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Am J Physiol Heart Circ Physiol 292: H751-H757, 2007. First published November 10, 2006; doi:10.1152/ajpheart.01016.2006
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Oxygen Sensing: Life and Death of a Cell

Acute intermittent hypoxia activates myocardial cell survival signaling

Ah-Mee Park, Hiroko Nagase, Shilpashree Vinod Kumar, and Yuichiro J. Suzuki

Department of Pharmacology, Georgetown University Medical Center, Washington, DC

Submitted 16 September 2006 ; accepted in final form 6 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Intermittent hypoxia (IH) with repeated episodes of hypoxia-normoxia cycle has been shown to exert preconditioning-like cardioprotective effects. To understand the mechanism of these events, we investigated the changes in cardiac gene expression in response to acute IH. Mice were subjected to five cycles of 2 min of 10% O2 plus 2 min of 21% O2. RNA was isolated, and gene array analysis was performed. Results show that the expression of antiapoptotic genes, such as Bcl-2 and Bcl-xL, were increased after acute IH. GATA-4 regulates transcription of these genes, and, consistently, GATA-4 activity was increased by acute IH. Although the phosphorylation of GATA-4 has been shown to regulate its activity, no changes in GATA-4 phosphorylation status by acute IH were noted. Gene transcription of gata4 was increased by acute IH, and this might be responsible for the enhanced GATA activity. To understand the mechanism of acute IH activation of gata4 gene transcription, we identified a promoter region of the mouse gata4 gene that is 1,000 bp immediately upstream from the transcriptional start site. In cardiac muscle cells, truncation of 1,000 to 250 bp did not alter the transcriptional activity, suggesting that the proximal 250-bp region contains important transcriptional regulatory sites. We further found that acute IH activates factors which bind to the proximal 100-bp region. Thus acute IH activates not yet identified factors that bind to the proximal 100-bp region of the gata4 promoter and, in turn, increases gata4 gene transcription, leading to enhanced expression of Bcl-2 and Bcl-xL.

Bcl; cardioprotection; GATA-4; heart; promoter

ISCHEMIC PRECONDITIONING is a powerful form of endogenous protection against myocardial infarction (18). A brief period of ischemia makes the heart more resistant to subsequent ischemic insult. Murry et al. (13) demonstrated in a dog model that four cycles of 5 min of ischemia and reperfusion reduced the infarct size induced by subsequent prolonged ischemia. Similarly, patients who have angina shortly before a heart attack have smaller infarcts than patients without prior angina (termed "warm-up phenomenon"), and preconditioning mechanisms found in animal models may play a role in the human warm-up phenomenon (18). Mechanisms underlying ischemic preconditioning and the warm-up phenomenon are under intense investigations. Identifications of endogenous cardioprotective factors and understanding their mechanisms should have significant impact on therapeutic strategies against ischemic heart disease.

Intermittent hypoxia (IH), with repeated episodes of hypoxia and normoxia, resembles repeated episodes of ischemia and reperfusion, which cause ischemic preconditioning. Consistently, in vivo IH exposure was found to exert cardioprotection in animal models and in humans. Cai et al. (4) reported that 24 h after treatment of mice with five cycles of 6 min of 6% O2 plus 6 min of 21% O2, the heart was protected against myocardial injury induced by 30 min of global ischemia followed by reperfusion (as determined by monitoring left ventricular developed pressure and myocardial infarction). This protection was not detected in hypoxia-inducible factor-1 (HIF-1) knockout mice, suggesting the importance of HIF-1 in cardioprotective mechanisms. More frequent episodes of IH have also been found to afford cardioprotection. Beguin et al. (2) found that rats treated with 40 s of 10% O2 plus 20 s of 21% O2 for 4 h had reduced myocardial infarction induced by subsequent global ischemia-reperfusion after 24 h of IH treatment. Cai et al. (4) concluded that the protective mechanism induced by IH is different from that promoted by ischemic preconditioning which promotes both early and delayed phases of protection; in contrast, IH only activates delayed protection, indicating the importance of transcriptional regulation by factors such as HIF-1.

