Heart Preparation for Ischemia ex-situ

Surgical anesthesia was induced in rats with an intraperitoneal injection of 100 mg/kg ketamine (Narketan®, Vetoquinol AG, Bern, Switzerland), and 10 mg/kg xylazine (Xylapan®, Vetoquinol AG, Bern, Switzerland). Adequate depth of anesthesia was confirmed by absence of the hind-paw withdrawal reflex. Hearts were then rapidly excised, placed in ice-cold phosphate-buffered saline, and working- mode preparations were established. Isolated, working rat hearts were prepared as previously described (5, 6). Briefly, hearts were aerobically perfused with a modified Krebs-Henseleit buffer containing 1.25 mM Ca²⁺, 11 mM glucose, 1.2 mM palmitate bound to 3% BSA and 0.5 mM lactate oxygenated with 95% O₂-5% CO₂ for a pre-ischemic period of 20 min at a constant pressure of 80 mmHg. Ischemia of 27 min was initiated and terminated by clamping and unclamping of the pre- and after- load perfusion lines, respectively. During the ischemic period, the heart was immersed in a glucose- and palmitate-free Krebs-Henseleit buffer at 37°C.