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. 2010 Jun 30;151(9):4467–4476. doi: 10.1210/en.2010-0237

Molecular Signature of Mineralocorticoid Receptor Signaling in Cardiomyocytes: From Cultured Cells to Mouse Heart

Celine Latouche 1, Yannis Sainte-Marie 1, Marja Steenman 1, Paulo Castro Chaves 1, Aniko Naray-Fejes-Toth 1, Geza Fejes-Toth 1, Nicolette Farman 1, Frederic Jaisser 1
PMCID: PMC2940498  PMID: 20591974

Abstract

Excess mineralocorticoid signaling is deleterious for cardiovascular functions, as demonstrated by the beneficial effects of mineralocorticoid receptor (MR) antagonism on morbidity and mortality in patients with heart failure. However, the understanding of signaling pathways after MR activation in the heart remains limited. We performed transcriptomic analyses in the heart of double-transgenic mice with conditional, cardiomyocyte-specific, overexpression of the MR (MRcardio mice) or the glucocorticoid receptor (GR; GRcardio mice). Some of the genes induced in MRcardio mice were selected for comparative evaluation (real time PCR) in vivo in the heart of mice and ex vivo in the MR-expressing cardiomyocyte H9C2 cell line after aldosterone or corticosterone treatment. We demonstrate that chronic MR overexpression in the heart results in a limited number of induced (n = 24) and repressed (n = 22) genes compared with their control littermates. These genes are specifically modulated by MR because there is limited overlap (three induced, four repressed) with the genes that are regulated in the heart of GRcardio mice (compared with control mice: 70 induced, 73 repressed). Interestingly, some MR-induced genes that are up-regulated in vivo in mice are also induced by 24-h aldosterone treatment in H9C2 cells, such as plasminogen activator inhibitor 1 and Serpina-3 (α1-antichymotrypsin). The signaling pathways that are affected by long-term activation of MR may be of particular interest to design novel therapeutic targets in cardiac diseases.

This study identifies target genes specifically modulated in the heart by long-term in vivo activation of the mineralocorticoid receptor.

Pathological activation of the mineralocorticoid receptor (MR) is involved in the adverse cardiovascular effects of aldosterone in humans and animal models (1). Deleterious consequences of enhanced mineralocorticoid signaling has been highlighted by clinical trials that showed a major benefit of addition of MR antagonists on top of standard treatment in patients with heart failure (2,3). The MR is a ligand-dependent transcription factor belonging to the superfamily of nuclear receptors that includes steroid receptors and a number of other receptors (4). The MR is always coexpressed with the closely related glucocorticoid receptor (GR) in epithelial cells of the distal nephron and also in cardiomyocytes, neurons, and other nonclassical targets like keratinocytes and adipocytes (5,6). The definition of MR-mediated vs. GR-mediated cellular events is often difficult because these two receptors share common ligands. In particular, MR binds, with similar high affinity (dissociation constant 0.5–1 nm), aldosterone as well as glucocorticoids, which are 100- to 1000-fold more abundant in the plasma than aldosterone. The GR has an equal (lower) affinity for both hormones (dissociation constant 10–30 nm) (5,6). In classical aldosterone target cells such as the renal collecting ducts, permanent MR occupancy by glucocorticoids is prevented because the hormone is metabolized by the enzyme 11β-hydroxysteroid dehydrogenase-2 (HSD2) (7). In cardiomyocytes, HSD2 is expressed at low levels and MR occupancy by glucocorticoids may predominate (5,6,8).

The effects of aldosterone on cardiac cells have been documented experimentally. A direct relationship between plasma aldosterone and cardiac L-type calcium current has been established (9), and up-regulation of such currents by corticosteroids is mediated by the MR, not the GR (10). It has recently been shown that aldosterone regulates intracellular calcium movements and activates the ryanodine receptor (11). To discriminate between MR- and GR-dependent pathophysiological roles in vivo, we produced double-transgenic mice with cardiomyocyte-specific MR (12) or GR (13) overexpression (MRcardio and GRcardio mice). These mice display distinct cardiac phenotypes: MRcardio mice present ventricular arrhythmias, whereas GRcardio mice have atrioventricular conduction defects, and sodium and potassium currents partly differ in cardiomyocytes isolated from these two mouse models. However, the signaling pathways and mechanisms whereby excess mineralocorticoids may lead to cardiac pathology are not yet elucidated. To address these questions, some studies searched for differentially expressed genes in response to aldosterone treatment (14,15,16,17). Among the up-regulated genes that were detected, some are related to extracellular matrix regulation, signaling, and regulation of vascular tone and inflammation, thus susceptible to interfere with cardiovascular functions. Whereas these analyses focused on the early response to the hormone (a few hours at most), much less is known about the genes that may be regulated by aldosterone/MR in the long term in vivo. Identification of genes that are involved in adaptation or compensatory events should enhance our understanding of the pathways that are altered after MR activation in pathological situations.

