After 20–30 minutes of washout, gradual warming after cold cardioplegia to 37°C, tissue recovery, and stabilization, the wedges were stained with 4 μM di-4-ANEPPS (Molecular Probes, Eugene, OR), a membrane potential-sensitive fluorescent dye having no known electrophysiological effects and used widely in optical mapping studies in hearts of many species.
We immobilized the wedges with 10 μM Blebbistatin (Tocris Bioscience, Ellisville, MO) which inhibits the adenosine triphosphatases (ATPases) associated with class II myosin isoforms in an actin-detached state, and thus successfully blocks cardiac contraction without any effect on electrical activity, including ECG parameters, atrial and ventricular effective refractory periods, and atrial and ventricular activation patterns in many mammalian species.21 (link), 22 (link) We used microelectrode recordings to validate the effect of Blebbistatin in the human ventricle (see Online Supplement).
The wedges were paced at the endocardium by 5–10 ms pulses at 2 × diastolic current thresholds at a pacing cycle length (CL) ranging from 4,000ms to the ventricular functional refractory period. Two Ag/AgCl electrodes were immersed into the superfusion solution, one at the epicardial and the other at the endocardial side, to document the transmural pseudo-ECG (Fig. 1C).
An optical mapping system23 with a 100×100 pixels resolution MiCAM Ultima-L CMOS camera (SciMedia, USA Ltd., CA) collected the fluorescent light from an area of 2–3 cm by 2–3 cm (Fig. 1B) on the cut-exposed transmural surface of the wedge. Optical action potentials (APs) were recorded from the transmural optical field of view (20×20 to 30×30 mm2) with a spatial resolution of 200–300 μm/pixel at a rate of 1,000 frames/s (Fig. 1C). The fluorescent signals were amplified, digitized, and visualized during experiment using specialized software (SciMedia, USA Ltd., CA)