Supplementary Methods

1. Supplementary Methods Benidipine Reduces Ischemia Reperfusion Induced Systemic Oxidative Stress through Suppression of Aldosterone Production in Mice Keisuke Ohtani1, Soichiro Usui1, Shuichi Kaneko1, Shin-ichiro Takashima1, Katsunori Kitano1, Kanako Yamamoto1, Masaki Okajima1, Hiroshi Furusho1, Masayuki Takamura1 1Department of Disease Control and Homeostasis, Graduate School of Medical Science, Kanazawa University, Running title: Benidipine inhibits ischemia reperfusion injury in hearts

2. Supplementary Methods Determination of myocardial infarct size At the end of the 24 h reperfusion period, after an overdose of 10% sodium pentobarbital (70 mg/kg i.p.), the chest was opened and the heart was re-exposed. The ligature around the coronary artery was retied and 1 ml 3% Evans blue dye (Sigma Chemical Co.) was injected into the left ventricular cavity. The dye was circulated and distributed uniformly except in the region of the heart that had been perfused previously by the occluded coronary artery. Slices were incubated individually with 2% triphenyl tetrazolium chloride (TTC) for 10 min at 37°C to distinguish the stained viable tissue from the unstained infarcted area. The area-at-risk (AAR) and the areas that were stained with TTC (ischemic but viable tissue) and not stained with TTC (infarct myocardium) were measured digitally using Image J software (NIH, USA). MI size was expressed as the percentage of infarct area (IA) relative to the total AAR1. Echocardiographic assessment of LV function Mice were lightly anesthetized at 7 days after operation with a half dose of 10% sodium pentobarbital2 (30 mg/kg i.p.). We performed transthoracic echocardiography on the LV using a 15-8L MHz transducer interfaced with a Sonos 5500 (Agilent Technology, Philips). The LV end-diastolic diameters (LVEDD), LV end-systolic diameters (LVESD), were assessed in all groups of mice. LV fractional shortening (LVFS) was calculated according to the following equation: LVFS = ([LVEDD + LVESD] / LVEDD) × 100. Western blot analysisTissue homogenates were prepared using RIPA buffer (Upstate). Antibodies against Bax, Bcl2 and GAPDH were purchased from Santa-Cruz Biotechnologies. Antibody against phospho-eNOS was obtained from Cell Signaling Technologies. The band density was quantified by using Image J soft ware (NHI). References 1. Takuwa N, Ohkura S, Takashima S, Ohtani K, Okamoto Y, Tanaka T, Hirano K, Usui S, Wang F, Du W, Yoshioka K, Banno Y, Sasaki M, Ichi I, Okamura M, Sugimoto N, Mizugishi K, Nakanuma Y, Ishii I, Takamura M, Kaneko S, Kojo S, Satouchi K, Mitumori K, Chun J, Takuwa Y. S1p3-mediated cardiac fibrosis in sphingosine kinase 1 transgenic mice involves reactive oxygen species. Cardiovasc Res. 2010;85:484-493 2. Kawahara Y, Tanonaka K, Daicho T, Nawa M, Oikawa R, Nasa Y, Takeo S. Preferable anesthetic conditions for echocardiographic determination of murine cardiac function. J Pharmacol Sci. 2005;99:95-104