In this study, we performed a transcriptomic analysis to search for cardiac genes that could be affected by chronic increase in MR expression. We used mice with conditional MR or GR overexpression in cardiomyocytes (12,13). The expression levels of some genes identified in our array analyses together with some of the early aldosterone-induced genes identified previously (16) were evaluated both in the heart of MRcardio and GRcardio mice as well as in a cardiomyocyte cell line treated with aldosterone. Real-time PCR analysis showed that some genes are up-regulated both in the initial phase of aldosterone action in the cell model and in the heart of MRcardio mice.

Materials and Methods

H9C2/MR+ cell culture

H9C2/MR+ cells represent a clonal cell line of cardiomyocytes derived from the embryonic rat ventricle that stably express the MR (16); H9C2/MR+ cells exhibit features of cardiomyocytes as they express the Ca channel ICaL and α-myosin heavy chain (MHC) (Supplemental Fig. 1, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). They were grown in DMEM/F12 medium supplemented with 10% heat-inactivated fetal bovine serum plus an antibiotic mixture (penicillin-streptomycin; Invitrogen, Carlsbad, CA; and 12.5 μg/ml tylosin; Sigma-Aldrich, St. Louis, MO) to approximately 40% of confluence. For experiments, cells were grown in steroid-free medium 2 d before stimulation. Cells were treated with 10−8 mol/liter aldosterone (Sigma), 10−8 mol/liter corticosterone (Sigma), or vehicle for 24 h. To investigate the specificity of action of these steroids, the MR antagonist RU28318 (10−6 mol/liter; Tocris Bioscience, Bristol, UK) or GR antagonist RU38486 (10−6 mol/liter; Sigma) was used.

Cardiac MR or GR conditional mouse models

The previously characterized tetO-human (h) MR and tetO-hGR mouse strains were crossed with the αMHC-tTA transactivator mouse strain to obtain double-transgenic αMHC-tTA/tetO-hMR (MRcardio) (12) or αMHC-tTA/tetO-hGR (GRcardio) (13) mice with conditional cardiomyocyte-specific hMR or hGR expression. Doxycycline (2 mg/ml in drinking water; Sigma) was given to the pregnant mothers until birth of the progeny to avoid hMR expression in the embryos and so prevent the embryonic lethality previously reported in MRcardio mice. Six-week-old mice were used for this study. To switch off the transgene expression in 3-month-old MRcardio mice, doxycycline was administered for 4 wk before the animals were killed. To antagonize the MR, potassium canrenoate (100 mg/kg·d in drinking water; Sigma) was provided to MRcardio mice and their controls (1 month old) for 8 wk. The use of animals was in accordance with the guidelines of the European Community and approved by our Institutional Animal Care and Use Committee.

DNA microarray analysis

Total RNA was isolated from ventricles from 10 mice of the hMR conditional model (five controls and five MRcardio) and 18 mice of the hGR conditional model (nine controls and nine GRcardio) using TRIZOL reagent (Invitrogen). mRNA was isolated using the Oligotex mRNA kit (QIAGEN, Courtaboeuf, France). RNA and mRNA quality was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies France, Massy, France). The mRNA from MRcardio mice was pooled and labeled with Cy5 in three separate reactions; mRNA from their controls was pooled and labeled with Cy3 in three separate reactions. The same procedure was used for GRcardio mice and their controls. Cy3- and Cy5-labeled cDNA was prepared using the CyScribe cDNA postlabeling kit (GE Healthcare Europe, Saclay, France). Using this kit, Cy3 and Cy5 are not incorporated during cDNA synthesis but are chemically coupled to aminoallyl-deoxyuridine 5-triphosphate that has been incorporated into all cDNA samples. This strategy leads to an even incorporation of both Cy3 and Cy5 and minimizes experimental variation caused by uneven incorporation of labels. Labeling reactions were performed separately for each microarray. Three microarray hybridizations were performed for the MR transgenic mice and six for the GR transgenic mice. The hybridization mixture was preincubated with human Cot-I DNA (i.e., the fraction of genomic DNA consisting largely of highly repetitive sequences), yeast tRNA, and polyA RNA and hybridized to microarrays.

