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Endocrinology Vol. 148, No. 4 1502-1510
Copyright © 2007 by The Endocrine Society

Early Aldosterone-Regulated Genes in Cardiomyocytes: Clues to Cardiac Remodeling?

Géza Fejes-Tóth and Anikó Náray-Fejes-Tóth

Dartmouth Medical School, Department of Physiology, Lebanon, New Hampshire 03756

Address all correspondence and requests for reprints to: Géza Fejes-Tóth, Department of Physiology, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, New Hampshire 03756-0001. E-mail: geza.fejes-toth{at}dartmouth.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent clinical studies demonstrated beneficial effects of mineralocorticoid receptor (MR) antagonists in patients with heart failure and other cardiovascular diseases. However, the underlying molecular mechanisms are poorly understood, and the genes that mediate direct effects of aldosterone in the cardiovascular system are yet to be identified. The goal of this study was to identify genes that are directly regulated by aldosterone in cardiomyocytes and thus potentially play a role in initiating MR-mediated effects in the heart. We generated clonal cell lines of cardiomyocytes (H9C2 cells) stably expressing the MR. Using these cell lines and Affymetrix microarrays, we determined the effects of physiological concentrations of aldosterone on the gene expression profile. In two independent microarrays we identified 48 genes that were induced more than 1.5-fold (27 known genes and 21 expressed sequence tags) and five (three known genes and two expressed sequence tags) that were suppressed by a 2-h aldosterone treatment. We focused on eight genes that have a potential function in cardiovascular regulation and verified their aldosterone regulation using quantitative RT-PCR. These include genes related to extracellular matrix regulation (tenascin-X, ADAMTS1, PAI-1, UPAR, and hyaluronic acid synthase-2), signaling, and regulation of vascular tone (RGS2, adrenomedullin) and inflammation (orosomucoid). Protein synthesis inhibitors did not prevent aldosterone induction of these genes. We conclude that in cardiomyocytes aldosterone rapidly and directly regulates the expression of several genes that are involved in cardiac remodeling and regulation of blood pressure and thus might be mediators of the physiological and pathophysiological effects of aldosterone on the cardiovascular system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ADVERSE CARDIOVASCULAR effects of exogenously administered mineralocorticoids, in the presence of high-salt diet, were first described more than 50 yr ago (1). More recent studies, in both human and experimental animals, indicate that inappropriate aldosterone for salt status leads to vascular injury and cardiac fibrosis as well as endothelial and baroreceptor dysfunctions (reviewed in Refs. 2, 3, 4). These observations prompted a renewed interest in the use of mineralocorticoid receptor blockers in the treatment of cardiovascular diseases. Two large clinical trials, Randomized Aldactone Evaluation Study (RALES) and Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS), demonstrated that the mineralocorticoid receptor (MR) blockers spironolactone and eplerenone significantly reduced morbidity and mortality in patients with heart failure (5, 6). The site of these beneficial actions and the underlying cellular and molecular mechanisms remain unknown. Several lines of evidence suggest a direct effect of aldosterone/MR on both cardiac and vascular functions (2, 3, 4).

A role for myocardial MRs in cardiac function is also underscored by the development of heart failure and cardiac fibrosis when the expression of MRs is down-regulated (7) and the susceptibility to arrhythmias with their overexpression (8). Similarly, heterologous expression of 11ß-hydroxysteroid dehydrogenase-2 in the myocardium of transgenic mice, which allows aldosterone to occupy cardiac MRs, also leads to dilated cardiomyopathy (9).

The genes that mediate either physiological or pathological effects of aldosterone in the cardiovascular system have not yet been identified, although several genes have been found to be modulated in the heart after long-term treatment (weeks) with mineralocorticoids and high salt (10, 11, 12). Whether aldosterone regulates these genes directly or secondarily via changes in blood pressure or by affecting the cellular composition of the heart is not known. To circumvent these limitations, several studies examined the effects of aldosterone added directly to cells in culture. The expression of a number of genes with possible involvement in mineralocorticoid-induced pathology was found to be altered by aldosterone [inducible nitric oxide synthase (13)], plasminogen activator inhibitor (PAI)-1 (14), nicotinamide adenine dinucleotide phosphate reduced oxidase (10), Na-K ATPase (15), sodium hydrogen exchanger-1 (NHE1) (16), cyclooxygenase-2, IL-6 (17), atrial natriuretic factor, and {alpha}-myosin heavy chain (18) and osteopontin (19). However, in most studies significant changes were seen only at supraphysiological concentrations of aldosterone (micromoles) that also activate the glucocorticoid receptors, suggesting that the bulk of these effects were probably not mediated via the MR.

