzed by two-way ANOVA. Post hoc comparisons were performed with Bonferroni t-tests. Statistical significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Acute IH-induced delayed myocardial preconditioning. Acute exposure to IH induced a delayed myocardial protection characterized by a significant decrease in infarct size (15.9 ± 5.6 in IH vs. 33.8 ± 5.0% in N rats) 24 h later (Figs. 3 and Go5). This was accompanied by a significant increase in CF during reperfusion (mean reperfusion value of 9.7 ± 0.4 ml·min–1·g–1 in IH vs. 6.7 ± 0.4 ml·min–1·g–1 in N rats) (Figs. 4A and 6A). In addition, the increase in LVEDP on reperfusion was significantly smaller in IH compared with N rats (mean reperfusion value of 27.3 ± 4.0 vs. 54.1 ± 3.5 mmHg) (Figs. 4B and 6B). For the other hemodynamic parameters, there was no significant difference in the response of the various groups to I/R (Table 1). Hemodynamic values at the end of stabilization were not statistically different between the various groups studied (data not shown).


Figure 3
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  		Fig. 3. Infarct size [I; expressed as a percentage of total ventricular area (V)] after 30 min of no-flow global ischemia and 120 min of reperfusion in rats exposed 24 h earlier to N or IH and the effect of Ag (100 µM) perfusion during 10 min before ischemia. Values are means ± SE. *P < 0.05.

 

Figure 4
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  		Fig. 4. A: coronary flow (ml·min–1·g of heart–1) at reperfusion. B: increase in left ventricular end-diastolic pressure (LVEDP) relative to baseline value during reperfusion in rats exposed 24 h earlier to IH or N and the effect of Ag (100 µM) perfusion during 10 min before ischemia. Values are means ± SE. *P < 0.05.

 

Figure 5
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  		Fig. 5. Infarct size (expressed as a percentage of total ventricular area) after 30 min of no-flow global ischemia and 120 min of reperfusion in rats exposed 24 h earlier to N or IH and the effect of CdCl2 (1 mg/kg ip) pretreatment 1 h before N or IH. Values are means ± SE. *P < 0.05.

 

Figure 6
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  		Fig. 6. A: coronary flow (ml·min–1·g of heart–1) at reperfusion. B: increase in LVEDP relative to baseline value during reperfusion in rats exposed 24 h earlier to IH or N and the effect of CdCl2 (1 mg/kg ip) pretreatment 1 h before IH or N. Values are means ± SE. *P < 0.05.

 

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  		Table 1. Hemodynamic parameters throughout ischemia-reperfusion

 
Abolition of the IH-induced cardioprotection by Ag. Selective inhibition of iNOS by Ag perfusion before I/R resulted in loss of IH-induced cardioprotection. Infarct sizes of Ag-perfused groups were increased compared with results shown in the IH group (33.1 ± 3.9 and 35.4 ± 3.9 vs. 15.9 ± 5.6% in AgN, AgIH, and IH groups, respectively) (Fig. 3). This was accompanied by a decrease in CF (mean reperfusion value of 5.8 ± 0.1 and 5.6 ± 0.1 vs. 9.7 ± 0.4 ml·min–1·g–1 in AgN, AgIH, and IH groups, respectively) (Fig. 4A). Finally, Ag perfusion increased LVEDP on reperfusion in both N and IH groups (mean reperfusion value of 68.3 ± 2.7 and 70.3 ± 3.0 vs. 27.3 ± 4.0 mmHg in AgN, AgIH, and IH groups, respectively) (Fig. 4B). Ag treatment did not significantly affect the other hemodynamic parameters (Table 1).

Prevention of the IH-induced cardioprotection by cadmium chloride pretreatment. Cadmium chloride administration 1 h before N or IH prevented the protective effect of IH on infarct size (27.4 ± 6.1 and 30.1 ± 5.4 vs. 15.9 ± 5.6% in CdN, CdIH, and IH rats, respectively) (Fig. 5). In addition, CF during reperfusion decreased compared with that shown in IH rats (mean reperfusion value of 6.6 ± 0.4 and 6.0 ± 0.4 vs. 9.7 ± 0.4 ml·min–1·g–1 in CdN, CdIH, and IH rats, respectively) (Fig. 6A). Finally, cadmium chloride pretreatment led to an increase in LVEDP during reperfusion (mean reperfusion value of 35.0 ± 4.0 and 43.0 ± 3.3 vs. 27.3 ± 4.0 mmHg in CdN, CdIH, and IH rats, respectively) (Fig. 6B). For all parameters, there was no aggravation in cadmium chloride-treated rats compared with nontreated N rats (Figs. 5 and 6). Cadmium chloride treatment did not significantly affect the other hemodynamic parameters (Table 1).

Myocardial HIF-1 activation during IH and its prevention by cadmium chloride pretreatment. IH induced a 1.6-fold increase in activated myocardial HIF-1{alpha} (linked with HIF-1β to form HIF-1) in nuclear extracts from IH rats compared with N rats (72.3 ± 4.0 vs. 44.8 ± 9.5 arbitrary units). This increase was abolished by cadmium chloride pretreatment 1 h before IH (47.4 ± 13.3 and 42.1 ± 9.7 arbitrary units in CdN and CdIH groups, respectively) (Fig. 7).