Microarrays were prepared in-house using 50-mer oligonucleotide probes (Eurofins MWG Operon, Ebersberg, Germany). These microarrays have been previously validated (18,19,20,21). The probes were spotted onto epoxy-silane-coated glass slides using the Lucidea array spotter (GE Healthcare Europe). The 5419 genes that were represented on the microarrays have been selected for their involvement in cardiovascular and/or skeletal muscle normal and pathological functioning. Selection was based on the following: 1) substrative hybridization experiments (22), 2) genome-wide microarray hybridizations, and 3) literature data (23). The total number of analyzed oligonucleotides was 3459. Each gene probe was spotted in triplicate (for more information, see http://cardioserve.nantes.inserm.fr/ptf-puce/spip.php?article59).

Hybridized arrays were scanned by fluorescence confocal microscopy (Scanarray 4000XL; GSI-Lumonics, Warwickshire, UK). Fluorescence signal measurements were obtained separately for each fluorochrome at a 10-μm/pixel resolution. Hybridization and background signal intensities and quality control parameters were measured using GenePix Pro 5.0 (Molecular Devices, Sunnyvale, CA). A Lowess normalization procedure (24) was performed to correct for technical biases. The procedure was applied channel by channel as described previously (25). For each microarray, Cy3 and Cy5 signal intensities were individually normalized to a prototype defined as the median profile of all Cy3 or Cy5 signal intensities. The data discussed in this publication have been deposited in the National Centre for Biotechnology Information’s Gene Expression Omnibus and are accessible through Gene Expression Omnibus series accession no. GSE20525 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE20525).

Quantitative RT-PCR

Total RNA was extracted from cells (four to 16 wells per condition) and frozen hearts (individual samples from three to eight mice per group from experimental series distinct from those used for microarrays) using TRIZOL reagent (Invitrogen), according to the manufacturer’s protocol, and deoxyribonuclease treated. Reverse transcription was performed with 2 μg of total RNA, random primers (Invitrogen), and Superscript II reverse transcriptase (Invitrogen). Transcript levels were analyzed by real-time PCR in an iCycler iQ apparatus (Bio-Rad Laboratories, Marnes La Coquette, France) with SYBR Green I detection. The reactions were performed in duplicate for each sample using a quantitative PCR master mix plus SYBR with fluorescein (Eurogentec, Angers, France) with 300 nmol/liter of each sense and antisense primer and 15 ng of cDNA in 25 μl total volume.

The thermal cycling parameters were: initial denaturation at 95 C for 10 min, followed by 40 cycles at 95 C for 15 sec and 60 C for 1 min. Relative expression of the mRNA was quantified using the equation described by Pfaffl (26): ratio = (Etarget)Ĉttarget(mean control-sample)/(Eref)Ĉtref(mean control-sample); E represents the efficiency of the PCR, and Ct represents the number of cycles at which the amplified product reach threshold. The mRNA levels were normalized for β-actin mRNA in samples obtained from culture cells or ubiquitin C (ubc) mRNA in mouse heart. Values in control conditions were set as 1 for each gene, and fold changes are provided in the figures. The sequences of the specific primers are detailed in Supplemental Table 1.

Western blot analysis

Heart samples were homogenized in lysis buffer (50 mm Tris, pH 8; 150 mm NaCl; 1% Nonidet P-40; 1% Na deoxycholate; 0.1% sodium dodecyl sulfate; 1 m EDTA; 1 mm Na3VO4; 50 mm NaF; 2 mm phenylmethylsulfonyl fluoride) containing a protease inhibitor cocktail (Roche Diagnostics, Meylan, France); lysates were subjected to SDS-PAGE, and after blockade with BSA, Western blot was performed using a polyclonal anti-Serpina-3 (1:500; AVIVA Systems Biology, San Diego, CA) followed by antirabbit IgG (1:10,000; GE Health Care Europe). Antibody was visualized by using ECL Plus reagents (GE Health Care) and signals were acquired in a Las4000 DarkBox (Fuji Photo Film Europe GMBH, Düsseldorf, Germany). β-Actin (1:500; Abcam, Paris, France) was used as internal control for protein loading.

Statistical analysis

Linear model for microarray data (27) was used to identify genes with statistically significant differential expression (adjusted P < 0.0001) between transgenic and control mice. High-Throughput GoMiner (28) was used to identify functional categories that were overrepresented among the differentially expressed genes compared with all analyzed genes.

Results of quantitative PCR are provided as mean ± sem. The nonparametric Mann-Whitney U test was used to assess statistical differences between two experimental groups. Differences among more than two experimental conditions were tested by the ANOVA one-way test and Newman and Keuls test (StatEl software; AdScience, Paris, France). P < 0.05 was considered significant.

