Preparation of Langendorff-perfused hearts

Animals were anesthetized with pentobarbital sodium (60 mg/kg, i.p.) and heparinized with heparin sodium (100 U, i.p.). Hearts were quickly isolated, excised and transferred to ice-cold (4°C) HEPES-buffered Tyrode’s solution (mM: NaCl 130; KCl 5.4; CaCl₂ 1.8; MgCl₂ 1; Na₂HPO₄ 0.3; HEPES 10; glucose 10; pH adjusted to 7.4 with NaOH), bubbled with 95% O₂-5% CO₂. Hearts were then rapidly cannulated with a tailor-made 21-gauge cannula in aorta and perfused with 37°C HEPES-buffered Tyrode’s solution at 2–2.5 ml/min using a Langendorff-perfusion system (AD Instruments, Australia). Each heart was perfused for 20 minutes before electrophysiology tests. Hearts that did not recover to the regular spontaneous rhythm or had irreversible myocardial ischemia were discarded [25].