Aldosterone Nongenomically Produces NADPH Oxidase–Dependent Reactive Oxygen Species and Induces Myocyte Apoptosis

363 Hypertens Res Vol.31 (2008) No.2 p.363-375 Original Article Aldosterone Nongenomically Produces NADPH Oxidase–Dependent Reactive Oxygen Species and Induces Myocyte Apoptosis Hironori HAYASHI1),2),*, Miyuki KOBARA2),*, Masaki ABE2), Nana TANAKA2), Eri GOUDA2), Hiroe TOBA2), Hiroyuki YAMADA1), Tetsuya TATSUMI1), Tetsuo NAKATA2), and Hiroaki MATSUBARA1) The roles of aldosterone in the progression of heart failure have not been fully elucidated. This study examined whether aldosterone nongenomically activates reactive oxygen species (ROS) production, causing myocyte apoptosis. Addition of aldosterone to neonatal rat cardiac myocytes caused the activation of NADPH oxidase and intracellular ROS production in a dose-dependent manner (10 – 9–10 – 7 mol/L). NADPH oxidase activation was evident as soon as 5 min after aldosterone treatment. Neither an inhibitor for nuclear transcription (actinomycin D) nor an inhibitor of new protein synthesis (cycloheximide) blocked this rapid activation, and specific binding of aldosterone to plasma membrane fraction was inhibited by eplerenone, suggesting a nongenomic mechanism. Aldosterone did not affect the mRNA or protein levels of NOX2, which is a major subunit of NADPH oxidase in myocytes, after 48 h. Nuclear staining with DAPI showed that aldosterone (10 – 7 mol/L) increased the myocyte apoptosis (2.3 fold, p < 0.001), coincident with the activation of caspase-3 (1.4 fold, p < 0.05), compared with the serum-deprived control after 48 h. Aldosterone also induced phosphorylation of apoptosis signal–regulating kinase 1 (ASK1). These effects of aldosterone on myocyte ROS accumulation, ASK1 activation, and apoptosis were abolished by eplerenone, a mineralocorticoid receptor (MR) antagonist, apocynin, an inhibitor of NADPH oxidase activation, and tempol, a free radical scavenger, but by neither RU486, a glucocorticoid receptor antagonist, nor butylated hydroxyanisol (BHA), a mitochondrial ROS scavenger. In conclusion, aldosterone-mediated ROS production is blocked by eplerenone and induced by the nongenomic activation of NADPH oxidase, leading to myocyte apoptosis associated with ASK1 activation. These proapoptotic actions of aldosterone may play a role in the progression of heart failure. (Hypertens Res 2008; 31: 363–375) Key Words: aldosterone, myocyte, apoptosis, heart failure, reactive oxygen species Introduction Aldosterone is a major mineralocorticoid hormone secreted by the adrenal cortex, and a neurohormonal mediator of the renin-angiotensin-aldosterone system. Classically, it regu- lates electrolyte homeostasis and fluid balance by promoting sodium retention and potassium excretion in the kidney. Recent studies, however, have suggested that aldosterone is synthesized locally in the heart (1, 2) and have revealed that aldosterone receptor (mineralocorticoid receptor [MR]) is present in rodent hearts (3, 4) and human hearts (5). From the 1)Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan; and 2)Department of Clinical Pharmacology, Kyoto Pharmaceutical University, Kyoto, Japan. *These authors contributed equally to this study. Address for Reprints: Miyuki Kobara M.D., Ph.D., Department of Clinical Pharmacology, Kyoto Pharmaceutical University, 5 Misasagi Nakauchi-cho, Yamashina-ku, Kyoto 607–8414, Japan. E-mail: Received January 11, 2007; Accepted in revised form September 6, 2007. 364 100 50 0 Cont 10-9 Aldo 10-8 (%) 250 200 (% of Cont) 150 NADPH-stimulated O2- production B (%) 200 (% of Cont) NADPH-stimulated O2- production A Hypertens Res Vol. 31, No. 2 (2008) 150 100 50 0 Cont 5 min 30 min 60 min 24 h 10-7 (mol/L) Aldo 10-7 mol/L C (%) 160 140 (% of Cont) NADPH-stimulated O2- production 48 h 120 100 80 60 40 20 0 Cont +EPL +RU +APO +BHA COR EPL RU APO BHA Aldo 10-7 mol/L Fig. 1. Effect of aldosterone (Aldo) on NADPH oxidase activity in myocytes. NADPH oxidase–dependent O2− production was measured by lucigenin-enhanced chemiluminescence. A: Myocytes were treated with the indicated concentrations of Aldo (10 − 9– 10 − 7 mol/L) for 30 min (n= 7–8). ‡p< 0.001 vs. control (Cont). B: Myocytes were treated with 10 − 7 mol/L Aldo for the indicated time periods (n= 5–7). *p < 0.05, ‡p< 0.001 vs. Cont. C: Myocytes were stimulated by Aldo (10 − 7 mol/L) or corticosterone (COR, 10 − 7 mol/L) for 30 min in the presence or absence of eplerenone (EPL, 10 μmol/L), RU486 (RU, 10 μmol/L), apocynin (APO, 300 μmol/L), or butylated hydroxyanisol (BHA, 10 μmol/L) (n = 5–7). *p < 0.01 vs. Cont; †p< 0.01 vs. Aldo. Data are expressed as the relative difference compared with Cont. Recently, two large clinical trials demonstrated that MR antagonists improve morbidity and mortality in patients with heart failure (6, 7). Local aldosterone production in the heart tissue (1, 8) as well as the plasma aldosterone level (9, 10) have been reported to be increased in patients with heart failure. The plasma aldosterone level also positively correlates with the mortality in patients with heart failure (9). Furthermore, recent reports provide evidence that excess aldosterone has direct adverse effects on the heart (11–14). However, the underlying mechanisms of these deleterious effects have not been fully defined. Reactive oxygen species (ROS) are exacerbating factors in the progression of heart failure (15). Experimental and clinical studies have demonstrated that excessive levels of ROS are produced in failing hearts, and are associated with the severity of heart failure (16–18). In addition, ROS are important apoptosis inducers (19), and progressive loss of cardiac myocytes by apoptosis has a critical role in the progression of heart failure (20). One of the major sources of ROS in the cardiovascular system is NADPH oxidase (21–23), and its activity is upregulated in failing hearts (22, 23). Aldosterone has been reported to activate NADPH oxidase in several kinds of cells in vitro (24, 25). Chronic aldosterone infusion has been shown to increase NADPH oxidase activity in the heart tissue in vivo, leading to enhancement of cardiac oxidative stress and cardiac fibrosis (11, 26). Aldosterone action is known to be induced via a classical genomic mechanism, whereas we have observed the nongenomic (rapid) action of aldosterone in cardiac myocytes (27). Aldosterone-induced NADPH oxidase activation has been reported to be a nongenomic action in renal mesangial cells and vascular smooth muscle cells (24, 25). It is not fully understood whether aldosterone nongenomically stimulates NADPH oxidase, followed by the promotion Hayashi et al: Aldosterone-Induced ROS Promotes Myocyte Apoptosis 365 A a b c Cont Aldo 10-8 10-9 B a b Cont c 5 min 10-7 30 min 300 200 100 0 10 -9 Aldo 10-8 10 -7 (mol/L) e 60 min D (%) 400 Cont (mol/L) d Relative DCF fluoresence (% of Cont) Relative DCF fluoresence (% of Cont) C d f 24 h 48 h (%) 700 600 500 400 300 200 100 0 Cont (...truncated)