Results

In the cardiomyocyte H9C2 cell line stably transfected with the MR (referred as H9C2/MR+), several early aldosterone-regulated genes have previously been identified after short-term (2 h) aldosterone treatment (16). To document the receptor specificity (MR vs. GR occupancy) of this hormonal response, we selected six gene products identified in this study: 1) the serum- and glucocorticoid-induced kinase Sgk1 that is regulated by corticosteroid hormones in kidney cells; 2) three genes of the extracellular matrix: the metalloprotease Adamts1, the plasminogen activator inhibitor (PAI)-1, and tenascin X (Tnx); 3) regulator of G protein signaling 2 (Rgs2); and 4) the FK506 binding protein (Fkbp5). Twenty-four hours of treatment of H9C2/MR+ cells with aldosterone increased Sgk1, Adamts1, PAI-1, Rgs2, Fkbp5, and Tnx mRNAs (Fig. 1), confirming our earlier observations for Adamts1, Rgs2, and PAI-1 (16). The up-regulation of all these genes was reduced by the MR antagonist RU28318 and the GR antagonist RU38486. Thus, these genes appear to be regulated via the MR and also via the endogenous GR (Fig. 1).

Figure 1.

Figure 1

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Effect of steroid receptor antagonists on aldosterone-induced genes in H9C2/MR+ cells. Cells were treated with 10−8 m aldosterone (Aldo) alone for 24 h or in association with MR antagonist RU28318 (10−6 m) or GR antagonist RU38486 (10−6 m). Values of mRNA levels (real time PCR) were normalized for β-actin expression. Values in control (nontreated) cells were set as 1 for each gene, and fold changes are shown on the figure; data are provided as mean ± sem (n = 4–16 wells per condition). *, P < 0.05, **, P < 0.01 treatment vs. control; #, P < 0.05, ##, P < 0.01 treatment vs. Aldo alone (ANOVA analysis, Newman and Keuls test).

To evaluate the effect of long-term receptor activation in vivo, we took advantage of our mouse models with cardiomyocyte-specific overexpression of the MR (12) or the GR (13). MRcardio and GRcardio mice exhibit a 5-fold increase in MR or GR mRNA expression, respectively, compared with their control littermates (Supplemental Fig. 2). These mice were studied in steady-state basal situation, i.e. in the presence of normal endogenous corticosteroid plasma levels. Interestingly, some of the gene products identified as early aldosterone-induced genes in the cell model were also up-regulated in vivo in the heart of MRcardio mice compared with their control littermates: Adamts1, PAI-1, Rgs2, and Fkbp5 mRNAs were significantly increased in the heart of mice with cardiac overexpression of MR, whereas Sgk1 and Tnx levels did not differ (Fig. 2). In contrast, cardiac GR overexpression did not modify these genes, except for Fkbp5 that was moderately induced in the heart of GRcardio mice (its induction was 10-fold lower than in MRcardio mice). To identify more specifically genes that are regulated in vivo by corticosteroids in the heart, we investigated MR- or GR-dependent changes in the cardiac transcriptome by microarray analyses of the two mouse models with cardiac overexpression of MR or GR compared with their respective controls. A list of MR- and GR-regulated genes in the heart of each mouse model (each compared with their control littermates) is provided in Table 1 (MR regulated genes) and Supplemental Table 2 (GR regulated genes). We found that cardiac MR overexpression resulted in less regulated genes than did GR overexpression. MR overexpression in cardiomyocytes resulted in 24 transcripts that were significantly up-regulated, and 22 that were down-regulated in the heart of MRcardio mice compared with their control littermates. These transcripts correspond mainly to structural proteins, regulatory enzymes, transcription factors, and stress response genes. Cardiac GR overexpression led to the up-regulation of 70 transcripts and down-regulation of 73 transcripts (Supplemental Table 2); however, the changes were smaller than those observed in the MRcardio mice. Interestingly, only few modified cardiac genes were common to the MRcardio and GRcardio mouse models (only three up-regulated genes and four down-regulated genes), indicating that the two receptors control different transcription networks (Fig. 3 and Supplemental Table 3).

Figure 2.

Figure 2

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Induced genes in the heart of mice overexpressing the MR (MRcardio) or the GR (GRcardio). Some genes that have been shown to be induced by aldosterone in H9C2/MR+ cells have been measured in the heart from transgenic mice. Values of mRNA levels (real time PCR) were normalized for ubc expression. Values in control mice were set as 1 for each gene, and fold changes are shown on the figure; data are provided as mean ± sem (n = 3–8 mice per condition). *, P < 0.05, **, P < 0.01 transgenic mice compared with their own controls (Mann-Whitney U test).

Table 1.

