Aldosterone Inhibits the Fetal Program and Increases Hypertrophy in the Heart of Hypertensive Mice

<div><h3>Background</h3><p>Arterial hypertension (AH) induces cardiac hypertrophy and reactivation of “fetal” gene expression. In rodent heart, alpha-Myosin Heavy Chain (MyHC) and its micro-RNA miR-208a regulate the expression of beta-MyHC and of its intronic miR-208b. However, the role of aldosterone in these processes remains unclear.</p> <h3>Methodology/Principal Findings</h3><p>RT-PCR and western-blot were used to investigate the genes modulated by arterial hypertension and cardiac hyperaldosteronism. We developed a model of double-transgenic mice (AS-Ren) with cardiac hyperaldosteronism (AS mice) and systemic hypertension (Ren). AS-Ren mice had increased (x2) angiotensin II in plasma and increased (x2) aldosterone in heart. Ren and AS-Ren mice had a robust and similar hypertension (+70%) versus their controls. Anatomical data and echocardiography showed a worsening of cardiac hypertrophy (+41%) in AS-Ren mice (P<0.05 vs Ren). The increase of ANP (x 2.5; P<0.01) mRNA observed in Ren mice was blunted in AS-Ren mice. This non-induction of antitrophic natriuretic peptides may be involved in the higher trophic cardiac response in AS-Ren mice, as indicated by the markedly reduced cardiac hypertrophy in ANP-infused AS-Ren mice for one month. Besides, the AH-induced increase of ßMyHC and its intronic miRNA-208b was prevented in AS-Ren. The inhibition of miR 208a (−75%, p<0.001) in AS-Ren mice compared to AS was associated with increased Sox 6 mRNA (x 1.34; p<0.05), an inhibitor of ßMyHC transcription. Eplerenone prevented all aldosterone-dependent effects.</p> <h3>Conclusions/Significance</h3><p>Our results indicate that increased aldosterone in heart inhibits the induction of atrial natriuretic peptide expression, via the mineralocorticoid receptor. This worsens cardiac hypertrophy without changing blood pressure. Moreover, this work reveals an original aldosterone-dependent inhibition of miR-208a in hypertension, resulting in the inhibition of β-myosin heavy chain expression through the induction of its transcriptional repressor Sox6. Thus, aldosterone inhibits the fetal program and increases cardiac hypertrophy in hypertensive mice.</p> </div

Effect of aldosterone on CREB expression.

<p>(<b>A</b>): quantitative RT-PCR analysis of CREB in 9 month-old mouse ventricles and (<b>B</b>) representative immunoblots of p-CREB, CREB and GAPDH levels in ventricle protein extracts. Abbreviations and symbols as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038197#pone-0038197-g001" target="_blank">Figure 1</a>.</p

Cardiac hyperaldosteronism inhibits hypertension-induced β-MyHC expression.

<p>The expression of β-MyHC was analysed in 9 month-old mice by: (<b>A</b>) quantitative RT-PCR in ventricles of control mice (WT, AS), of Ren mice and AS-Ren mice untreated or treated with eplerenone (100 mg/kg/day) for 30 days, (<b>B</b>) quantification of β-MyHC and total MyHC protein levels in ventricles after immunoblot (representative signals are shown), (<b>C</b>) quantitative RT-PCR in NMCM stimulated by 10⁻⁵M AngII in the absence or presence of aldosterone (data are mean of 3 independent experiments), Abbreviations and symbols as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038197#pone-0038197-g001" target="_blank">Figure 1</a>. n = 10–12/group.</p

Anatomy and cardiac function.

<p><u>Abbreviations:</u> LVPW: Left Ventricular Posterior Wall thickness; IVSd: InterVentricular Septum thickness in diastole; SF: Shortening Fraction; BW: Body Weight; HW: Heart Weight.</p>*<p>p<0.05.</p>**<p>p<0.01.</p>***<p>p<0.001 vs WT.</p>£<p>p<0.05 vs AS.</p>$<p>p<0.05 vs Ren.</p

Effect of combined cardiac aldosterone and hypertension on cardiac miR-208 and Sox6 expression.

<p>(<b>A</b>): scheme of the interplay of miR-208a and -208b on the regulation of <i>Myh7</i> expression in hypertensive heart, as drawn from literature data. (<b>B-D</b>): quantitative RT-PCR analysis of miR-208a, Sox6 and miR-208b expression in 9 month-old WT, AS, R and AS-R mice and in AS-R eplerenone treated mice (100 mg/Kg/day) for one month. (<b>E</b>): quantitative RT-PCR analysis of Sox6 expression in NMCM stimulated by 10⁻⁵M AngII in the presence or absence of aldosterone (10⁻⁹ M or 10⁻⁷ M). Abbreviations and symbols as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038197#pone-0038197-g001" target="_blank">Figure 1</a>; HT: hypertension; n = 10–14/group.</p