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Cardiovascular Research 2005 65(1):187-194; doi:10.1016/j.cardiores.2004.09.026
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Copyright © 2004, European Society of Cardiology

Egr-1 negatively regulates expression of the sodium–calcium exchanger-1 in cardiomyocytes in vitro and in vivo

Chunlei Wanga, Stevan Dostanica, Nicolas Servanta and Lorraine E. Chalifoura,b,c,*

aLady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 3755 chemin Côte Ste Catherine, Montréal, Québec, Canada, H3T 1E2.
bDivision of Experimental Medicine, Department of Medicine, McGill University, Montreal, Canada
cThe Bank of Montréal Research Center for the Study of Heart Disease in Women, Sir Mortimer B. Davis-Jewish General Hospital, Canada

* Corresponding author. Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 3755 chemin Côte Ste Catherine, Montréal, Québec, Canada, H3T 1E2. Tel.: +1 514 340 8222x4295; fax: +1 514 340 7502. Email address: lorraine.chalifour{at}mcgill.ca

Received 11 February 2004; revised 3 September 2004; accepted 28 September 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Increased expression of the transcription factor early growth response gene-1 (Egr-1) accompanies catecholamine infusion. Catecholamine-treated, Egr-1-deficient (–/–) mice show exacerbated cardiac damage when compared to similarly treated wild-type (+/+) mice, suggesting that Egr-1 reduces heart damage. We sought to identify Egr-1-mediated cardiac sparing genes.

Methods: Microarray analyses identified increased sodium calcium exchanger-1 (NCX1) expression in catecholamine-treated –/– mice. Immunoblots assessed NCX1 expression in +/+, –/–, and transgenic mice overexpressing Egr-1 in heart and cardiac differentiated H9c2 cells harboring wild-type Egr-1 (wtEgr-1) or NAB-binding ablating mutations. Chromatin immunoprecipitation (ChIP) used anti-Egr-1 antibody coupled to amplification of purified Egr-1/associated DNA.

Results: Immunoblots revealed a two- to threefold increase in NCX1 in catecholamine-stimulated and naïve –/– versus +/+ mice. In contrast, transgenic mice overexpressing Egr-1 in heart had 30% of normal NCX1 protein. Thus, the in vivo data indicate that Egr-1 negatively controls NCX1 expression. In vitro cardiac differentiated H9c2 cells overexpressing wtEgr-1 also showed 30% NCX1 expression. However, cells overexpressing NAB-ablating Egr-1 mutations showed four- to fivefold increased NCX1 expression. NCX1 promoter DNA was specifically amplified from Egr-1/associated DNA. Thus, the in vitro results indicate that Egr-1/NAB interactions are critical for NCX1 repression at the NCX1 promoter.

Conclusions: NCX1 is responsible for calcium exit from cardiomyocytes, and continued overexpression is thought to be detrimental. We propose that one way Egr-1 action is cardiac sparing is by promoting a reduction in NCX1 expression.

KEYWORDS Adrenergic agonist; Heart failure; Gene expression; Remodelling; Sodium calcium exchanger


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
The transcription factor early growth response gene-1 (Egr-1) contains transcriptional activation (a.a. 1–218), repressor (R1; a.a. 281–314), and sequence-specific DNA binding domains (a.a. 332–419; reviewed in Ref. [1]). NGFI-A-binding proteins 1 and 2 (NAB1, NAB2) bind to Egr-1 at the R1 site to reduce transactivation. Egr-1 is increased after receptor activation, hypoxia, and mechanical stresses in many animal models of heart disease [2–6], and Egr-1 binding sites are present in such genes as ANF, {alpha}-MHC, ß-MHC, and skeletal actin [2,3]. Altered expression of these proteins is a hallmark feature of rodent hypertrophy, and their change is thought to aid contraction. The data are consistent with the idea that Egr-1 integrates stimulus/transcription coupling in cardiac remodeling and, furthermore, that the products of its target genes are responsible for the compensatory physiological alterations necessary for continued heart function.