List of cardiac transcripts that differ between mice overexpressing the MR (MRcardio) and their control littermates

Symbol Accession no. Full name Fold
Up-regulated genes
 Serpina-3n NM_009252 Serine (or cysteine) proteinase inhibitor, clade A, member 3N 8.57
 Tnnt3 L49018 Troponin T3, skeletal, fast 3.43
 Nr4a2 NM_013613 Nuclear receptor subfamily 4, group A, member 2 2.83
 MT1H NM_005951 Metallothionein 1H 2.81
 Sepr NM_013759 Selenoprotein R 2.77
 Gsta4 NM_010357 Glutathione S-transferase, α 4 2.60
 Cnn3 BC005788 Calponin 3, acidic 2.58
 GPR72 AF236081 G protein-coupled receptor 72 2.43
 COL1A1 NM_000088 Collagen, type I, α1 2.38
 Apoc1 NM_007469 Apolipoprotein C-I 2.17
 COL1AOI M32790 α-1 Collagen type 1 (osteogenesis imperfecta allele) 2.08
 Gpx3 AK002219 Glutathione peroxidase 3 2.03
 TACC2 NM_021314 Transforming, acidic coiled-coil containing protein 2 1.83
 Nrip3 NM_020610 Nuclear receptor interacting protein 3 1.82
  BC017540 RIKEN cDNA 2610102M01 gene 1.82
 Fn1 XM_129845 Fibronectin 1 1.80
 H2-Ab1 M13539 Histocompatibility 2, class II antigen A, β 1 1.80
 Des AJ250633 Desmin 1.77
 Hmox2 NM_010443 Hemeoxygenase (decycling) 2 1.74
  AC167659 Mus musculus chromosome 7, clone RP23-165I8, complete sequence 1.68
 S3–12 NM_020568 Plasma membrane-associated protein, S3-12 1.60
 Nfia D90173 Nuclear factor I/A 1.58
 Ptprcap NM_016933 Protein tyrosine phosphatase, receptor type, C Polypeptide-associated protein 1.43
 Gpx1 NM_008160 Glutathione peroxidase 1 1.42
Down-regulated genes
 Abcc9 NM_021041 ATP-binding cassette, subfamily C (CFTR/MRP), member 9 0.40
 Mlf1 NM_010801 Myeloid leukemia factor 1 0.41
 FLJ10948 AK001810 Hypothetical protein FLJ10948 0.42
 Reg3g NM_011260 Regenerating islet-derived 3γ 0.43
  AK018837 RIKEN cDNA 1700016D08 gene 0.45
 CYFIP2 AF160973 Cytoplasmic FMR1 interacting protein 2 0.55
 SEMA3C NM_006379 Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3C 0.55
  AK010194 RIKEN cDNA 2310076E16 gene 0.56
 Fkbp4 NM_010219 FK506 binding protein 4 0.57
 Hrc AF132218 Histidine-rich calcium binding protein 0.57
 Pdhb NM_024221 Pyruvate dehydrogenase (lipoamide)-β 0.60
 VLDLR S73849 Very low-density lipoprotein receptor 0.60
 Idh3b NM_130884 Isocitrate dehydrogenase 3 (NAD+)-β 0.62
  AK004874 RIKEN cDNA 5730402K07 gene 0.63
 Ghr NM_010284 GH receptor 0.63
 PPAT U00238 Phosphoribosyl pyrophosphate amidotransferase 0.63
 Hspa9a NM_010481 Heat shock protein, A 0.68
 PPIB X58990 Peptidylprolyl isomerase B (cyclophilin B) 0.68
 Tuba4 NM_009447 Tubulin-α4 0.68
 VEGFA AF022375 Vascular endothelial growth factor 0.70
 Gucy1a3 NM_021896 Guanylate cyclase 1, soluble, α3 0.71
 Rrad NM_019662 Ras-related associated with diabetes 0.71
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Figure 3.

Figure 3

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Comparison of cardiac transcriptomes in the heart of mice overexpressing the MR (MRcardio) or the GR (GRcardio). For each mouse model (MRcardio or GRcardio), the circles refer to genes that are up- or down-regulated in the heart of transgenic mice compared with their own control littermates. The lists of differentially expressed genes in each mouse model are provided in Table 1 and Supplemental Tables 2 and 3. See tables for the abbreviations.

