2.2. Myocardial ischemia/reperfusion injury protocol

Isolated mouse hearts were Langendorff-perfused as previously described [8,9]. In brief, mice were anesthetized with pentobarbitone sodium and sodium heparin (140 mg/kg body weight and 200 IU, respectively, intraperitoneally). Hearts were rapidly excised and the aorta was cannulated. The hearts were then perfused with oxygenated (95% O₂ + 5% CO₂) Krebs-Henseleit (K-H) buffer at 37 °C (pH7.4). The K-H buffer contained 118.5 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 1.2 mM MgSO₄, 1.4 mM CaCl₂, 11 mM glucose and 2.0 mM sodium pyruvate. Perfusion was in the non-recirculating Langendorff mode at a constant pressure (80 mmHg) and hearts were paced at 540 bpm. A left atrial resection was performed before insertion of a water-filled balloon through the left atrium into the left ventricle. Left ventricular pressure measurements were performed using the balloon inflated to give an end-diastolic pressure (EDP) of 5 − 10 mmHg. The EDP was adjusted to 5–10 mmHg at 5 min before ischemia. Langendorff-perfused hearts were stabilized for 20 min and subjected to 35 min of global ischemia, followed by 20 or 60 min of reperfusion.