Figure 7
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  		Fig. 7. Myocardial hypoxia-inducible factor (HIF)-1 activation, expressed in arbitrary units of optical density (OD450nm), measured in nuclear extracts from hearts collected immediately after IH or N, with or without CdCl2 (1 mg/kg ip) pretreatment 1 h before IH or N. Values are means ± SE. *P < 0.05.

 
Interaction between HIF-1{alpha} and myocardial iNOS gene in IH-induced cardioprotection and its prevention by cadmium chloride pretreatment. The ChIP assay allows the direct evaluation of transcription factor binding on gene promoters. This technique enabled us to cross-link and immunoprecipitate HIF-1{alpha} bound on the myocardial iNOS promoter at the end of IH exposure with or without cadmium chloride pretreatment. Figure 8, A and B, shows the PCR amplification of a DNA sequence containing the HRE of the myocardial iNOS gene. PCR amplification was obtained only in the immunoprecipitated DNA from the IH group, indicating a binding of HIF-1 to the myocardial iNOS gene promoter in our cardioprotective conditions and demonstrating that cadmium chloride pretreatment specifically prevented HIF-1 targeting of the iNOS gene (Fig. 8B). Lack of amplification in mock conditions confirmed the specificity of the immunoprecipitation technique. PCR signals from DNA fragments before IP (input) show that the IP results are not due to differences in sample DNA content. Finally, lack of amplification with control PCR primers specific for intron 1 of the rat iNOS gene (~3 kb from the HRE site) ensured that results were not due to genomic DNA contamination of the immunoprecipitate (data not shown).


Figure 8
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  		Fig. 8. Myocardial in vivo chromatin immunoprecipitation of HIF-1{alpha} linked to the inducible nitric oxide synthase (iNOS) hypoxia response element (HRE; shown in A). B: PCR amplification of samples after immunoprecipitation without (Mock) and with (IP) anti-HIF-1{alpha} antibody and of fragmented DNA before IP (Input). DNA was extracted from fresh hearts collected immediately after IH or N, with or without CdCl2 (1 mg/kg ip) pretreatment. C: histogram representing PCR performed on serial input dilutions confirming that amplification is proportional to DNA quantity.

 
Myocardial iNOS gene expression after IH and its prevention by cadmium chloride pretreatment. Western blot analysis confirmed that HIF-1 binding to promoter of the iNOS gene induced by IH resulted in a significant increase in myocardial iNOS content and that prevention of HIF-1{alpha} stabilization by cadmium chloride pretreatment abolished iNOS gene expression (Fig. 9).


Figure 9
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  		Fig. 9. Western blot analysis of myocardial iNOS protein content in cytosolic extracts from hearts collected 24 h after IH or N, with or without CdCl2 (1 mg/kg ip) pretreatment. Values are means ± SE. *P < 0.05 vs. other groups.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The principal findings of this study are the following: first, it provides a direct demonstration of the interaction of HIF-1 with the iNOS gene and their role in the delayed myocardial preconditioning induced by IH in rat; second, it shows that acute administration of a low dose of cadmium can be used as a pharmacological tool to prevent HIF-1{alpha} stabilization in vivo; and third, it brings new insight into the cardiovascular consequences of cadmium exposure.

In accordance with the results of the present study, we have previously shown that acute IH induces a delayed myocardial preconditioning in the rat with a reduction of infarct size (4) and an increase in CF and a decrease in LVEDP during reperfusion (3). The improved recovery in postischemic CF brought about by this form of preconditioning could account for the myocardial tissue salvage and reduced myocardial stunning. The relationship between these parameters is underlined by the results of the present study showing their concomitant abolition by Ag and cadmium chloride pretreatment. The improvement in CF could be explained by an increase in cardiac NO availability. Indeed, many studies have demonstrated that iNOS is an essential mediator of the delayed phase of myocardial preconditioning induced by brief episodes of I/R (7), acute sustained hypoxia (32), heat stress (1), and a number of pharmacological agents (18). We have previously shown that NOS was involved in the delayed preconditioning induced by acute IH (4). In the present study, the abolition of the cardioprotective and CF-sparing effects by perfusion with the selective iNOS inhibitor Ag, before ischemia, confirms that iNOS is the isoform involved. Interestingly, the CF improvement was seen on reperfusion only because baseline values did not differ between the various groups studied. This has been reported with other forms of iNOS-related preconditioning. Thus Tosaki et al. (29) have shown that iNOS overexpression was without effect on baseline CF but that the latter was significantly increased during reperfusion in preconditioned groups. It has been proposed that the iNOS protein is induced by preconditioning in an inactive form, which requires an activation stimulus, such as that provided by ischemia, to obtain maximal enzyme activity. The activation of kinases and/or inhibitors of phosphatases by ischemia can thus promote phosphorylation of the inactive form of iNOS induced (34, 14).