In humans, a hypobaric IH protocol was employed to treat patients with coronary heart disease and dyslipidemia. After 10 mo, none of the 37 patients developed myocardial infarction (17). Burtscher et al. (3) reported that two to four cycles of 10–14% O2 (3–5 min) plus 21% O2 (3 min) IH increased exercise tolerance in elderly men with and without coronary artery disease (3). Although further investigations are needed to determine the possible clinical use of IH, some of the human and animal results have shown that IH, as well as signaling mechanisms involved in IH, might have therapeutic potential against myocardial injury.

To understand the mechanism of IH-mediated cardioprotection, the present study investigated initial gene expression changes in the heart in response to treating mice with short-term IH. Five episodes of 2 min of hypoxia (10% O2) plus 2 min of normoxia (21% O2) triggered upregulation and downregulation of various genes. Among them, antiapoptotic bcl-xL and bcl-2 mRNA levels were found to be increased in response to IH. A known transcriptional regulator of bcl-xL (1, 9) and bcl-2 (10), GATA-4 was found to be activated by IH through the promotion of gata4 gene transcription. We cloned the mouse gata4 promoter and identified an acute IH responsive region.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male C57BL/6 mice (12–14 wk old) were placed in a chamber for the OxyCycler oxygen profile controller (BioSpherix, Redfield, NY) that was set to cycle between 10% and 21% O2 every 2 min (see Fig. 1A). Normoxia controls were subjected to ambient 21% O2 in the separate OxyCycler chamber. Animals were fed normal rat chow, and all protocols involving animals were approved by the Georgetown University Animal Care and Use Committee and abide by the National Institutes of Health guidelines.


Figure 1
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  		Fig. 1. Effects of acute intermittent hypoxia (IH) on gene expression. A: IH profile. Actual changes in O2 tension in the hypoxic chamber are shown. B: mice were subjected to IH with 5 cycles of 2 min of hypoxia (10% O2) plus 2 min of normoxia (21% O2) and then maintained in the normoxic condition for 6 h. Hearts were excised, total RNA was isolated from left and right ventricles, and gene expression patterns were determined by GE Array Signal Transduction Pathway Finder (MM-008). The numbers 4 and 6 indicate positions for bcl-2 and bcl-xL. C: values represent means ± SE of the intensity of bcl-2 and bcl-xL mRNA levels determined by densitometry.

 
Gene array analysis. To identify the effects of IH on gene expression in the heart, we performed gene array. Animals were treated with IH, and total RNA was isolated from left and right ventricles using TRIzol (Invitrogen, Carlsbad, CA) and was subjected to mRNA expression analysis using the GE Array Q series Mouse Signal Transduction Pathway Finder Array (MM-008; SuperArray Bioscience, Frederick, MD). RT labeling was used for synthesizing and labeling cDNA probes, and the chemiluminescent detection method was utilized. The complete gene table can be obtained at www.superarray.com.

Reverse transcription-polymerase chain reaction. Total RNA (1 µg) extracted from left and right ventricles by using TRIzol was reverse-transcribed by oligo(dT) priming and MMLV RT (Applied Biosystems, Foster City, CA). The resultant cDNA was amplified using Taq DNA polymerase (Invitrogen) and resolved on a 1.5% agarose gel containing ethidium bromide (7). PCR primers for mouse gata4 were 5' primer, 5'-GAT GGG ACG GGA CAC TAC CTG-3'; and 3' primer, 5'-ACC TGC TGG CGT CTT AGA TTT-3', which produce a 309-bp product. PCR primers for mouse bcl-xL were as follows: 5' primer, 5'-CAT CCA AAC TGC TGC TGT GG-3'; and 3' primer, 5'-TTA TCT TGG CTT TGG ATC CTG -3', which produce a 337-bp product. PCR primers for mouse bcl-2 were as follows: 5' primer, 5'-GCG CAA GCC GGG AGA ACA-3'; and 3' primer, 5'-AGA CGT CCT GGC AGC CAT-3', which produce a 207-bp product. Denaturing was performed at 94°C for 45 s; annealing for 45 s at 58°C (for gata4), 53°C (for bcl-xL), and 58°C (for bcl-2); and polymerase reactions for 2 min at 72°C (30 cycles). The gapdh mRNA level was also monitored by using primers from BD Biosciences Clontech (Palo Alto, CA) as an internal control.

