The mineralocorticoid receptor promotes fibrotic remodeling in atrial fibrillation

Abstract

We studied the role of the mineralocorticoid receptor (MR) in the signaling that promotes atrial fibrosis. Left atrial myocardium of patients with atrial fibrillation (AF) exhibited 4-fold increased hydroxyproline content compared with patients in sinus rhythm. Expression of MR was similar, as was 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which also increased. 11β-HSD2 converts cortisol to receptor-inactive metabolites allowing aldosterone occupancy of MR. 11β-HSD2 was up-regulated by arrhythmic pacing in cultured cardiomyocytes and in a mouse model of spontaneous AF (RacET). In cardiomyocytes, aldosterone induced connective tissue growth factor (CTGF) in the absence but not in the presence of cortisol. Hydroxyproline expression was increased in cardiac fibroblasts exposed to conditioned medium from aldosterone-treated cardiomyocytes but not from cardiomyocytes treated with both cortisol and aldosterone. Aldosterone increased connective tissue growth factor and hydroxyproline expression in cardiac fibroblasts, which were prevented by BR-4628, a dihydropyridine-derived selective MR antagonist, and by spironolactone. Aldosterone activated RhoA GTPase. Rho kinase inhibition by Y-27632 prevented CTGF and hydroxyproline, whereas the RhoA activator CN03 increased CTGF expression. Aldosterone and CTGF increased lysyl oxidase, and aldosterone enhanced miR-21 expression. MR antagonists reduced the aldosterone but not the CTGF effect. In conclusion, MR signaling promoted fibrotic remodeling. Increased expression of 11β-HSD2 during AF leads to up-regulation of collagen and pro-fibrotic mediators by aldosterone, specifically RhoA activity as well as CTGF, lysyl oxidase, and microRNA-21 expression. The MR antagonists BR-4628 and spironolactone prevent these alterations. MR inhibition may, therefore, represent a potential pharmacologic target for the prevention of fibrotic remodeling of the atrial myocardium.

Keywords: CTGF; Fibrosis; Heart; Lysyl Oxidase; MicroRNA; Mineralocorticoid Receptor.

FIGURE 1.

Fibrosis and the mineralocorticoid receptor in human left atrium. Samples from patients with AF or SR are matched for atrial size. A, representative Sirius red staining of SR and AF patients; 10- and 100-fold magnification. B, quantification of fibrosis by Sirius red staining. C, content of hydroxyproline assessed by hydroxyproline assay in LA. Shown are mRNA expression (RT-PCR) (D) and protein expression (Western blot) (E) of mineralocorticoid receptor in LA of SR and AF patients. F, protein expression of 11β-HSD2 in LA. *, p < 0.05.

FIGURE 2.

SPARC expression in human left atria and correlations with mediators of fibrosis. A, immunohistochemical staining for SPARC (TRITC, red) and vimentin (lower panel, green, FITC) or α-sarcomeric actin (upper panel, green, FITC) in LA (10-fold magnification). Both cardiac myocytes (α-sarcomeric actin-positive) and fibroblasts (vimentin-positive) express SPARC. B, quantification of SPARC protein expression in LA. C–H, correlations between hydroxyproline expression in human LA and 11β-HSD2 (C), CTGF (D), SPARC (E), miR-21 (F), Sprouty-1 (G), and RhoA (H). *, p < 0.05.

FIGURE 3.

Expression of hydroxyproline, MR and CTGF in cultured rat cardiac fibroblasts. A, MR mRNA expression in cells treated with aldosterone (Aldo, 10⁻⁸ m, 24 h), TGF-β (5 ng/ml, 24 h), or vehicle as control. B, hydroxyproline concentration in supernatants of fibroblasts using hydroxyproline assay. Spirono, spironolactone. C, aldosterone concentration curve (1, 10, and 100 nm, all 24 h) regarding CTGF expression. D, CTGF expression in fibroblasts treated with either with BR-4628, spironolactone, or vehicle as control. Shown are CTGF protein expression in fibroblasts treated with aldosterone (10⁻⁸ m, 24 h) and preincubation with BR-4628 (500 nm, 25 h) (E) or spironolactone (500 nm, 25 h) (F). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 4.

RhoA/Rho kinase pathway mediates fibrotic remodeling downstream of MR in cardiac fibroblasts. A, CTGF protein expression in fibroblasts treated with TGF-β (5 ng/ml, 24 h) with and without preincubation with BR-4628. B, CTGF protein expression in fibroblasts treated with TGF-β (5 ng/ml, 24 h) with and without preincubation with Y-27632 (10 μm, 25 h), a Rho kinase inhibitor. C, CTGF protein expression after aldosterone (Aldo) treatment with and without preincubation of Y-27632. D, CTGF protein expression in fibroblasts treated with RhoA activator CN03 (1 μg/ml, 3 h) only and with preincubation with BR-4628 or spironolactone (Spirono). RhoA activity (pulldown assay) (E) and RhoA total protein expression (Western blot) (F) in aldosterone treatment in cardiac fibroblasts. G, hydroxyproline concentration in aldosterone-treated cardiac fibroblasts with and without preincubation with Y-27632. H, CTGF mRNA expression (RT-PCR) in cardiac fibroblasts incubated with aldosterone and BR-4628, spironolactone, or Y-27632 pretreatment, respectively. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 5.

Regulation of LOX and miR-21 via MR in cardiac fibroblasts. A, LOX expression in CTGF-treated (1 ng/ml, 1 h) fibroblasts with or without pretreatment with BR-4628 or spironolactone (Spirono). LOX expression in cardiac fibroblasts incubated with aldosterone (Aldo) and pretreated with either BR-4628 (B) or spironolactone (C) is shown. D, LOX mRNA expression (RT-PCR) in cardiac fibroblasts incubated with aldosterone and BR-4628, spironolactone, or Y-27632 pretreatment. E, microRNA-21 (miR-21) expression quantified by TaqMan-PCR in fibroblasts treated with aldosterone, aldosterone and BR-4628 or aldosterone and spironolactone. F, protein expression of the miR-21 downstream target Sprouty-1 in cells treated with aldosterone, aldosterone and BR-4628, or aldosterone and spironolactone. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 6.

Role and regulation of 11β-HSD2 in neonatal cardiomyocytes and transgenic RacET mice. A, CTGF protein expression in myocytes treated with aldosterone (Aldo, 10⁻⁸ m, 24 h), hydrocortisone (127 ng/ml, 24 h), or both. B, hydroxyproline concentration in cardiac fibroblasts 24 h after the addition of conditioned medium from cardiomyocytes, which were cultured with aldosterone (10⁻⁸ m, 24 h), hydrocortisone (12.7 μg/ml, 24 h), or both. MR (C) and 11β-HSD2 (D) protein expression in neonatal cardiac myocytes paced rhythmic or arrhythmic at 3 Hz for 24 h. MR (RT-PCR) (E) and 11β-HSD2 (F) mRNA expression (TaqMan-PCR) in neonatal cardiac myocytes paced rhythmic or arrhythmic (3 Hz, 24 h). Protein expression of MR (G) and protein expression of 11β-HSD2 (H) in atria of 12-month-old transgenic RacET compared with wild type control mice is shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001.