Preparation of Langendorff-perfused Hearts, Monophasic Action Potential Recordings, and Ventricular and Atrial Arrhythmia Inducibility

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 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, and a pseudoelectrogram was monitored. Hearts that did not recover to the regular spontaneous rhythm or had irreversible myocardial ischemia were discarded.¹⁵ A pair of platinum-stimulating electrodes were positioned on the basal surface of the right ventricle to deliver regular pacing and a custom-made Ag–AgCl electrode consisting of two 0.25-mm, Teflon-coated, silver wires was located at LV to record the monophasic action potential (MAP). The pacing cycle length (PCL) ranged from 150 to 30 ms, with successive 10 ms decreases. The pacing at each PCL lasts for 30 seconds, followed by a 30-second resting period to avoid pacing memory. The 90% action potential duration (APD90) was defined as the average 90% repolarization time for at least 6 successive MAPs. The APD alternans (ALT) was determined by 2 consecutive beats whose APD90 differed by at least 5% over 10 beats. The APD ALT threshold was defined as the maximal PCL (PCLmax) that induced an APD ALT. Burst pacing (2 ms pulses at 50 Hz, 2 seconds burst duration), which was repeated for 20 times separated by 2-second intervals, was used to induce VA. VA was defined as continuous rapid ventricular contractions of 2 seconds or more.¹⁶ Inducibility of atrial arrhythmias (AAs) was tested using the burst-pacing protocol described by Verheule et al.¹⁷ Burst pacing was applied in the right atrial locations, and MAP was recorded from left atrial epicardium. AAs were defined as a period of rapid atrial rhythm lasting for at least 2 seconds. The episodes of AA or VA were analyzed and quantified. All the electrophysiological studies were performed and analyzed with randomization and blinding.