Egr-1 –/– mice are viable and display no life-threatening phenotypes [7]; however, under catecholamine infusion, they show exaggerated gene expression and histological changes, and physiological evidence of cardiac failure [8]. Egr-1 –/– mice are also more susceptible to the cardiotoxin doxorubicin than Egr-1 replete mice [9]. Furthermore, when male and female neonate rats were injected with doxorubicin and were then swim-exercise trained as adults, we found doxorubicin-damaged and exercised female rats had more extensive cardiac damage than the similarly treated male rats and had reduced Egr-1 levels [10]. These results link low Egr-1 expression to increased cardiac damage and support the notion that Egr-1-mediated gene expression changes reduce cardiac damage.

In this report, we show that Egr-1 negatively regulates the expression of the cardiac sodium calcium exchanger-1 (NCX1) in vivo and in cardiac differentiated cells in vitro. NCX1 reduces intracellular calcium by exchanging it for extracellular sodium (reviewed in Ref. [11]). Increased NCX1 is thought to be initially beneficial; however, its high expression in end-stage failure suggests that continued expression is detrimental. We found NCX1 expression increased in Egr-1-deficient mice and decreased in transgenic mice overexpressing Egr-1 in heart when compared to wild-type mice. Using mutants of Egr-1 in in vitro studies, we found that the ability of Egr-1 to interact with its negative regulator NAB was critical for NCX-1 repression. We conclude that Egr-1, as part of its cardioprotection action, functions to reduce NCX-1 expression.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Materials
Immobilon P membrane was purchased from Millipore (Bedford, MA). NCX1 R3F1 monoclonal antibody was a gift from Dr. Ken Philipson, University of California, Los Angeles, CA. NCX1 C2C12 monoclonal antibodies (two different lots) were purchased from Affinity BioReagents (Golden, CO). These antibodies recognize primarily the 120-kD NCX1 protein at different epitopes, were raised against purified canine cardiac NCX1, and have been extensively characterized [12]. Egr-1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and recognizes the 82-kD protein. Monoclonal anti-tubulin antibodies were purchased from StressGen (Victoria, Canada) and Sigma (St. Louis, MI) and recognized a 55-kD protein. Secondary antibodies and enhanced chemiluminescent detection kits were purchased from Pierce (Rockford, IL). Oligonucleotides were prepared by the Sheldon Biotechnology Centre, McGill University (Montreal, Canada). Herculase DNA polymerase was purchased from Stratagene (La Jolla, CA). SuperScript II reverse transcriptase, random primers, and dNTPs were purchased from InVitrogen (Carlsbad, CA). H89, PD98059, and SB202190 were purchased from Calbiochem (San Diego, CA). Other chemicals were from Sigma.

2.2. Animal manipulation
Experimental studies used 4- to 6-month-old adult male wild-type (+/+) C57Bl/6 or Egr-1-deficient (–/–) mice, n=4 group. Egr-1 –/– mice have a C57Bl/6 genetic background.

The –1256 to +22 muscle creatine kinase (MCK) promoter, gift of Dr. Stephen D. Hauschka, University of Washington, Seattle, WA, was ligated to wild-type Egr-1 cDNA. MCK-Egr-1 transgenic mice were generated by the University of Toronto Transgenic and Knockout Facility, Toronto, Canada. Gene-modified mice were identified by standard means [18]. All experiments were performed according to the guidelines of the Canadian Council of Animal Care which conform to those of the National Institutes of Health.

Agonists, dissolved in vehicle (phosphate-buffered saline containing 0.5 mM ascorbic acid), were administered for 7 days via osmotic minipumps (Alza, Palo Alto, CA). Pumps delivered a mix of isoproterenol (ISO)+phenylephrine (PE; ISO+PE; 6 mg ISO+6 mg PE/kg/day), isoproterenol+phenylephrine+propranolol (P; ISO+PE+P; 6 mg ISO+6 mg PE+6 mg P/kg/day), dobutamine (DOB; 6 mg/kg/day), or metaproterenol (META; 6 mg/kg/day). Controls received vehicle-loaded minipumps. Animals, n=4 per group, were anesthetized with Avertin (0.015 ml/gm body weight, 2.5% solution in phosphate-buffered saline) and minipumps implanted dorsally. Animals were given food and water ad libitum.