To begin to identify genes that are directly regulated by the MR, we generated a cardiomyocyte cell culture model that stably expresses MRs and exhibits robust transcriptional responses to physiological concentrations of aldosterone. Using this system, we report here the identification of several genes whose role in cardiovascular function had been established but their regulation by aldosterone was previously unknown.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of H9C2 cell lines stably expressing the MR
A full-length rat MR was subcloned into the HindIII and Bsp1201 sites of the retroviral vector pLNCX. As a control, we used the empty vector pLNCX. Retroviri were generated by transient transfection of these constructs into amphotropic Phoenix cells (American Type Culture Collection, Manassas, VA), grown in DMEM/F12 medium (1:1) supplemented with 10% heat-inactivated fetal bovine serum plus an antibiotic mixture (75 µg/ml penicillin, 100 µg/ml streptomycin, and 12.5 µg/ml tylosin), using Lipofectamin PLUS reagent (Invitrogen, Carlsbad, CA). H9C2 cells were cultured in the same medium to approximately 40% of confluence and infected with retroviri as described previously (20). Individual clones were selected with the neomycin analog G418 (500 µg/ml). These cells are referred to as H9C2/MR+ cells.

Determination of 3H-aldosterone binding
Four individual H9C2/MR+ clones were expanded and tested for 3H-aldosterone binding along with the control cell line expressing the empty retroviral vector. In pilot experiments Scatchard analysis of saturable binding was performed using various concentrations of [1,2-3H]aldosterone (specific activity 36 Ci/mmol; Amersham, GE Healthcare Bio-Sciences Corp., Piscataway, NJ). Cells (500,000 cells/sample) were incubated at 37 C with 0.05–4 nM 3H-aldosterone. To determine nonsaturable binding, aliquots of cells were incubated with a 1000-fold excess of unlabeled aldosterone. To determine a number of MR/cell, parent H9C2 cells and H9C2-MR+ clones were incubated with 2 nM 3H-aldosterone (a concentration that was found to be saturating the MR) in the presence (nonspecific binding) or absence (total binding) of a 1000-fold excess of unlabeled aldosterone. Specific binding of aldosterone was defined as the difference between the radioactivity measured in samples with and without unlabeled aldosterone and was normalized for cell number. Specific binding of 3H-aldosterone was also tested in the presence of 100-fold excess of RU486, a glucocorticoid receptor antagonist.

Western blotting
H9C2/MR+ cells were grown in DMEM/F12 medium with 10% fetal bovine serum in monolayers. Western blot analysis was performed on protein samples from parent H9C2 cells and from H9C2/MR+ cells using a mouse monoclonal antibody against the N-terminal 1–18 amino acid of the rat MR, kindly provided by Dr. Celso Gomez-Sanchez [University of Mississippi, Jackson, MS; (21)]. Protein samples (10 µg) were electrophoresed in 10% SDS-PAGE gels and transferred to Immobilon-P membranes (Millipore, Billerica, MA). Ponceau S dye (Sigma, St. Louis, MO) was used to confirm equal loading of the protein. The membranes were then blocked in SuperBlock blocking buffer (Pierce Chemical, Rockford, IL) containing 0.5% Tween 20 or 5% dry milk in TBST [10 mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween 20, 124 µM thimerosal] for 1 h at room temperature and then incubated with the primary antibody diluted 1:1000 in blocking reagent for 2 h at room temperature. After a series of washes with TBST, the membranes were incubated for 1–2 h at room temperature with an antimouse horseradish peroxidase-linked antibody (Zymed Laboratories, Inc., San Francisco, CA) diluted to 1:5000. Finally, after a series of washes with TBST, blots were incubated in ECL chemiluminescence substrate (Amersham Pharmacia Biotech, Piscataway, NJ). The membranes were imaged using a ChemiImager 5500 (Alpha Innotech, San Leandro, CA).

DNA microarray analysis
Cells, grown on steroid-free medium for 2 d, were treated with 1 nM aldosterone or vehicle for 2 h. Total RNA was isolated using TRI-Reagent (Molecular Research Center, Cincinnati, OH), according to the manufacturer’s protocol, DNase treated, and further purified using the RNeasy minikit (QIAGEN, Valencia, CA). Integrity of RNA was determined using formaldehyde agarose gel electrophoresis. RNA samples from three separate experiments receiving the same treatment were pooled. Gene expression profiles were determined using Affymetrix Rat 230 2.0 expression arrays (Affymetrix, Santa Clara, CA). Preparation of the labeled cDNA and microarray hybridization were performed by the Microarray Core Facility at Dartmouth. The samples of total RNA were processed following standard one-cycle eukaryotic target preparation protocol from Affymetrix. Briefly, total RNA was first reverse transcribed using T7-oligo(dT) promoter primer in the first-strand cDNA synthesis reaction. After RNase H-mediated, second-strand cDNA synthesis, the double-stranded cDNA was purified and served as a template in the subsequent in vitro transcription reaction. The in vitro transcription reaction was carried out in the presence of T7 RNA polymerase and a biotinylated nucleotide analog/ribonucleotide mix for cRNA amplification and biotin labeling. Fifteen micrograms of biotinylated cRNA targets were then cleaned up, fragmented, and hybridized to GeneChip (Affymetrix) array during the overnight incubation at 45 C. After hybridization the arrays were stained with streptavidin-phycoerythrin in the GeneChip Fluidics station. After staining the arrays were scanned using Affymetrix GeneChip scanner with the laser filter set at 570 nm, pixel size 2.5 µm. The array image data were acquired, and the fluorescent signal intensities were quantified using Affymetrix GCOS version 1.2 software with the following settings of quantitation parameters: alpha 1 = 0.05, alpha 2 = 0.065, tau = 0.015, gamma 1H = 0.0045, gamma 1L = 0.0045, gamma 2H = 0.006, gamma 2L = 0.006, perturbation = 1.1, target intensity = 150. The generated cell intensity files (*.cel) and analysis output files (*.chp) were then imported into the GeneTraffic-Uno software (Iobion Inc., La Jolla, CA) for further analysis.

