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. 2010 Sep;151(9):4467-76.
doi: 10.1210/en.2010-0237. Epub 2010 Jun 30.

Molecular signature of mineralocorticoid receptor signaling in cardiomyocytes: from cultured cells to mouse heart

Celine Latouche  1 , Yannis Sainte-Marie, Marja Steenman, Paulo Castro Chaves, Aniko Naray-Fejes-Toth, Geza Fejes-Toth, Nicolette Farman, Frederic Jaisser
Affiliations

Molecular signature of mineralocorticoid receptor signaling in cardiomyocytes: from cultured cells to mouse heart

Celine Latouche et al. Endocrinology. 2010 Sep.

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 (alpha1-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.

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Figures

Figure 1
Figure 1
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).
Figure 2
Figure 2
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).
Figure 3
Figure 3
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.
Figure 4
Figure 4
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
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).
Figure 6
Figure 6
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
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.
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