At sacrifice, animals were killed by cervical dislocation, their hearts excised, rinsed in phosphate-buffered saline, blotted dry, and weighed. Heart weight to body weight ratios (HW/BW) were calculated and are expressed as mg/gm (Table 1).


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  		Table 1 Heart weight, body weight, and heart weight to body weight ratio

 
2.3. Microarray analysis
Total RNA from 4 ISO+PE-infused +/+ or –/– mice was pooled, labeled, and hybridized to Affymetrix Mu74Av2 Gene Chips (Affymetrix, Santa Clara, CA) [13]. To verify NCX1 differential expression DNA-free RNA from a second series of catecholamine-treated mice was purified and semiquantititve RT-PCR performed using either tubulin or NCX1-specific primers [9,10].

2.4. Cell culture and manipulation
H9c2 cells (ATCC CRL 1446) were cultured in DMEM plus 10% fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin under standard conditions. Plasmids containing cytomegalovirus immediate-early (CMV) promoter-driven wild-type Egr-1 (wtEgr-1) or a WT1-Egr-1 fusion protein [WT-1 5' region (a.a. 1–307) linked to the zinc finger and 3' region of Egr-1 (a.a. 337–533; WT1-Egr-1)] [14] were gifts from Dr. Frank Rauscher III, Wistar Institute of Anatomy and Cell Biology, Philadelphia, PA. CMV-driven I293F Egr-1, harbouring a mutation in the R1 region, was a gift of Dr. Jeffrey Milbrandt, Washington University School of Medicine, St. Louis, MO.

H9c2 cells were transfected with SV2Neo alone or with the CMV-Egr-1 plasmids in a 1:10 ratio using Superfect according to the manufacturer's instructions. Individual transfected cell clones were selected in 400 µg/ml G418 and maintained in 200 µg/ml G418. Three Egr-1 overexpressing cell clones from each transfection all expressing relatively equivalent Egr-1 levels were chosen for comparisons.

H9c2 cells were treated with reduced serum (1%) and daily retinoic acid (RA), 10–8 M, additions to induce cardiac differentiation [15]. Drugs were added after 3 days of differentiation.

2.5. Protein isolation and analysis
Immunoblot analyses were conducted on unfractionated homogenates to avoid the issue of different recoveries of membranes from different samples. For heart samples, the entire isolated ventricle was homogenized to completion in 1 ml of 50 mM Tris (pH 7.4), 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS, 150 mM NaCl, 1 x complete protease inhibitors (Roche, Indianapolis, IN), 1 mM Na vanadate, and 1 mM PMSF. Homogenates were clarified by centrifugation at 10,000 x g for 5 min at 4 °C and used in immunoblot assays without further manipulation. Confluent cultures in 10 cm2 dishes were homogenized in 200 µl of RIPA buffer (10 mM Na phosphate, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS, 1 mM Na vanadate, 1 x complete protease inhibitors, and 1 mM PMSF) to create a whole cell lysate. Protein concentrations were determined using the BioRad Protein Determination Assay (BioRad Hercules, CA).

Measurement of Egr-1, NCX1, and tubulin protein expression was performed by semiquantitative immunoblots using standard methods. For immunoblots, 40 µg protein from whole cell lysates or 100 µg protein from heart homogenates was electrophoresed through SDS-PAGE and electrophoretically transferred to Immobilon P membranes. Membranes were stained with Ponceau S to confirm equivalent protein loading and transfer. Individual membranes were placed in plastic bags, solutions added, and the bag edges heat sealed (Philips Food Saver). Egr-1 or NCX1 antibodies were diluted 1:200, antitubulin antibody was diluted 1:2000, and horseradish-peroxidase-coupled secondary antibodies diluted 1:20,000. Each series of immunoblots was repeated at least three times. For semiquantification of immunoblots, the linearity of the signal as a function of the amount of protein loaded was verified by testing several amounts of protein (range 20–100 µg). The densitometry was linear with a regression coefficient of 0.92.