To determine differentially expressed genes in H9C2/MR+ cells after aldosterone treatment, raw data were first analyzed using Affymetrix Microarray Suite 5.0 software. The signal values and the present, absent, or marginal calls were computed for all probe sets.

Quantitative RT PCR
H9C2/MR+ cells were maintained in steroid-free medium for 48 h, treated with 1 nM aldosterone for 30 min to 24 h, and then lysed in TRI reagent. Total RNA was prepared and cDNA was synthesized using 2 µg total RNA as described (22). Sense and antisense PCR primers for were selected based on the published rat sequences. The sequences of the primers used to detect specific genes are available on request.

For the determination of the expression levels of tenascin-X (TNX), a disintegrin, and metalloprotease with thrombospondin motifs (ADAMTS1), and orosomucoid-1 (Orm-1) mRNAs, we used real-time quantitative PCR. PCRs were performed using iTaq SYBR Green supermix with Rox (Bio-Rad, Hercules, CA). The reactions were performed in triplicate, according to the manufacturer’s protocol with 200 nM of each sense and antisense primer and 30–40 ng of cDNA in a final reaction volume of 25 µl. The thermal cycling parameters were: initial denaturation at 95 C for 3 min, followed by 40 cycles at 95 C for 15 sec and 57 C for 30 sec. The PCR products and threshold cycle were determined and analyzed using AB 7300 (Applied Biosystems, Foster City, CA). Changes between the untreated and hormone-treated mRNA levels were based on the number of cycles at which the amplified product reached threshold (Ct). Relative expression of the mRNA was quantified using the equation: relative expression = 2-(Ct gene of interest – Ct ß-actin). To verify that the real-time PCR products were not contaminated from SYBR green binding to nonspecific double stranded DNA, a dissociation stage was performed: 95 C for 15 sec, 57 C for 30 sec, and 95 C for 15 sec.

To determine the relative abundance of the regulator of G protein signaling-2 (RGS2), adrenomedullin (AM), PAI-1, the urokinase plasminogen activator receptor (UPAR), and hyaluronic acid synthase-2 (HAS2) mRNAs in control and aldosterone-treated H9C2-MR+ cells, we used quantitative RT-PCR methods as described earlier (22). PCRs were performed under standard conditions with four different amounts (20, 5, 1.25, and 0.3125 ng) of cDNA originating from control or aldosterone-treated cells. The sequences of the specific primers and specific PCR amplification conditions are available on request. ß-Actin mRNA in each sample was determined using primers and conditions as described (22). cDNA samples derived from control and aldosterone-treated cells were always amplified simultaneously. The PCR products were separated on a 5% polyacrylamide gel and quantitated by densitometry using a FluorImager 575 (Molecular Dynamics, Sunnyvale, CA). The slope of the amount of PCR products vs. amount of template cDNA was derived by linear regression. These values were normalized for the amount of ß-actin mRNA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of cardiomyocyte cell lines stably expressing MR
The H9C2 cell line originates from rat neonatal cardiomyocytes. Although cardiomyocytes in vivo express MRs, cell lines originating from MR-expressing tissues typically lose functional receptors; therefore, we first tested for expression of MR in these cells by determining 3H-aldosterone binding. These experiments revealed that parent H9C2 cells exhibit very low levels of 3H-aldosterone binding (at 2 nM of 3H-aldosterone, which saturates the MRs), and the binding was not specific for the MR because it could be displaced by a 100-fold excess of the glucocorticoid receptor antagonist, RU 486 (Fig. 1Go). Therefore, to facilitate a comprehensive study of corticosteroid-regulated gene expression, we stably expressed the rat MR in H9C2 cells using a retroviral technique. We generated several clonal lines stably expressing MRs. The level of MR expression in these clonal cell lines ranged between 8,000 and 72,000 MR/cell. For further studies, we chose two clonal cell lines with different levels of expression, clones 2 and 3. We confirmed the expression of MR in these clones and the lack of it in H9C2 parent cells using an antibody specific for the MR (21). Western blot analysis reveled that whereas the parent H9C2 cells had no detectable level of MR proteins, clones 2 and 3 expressed a strong immunoreactive band, whereas clone 4 expressed a much weaker band, with the appropriate size of the MR (Fig. 1Go, insert). In addition, using the same anti-MR antibody and immunocytochemistry, we observed homogenous expression of the MR in both cell lines (data not shown).


