The present study protocol was approved by the Animal Care Committee of Tel Aviv University, Tel Aviv, Israel. Male Wistar rats (weight, 350 to 400 g) were anesthetized by intraperitoneal injection of pentobarbital sodium (30 mg/kg). The hearts were rapidly excised, immersed in cold saline solution (4°C), and mounted on the stainless steel cannula of a modified Langendorff perfusion apparatus. Retrograde aortic perfusion was initiated at a perfusion pressure of 85 mm Hg with an oxygenated modified Krebs-Henseleit (KH) buffer solution composed of the following: NaCl, 118 mmol/L; KCl, 4.7 mmol/L; CaCl, 2.0 mmol/L; MgSO4 7H2O, 1.2 mmol/L; KH2Po4, 1.2 mmol/L; glucose, 11.1 mmol/L; NaHCO3, 25 mmol/L. The perfusate was bubbled continuously with 95% O2 and 5% CO2, maintaining a pH of 7.4 to 7.5. The Po2 and Pco2 values in the perfusion solution were 450 to 550 mm Hg and 25 to 30 mm Hg, respectively. The temperature of the heart was monitored by a thermistor implanted in the right ventricular wall, and was carefully maintained at 37°C or 31°C (at the time of ischemia) by wrapping the perfusate reservoir and the isolated heart in a water jacket. The right atrium was removed, and the heart was paced to 300 beats/min at 4 V using an external pacemaker (type E4162; Devices Limited, Implants Division; Garden City, UK), ensuring an identical heart rate for all of the hearts studied. A water-filled latex balloon was placed in the LV cavity through a small incision in the left atrium and was connected to a pressure transducer (model PI 32284; Mennen Medical; Hamburg, Germany). The balloon was tied and inflated to a volume that produced 5 mm Hg diastolic pressure. Zero calibration of the pressure transducer was maintained throughout the experiment.