Real-time PCR validation was performed on selected MR-specific regulated genes that are involved in extracellular matrix remodeling and that may interfere with the balance between pro- and antifibrotic processes occurring during the development of cardiac diseases. Analyses were performed on hearts originating from experimental series distinct from those used for microarray analyses (Fig. 4) and on H9C2/MR+ cells treated with aldosterone or corticosterone (Fig. 5). In the heart of MRcardio mice, the serine-protease inhibitor Serpina-3 (or α1-antichymotrypsin), the fast skeletal muscle troponin T3 (Tnnt3), and two extracellular matrix-related genes fibronectin 1 (Fn1) and collagen 1a (Col1a) were increased, whereas they were not modified in the GRcardio model (Fig. 4). Specific down-regulation of genes in MRcardio mice, but not in GRcardio mice, was confirmed for pyruvate dehydrogenase, isocitrate dehydrogenase, vascular endothelial growth factor, and myeloid leukemia factor 1 (Supplemental Fig. 3).

Figure 4.

Figure 4

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Validation of differentially expressed genes identified in microarray analysis in the heart of mice with cardiac overexpression of MR. Values of mRNA levels (real time PCR) were normalized for ubc expression. Values in control mice were set as 1 for each gene, and fold changes are shown on the figure; data are provided as mean ± sem (n = 3–8 mice per condition). *, P < 0.05, **, P < 0.01 transgenic mice compared with their own controls (Mann-Whitney U test).

Figure 5.

Figure 5

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Differentially expressed genes identified in the heart of mice with cardiac overexpression of MR are also expressed in H9C2/MR+ cells. Cells were treated with 10−8 m aldosterone (Aldo) (A) or 10−8 m corticosterone (Cortico) (B) alone for 24 h or in association with MR antagonist RU28318 (10−6 m) or GR antagonist RU38486 (10−6 m). Values of mRNA levels (real time PCR) were normalized for β-actin expression. Values in control (nontreated) cells were set as 1 for each gene, and fold changes are shown on the figure; data are provided as mean ± sem (n = 4–12 wells per condition). *, P < 0.05, **, P < 0.01 treatment vs. control; #, P < 0.05, ##, P < 0.01 treatment vs. Aldo or Cortico alone (ANOVA analysis, Newman and Keuls test).

In H9C2/MR+ cells, the expression of Serpina-3 was enhanced by treatment with aldosterone, whereas Tnnt3, Fn1, and Col1a were unaffected (Fig. 5). Serpina-3 is a mineralocorticoid-specific regulated transcript because its induction by aldosterone is reduced in the presence of the specific MR antagonist RU28318 but not in the presence of the GR antagonist RU38486. Corticosterone had comparable effects, indicating that Serpina-3 may be regulated by the MR occupied by aldosterone as well as by a glucocorticoid hormone. In this cell model, Serpina-3 up-regulation by aldosterone occurred after 10 h exposure to aldosterone, i.e. not in the initial phase of hormonal response (late response gene); Adamts1 expression was enhanced much earlier (after 2 h of hormone addition), suggesting that it may be a primary MR target (Fig. 6). Corticosterone also increased Serpina-3 and Adamts1 with the same time course (Fig. 6). Thus, Serpina-3 mRNA is induced 10–24 h after MR activation by aldosterone in H9C2/MR+ cells as well as in vivo after chronic MR overexpression in cardiomyocytes. In MRcardio mice, both doxycycline treatment, which blunts MR overexpression, and canrenoate treatment, which blocks MR activity, prevented the rise in cardiac Serpina-3 expression (Fig. 7A). The up-regulation of Serpina-3 was also observed at the protein level in the heart of MRcardio mice (Fig. 7B).

Figure 6.

Figure 6

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Time course of aldosterone and corticosterone effects on the expression of Serpina-3 and Adamts1 in H9C2/MR+ cells. Cells were incubated with 10−8 m aldosterone (Aldo; closed symbols) or corticosterone (open symbols); Serpina-3 (A) and Adamts1 (B) mRNA were measured by real-time PCR. Values of mRNA levels were normalized for β-actin expression. Values in control (nontreated) cells were set as 1 for each gene, and fold changes are shown on the figure; data are provided as mean ± sem (n = 4 wells per condition). *, P < 0.05, **, P < 0.01 aldo vs. control (ANOVA analysis, Newman and Keuls test).

Figure 7.

Figure 7

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Serpina-3 expression in the heart of control-MR and MRcardio mice. A, Animals were treated by doxycycline (DOX) to switch off the transgene or by potassium canrenoate (CANRE), a MR antagonist. Values of Serpina-3 mRNA levels (real time PCR) were normalized for ubc expression. Values in control mice were set as 1, and fold changes are shown on the figure; data are provided as mean ± sem (n = 5–6 mice per condition). B, Western blot analysis shows increased Serpina-3 expression in the heart of 1.5-month-old MRcardio mice compared with control littermates. β-Actin was used as internal control. The signal was quantified and values are provided as mean ± sem (n = 6 mice per group). *, P < 0.05; **, P < 0.01. MRcardio vs. control-MR mice.

