2. MATERIALS AND METHODS

2. MATERIALS AND METHODS 2.1 Materials [3H]-Digoxin (37Ci/mmol) ... of infusion treatment, animals were anaesthetized with sodium pentobatbital (50 mg...

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2. Materials and Method

2. MATERIALS AND METHODS 2.1 Materials [3H]-Digoxin (37Ci/mmol) was purchased from Perkin-Elmer Life Sciences Inc. (Boston, USA) and [U-14C]-Sucrose (660mCi/mmol) was purchased from Amersham Bioscience UK Limited. Digoxin (12β-hydroxydigitoxin, C41H64O14), (-)-Isoproterenol (+)-bitartrate salt (C11H17NO3·C4H6O6), Lipopolysaccharides (from Escherichia coli: serotype 055:B5) were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). KB-R7943 was kindly donated from Nippon Organon KK and all other chemicals and solvents were of the highest grade available. ALZET micro-osmotic pump (DURECT Corp. USA) was used for inducement of hypertrophy; ALZET model 1003D (1.0 ± .15µl/hr, 3 days, 90 ± 10µl).

Perfused rat heart experiment system configuration. HSE-Harvard Isolated Heart Size 3 & ISOHEART software ISOTEC pressure transducer and HSE-Harvard PLUGSYS system M3 LAUDA pump (type MS) LAUDA DR.R. WOBSER GmbH & Co., KG IVAC-50ml syringe pump (IVAC P4000) MEDIZINTECHNIK GMBH ISMATEC MS-REGLO pump Graphtec Linearecorder mark 8 (WK3500)

Krebs-Henseleit buffer solution composition. NaCl KCl CaCl2·2H2O MgSO4·7H2O NaHCO3 KH2PO4 Glucose Sod. pyruvate BSA Aerating gas pH

M.W. (g/mol) 58.44 74.56 147.02 246.48 84.01 136.09 198.17 110.00 N.A.

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Concentrations (mmol/l) 118.00 4.70 1.50 1.66 24.88 1.18 5.55 2.00 0.1 % 95%O2-5% CO2 7.4

2. Materials and Method 2.2 Isolated perfused heart Wistar rats (280 – 320 g) were used in this study. Surgery on the rat was performed using general anesthesia and artificial ventilation. Sodium pentobarbital was injected as a bolus at concentration of 50 mg/kg. Following the onset of anesthesia, rats were fixed on an appropriate operating table and 500 IU of heparin was pre-treated with i.v. injection to prevent blood coagulation in blood vessels. A cannula wass bound into the trachea for ventilation, and the skin is incised by a longitudinal cut from the middle of abdomen up to the throat. Then, the abdomen is opened up to the diaphragm. The diaphragm is cut off the ribs following the anterior part of the inferior thoracic aperture. The thorax is cut open on the left and right side following the bone-cartilage-border on a line parallel to the sternum starting at the diaphragm and proceeding as far cranial as to the first rib. The complete anterior thoracic wall is turned upwards over the rat’s head and the sternum is split from the xiphoid process exactly in the middle. The ribs are cut as far lateral as possible and the two thorax halves are turned upwards. The pericardium is removed as far as its attachment at the vascular system and any connective tissue around the ascending aorta is discarded. An aortic cannula filled with perfusate was rapidly inserted into the aorta and the pulmonary artery was incised to allow outflow of perfusate. Retrograde perfusion was started with an oxygenated perfusate consisted of Krebs-Henseleit buffer solution, pH 7.4. A latex balloon attached to the end of a steel catheter was placed in the left ventricular through mitral valve. The catheter and the balloon are filled with a mixture of ethanol and water (50:50), and the other end is linked to the amplifier module (HSE-Harvard PLUGSYS) via a pressure transducer. The balloon is inflated with water to create a diastolic pressure of 5 to 6 mmHg. Langendorff apparatus is depicted in Figure 8. The heart is perfused with a Krebs-Henseleit buffer at 37°C with a 60 mmHg pressure. After stabilization, the system is changed to constant flow condition maintaining a coronary flow of 9.5 ± 0.4 ml/min. The hearts are beating spontaneously at an average rate of 270 ± 20 beats/min. Left ventricular (LV) pressure and heart rate (HR) are continuously monitored by means of the balloon. Coronary perfusion pressure (CPP) is regulated by a perfusion pressure control module and measured by a pressure transducer connected to the aortic infusion cannula. A physiological recording system (Hugo Sachs Elektronik, March, Germany) 17