Western blot analysis. To prepare total tissue homogenates, heart ventricles were washed in PBS and solubilized with 50 mM HEPES solution (pH 7.4) containing 1% (vol/vol) Triton X-100, 4 mM EDTA, 1 mM sodium fluoride, 0.1 mM sodium orthovanadate, 1 mM tetrasodium pyrophosphate, 2 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Equal protein amounts of cell lysates were electrophoresed through a reducing SDS polyacrylamide gel and electroblotted onto a nitrocellulose membrane. The membrane was blocked and incubated with the polyclonal IgG for Bcl-xL (Santa Cruz Biotechnology, Santa Cruz, CA) and the detection made with horseradish peroxidase-linked secondary antibodies and enhanced chemiluminescence system (Amersham Life Science, Arlington Heights, IL).

Electrophoretic mobility shift assay. Left and right ventricles were homogenized by Polytron (Kinematica, Switzerland) in 4 vol of ice-cold homogenization buffer containing 10 mM HEPES (pH 7.5), 0.5 M sucrose, 0.5 mM spermidine, 0.15 mM spermin, 2 mM EDTA, 2 mM EGTA, 1 mM sodium fluoride, 0.1 mM sodium orthovanadate, 1 mM tetrasodium pyrophosphate, 1 mM PMSF, 5 µg/ml leupeptin, and 10 µg/ml aprotinin. Nuclear-rich fractions were prepared as previously described (15) with modifications. Homogenates were centrifuged at 12,000 g for 30 min at 4°C. Pellets were lysed in 2 vol of ice-cold homogenization buffer containing 0.1% Igepal and 0.5 M sucrose by homogenizing with Polytron and then centrifuged at 12,000 g for 30 min at 4°C and washed with ice-cold homogenization buffer containing 0.35 M sucrose. After being washed, the nucleus was extracted with 1 vol of ice-cold homogenization buffer containing 0.3 M NaCl and 10% glycerol for 60 min at 4°C with mixing at 1,400 rpm. Nuclear extracts were collected by centrifugation at 16,000 g for 30 min at 4°C.

For EMSA, the binding reactions were performed for 20 min in 5 mM Tris·HCl (pH 7.5), 37.5 mM KCl, 4% (wt/vol) Ficoll-400, 0.2 mM EDTA, 0.5 mM DTT, 1 µg poly(dI-dC)·poly(dI-dC), 0.25 ng (>20,000 counts/min) 32P-labeled double-stranded oligonucleotide, and 5- to 10-µg protein of nuclear extract. Electrophoresis of samples through a native 6% polyacrylamide gel was followed by autoradiography. The double-stranded oligonucleotide probes used in this study include the proximal GATA element from bcl-xL promoter with a sequence of 5'-AAG CCA AGA TAA GGT TCT or the GATA consensus elements 5'-CAC TTG ATA ACA GAA AGT GAT AAC TCT-3' (Santa Cruz Biotechnology). The double-stranded oligonucleotides, which represent seven regions within the gata4 promoter, as shown in GoGoGoGoFig. 6C, were also labeled with 32P. Supershift experiments were performed by incubating nuclear extracts with 2 µg of GATA-4 antibody (Santa Cruz Biotechnology) or the antibody against phosphorylated GATA-4 at serine-105 (Abcam, Cambridge, MA).


Figure 2
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  		Fig. 2. Effects of acute IH on bcl-2 and bcl-xL mRNA expression. Mice were subjected to IH with 5 cycles of 2 min of hypoxia (10% O2) plus 2 min of normoxia (21% O2) and maintained in the normoxic condition for durations indicated. C, control mice without IH treatment. A and B: hearts were excised, total RNA was isolated from left and right ventricles, and mRNA levels of bcl-2, bcl-xL, and gapdh were monitored by RT-PCR. Line graphs represent means ± SE of the intensity of bcl-2 and bcl-xL mRNA levels determined by densitometry (n = 10 animals). *P < 0.05, significantly different from normoxic control values. C: ventricular homogenates were subjected to Western blot analysis with the Bcl-xL antibody.