Discussion

This study was designed to identify cardiac genes that are specifically regulated through the MR pathway. In the literature, previous searches for cardiac aldosterone-responsive genes focused on the relatively early effects of the hormone. After in vivo injections of aldosterone to uninephrectomized salt-loaded mice, Turchin et al. (14) showed that a group of genes were transiently down-regulated, whereas no induced genes were identified. After incubation of neonatal mouse cardiomyocytes in primary culture with high doses of aldosterone (occupying both the MR and GR), about 50 up-regulated and 50 down-regulated genes were revealed (15). Modulated genes were more likely GR rather than MR dependent, as shown for the ectoribosyltransferase 3 (15). Early aldosterone-regulated genes have also been investigated using a clonal cardiomyocyte cell line stably expressing MR [H9C2/MR+ cells (16)]; 48 up-regulated and five down-regulated genes have been identified in the presence of physiological concentration of aldosterone (1 nm) for 2 h. Yoshikawa et al. (17) also used neonatal cardiomyocytes (from rat) and identified a large series of glucocorticoid- and aldosterone (100 nm)-regulated genes. These results showed limited or no overlap between studies, and this was also the case when compared with our own transcriptomic approach. This is probably due to the different hormonal conditions used in different cell or animal models, including comparison of relatively short-term responses (few hours hormone treatment) with long-term enhanced MR expression.

We used two strategies to assess MR- vs. GR-specific responses. The use of pharmacological MR/GR agonists/antagonists to analyze gene regulation and specificity in cultured cells is a classical and relatively easy approach, but it is limited by the cross-reactivity of these pharmacological agents with MR and GR. As a matter of fact, aldosterone treatment may result in both MR and GR activation when used at 10 nm; glucocorticoids can also bind to the MR (in the absence of the MR protecting enzyme HSD2). Classical MR antagonists like spironolactone may also block the GR when used at high doses (29). In vivo, the use of cardiomyocyte-specific MR vs. GR overexpression in mice has the major advantage of selectively modifying one receptor in a chosen cell type without affecting the endogenous ligands. Although the nature of the ligand that occupies cardiac MR in vivo cannot be ascertained, it is conceivable that the gene regulation observed in MRcardio mice depend on MR occupied by glucocorticoids because HSD2 is expressed at low level in cardiomyocytes. It should be noted that the level of receptor mRNA overexpression in these two mouse models is moderate. Interestingly, enhanced receptor mRNA levels were reported in some pathological situations: in animal models, and humans (30,31,32,33). It is difficult to compare directly the expression levels of MR or GR in our models with those described in the literature because the protein level or binding activity could not be assessed in our study. In addition, the translation efficiency or stability of the recombinant receptor may be different from that of the endogenous transcripts. The mostly distinct transcriptional regulation induced by MR vs. GR in the same cellular context raise the question of additional mechanisms of specificity such as differential interactions of these two receptors with nuclear coregulators such as protein inhibitor of activated signal transducer and activator of transcription (PIAS1) or eleven-nineteen lysine-rich leukemia (ELL) (6). Indeed, PIAS1 was shown to be a corepressor of MR transactivation, and ELL is a MR-specific (not GR) coactivator. Whether and how such coregulators participate in vivo to the distinct MR/GR effects remain to be established.

Two main findings arose from this study: 1) in the heart, most MR-regulated genes differ from GR-regulated ones, with a limited overlap, suggesting a specific role of MR activation in the heart, and 2) some MR/aldosterone-regulated genes are found in both the early hormonal response in the cell model and after long-term MR overexpression in the mouse model.

Table 2 compares expression patterns that are modified by aldosterone in H9C2/MR+ and the heart of mice after MR overexpression. Transcripts of the early cellular response phase to aldosterone, Sgk1 and Tnx, are not enhanced in MRcardio mouse model, indicating that these genes do not participate in the long-term adaptation to receptor activation. Sgk1 is the best-characterized aldosterone-induced protein in epithelial cells, such as the renal collecting duct, in which its expression is transiently induced by corticosteroids bound to the MR and GR (34,35,36). Tenascins are glycoproteins that modulate cell adhesion to the extracellular matrix (37). Tenascin X is expressed at high levels in the heart and other tissues and has been shown to be down-regulated by glucocorticoids in fibroblasts but not carcinoma cells (38). The in vivo increase of Tnnt3 (a fast skeletal muscle protein) and Fn1/Col1a (encoding for proteins of the extracellular matrix) may correspond to structural adaptation to enhanced cardiac MR signaling. One limitation of using the H9C2/MR+ cell line is that it exhibits features of cardiomyocytes but also skeletal muscle cells. However, in the absence of cardiac cell line with all properties of cardiomyocytes, the H9C2-MR+ model appears as a useful tool.