2.6. Chromatin immoprecipitation (ChIP)
Cardiac differentiated H9c2 cells were washed twice and 2 ml/10 cm2 dish of serum-free media containing 1% formaldehyde added for 30 min. Fixed cells were collected by brief centrifugation, lysed, and sonicated. ChIP used Egr-1 antibody or no antibody control according Upstate Biotechnology (Lake Placid, NY) protocol. Egr-1/DNA formaldehyde cross-linkages were reversed by heating at 65 °C for 4 h, DNA purified by phenol/chloroform, chloroform and ethanol precipitation then resuspended in 100 µl TE (10 mM Tris, pH 7.8, 1 mM EDTA). PCR was performed using DNA isolated prior to antibody incubation (input DNA) or after immunoprecipitation (IP DNA). NCX1 promoter-specific primers sequences were forward 5'-GGAGCACCCTCGTGGAAGCAG-3' and reverse 5'-CCTTCCAGCCAGCCCATTTCCC-3'. The NCX1 exon 2 coding sequence primers were forward 5'-CCAATGTTTCAATGGGATTTCG-3' and reverse 5'-CGGTGAAGTTATGGCCACACAC-3'. Tubulin forward primer was 5'-TCCATCCACGTCGGCCAGGCT-3' and reverse 5'-CTAGGGCTCAACCACAGCAGT-3. Amplification used Herculase Enhanced DNA polymerase and 31 cycles. Positive control reactions contained mouse genomic DNA, and negative controls contained all reagents except DNA.

2.7. Statistical analysis
X-ray films from several exposures were scanned using an HP ScanJet 5100 C and HP Precision Scan Software (Hewlett-Packard, Palo Alto, CA, USA). The areas under the peaks were quantified using ScionImage Release Beta 3 software (National Institutes of Health, Bethesda, MD, USA). Values are expressed as the mean ± the standard deviation. Comparisons were made using ANOVA followed by Dunnett's modified t-test. A P-value <0.05 was considered significant. NCX1 or Egr-1 signal was standardized to the signal of tubulin from the same blot for each individual sample.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Identification of discordantly expressed RNAs
We sought to identify RNAs differentially expressed in catecholamine-stimulated wild-type (+/+) versus Egr-1-deficient (–/–) hearts. We treated +/+ and –/– male mice (n=4) with ISO+PE for 7 days. The HW/BW (milligram wet heart weight per gram body weight) of ISO+PE-treated –/– and +/+ mice (6.78 ± 0.22 and 6.5 ± 0.4, respectively, P>0.05) was similar (Table 1). Ventricular RNA was purified, and equal amounts of RNA from the four mice in each cohort pooled, and mouse Affymetrix Mu74Av2 Gene Chips probed. NCX1 mRNA was 2.15-fold overexpressed in the catecholamine-infused –/– mice versus +/+ mice. A second set of +/+ and –/– mice (n=3 per genotype) were infused with ISO+PE and ventricle RNA isolated. Semiquantitative RT-PCR amplification using NCX1-specific compared to tubulin-specific primers confirmed a twofold (2 ± 0.3 vs. 1 ± 0.25, P<0.05) increase in NCX1 RNA in the –/– mice (Fig. 1A).