2. Materials and Method is used to monitor left ventricular systolic pressure (LVSP), left ventricular enddiastolic pressure (LVEDP), maximum and minimum values of rate of left ventricular pressure development (LVdP/dtmax and LVdP/dtmin), heart rate and coronary perfusion pressure.

Figure 8. Langendorff apparatus for isolated perfused heart. (modified from Operating Instructions for the experimental apparatus isolated heart size 3 type 830, Hugo Sachs Elektronic).

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2. Materials and Method 2.3 Experimental protocol The following experiments were performed in two groups of hearts (n = 5 in each) with calcium concentrations in perfusate of 0.5 and 1.5 mM, respectively. After 20-min periods of equilibration, three doses (15, 30, and 45 µg) of [3H]-digoxin were administered as 1-min infusions, permutating the sequence of doses with an interval of 15 min. Infusion was performed into the perfusion tube close to the aortic cannula using an infusion device. Outflow samples were collected every 5 s for 2 min and every 30s for the next 5 min (total collection period, 7 min) and the cardiac response was measured. After an equilibration period of 10 min, these experiments were repeated in the presence of KBR (0.1 µM) in perfusate (starting 15 min after perfusion with KBRcontaining buffer). In selected experiments, [U-14C]-Sucrose (2 µCi/10 ml, 0.3 µM) was simultaneously administered as 1-min infusion with digoxin. Outflowing perfusate were collected as same protocol that of digoxin for 7 mins, and it was analyzed with Liquid Scintillation Counter. The outflow samples were kept frozen at -20°C until analysis.

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2.4 Hypertrophy inducement Cardiac hypertrophy was induced in male Wistar rats weighing 280 to 330 g (n = 5) by treatment with isoprenaline for 4 days. The control group received vehicle infusion (n = 5). Delivery of isoprenaline was achieved by implanting a mini-osmotic pump filled with sterilized isoprenaline solution or vehicle (0.1 % ascorbic acid). The mean pumping rate was 1.06 ± 0.04 µl/hr and mean fill volume was 93.8 ± 4.5 µl. The miniosmotic pump was implanted underneath of the neck skin under ether anesthesia.

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2. Materials and Method Isoprenaline was continuously infused at a rate of 2.4 mg/kg/day over 4 days. At the end of infusion treatment, animals were anaesthetized with sodium pentobatbital (50 mg/kg, i.p.) and the hearts were excised under the condition of trachea ventilation. After finishing the perfusion experiment, the heart was separated into atrial (AT), right ventricular (RV), and left ventricular (LV) sections. Hypertrophy was monitored by the ratio of myocardial wet weight (each section) to body weight.

2.5 Sepsis inducement Sepsis was carried out by a single intra peritoneal injection of 4 mg/kg LPS (Lipopolysaccharides from Escherichia coli: serotype 055:B5), and normal saline (0.9 % NaCl) was injected to the other animal group as a control (sham). 4 hours after injection, the heart was excised for isolated heart experiment. Before excision of the heart, rectal temperature of rat was measured with electronic thermometer.

2.6 Determination of digoxin in perfusate The outflow samples were kept frozen at -20 °C until analysis. For determination of [3H]-digoxin concentration in the perfusate, 200 µl of collected outflow sample was transferred to a scintillation vial and 2 ml of cocktail (Lumasafe™ Plus) was added. After vigorous mixing, the radioactivity was measured with a liquid scintillation counter (Perkin-Elmer Instruments, Shelton, CT).

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