Table 2.

Comparison of gene expression in H9C2/MR+ cells and in the heart of mice overexpressing the MR (MRcardio)

H9C2/MR+ cells MRcardio mice
Ex vivo In vivo
↑ Sgk1 →
↑ Adamts1 ↑
↑ PAI-1 ↑
↑ Rgs2 ↑
↑ Fkbp5 ↑
↑ Tnx →
↑ Serpina-3 ↑
→ Troponin-T3 ↑
→ Fibronectin1 ↑
→ Collagen 1a ↑
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Among the mineralocorticoid-specific selected transcripts, five are up-regulated in both the cell line and the MRcardio mouse model. Some of these (Rgs2, Adamts1, and PAI-1) were previously found to be up-regulated by aldosterone in H9C2/MR+ cells (16). Rgs2 is a multiple regulator and is involved in cardiovascular physiopathology (39). Fkbp5 is a protein that can bind immunosuppressive drugs used to improve efficiency of organ transplant. Fkbp5 is a cochaperone of several steroid receptors including the MR and thus may influence its functions (40). Three genes have close connections with the regulation of the extracellular matrix. Adamts1 (41) is a zinc metalloprotease involved in tissue remodeling and angiogenesis as well as a number of other pathological processes. An increase in its expression has been documented in the heart in the acute phase after coronary artery ligation, preceding the changes in matrix metalloproteinases (42). PAI-1, a member of the serpin family, is the major physiological inhibitor of tissue-type plasminogen activator and urokinase-type plasminogen activator, which activate plasminogen to its active form plasmin and lead to fibrinolysis. PAI-1 is involved in numerous pathophysiological processes, including cardiovascular injury, cell-matrix interactions, and aldosterone-related cardiovascular damage (43,44). Serpina-3 (also named α1-antichymotrypsin) is a serine protease inhibitor with multiple functions including maturation of pro-matrix metalloproteinase 9 and wound healing, regulation of apoptosis, infectious diseases, and inflammation (45). Serpina-3, as a member of acute-phase reaction, is also regulated by many stimuli including insulin and growth factors; it belongs to the genes that have been identified previously in different microarrays in the failing myocardium as reviewed by Asakura and Kitakaze (46). Because we show that Serpina-3 expression in the heart is linked to MR activation, it will be interesting to determine whether Serpina-3, which can be secreted in the blood (47), could serve as a biomarker of mineralocorticoid-related cardiac damage. Several genes linked to extracellular matrix remodeling are up-regulated in MRcardio mice. This finding contrasts with the absence of major morphological alterations in these mice (12). However, fibrosis has been evidenced when these mice are challenged with angiotensin II, suggesting that MR activation on top of a pharmacological challenge may precondition the cardiac tissue and favor cardiac remodeling and dysfunction (48).

Of note is that our transcriptomic screening did not evidence any significant change in cardiac ion channel expression between control and MRcardio mice that have been shown to display abnormal Ca transients and ion channel activities (11,12); this may be due to the low level of expression of these channels. Alternatively, channel activities may be linked to posttranscriptional events regulated by MR.

In conclusion, we have identified genes that are up-regulated in the heart after chronic MR overexpression. The signaling pathways that are affected by long-term stimulation of MR may be of particular interest to design novel therapeutic targets in cardiac diseases.

Supplementary Material

[Supplemental Data]
en.2010-0237_index.html (770B, html)

Footnotes

This work was supported by the Institut National de la Santé et de la Recherche Médicale, the Agence Nationale pour la Recherche Grants ANR 005-PCOD005 and ANR-09-BLANC0156-01, the Fondation de France, the Centre de Recherche Industrielle et Technique, the European Section of the Aldosterone Council, and National Institutes of Health Grants DK41841 and HL085024. C.L. was recipient of a PhD grant from the Ministere de la Recherche; Y.S.-M. was supported by the Convention Industrielle de Formation per la Recherche PhD program of Agence Nationale de Valorisation de la Recherche and Nucleis Société Anonyme (Lyon, France).

Disclosure Summary: The authors have nothing to declare.

First Published Online June 30, 2010

Abbreviations: Col1a, Collagen 1a; Fkbp5, FK506 binding protein; Fn1, fibronectin 1; GR, glucocorticoid receptor; h, human; HSD2, 11β-hydroxysteroid dehydrogenase-2; MHC, myosin heavy chain; MR, mineralocorticoid receptor; PAI, plasminogen activator inhibitor; Tnnt3, troponin T3; ubc, ubiquitin C.

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