Figure 1
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  		Fig. 1 NCX1 and tubulin expression in naïve or catecholamine-treated +/+ and –/– mice. Male mice, n=4, were implanted with osmotic minipumps loaded with either vehicle (–), isoproterenol plus phenylephrine (ISO+PE), isoprotenol plus phenylephrine plus propranolol (ISO+PE+P), dobutamine (DOB), or metaproterenol (META). Ventricles from naïve mice or mice 7 days postpump implantation were separated and RNA or protein prepared. DNA-free RNA was isolated from ISO+PE- stimulated mice and RT-PCR performed using NCX1- or tubulin-specific primers. Equal portions of the PCR reactions were separated by agarose electrophoresis, transferred to membrane, and probed with authentic radioactive NCX1 or tubulin DNA. Positive control (+c) containing mouse genomic DNA and negative control (-c) containing all components except DNA were amplified concurrent with test samples. Protein, 100 µg, was separated on SDS-PAGE, transferred to Immobilon P, and stained with Ponceau S to confirm equal protein loading and transfer. Blots were incubated with antisera to NCX1 or tubulin as indicated on the left with the arrows on the right indicating the migration of the 120-kD NCX1 and 55-kD tubulin. Tubulin expression is shown to confirm relatively equal loading of protein in each lane. Representative blots are shown. (A) Comparison of RNA and protein expression after ISO+PE infusion with genotype; (B) comparison of protein expression in naïve mice; (C) comparison of protein expression in catecholamine infused mice.

 
3.2. NCX1 protein is overexpressed in catecholamine-infused –/– versus +/+ mice
We found NCX1 protein increased 2.5-fold (2.5 ± 0.9 vs. 1 ± 0.4, n=4, P<0.05) in ISO+PE-infused –/– mice compared to ISO+PE-infused +/+ mice (Fig. 1). To determine the level of NCX1 in unstimulated hearts, we measured NCX1 expression in naïve –/– and +/+ mice (Fig. 1B). We found NCX1 expression was increased 2.4-fold (2.4 ± 0.7 vs. 1 ± 0.3, n=4, P<0.05) in naïve –/– versus +/+ mice.

ISO is a nonspecific β-adrenergic agonist activating both β1-AR and β2-AR. To determine the specificity of the β-AR response, we treated –/– and +/+ mice with minipumps loaded with ISO+PE plus the nonspecific β-blocker, propranolol (P). The HW/BW of ISO+PE+P-treated mice was similar to that of mice harboring vehicle loaded pumps (5.2 ± 0.28 and 5.0 ± 0.22, respectively, P>0.05; Table 1). This result is in agreement with a previous study where we showed minimal increases in HW/BW in response to PE alone [5]. Propranolol addition reduced NCX1 in both genotypes to levels similar to that present in vehicle-treated mice (Fig. 1C). This suggests that increased NCX1 expression is primarily a β-AR response. To determine the specificity of the β-AR response, we included dobutamine, a β1-AR-specific agonist or metaproterenol, a β2-AR-specific agonist in the minipumps. We found increased NCX1 protein in –/– versus +/+ mice regardless of catecholamine (Fig. 1C). Thus, the increase in NCX1 protein with catecholamines is a nonspecific β-AR response.

3.3. Egr-1 overexpression in heart reduced NCX1 expression in vivo
The data suggest that Egr-1 negatively regulates NCX1 expression. By corollary, transgenic mice that overexpress Egr-1 in heart should express decreased NCX1. We isolated transgenic mice using the muscle creatine kinase (MCK) promoter to drive Egr-1 overexpression in heart. Transgenic MCK-Egr-1 mouse line C displayed a physiological increase (3.2 ± 1- vs. 1 ± 0.4-fold, n=5, P<0.05) in Egr-1 protein in heart and not in smooth or skeletal muscle (Fig. 2A and B). MCK-Egr-1 transgenic mice have 34 ± 12%, P<0.05, the NCX1 expression compared to nontransgenic littermates (1 ± 0.3; Fig. 2A). The in vivo data suggest that Egr-1-mediates a reduction in NCX1 transcription and reduced NCX1 protein.