Figure 1
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  		FIG. 1. Aldosterone (aldo)-binding in parent H9C2 cells and three independent clones stably expressing MRs. Cells (500,000/sample) were incubated with 2 nM 3H-aldosterone with (specific binding) or without (total binding) a 1000-fold excess of unlabeled aldosterone at 37 C for 60 min. To prevent binding of labeled aldosterone to glucocorticoid receptors, a separate groups of cells was also incubated with 0.2 µM RU486. The insert shows Western blot analysis of stably expressed MRs in H9C2/MR+ cells. Protein samples from parent and three independent clonal lines of H9C2/MR+ cells have been separated and blotted with an antibody against the MR as described in detail in Materials and Methods. Lane 1, Parent H9C2 cells; lanes 2–4, clonal H9C2/MR+ lines 2–4, respectively. A strong immunoreactive band with the expected size was observed in clones 2 and 3, whereas clone 4 expressed MR at low levels, and in the parent cells, MR expression was undetectable.

 
To test whether the expressed MRs are transcriptionally active, we determined the effect of 1 nM aldosterone on the induction of serum- and glucocorticoid-induced kinase-1 (SGK1), a well-known early aldosterone-regulated gene in other cell types (23, 24). The levels of SGK1 mRNA were significantly higher after aldosterone treatment in all clonal cell lines tested but not parent cells (Fig. 2Go), indicating that the stably expressed MRs are indeed functional in H9C2/MR+ cells. To demonstrate that SGK1 can be induced in cardiomyocytes by steroids if the appropriate receptor is expressed, we tested the effect of activating glucocorticoid receptors by dexamethasone, which indeed resulted in the expected increase in SGK1 mRNA levels (Fig. 2Go). Currently some of the H9C2/MR+ cell lines are at passage number greater than 25 and show continued stable MR expression.


Figure 2
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  		FIG. 2. Effect of aldosterone on SGK1 mRNA expression in H9C2 cell lines stably expressing MR or the empty vector pLNCX. One nanomole aldosterone significantly induced SGK1 expression in H9C2/MR+ clones 2 and 3 but not H9C2 cells expressing the empty vector (n = 3 for each cell type). Cells were grown for 48 h in steroid-free medium, and then medium was changed to the same (control) or supplemented with 1 nM aldosterone (Aldo) for 2 h. As a control, we induced SGK1 in H9C2/vector cells by 10 nM dexamethasone (Dex). SGK1 mRNA values were normalized for ß-actin mRNA; values shown are relative values vs. control (untreated cells). *, P < 0.05 vs. control (no steroid), using one-tailed unpaired t test.

 
Identification of aldosterone-regulated genes in H9C2/MR cells by microarray analysis
To identify early, aldosterone-regulated genes in cardiomyocytes, two independent clonal cell lines of H9C2/MR+ cells were treated with a physiological concentration (1 nM) of aldosterone or vehicle for 2 h, and the gene expression profiles were determined by microarray analyses. The complete list of aldosterone-regulated genes identified in two independent experiments is shown in supplementary Table 1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://endo.endojournals.org. We listed only those genes that were regulated in both microarray analyses by aldosterone. These include 12 known genes and three expressed sequence tags (ESTs) that were up-regulated more than 2-fold and 27 known genes and 21 ESTs that were induced more than 1.5-fold. These aldosterone-induced genes included ones that have been previously shown to be enhanced by aldosterone in other cell types, such as SGK1 (23, 24) and glucocorticoid-induced leucine zipper (GILZ) (25), supporting the validity of our microarray analysis. The number of genes suppressed by aldosterone was much smaller: three known genes and two ESTs.

For RT-PCR verification and further studies, we selected three groups of eight genes based on their potential relevance to the regulation of cardiovascular function by aldosterone.

Aldosterone regulation of extracellular matrix-related genes
The microarray experiments revealed that several genes that are potentially important for the structure or regulation of extracellular matrix were rapidly regulated by aldosterone. Aldosterone-induced genes in this pathway include TNX (26), ADAMTS1 (27), and PAI-1 (14). TNX is a large extracellular matrix protein, highly expressed in the heart in vivo (28). Our real-time PCR results verified the rapid aldosterone induction of TNX transcript (approximately 12-fold increase at 2 h after hormone addition; Fig. 3Go). Interestingly, in the continuous presence of 1 nM aldosterone, TNX mRNA levels increased further, reaching greater than 100-fold higher levels than control by 24 h. A representative time course of aldosterone induction is shown in Fig. 4AGo. This large, sustained induction of TNX mRNA was observed in both clonal H9C2/MR+ cell lines.