 

Figure 3
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  		Fig. 3. Effects of acute IH on GATA-4 DNA binding activity. A: mice were subjected to IH with 5 cycles of 2 min of hypoxia (10% O2) plus 2 min of normoxia (21% O2) and then maintained in the normoxic condition for durations indicated. Ventricular nuclear extracts were subjected to EMSA using the 32P-labeled oligonucleotide containing the proximal GATA element from the bcl-xL promoter. Nuclear extract sample from 6-h time point was also incubated with the GATA-4 antibody (+ab) for supershift analysis; the arrow indicates the supershifted band. Values in the bar graph represent means ± SE (n = 6 animals). *P < 0.05, significantly different from the control. B: mice were subjected to IH with 1, 3, or 5 cycles of 2 min of hypoxia plus 2 min of normoxia. Six hours after IH, ventricular nuclear extracts were prepared. The GATA DNA binding activity was monitored by EMSA. Values represent means ± SE (n = 5–6 animals) of fold increase in GATA binding activity relative to normoxic control animals. *P < 0.05, significantly different from the control (Con).

 

Figure 4
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  		Fig. 4. Effects of acute IH on GATA-4 phosphorylation. Mice were subjected to IH with 5 cycles of 2 min of hypoxia (10% O2) plus 2 min of normoxia (21% O2) and maintained in the normoxic condition for durations indicated. Ventricular nuclear extracts were subjected to EMSA with the 32P-labeled oligonucleotide containing the consensus GATA sequence in the absence and presence of the antibody (ab) for phosphorylated GATA-4 at serine-105 (2 µg) in the binding reaction mixtures. Percentage of phosphorylated GATA-4 was calculated by using the GATA band intensity in the absence (x) and presence (y) of the phosphorylated GATA-4 antibody using an equation, 100(x – y)/x. The values in the bar graph represent means ± SE (n = 6 animals). Cont, control.

 

Figure 5
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  		Fig. 5. Effects of acute IH on gata4 mRNA expression. Mice were subjected to IH with 5 cycles of 2 min of hypoxia (10% O2) plus 2 min of normoxia (21% O2) and maintained in the normoxic condition for durations indicated. Total RNA was isolated from left and right ventricles and analyzed by RT-PCR using primers for mouse gata4 or gapdh mRNA. The line graph represents means ± SE of the fold increase in gata4 mRNA relative to control (n = 10 animals). *P < 0.05, significant difference.

 

Figure 6
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  		Fig. 6. Effects of acute IH on DNA binding activity toward proximal gata4 promoter. A: structure of mouse gata4 gene with transcriptional start site that was identified via 5' rapid amplification of cDNA ends. The black rectangular region indicates transcribed regions, which span a 3.5-kb intron. The grey area denotes the putative promoter region that was cloned into the luciferase reporter vector. B: HL-1 cardiac muscle cells were transfected with the luciferase construct controlled by the 1,000-, 500-, or 250-bp proximal region of the putative gata4 promoter. Values represent means ± SE of the luciferase activity. RLU, relative light units. C: various double-stranded oligonucleotide probes were constructed within the proximal 250-bp putative gata4 promoter region. The numbers indicate the designated probe numbers. D: nuclear extracts from control mouse heart ventricles were subjected to EMSA using 32P-labeled oligonucleotide probes 1–7. E: mice were subjected to IH with 5 cycles of 2 min of hypoxia (10% O2) plus 2 min of normoxia (21% O2) and then maintained in the normoxic condition for durations indicated. Nuclear extracts were subjected to EMSA using 32P-labeled oligonucleotide probes 2 and 3. The arrows indicate the bands that were increased in response to acute IH.

 
5' Rapid amplification of cDNA ends. Total RNA was isolated from C57BL/6 mouse left and right ventricles by TRIzol. Antisense primer (5'-CAG CAT CAA AGC AGA AAC-3'), located within exon 2, was used for first-strand synthesis. Subsequent amplification was performed by using the 5' rapid amplification of cDNA ends (5'RACE) system as well as GeneRacer kit (Invitrogen). In brief, first-strand cDNA was tailed with recombinant TdT and a linker (dC) oligonucleotide. 5'RACE was performed by incubating with a RACE primer located upstream of the antisense primer (5'-AGG CTC TGG TTT GCT CAG GAA AAA-3') and with Abridged Anchor Primer using Platinum Taq High-Fidelity DNA polymerase (Invitrogen). Subsequently, nested PCR was performed with a nested primer designed upstream of RACE primer (5'-CCA AAT TGG ATT TGC GGT TGC T-3') and Abridged Universal Amplification Primer. The nested primer was used to sequence the PCR product to determine the transcriptional start site.