Figure 2
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  		Fig. 2 NCX1 expression in MCK-Egr-1 transgenic mice. Male nontransgenic (NT) or transgenic (T) mice (n=4) were sacrificed at 4 months of age, and protein homogenates prepared from cardiac ventricle, stomach, or skeletal muscle. Protein, 100 µg, was separated on SDS-PAGE, transferred to Immobilon P, and stained with Ponceau S to confirm equal protein loading and transfer. Blots were incubated with antisera to Egr-1, NCX1, or tubulin as indicated on the left with the arrows on the right indicating the 82-kD Egr-1, 120-kD NCX1, and 55-kD tubulin protein migration. Tubulin expression is shown to confirm relatively equal loading of protein at each lane. Representative blots are shown from three independent experiments. (A) Egr-1 and NCX1 expression in nontransgenic and transgenic heart; (B) Egr-1 expression in noncardiac muscled from nontransgenic and transgenic mice.

 
3.4. Egr-1 is increased with catecholamine activation via a p38 MAP kinase pathway in differentiated H9c2 cells
H9c2 cells will arrest proliferation and differentiate to a cardiac phenotype when cultured in media with reduced serum and daily 10–8 M retinoic acid (RA) additions [15]. We treated cardiac differentiated H9c2 cells with saline, ISO (2 µM), or ISO plus inhibitors and measured Egr-1 by immunoblots (Fig. 3). We found increased (2.8 ± 0.9- vs. 1 ± 0.5-fold, n=3, P<0.05) Egr-1 after 4 h of ISO stimulation that was reduced to control levels by the β-AR blocker propranolol, the PKA inhibitor H89, or the p38 MAP kinase inhibitor SB202190, but not by the MAP kinase kinase inhibitor PD98059 (Fig. 3). The data indicate that Egr-1 protein is increased with β-AR activation and suggest that stimulation occurs via the β-AR-PKA-p38MAP kinase pathway.


Figure 3
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  		Fig. 3 Egr-1 expression cardiac differentiated H9c2 cells in vitro. After 3 days in the differentiation media, vehicle (c) or ISO (2 µM) was added with lysis in RIPA buffer 6 h later. Propranolol (P, 2 µM), H89 (1 µM), PD98059 (PD, 5 µM), or SB202190 (SB, 5 µM) were added 30 min prior to ISO. Protein, 40 µg, was separated by SDS-PAGE and transferred to Immobilon P. Blots were incubated with anti-Egr-1 or tubulin as indicated on the left with arrows on the right indicating the migration of the 82-kD Egr-1 and 55-kD tubulin protein. Representative blots from three independent experiments are shown.

 
3.5. Egr-1 protein is located on the NCX1 promoter
To determine if Egr-1 is bound to the NCX1 promoter, we performed chromatin immunoprecipitation using anti-Egr-1 antibodies to collect Egr-1/DNA complexes from cardiac differentiated H9c2 cells (Fig. 4). We established that the Egr-1 antibody recognized formaldehyde fixed and sonicated Egr-1/DNA complexes and that input DNA was similar in all samples (Fig. 4A, B, and C). DNA associated with the IP reactions was purified. We found NCX1 promoter DNA was amplified in H9c2 samples incubated with anti-Egr-1 antibody, but not in no-antibody control reactions (Fig. 4D). NCX1 Br2/1d promoter and exon 2 are separated by >50 kbp [16]. No amplification was detected in any sample when exon 2-specific primers were used. The data suggest Egr-1 protein binds to NCX1 promoter DNA.


Figure 4
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  		Fig. 4 Chromatin immunoprecipitation using anti-Egr-1 antibodies. Cardiac differentiated H9c2 cells were prepared and the chromatin fixed. (A) Protein homogenates were incubated with no-antibody, 2 or 5 µl anti-Egr-1 antibody as indicated, the Egr-1/antibody complex collected by protein A-Sepharose and solubilized. Equal aliquots were electrophoresed through SDS-PAGE and immunoblotted with anti-Egr-1 sera. (B) Chromatin from fixed H9c2 cells was sonicated and DNA, 1 µl (1) or 2 µl (2), electrophoresed through 1% agarose gels then stained with ethidium bromide. DNA size markers are indicated on the left. Two preparations are shown. (C) A portion of B was kept to measure input DNA prior to antibody incubation. Input DNA was purified and resuspended in TE. Equal volumes, 1 µl (1) or 2 µl (2), were amplified from two different preparations using tubulin-specific primers. The negative control contains all reagents except DNA. (D) Equal portions of B were incubated with no-antibody (–) or 5 µl anti-Egr-1 (+) antibody and the DNA associated with Egr-1 isolated and purified. Purified DNA, 1, 2, or 3 µl, was amplified using either NCX1 promoter-specific or exon 2-specific primers as indicated. Negative controls included all reagents except DNA. Positive controls included all reagents plus purified mouse DNA. DNA size markers are included on the left. The arrow points to the amplified NCX1 DNA.