Figure 3
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  		FIG. 3. Aldosterone-induced early genes in H9C2/MR+ cells. Cells were treated or not with 1 nM aldosterone (Aldo) for 2 h, then RNA extracted, reverse transcribed, and quantitative RT-PCR performed as described in Materials and Methods. Aldosterone significantly increased the expression of TNX, ADAMTS1, and PAI-1 (A), whereas it significantly decreased the expression of UPAR and HAS2 (B). Values of mRNA levels were normalized for ß-actin mRNA levels in each sample. These values in control (nontreated cells) were set as 1 for each gene, and fold changes are shown on the figure. *, P < 0.05, **, P < 0.01 vs. control (no steroid), using two-tailed unpaired t test.

 

Figure 4
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  		FIG. 4. Time course of aldosterone (Aldo) effect on the expression of TNX (A), ADAMTS1 (B), PAI-1, and UPAR (C). Note logarithmic scale in A. Aldosterone induced TNX mRNA levels approximately 100-fold at 24 h. Note the simultaneous increase in the expression of the inhibitor, PAI-1, and the decrease in the expression of the receptor, UPAR (C). *, P < 0.05 vs. control, using two-tailed unpaired t test.

 
As illustrated on Fig. 3Go, RT-PCR analysis verified the rapid induction of ADAMTS1, a metalloprotease that is also highly expressed in the heart (27), and PAI-1, a known steroid regulated gene in other tissues (14, 29, 30). Time-course analysis revealed that the induction of ADAMTS1, similarly to that of TNX, continued up to 24 h (which was the longest time point we studied) (Fig. 4BGo). On the other hand, the induction of PAI-1 reached maximum at 2 h after aldosterone and decreased slightly thereafter (Fig. 4CGo).

Interestingly, our microarray analyses also revealed two extracellular matrix-related genes that were down-regulated by aldosterone: UPAR (also called Plaur) (31) and HAS2 (32). UPAR, the cellular receptor for urokinase plasminogen activator, is involved in extracellular matrix degradation and tissue remodeling (33) of the plasminogen/plasmin system and was shown to play a role in cell adhesion, migration, and angiogenesis (34). As illustrated in Fig. 4CGo, the mRNA levels for UPAR were decreased rapidly by aldosterone, reaching a nadir at 2 h and then returned to control levels at 24 h. HAS2 is the enzyme that is responsible almost exclusively for the synthesis of hyaluronic acid (HA) in the cardiovascular system (32). RT-PCR results verified that in H9C2/MR+ cells the levels of HAS2 mRNA are significantly decreased 2 h after 1 nM aldosterone treatment (Fig. 3Go).

Aldosterone induction of Orm-1, an inflammation/acute phase-related gene
The gene with highest induction by aldosterone in both microarray experiments was Orm-1 or {alpha}-acidic glycoprotein, an acute phase-related gene. Elevated levels of plasma Orm-1 are considered a cardiovascular risk factor (35). Interestingly, our quantitative RT-PCR results revealed an extremely pronounced induction of Orm-1 mRNA by aldosterone. Orm-1 mRNA levels were approximately 100-fold higher 2 h after aldosterone treatment than in control cells and reached 1000-fold higher levels by 24 h (Fig. 5AGo). The dose-response relationship of the effect of aldosterone on Orm-1 mRNA expression is illustrated in Fig. 5BGo. A significant, approximately 10-fold induction was observed with 0.1 nM aldosterone, a concentration corresponding to the lower end of the physiological range, and the effect was almost maximal at 1 nM, indicating that it is mediated via the MR, and most likely it has physiological significance.


Figure 5
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  		FIG. 5. The effect of aldosterone on the induction of Orm-1 is time and concentration dependent. Note logarithmic scale. Orm-1 mRNA expression levels (normalized for ß-actin mRNA) were significantly elevated by treatment with 1 nM aldosterone at every time point after 30 min (A). Orm-1 mRNA induction by aldosterone was statistically significant at concentrations above 0.1 nM (B). P > 0.05 vs. control, using two-tailed unpaired t test.

 
Aldosterone-regulated genes with potential role in signaling and regulation of vascular tone
In addition to SGK1, which is involved in cellular signaling pathways, our microarray identified RGS2 (36) and adrenomedullin (37) as aldosterone-induced early genes in cardiomyocytes. RGS proteins play a key role in the regulation of G protein-coupled receptor signaling. G protein-coupled receptors mediate many extracellular signals that regulate cardiomyocyte growth and differentiation. The heart expresses several RGS proteins (38) but their individual roles in regulating cardiovascular function is still not entirely clear. Adrenomedullin is a vasodilator produced by cardiomyocytes, vascular smooth muscle cells, and endothelial cells (37). As shown in Fig. 6Go, our RT-PCR experiments verified that the expression of both RGS2 and adrenomedullin was rapidly induced by 1 nM aldosterone in H9C2/MR+ cells. The induction was statistically significant as early as 30 min after addition of the hormone (Fig. 6Go) and remained elevated at 24 h.