The iNOS gene is one of the numerous genes upregulated by HIF-1 in the adaptive response of the cardiovascular system to hypoxia (27). In the present study, we were able to measure nuclear HIF-1 activity in tissue extracts, thus providing the first in vivo demonstration of its increase in the myocardium in response to hypoxia. This suggests a more important cytosolic stabilization of HIF-1{alpha} after IH since its transcriptional activity is directly related to its oxygen-mediated degradation (5). In accordance with our results, an increase in myocardial HIF-1{alpha} measured by electrophoretic mobility gel shift assay has previously been reported after simulated hypoxia with cobalt chloride in mice (31).

Our study also provides the first in vivo evidence for the targeting of the myocardial rat iNOS promoter by HIF-1{alpha} in response to IH. Other studies on the regulation of the iNOS gene by HIF-1 in the heart have been based on indirect observations using knockout iNOS mice (24, 31), overexpression of constitutively active HIF-1{alpha} (11), or prolyl-4-hydroxylase gene silencing (24). Using ChIP analysis, we demonstrate that HIF-1 directly interacts with the iNOS promoter in myocardial extracts after intermittent hypoxic preconditioning. The resulting increase in myocardial iNOS content confirmed that this interaction resulted in a transcription of the gene.

Cadmium chloride has been shown to inhibit HIF-1{alpha} stabilization in Hep3B cells by enhancing its proteasome-dependent degradation (10). In the same cell line, the ability of cadmium to suppress erythropoietin production has been linked to its inhibitory effect on HIF-1 DNA binding and erythropoietin promoter activity (16, 25). Here, we report that in vivo administration of a single low dose of cadmium before IH prevented myocardial HIF-1 activation as well as its binding to the iNOS gene, as shown by the ChIP assay, and the resultant iNOS expression. Cadmium chloride pretreatment thus abolished the delayed cardioprotection in a manner similar to that observed with the iNOS inhibitor Ag. Cadmium chloride appears to stimulate the proteasomal degradation of HIF-1{alpha} via an action on the ubiquitin system (20). Indeed, DNA microarray analysis of gene expression induced by a nonlethal dose of cadmium in human HeLa cells shows that the ubiquitin pathway is activated (33). Cadmium has been shown to promote nuclear translocation of metal-regulatory transcription factor-1 interacting with metal-responsive elements, which have been identified in promoter regions of genes involved in heavy metal homeostasis (28). Whether this adaptative response is responsible for the upregulation of the ubiquitin system and the increase in HIF-1{alpha} degradation by cadmium remains to be determined.

Cardiovascular tissues are known to be highly vulnerable to environmental chemicals and pollutants, the most well known being cadmium-containing tobacco smoke (6). Smoking is among the strongest independent predictor of premature heart disease. Second-hand smoke, recently classified as a pollutant, increases the risk of heart disease by 30%, in particular through decreased nitric oxide synthesis (2). The results of the present study, showing that a low dose of cadmium abolishes myocardial preconditioning by preventing iNOS upregulation by HIF-1, bring into light the cardiovascular risk of cadmium exposure. By linking a specific constituent of tobacco smoke such as cadmium to cardiovascular toxicity, this could help understand the molecular mechanisms by which smoking, in particular second-hand smoke, causes heart disease.

In conclusion, the abolition of the delayed myocardial preconditioning by cadmium chloride demonstrates the beneficial role of HIF-1 activation by acute IH. Whether HIF-1 also plays a role in chronic IH-related conditions, such as obstructive sleep apnea (OSA), remains to be investigated. Indeed, OSA patients are at increased risk for cardiovascular disease (30), but there is a dramatic decline in relative mortality after the age of 50 yr that could be related to activation of cardioprotective genes, such as the iNOS gene, by the nocturnal cycles of hypoxia-reoxygenation characteristic of OSA (22).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by research grants from the Institut National de la Santé et de la Recherche Médicale, the Région Rhône-Alpes, and the Association Grenobloise des Insuffisants Respiratoires. E. Belaidi and P. C. Beguin are recipients of doctoral fellowships from the Ministère de l'Enseignement Supérieur et de la Recherche.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Etienne Lefai for invaluable contributions to the ChIP assays, Nolwenn Miguet for help with the Western blot assays, Dr. Jean Gagnon for helpful advice and discussions, and Bruno Chapuis for the conception of the device monitoring the IH apparatus.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Godin-Ribuot, Laboratoire HP2, Université Grenoble 1, Institut Jean Roget, BP 170, 38042 Grenoble Cedex 9, France (e-mail: diane.ribuot{at}ujf-grenoble.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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E. Belaidi, M. Joyeux-Faure, C. Ribuot, S. H. Launois, P. Levy, and D. Godin-Ribuot
Major role for hypoxia inducible factor-1 and the endothelin system in promoting myocardial infarction and hypertension in an animal model of obstructive sleep apnea.
J. Am. Coll. Cardiol., April 14, 2009; 53(15): 1309 - 1317.
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