Role of Rho kinase and oxidative stress in cardiac fibrosis induced by aldosterone and salt in angiotensin type 1a receptor knockout mice

Large clinical trials have shown that mineralocorticoid receptor (MR) antagonists improve cardiovascular or total mortality in patients with heart failure or myocardial infarction even though the patients were taking angiotensin-converting enzyme inhibitors or angiotensin II receptor (AT1R) antagonists. We previously reported that cardiac fibrosis induced by aldosterone and salt (Ald-NaCl) was exaggerated in AT1aR knockout mice (AT1aR-KOs). As the association of Rho kinase and oxidative stress was reported in Ald-NaCl-induced hypertension of rats, we investigated the effects of an MR antagonist (eplerenone) and a Rho kinase inhibitor (fasudil) on Ald-NaCl-induced cardiac fibrosis in AT1aR-KOs. AT1aR-KOs were administered aldosterone (0.15 μg/h) subcutaneously using an osmotic minipump and were provided with 1% NaCl drinking water for 4 weeks. AT1aR-KOs receiving Ald-NaCl were treated with a low (30 mg/kg/day) or high (100 mg/kg/day) dose of eplerenone or a fasudil (100 mg/kg/day). Systolic blood pressure (SBP), left ventricular weight/body weight (LVW/BW), histological examination and cardiac gene expression were evaluated on day 28. Ald-NaCl treatment caused increases in SBP and LVW/BW in AT1aR-KOs, and eplerenone dose-dependently decreased SBP, LVW/BW and cardiac fibrosis. Fasudil decreased LVW/BW and cardiac fibrosis without affecting SBP. The expressions of connecting tissue growth factor (CTGF) and nicotinamide adenine dinucleotide phosphate (NADPH) components (p22phox, p47phox and p67phox) were increased in Ald-NaCl-treated AT1aR-KOs, and eplerenone or fasudil decreased the expression of CTGF and NADPH components. Phosphorylated ERM (a marker of the phosphorylation of Rho kinase) was increased in Ald-NaCl-treated AT1aR-KOs and was decreased by eplerenone. Nitrotyrosine and 4-hydroxy-2-nonenal, which indicate tissue damage via oxidative stress, were increased in AT1aR-KO and were apparently attenuated by eplerenone or fasudil. These results suggested that the Rho kinase pathway was activated to induce cardiac fibrosis by Ald-NaCl via MR in AT1aR-KOs. A Rho kinase inhibitor as well as eplerenone might be useful for cardiac damage by Ald-NaCl.