Cloning of gata4 gene promoter. Fragments (1,000, 500, and 250 bp) of the gata4 gene promoter were cloned by PCR cloning using mouse genomic DNA obtained from Promega (Madison, WI). Primers for each of the PCR fragments were as follows: 5'-TGA CAT GGT ACC AAA AGT TTA GCC CAA AGC GCG A-3' (1,000 bp forward), 5'-TGA CAT GGT ACC AAG GGC CAG TTC AGG TTT TAG TG-3' (500 bp forward), 5'-TGA CAT GGT ACC AAG GAC GTC GGG CTG CAC TGA-3' (250 bp forward), and 5'-CGG AAA GCT TCT CCG GCT TGT CCC CTG CTC-3' (reverse). The primers encode restriction digest sites (underlined) for cloning into the pGL3 basic luciferase reporter vector (Promega); forward primer encodes a Kpn I site and the reverse primer encodes a HindIII site. PCR was performed with Platinum Taq High-Fidelity DNA polymerase (Invitrogen). PCR reactions were performed for 40 cycles using a 30-s denaturation at 95°C, 1 min annealing at 65°C (for 1,000 bp) or 68°C (for 500 and 250 bp), and 6-min extension at 72°C. After electrophoresis on an agarose gel, PCR products were purified by using the QIAquick Gel Extraction kit (Qiagen, Valencia, CA). Purified PCR fragments were digested overnight at 37°C with Kpn I and HindIII (New England Biolabs, Beverly, MA) and ligated into the pGL3 luciferase reporter vector (Promega) by T4 DNA Ligase (New England Biolabs). Vectors positive for inserts were screened by digestion and subjected to bidirectional sequencing.

Luciferase assay. The day before transfection, HL-1 adult mouse cardiac muscle cells (8) were plated at 1.4 x 105 cells per well in a 12-well plate. DNA (1 µg) per well was transfected using the Fugene 6 transfection reagent (Roche Diagnostics, Indianapolis, IN) in serum-free, antibiotic-free RPMI. Cotransfection of the Renilla reporter was performed to normalize for transfection differences between wells. Cells were transfected for 6 h, and medium was then replaced with RPMI containing 0.1% FBS with antibiotics.

Luciferase assays were performed using the Dual Luciferase Assay kit (Promega). Transfected cells were washed in PBS and lysed. Cellular debris was removed by centrifugation at 14,000 g for 30 s. Cell lysates were added to Luciferase Assay Reagent II, and the firefly luciferase activities were read in a Model TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). An equal volume of Stop and Glow was added, and the Renilla reading was taken. The ratio of firefly luciferase to Renilla luciferase was calculated for each well of transfection.

Statistical analysis. Values are means ± SE. Significant differences between all groups were computed by one-way analysis of variance using F statistics. Statistically significant differences between two groups were determined by the Student's t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
IH activates gene expression of antiapoptotic bcl-2 and bcl-xL. To understand the initial events that might occur in the heart in response to IH exposure, we monitored myocardial gene expression patterns in mice treated with five episodes of 2 min of hypoxia (10% O2) plus 2 min of normoxia (21% O2). Mice were placed in an OxyCycler oxygen profiling chamber and subjected to O2 tension changes as depicted in Fig. 1A. After 20 min of IH treatment, mice were maintained in a normoxic condition. Six hours after the end of the IH cycle, the hearts were excised from mice and ventricular tissues were homogenized in TRIzol to isolate total RNA. RNA samples from IH-treated mice and normoxic controls were subjected to GE Array Signal Transduction Pathway Finder. The array contained a total of 96 genes, including 16 housekeeping genes. Among them, mRNA expression levels of Bcl-2, Bcl-xL, Gadd45, c-jun, Cdk2, Cdkn2b, Fn1, Hsf1, and N4wbp4T/MEPAI were increased greater than twofold, whereas mRNA levels of ATF-2, IAP2, and IL-2 were decreased by at least 25% (Fig. 1, B and C).

