Inhibition of aldosteronsynthase regulates miR-21 during atrial fibrillation in mice

Abstract

Introduction: Early studies have suggested that torasemide may influence fibrosis in the left ventricle by antialdosteronergic effects. The role of torsemide for structural remodeling during atrial fibrillation (AF) is unknown. Methods and results: 8 month old transgenic mice with cardiac overexpression of Rac1 GTPase (RacET) which develop spontaneous AF are characterized by increased atrial fibrosis, increased protein expression of the profibrotic cytokine connective tissue growth factor (CTGF, 257±77%), the key enzyme of collagen crosslinking lysyl-oxidase (LOX, 195±24%) and the fibrosis regulator microRNA-21 (miR-21, 252±43%) compared to wildtypes (WT). Long-term treatment with torsemide (10mg/kg/day) for 8 months prevented atrial fibrosis in RacET as well as the upregulation of CTGF (62±18%), LOX (124±23%) and miR-2 (68±7%) compared to vehicle, whereas Rac1 expression and activity was unaffected. Mineralcorticoide receptor expression was not altered by torsemide. There was no change of blood pressure but a significant reduction of heart rate in torsemide treated RacET associated with a reduced prevalence of atrial fibrillation (38% RacET+Tora vs. 70% RacET). Interestingly, in vitro studies of V97MZ cells (lung fibroblasts without endogenous aldosteronsynthase) transfected with humam aldosteronsynthase (CYP11B2) showed that torsemide inhibited CYP11B2 activity by 75±1.8%, most likely through a competitive inhibition of CYP11B2 by binding of torsemide to the heme binding site of CYP11B2 through its nitrogen ring. In order to test the underlying mechanism, primary neonatal cardiac fibroblasts were stimulated with angiotensin II (Ang; 1μM; 3 hours) and preincubated with or without the CYP11B2 specific inhibitor SL 242. SL 242 (1μM; 24 hours) reduced the expression of CTGF, LOX and miR-21 whereas Rac1 expression and activity was unaffected. All effects are significant with p<0.05. Conclusion: Torsemide treatment was associated with inhibition of aldosteronsynthase (CYP11B2), preventions of atrial fibrosis and reduced the prevalence of atrial fibrillation in RacET mice. These effects were mediated through reduced expression of the profibrotic regulators CTGF, LOX and miR-21.