Transcript Supplementary Information (ppt 104K)

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
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 AAR 1.
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 analysis
Tissue 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