Increases in antiapoptotic genes, such as Bcl-2 and Bcl-xL, are of great interest because acute IH has been shown to promote cardioprotective activity. To confirm the gene array results, RT-PCR analyses were performed to measure bcl-2 and bcl-xL mRNA expression. As shown in Fig. 2, bcl-2 and bcl-xL mRNA levels were increased after the IH treatment. Furthermore, the time-course study revealed that mRNA levels of bcl-2 (Fig. 2A) and bcl-xL (Fig. 2B) were upregulated as early as 30 min after the IH treatment. To determine whether the mRNA induction by acute IH is associated with protein expression, Western blot analysis was used to detect Bcl-xL protein levels. As shown in Fig. 2C, acute IH also rapidly increased Bcl-xL protein expression.

IH activates GATA-4 by enhancing its gene transcription. Since GATA-4 has been shown to regulate gene expression of antiapoptotic genes such as Bcl-xL (1, 9) and Bcl-2 (10) in cardiac myocytes, the effects of IH on the DNA binding activity of GATA-4 were monitored. EMSA revealed that the GATA DNA binding activity was increased as early as 30 min after the acute IH treatment (5 cycles of 2 min of hypoxia plus 2 min of normoxia) (Fig. 3A). Supershift assay demonstrated that the majority of GATA binding activity in the mouse heart is due to GATA-4. An increase in GATA-4 DNA binding activity did not occur with one or three cycles of 2 min of hypoxia plus 2 min of normoxia (Fig. 3B).

Since the phosphorylation of GATA-4 has been attributed to the activation of this transcription factor (8, 9, 12), effects of IH on GATA-4 phosphorylation were monitored via supershift analysis using the antibody against phosphorylated GATA-4 at the serine-105 position. As shown in Fig. 4, no significant differences in the phosphorylation status of GATA-4 were noted up to 24 h after the acute IH treatment.

The increased GATA-4 DNA binding activity may be due to the activation of its gene transcription, since we found that gata4 mRNA expression was increased in response to acute IH. A significant increase in gata4 mRNA expression was observed as early as 2 h after 20 min of acute IH treatment with five cycles of 2 min of hypoxia plus 2 min of normoxia (Fig. 5). The increased gata4 mRNA expression continued for 24 h after acute IH treatment. An increase in gata4 mRNA expression did not occur with one or three cycles of 2 min of hypoxia plus 2 min of normoxia (data not shown).

Effects of IH on gata4 promoter. To understand the mechanism of acute IH-mediated activation of gata4 gene transcription, we cloned the mouse gata4 promoter (14). We first performed the 5'RACE analysis to identify the transcriptional start site of the gata4 gene using adult mouse heart mRNA. 5'RACE results along with the sequence analysis revealed that the 5' mRNA end occurs 0.6 kb upstream of the ATG translational start site, with 3.5 kb untranscribed intron existing 0.5 kb upstream of the ATG codon. Thus the transcriptional start site was found to be 4.1 kb upstream from the ATG translational start site within the gata4 DNA (Fig. 6A). The 1,000-bp region immediately upstream from the identified transcriptional start site, which is conserved and shares 90% homology with the promoter of the rat gata4 gene, was cloned into the pGL3 luciferase reporter vector. Transfection of this luciferase construct in HL-1 adult mouse cardiac muscle cells resulted in the expression of a luciferase reporter controlled by the 1,000-bp fragment (Fig. 6B). The 1,000-bp region was truncated further to 500- and 250-bp regions immediately proximal to the transcriptional start site. As shown in Fig. 6B, the truncation of the 1,000-bp region did not significantly alter the basal transcriptional activity, suggesting that the 250 proximal region contains the key regulatory elements for the basal expression.