 
3.6. Egr-1/NAB protein/protein binding represses NCX1 expression
NAB proteins bind Egr-1 and reduce transactivation [17,18]. We transfected H9c2 cells with CMV-driven wild-type Egr-1 (wtEgr-1) or the NAB binding–ablating Egr-1 mutants, I293F, or WT1-Egr-1 DNA [27]. We selected three cell clones with similar levels of Egr-1 expression from each transfection for analysis. The results were consistent within each group, hence results from a single clone is shown (Fig. 5A). Cells overexpressing wtEgr-1 expressed 26% ± 13, P<0.05, the NCX1 protein present in controls (Fig. 5B). In contrast, cells expressing I293F or WT1-Egr-1 protein had fourfold (4.0 ± 1.2) and threefold (3.1 ± 0.9) elevated NCX1 expression, respectively, compared to that detected in H9c2 cells (1 ± 0.4). Thus, cells overexpressing Egr-1 had decreased NCX1, whereas cells expressing NAB-ablating mutations showed increased NCX1.


Figure 5
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  		Fig. 5 NCX1 expression in cardiac differentiated H9c2 cells overexpressing wild-type or mutant forms of Egr-1. Individual H9c2 clones overexpressing CMV-driven wild-type, I293F or WT1-Egr-1 forms of Egr-1 were selected with G418, differentiated and lysates prepared. Protein, 40 µg, was separated through SDS-PAGE and transferred to Immobilon P. Blots were incubated with anti-Egr-1, NCX1, or tubulin antisera as indicated on the left with the arrow on the right indicating the migration of the 82-kD Egr-1, 120-kD NCX1, and 55-kD tubulin proteins. Representative blots from three independent experiments are shown. SV2Neo (H9), CMV-I293F (I293F), CMV-WT1-Egr-1 (WT1-Egr-1), or CMV-wild-type Egr-1 (wtEgr-1). (A) Comparison of Egr-1 expression in cardiac differentiated H9c2 clones; (B) comparison of NCX1 expression in cardiac differentiated H9c2 clones.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Ca2+ regulation plays a central role in cardiac performance (reviewed in Refs. [19,20]) with altered calcium handling present in cardiac muscle undergoing prolonged catecholamine stimulation. Ca2+ entering cardiomyocytes to activate contraction must be removed to permit relaxation. NCX1 is a Na+ and Ca2+ exchanger moving three extracellular Na+ ions into the cell for every intracellular Ca2+ ion transferred to the cell exterior (forward mode). NCX1 might also increase Ca2+ influx (reverse mode) to load the SR with Ca2+ and directly activate contraction [21], although the physiological significance of this action is controversial. Changes in NCX1 expression and thus Ca2+ extrusion contribute to contractile dysfunction, alter the action potential, and play a role arrhythmogenesis. Therefore, mechanisms to reduce NCX1 in end-stage disease might be cardiac sparing, and NCX1 is proposed as a target for drug development [22]. Our in vivo results using Egr-1 knockout and Egr-1 transgenic mice and in vitro results using cardiac differentiated H9c2 cells show that NCX1 is reduced when Egr-1 is increased. Together, the results indicate that Egr-1 is a negative regulator of NCX1 expression.