Figure 6
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  		FIG. 6. Time course of aldosterone effect on the expression of RGS-2 (solid line) and AM (dashed line). *, P < 0.05 vs. control, using two-tailed unpaired t test.

 
Protein synthesis inhibition does not prevent induction of early aldosterone-regulated genes
The rapid time course of aldosterone regulation of the above genes suggests that the induction is direct, probably mediated via glucocorticoid response element (GRE)/mineralocorticoid response element in the promoter region and does not require de novo synthesis of additional aldosterone-regulated proteins. Indeed, the promoter of several of the induced genes, such as Orm-1 and PAI-1 contains a functional GRE (39, 40). To test this hypothesis more directly, we pretreated H9C2/MR+ cells with two structurally different inhibitors of protein synthesis, anisomycin and cycloheximide, and then studied the effect of a 2-h aldosterone treatment on the mRNA levels of three genes, TNX, RGS2, and ADM. Protein synthesis inhibitors did not prevent the effect of aldosterone for any of the early-induced genes we tested; results obtained with anisomycin are shown in Fig. 7Go, and similar results were obtained with cycloheximide. These results suggest a direct steroid effect, and indeed Nubiscan analysis indicated that in addition to the known functional GREs in the promoter of Orm-1 and PAI-1 (39, 40), the rat TNX gene also has a putative GRE/mineralocorticoid response element sequence (gagcacatgttgtacc) in the promoter, located –2.5 kb upstream from the first ATG codon.


Figure 7
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  		FIG. 7. Protein synthesis inhibition does not prevent induction of early aldosterone-regulated genes. H9C2/MR+ cells were grown in steroid-free medium for 2 d and then preincubated for 30 min with vehicle (control) or the protein synthesis inhibitor (inh.) anisomycin (aniso; 10 µM). One nanomole aldosterone (Aldo) was given (or not) at 30 min, and cells were incubated for an additional 2 h before RNA was prepared. RGS-2, AM, TNX, and ß-actin mRNA levels were determined by quantitative RT-PCR. Solid bars, no aldosterone; striped bars, 1 nM aldosterone. Values of mRNA levels were normalized for ß-actin mRNA levels in each sample. Comparisons were made between values for each treatment obtained with and without aldosterone; P < 0.05 for vehicle vs. aldosterone using two-tailed unpaired t tests for each gene.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Aldosterone, in addition to its well-established effects on epithelial cells, has been suggested to exert direct effects on the cardiovascular system (41, 42). Although renal and cardiovascular effects are difficult to dissociate in vivo, the observation that MR antagonists exert effects without a parallel change in blood pressure supports a direct role for MR/aldosterone in the regulation of cardiovascular functions (43).

The main known effects of aldosterone in classical target tissues are mediated via changes in transcription. On the other hand, genes that mediate direct cardiovascular effects of aldosterone in vivo are yet to be identified, and only a few genes that are clearly regulated via the MR have been described in cardiac or vascular tissues in cell culture. Among these is SGK1, which similarly as in renal epithelia (23, 24) is induced by aldosterone in cardiomyocytes (Ref. 44 and present data). A recent study described in vivo aldosterone-regulated genes in mouse heart tissue (45). However, the animals in that study were not adrenalectomized, and the plasma levels of corticosterone were greater than 100-fold higher in all mice than that of aldosterone in the treatment group (45). Because cardiac expression of 11ß-hydroxysteroid dehydrogenase-2 is thought to be quite low (17), it seems very likely that in the above study most if not all cardiac MRs were occupied by corticosterone and not aldosterone. In addition, none of the established aldosterone-regulated genes such as SGK1 or GILZ (25) was differentially expressed after aldosterone treatment (45). Thus, the relationship between aldosterone occupancy of the MRs and the genes that were found to be regulated in that study (45) remains to be established.

The main finding of the present study is the identification of early, aldosterone-regulated genes in cardiomyocytes that have the potential to mediate aldosterone’s direct effects on the cardiovascular system in vivo. Some of these genes were already known to be regulated by aldosterone in other cell types, such as SGK1 (23, 24) or PAI-1 (14, 46). Thus, it seems that some of the aldosterone-regulated genes in the cardiovascular system overlap with those regulated in epithelial cells. Nevertheless, it seems likely that the majority of aldosterone-regulated genes in cardiomyocytes are different from those regulated in epithelia. Importantly, our experiments identified a number of genes whose role in cardiovascular function has been established but their aldosterone-regulation was previously unknown as well as other genes that are likely to have a function in cardiac pathology.

In this study we focused on three groups of aldosterone-regulated genes that have the greatest potential to mediate the physiological or pathological cardiovascular effects of mineralocorticoids. In the first group, we assigned those genes that have some obvious or putative connection to the regulation of extracellular matrix or cardiac remodeling. These include PAI-1, UPAR, ADAMTS1, and TNX.