This 250-bp promoter region of the gata4 genes was further truncated into seven short, double-stranded oligonucleotide fragments (Fig. 6C). These fragments were 32P labeled and used for EMSA experiments with myocardial nuclear extract samples from acute IH-exposed mice and normoxic controls. Fig. 6D shows DNA binding patterns exhibited by various probes within the 250-bp gata4 promoter in control mouse heart nuclear extracts. We compared these patterns in control normoxic mouse hearts with hearts from mice subjected to acute IH (5 cycles of 2 min of hypoxia plus 2 min of normoxia). Although we did not note any IH-mediated changes in binding patterns of probes 1, 4, 5, 6, and 7, acute IH increased the DNA binding activity toward probes 2 and 3 (Fig. 6E). Thus, probes 2 and 3 regions within the gata4 promoter contain binding elements for factors that are activated in response to acute IH, and these factors may play important roles in the activation of gata4 gene transcription.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
IH with repeated episodes of brief periods of hypoxia and normoxia resembles ischemic preconditioning and has cardioprotective ability. Indeed, our gene array analysis revealed that acute IH with five cycles of 2 min of hypoxia plus 2 min of normoxia increases mRNA expression of antiapoptotic Bcl-2 and Bcl-xL. These results are consistent with the ability of IH to elicit cardioprotection (2, 4). To our knowledge, this is the first demonstration of the induction of cardiac gene expression in response to acute IH.

GATA-4 transcription factor has been identified as a cardiac muscle cell survival factor (7). Apoptotic agents such as anthracyclines can downregulate GATA-4, and overexpression of GATA-4 can attenuate the incidence of apoptosis (7). Major antiapoptotic proteins such as Bcl-xL (1, 9) and Bcl-2 (10) are regulated by GATA-4. Since acute IH can promote transcription of these antiapoptotic genes, we tested the hypothesis that GATA-4 might be activated by acute IH. Indeed, we found that the DNA binding activity of GATA-4 was upregulated in response to acute IH.

The activity of GATA-4 can be regulated by posttranslational modifications, including phosphorylation, acetylation, and sumoylation. Phosphorylation of GATA-4 was originally described to occur in response to the activation of MEK/ERK pathway (8, 11, 12) as well as p38 MAPK (5, 6). GATA-4 can also be modified at its lysine residues through acetylation (16, 20) and small ubiquitin-related modifier-1 (19). Acute IH, however, failed to elicit either GATA-4 phosphorylation (Fig. 4), acetylation, or sumoylation (data not shown). Instead, we found that gene expression of GATA-4 was enhanced in response to acute IH. Significant increase in gata4 mRNA expression was noted as early as 2 h after acute IH.

We identified the transcriptional start site of the mouse gata4 gene and cloned the putative promoter region immediately upstream from the transcriptional start site (14). The 1,000-bp region immediately upstream from the transcriptional start site is conserved among species and exhibited a strong transcriptional activity in cardiac muscle cells. Luciferase experiments in cardiac muscle cells demonstrated that the proximal 250-bp region contains important regulatory elements. We thus tested to see whether the DNA binding activities toward this region might be affected by acute IH. We found a region within the proximal 100 bp can bind to factors that are activated by acute IH. Further study is needed to identify these acute IH-responsive transcription factors.

In conclusion, in our experiments using a mouse model, acute IH consisting of five cycles of 2 min of hypoxia plus 2 min of normoxia promoted gene expression of antiapoptotic bcl-2 and bcl-xL. Acute IH also activated GATA-4, a known regulator of these genes. The promotion of gata4 gene transcription, rather than posttranslational modifications of GATA-4, seems to play important roles in the mechanism of acute IH-mediated GATA-4 activation. We found acute IH-responsive factors in the heart, which bind to the proximal 100-bp region of the gata4 promoter.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-67340 and HL-72844 (to Y. J. Suzuki).


   ACKNOWLEDGMENTS
 
We thank Dr. Regina Day (Uniformed Service University of the Health Sciences) for invaluable suggestions for cloning the gata4 promoter.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. J. Suzuki, Dept. of Pharmacology, Georgetown Univ. Medical Center, NW403 Medical-Dental Bldg., 3900 Reservoir Rd. NW, Washington, DC 20057 (e-mail: ys82{at}georgetown.edu)

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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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A.-M. Park and Y. J. Suzuki
Effects of intermittent hypoxia on oxidative stress-induced myocardial damage in mice
J Appl Physiol, May 1, 2007; 102(5): 1806 - 1814.
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