NCX1 was increased in –/– compared to +/+ mice. Phospholamban, ryanodine receptor-2, or sarcoplasmic reticulum calcium ATPase (SERCA) mRNAs were not significantly different in the microarray analyses. This result is similar to studies showing no increase in SERCA, calsequestrin, or phospholamban in NCX1 overexpressing transgenic mice [23,24]. The sodium–potassium ATPase (Na,K-ATPase) removes three Na+ ions from the cytosol and introduces two K+ ions from the extracellular space [25]. Primary decreases in Na,K-ATPase are associated with secondary increases in NCX1 activity as a homeostatic mechanism to maintain Na balance [25,26]. However, no change in Na,K-ATPase expression is found in NCX1 transgenic mice [25]. Thus, whereas increased Na,K-ATPase activity drives a decrease in NCX1, an increase in NCX1 does not decrease Na,K-ATPase. It appears that NCX1 expression can be increased without concomitant changes in other calcium- or sodium-regulating proteins.

NCX1 is increased in most human and animal cardiomyopathic models and especially in end-stage failure models [20,27–29]. Increased NCX1 activity is likely beneficial initially; however, its high expression in end-stage failure suggests that continued expression is detrimental. Egr-1 –/– male and female mice, with a roughly two-fold increase in NCX1 protein, have a normal life span, but have a reduced capacity to withstand catecholamine or doxorubicin damage to the heart [5,9,10]. Similarly, male {alpha}-MHC directed NCX1 overexpressing transgenic mice with two-fold increase in NCX1 protein have an increased susceptibility to ischemia/reperfusion injury and spontaneous death [30,31]. Female homozygous NCX1 transgenic mice show increased postpartum mortality from heart failure. Female Egr-1 –/– mice are infertile, hence it is unclear if the effect of the increase NCX1 expression during pregnancy and postpartum would be recapitulated. Targeted disruption of NCX1 caused an embryonic lethality in the homozygous mice, whereas heterozygous mice are viable [32,33]. The MCK-Egr-1 transgenic mice show no increase in mortality and have normal litter sizes with predicted numbers of transgene positive pups. This suggests that the reduced level of cardiac NCX1 in these mice is not overtly harmful. The accumulated data indicate that NCX1 expression must remain within a relatively narrow window in either basal or stimulated conditions.

The NCX gene, >80 kb, uses tissue-specific splicing to link noncoding exon 1d to the first coding exon, exon 2 in heart [16]. Amplification of NCX1 promoter DNA in ChIP assays using anti-Egr-1 antibody suggests Egr-1 occupancy of NCX1 promoter DNA. Egr-1 can bind to the SP1 GC-box recognition sequences particularly in promoters, such as NCX1, that contain multiple SP1 sites [34,35]. We conclude that Egr-1 negatively regulates NCX1 expression by binding to these overlapping SP1 sites. NAB binding, but not DNA binding, is ablated in the I293F point mutation and the WT1-Egr-1 fusion proteins [36,37]. Cells expressing these proteins showed increased NCX1 expression, suggesting that NAB binding is crucial for Egr-1-mediated NCX1 repression. This leads to the concept that Egr-1/NAB binding alters the ability and/or affinity of Egr-1 for other proteins at the promoter, and that it is the interaction with these ancillary proteins that is responsible for the negative regulation. Egr-1 itself positively controls NAB expression, and NAB is increased by stimuli that also increase Egr-1 expression. Our data are consistent with the accumulated data indicating that the main function of NAB is to limit Egr-1 transactivation [38,39]. In other work, we found increased NAB expression with catecholamine infusion [9] leading credence to the notion that NAB and Egr-1 interactions are important to the regulation of gene expression in catecholamine-stimulated hearts. Based on our in vitro and in vivo results, we hypothesize that Egr-1 reduces NCX1 expression and that this reduction is a part of its cardiac sparing mechanism.


   Acknowledgements
 
We thank Linda Wei of the University of Toronto Transgenic and Knockout Facility for generation of the MCK-Egr-1 transgenic mice, and Rob Sladek and Thomas J. Hudson of the Montreal Genome Centre for the microarray analysis. This study was supported by grants from the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Quebec, and The Bank of Montréal Research Center for the Study of Heart Disease in Women to LEC.


   Notes
 
Time for primary review 26 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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