PAI-1 is a multifunctional protein. Its best-studied function is the inhibition of tissue-type plasminogen activator in plasma, which consequently suppresses the activity of plasmin (for review see Ref. 30). In addition to its serine protease inhibitor function, PAI-1 also alters cell/matrix interactions by binding to vitronectin (47) and increases migration and proliferation of vascular smooth muscle cells (48). Increased PAI-1 expression (or activity) is associated with greater risk of cardiovascular disease (30). Recently it was also shown that PAI-1 has an important role in the development of thrombosis and atherosclerosis and other cardiovascular diseases in diabetics (49). Although the results obtained with PAI-1 knockout mice are somewhat contradictory, recent data show that PAI-1 deficiency protects against aldosterone-induced vascular injury (50). Congruent with our present findings, PAI-1 was shown to be induced by glucocorticoids and aldosterone in a number of cell types (14, 29, 46).

UPAR, the receptor for urokinase-type plasminogen activator (UPA), plays a role in cell adhesion, migration, pericellular proteolysis, and angiogenesis, and uPA/UPAR-related signaling mediates cross talk between monocytes and vascular smooth muscle cells (34). The fibrogenic response is accentuated in the absence of UPAR (34). The coordinated regulation by aldosterone of two important genes in the plasminogen/plasmin pathway, i.e. up-regulation of the inhibitor, PAI-1, and simultaneous down-regulation of the receptor, UPAR, is highly significant. Both of these changes are expected to result in the same biological effect and could very well be involved in the adverse effects of aldosterone, resulting in increased collagen deposition and cardiac fibrosis.

Similarly, ADAMTS1 could play a role in aldosterone’s fibrotic effects. ADAMTS1, a metalloproteinase and disintegrin, was originally cloned as an inflammation-associated gene (27). Later it was shown that ADAMTS1 is induced by myocardial infarction in cardiomyocytes and endothelium within the infarct zone (51). Furthermore, ADAMTS1 can disrupt extracellular matrix remodeling (52), and its overexpression accelerates atherosclerosis and neointima formation (53).

Finally, in the extracellular matrix-related gene group, we identified TNX as one of the genes showing the strongest induction by aldosterone. TNX belongs to the family of matricellular proteins. These are extracellular matrix proteins that modulate cell-matrix interactions and collagen accumulation and organization but do not have a direct structural role (26). In addition to tenascins, this family includes osteonectin, osteopontin, thrombospondin-1, and thrombospondin-2. TNX is highly expressed in the heart (54). TNX is an essential regulator of collagen deposition by fibroblasts (55) and is up-regulated during fibrosis after tissue injury (26). TNX deficiency in humans (Ehlers-Danlos syndrome), and knockout mice leads to a generalized connective tissue disorder due to dysfunction of fibrillar collagens (56). Thus, an aldosterone-induced expression of TNX in cardiomyocytes could lead to disturbed collagen deposition and extracellular matrix remodeling as well as cardiac fibrosis, which have been demonstrated to occur after prolonged mineralocorticoid and high salt treatment (57).

HAS2 is the enzyme that generates HA in the cardiovascular system. HA is a major component and an important organizer of the extracellular matrix (58). The hormonal regulation of HAS2 expression or activation has not been well established. Targeted deletion of the HAS2 gene in mice results in an absence of cardiac jelly and endocardial cushions, a loss of vascular integrity, and early embryonic death (59). The function of the aldosterone-induced suppression of HAS2 expression in cardiomyocytes is unclear, but it is logical to surmise a role in the remodeling of extracellular matrix that accompanies heart failure.

The second group of genes rapidly up-regulated by aldosterone in H9C2/MR+ cells includes genes that have been implicated in inflammation and acute phase reaction. Orm-1 (also called {alpha}-1 acid glycoprotein) is an acute phase protein that is induced by proinflammatory cytokines and glucocorticoids (60). Interestingly, elevated plasma level of Orm-1 is considered a cardiovascular risk factor. Patients with diabetes and metabolic syndrome have high plasma Orm-1 levels that may be responsible for the increased incidence of cardiovascular problems in this syndrome (35). Our data revealed an astonishing level of Orm-1 mRNA induction (approximately 1000-fold) by aldosterone in cardiomyocytes. As to our knowledge, this is the first observation that aldosterone, which has been proposed to cause inflammation in vascular tissues (11, 17), increases the gene expression of acute phase proteins directly in heart cells.

Intriguingly, it seems that some of the aldosterone-induced proteins might exert synergistic effects because Orm-1 forms a function-stabilizing complex with PAI-1 (61), and PAI-1 itself is recognized as an acute-phase reactant protein (62). A recent hypothesis suggested that PAI-1 and other acute-phase proteins might provide a link between the proinflammatory features and hypercoagulability that are characteristics of the metabolic syndrome (62). Interestingly, GILZ, which we found to be up-regulated by aldosterone (see supplemental table) is also an acute-phase protein (63).

The third group of genes rapidly induced by aldosterone in H9C2 cells contains genes involved in signal transduction and regulation of vascular tone. Within this group we focused on adrenomedullin and RGS2.

Adrenomedullin is a vasodilator produced by cardiomyocytes, vascular smooth muscle cells, and endothelium and in cultured vascular smooth muscle cells is up-regulated by glucocorticoids (64) and superphysiological (1 µM) concentration of aldosterone, which saturates the glucocorticoid receptor (65). Adrenomedullin levels are elevated in patients with various cardiovascular diseases such as essential hypertension (66), but it is unclear whether this is a direct or secondary event. Ventricular adrenomedullin levels correlate with the extent of cardiac hypertrophy in rats (67). Adrenomedullin has diverse biological effects, including cardioprotective actions (68). Adrenomedullin gene delivery was shown to attenuate vascular lesions caused by hypertension (69) and oxidative stress (70). Thus, an aldosterone-induced increase in adrenomedullin expression might be protective rather than mediating adverse effects on the cardiovascular system. In addition, adrenomedullin, just like aldosterone, has a positive inotropic effect (71), which would be beneficial in a healthy or diseased heart. Recently it was proposed that increased adrenomedullin production in response to aldosterone might induce vasodilation to counteract the systemic vasoconstriction of aldosterone (65), and thereby adrenomedullin might serve as a negative feedback mechanism protecting against an increase in blood pressure and volume retention in patients with inappropriate aldosterone excess (such as in primary aldosteronism or chronic heart failure).

RGS proteins are multifunctional signaling regulators and exert GTPase-activating protein activities. Many signals that regulate cardiomyocyte growth, differentiation, and function are mediated via heterotrimeric G proteins, which are controlled by RGS proteins. RGS proteins are necessary for normal cardiovascular function and are involved in hypertrophy and development of heart failure (72). Changes in cardiac RGS2 levels and in G(q/11) signaling produce cardiovascular phenotypes (38). Furthermore, haploinsufficiency and/or elimination of the RGS2 gene lead to hypertension in mice (73). Interestingly, certain RGS2 haplotypes also cosegregate with human hypertension (74). RGS2 is activated via the nitric oxide-cGMP pathway, which might be part of the mechanism by which it regulates blood pressure (75). Thus, RGS2, which we found to be rapidly and directly up-regulated by physiological concentrations of aldosterone, might mediate many cardiovascular effects of aldosterone, including cardiac hypertrophy. On the other hand, RGS2 was shown to selectively inhibit G(q/11) signaling and thereby attenuate/terminate the action of vasoconstrictors (76). A recent study showed that transgenic mice with activated Gq signaling develop heart failure, but the contractile dysfunction and structural abnormalities improved significantly after termination of the Gq signal (77), suggesting that RGS2 could be beneficial in heart failure. Such an effect is difficult to reconcile with a predominantly constrictor action of aldosterone and with the current dogma that aldosterone is detrimental in heart failure. On the other hand, induction of RGS2, and probably other proteins that are cardioprotective, might be part of a compensatory feedback mechanism initiated by aldosterone.

In summary, our results demonstrate significant effects of aldosterone on the gene expression profile of cardiomyocytes. All these genes are rapidly induced by physiological concentrations of aldosterone. Interestingly, although some of these genes are in the same pathway as genes identified in transgenic mice overexpressing 11ß-hydroxysteroid dehydrogenase-2 in cardiomyocytes (i.e. genes whose elevated expression could lead to cardiac fibrosis and/or inflammation), none of the genes we found to be regulated acutely by aldosterone overlapped with the ones elevated in the study by Qin et al. (9).

We conclude that aldosterone regulates the expression of several genes that may contribute to the development of cardiovascular damage (fibrosis, vasculitis) under pathological conditions. Blocking aldosterone regulation of these genes by MR antagonists such as epleronone or spironolactone might contribute to the beneficial effects observed in patients with chronic heart failure. However, it seems that activation of the MR by aldosterone also induces genes, such as adrenomedullin and RGS2, that are likely to exert cardioprotective effects and thus might be beneficial in heart failure.


   Acknowledgments
 
We thank Donna Morneau for excellent technical assistance and Dr. Celso Gomez-Sanchez for the generous gift of the MR antibodies.


   Footnotes
 
This work was supported by National Institutes of Health Grants DK41841 and DK58898.

First Published Online January 18, 2007

Abbreviations: ADAMTS1, A disintegrin, and metalloprotease with thrombospondin motifs; AM, adrenomedullin; Ct, threshold; EST, expressed sequence tag; GLIZ, glucocorticoid-induced leucine zipper; GRE, glucocorticoid response element; HA, hyaluronic acid; HAS2, hyaluronic acid synthase-2; MR, mineralocorticoid receptor; Orm-1, orosomucoid-1; PAI, plasminogen activator inhibitor; RGS2, regulator of G protein signaling-2; SGK1, serum- and glucocorticoid-induced kinase-1; TBST, Tris, NaCl, Tween 20, and thimerosal; TNX, tenascin-X; UPAR, urokinase plasminogen activator receptor.

Received October 26, 2006.

Accepted for publication January 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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