Electrophysiological Consequences of Acute Regional Ischemia/Reperfusion in Neonatal Rat Ventricular Myocyte Monolayers

Abstract

Background-Electrophysiological changes promoting arrhythmias during acute regional ischemia/reperfusion are challenging to study in intact cardiac tissue because of complex 3-dimensional myocardial and vascular geometry. We characterized electrophysiological alterations and arrhythmias during regional ischemia/reperfusion in a simpler 2-dimensional geometry of cultured neonatal rat ventricular myocyte monolayers. Methods and Results-Optical mapping of intracellular Ca (Ca i ) and voltage was performed with the use of Rhod 2-AM and Rh-237, respectively. Regional ischemia was mimicked by covering the central portion of monolayer with a glass coverslip, and reperfusion was mimicked by removing the coverslip. Monolayers were stained with fluorescent antibodies to detect total and dephosphorylated connexin-43 at various time points. During coverslip ischemia, action potential duration shortened, Ca i transient duration was prolonged, and local conduction velocity (CV) slowed progressively, with loss of excitability after 10.6Ϯ3.6 minutes. CV slowing was accompanied by connexin-43 dephosphorylation. During ischemia, spontaneous reentry occurred in 5 of 11 monolayers, initiated by extrasystoles arising from the border zone or unidirectional conduction block of paced beats. On reperfusion, excitability recovered within 1.0Ϯ0.8 minutes, but CV remained depressed for 9.0Ϯ3.0 minutes, promoting reentry in the reperfused zone. As connexin-43 phosphorylation recovered in the reperfused zone, CV normalized, and arrhythmias resolved. Conclusions-Acute regional ischemia/reperfusion in neonatal rat ventricular myocyte monolayers recapitulates electrophysiological alterations and arrhythmias similar to those observed during acute coronary occlusion/reperfusion in intact hearts. During early reperfusion, slow recovery from connexin-43 dephosphorylation leads to persistent CV slowing, creating a highly arrhythmogenic substrate.

Anisotropic conduction block and reentry in neonatal rat ventricular myocyte monolayers

“…NRVM monolayers were prepared as previously described (4,5) and plated on 22 ϫ 22 mm 2 plastic cover slips. Briefly, hearts from 2-to 3-day-old Sprague-Dawley rats were extracted, trimmed of atrial and fat tissue, and digested with collagenase (0.02%; Worthington) and pancreatin (0.06%; Sigma).…”

“…Briefly, hearts from 2-to 3-day-old Sprague-Dawley rats were extracted, trimmed of atrial and fat tissue, and digested with collagenase (0.02%; Worthington) and pancreatin (0.06%; Sigma). Myocytes were isolated from fibroblasts and other cells using a Percoll (Pharmacia) gradient (4,5). Cells were plated with a density of one million cells per cover slip.…”

“…As previously described (4,5), monolayers were stained by immersion in oxygenated Tyrode's solution at 37°C, containing the fluorescent voltage dye RH-237 (5 M for 5 min) and/or the Ca dye rhod 2-AM (5 M for 40 min) plus 0.016% (wt/wt) pluronic (Molecular Probes) and then placed in a perfusion bath at 37°C. Fluorescence was excited by two light sources (each with 4 light-emitting diodes; Luxeon, Ontario, Canada) filtered at 540 Ϯ 20 nm.…”

“…Raw data were processed with custom-written software as previously described (3,4). Action potential duration (APD) was measured at 80% repolarization.…”

Spontaneous high-frequency action potential

“…Peters [12] detailed that in myocardium distant from infarction Cx43 had a 47% reduction in gap junction surface area per unit cell volume. de Diego et al [61] detailed that during coverslip ischemia, local CV slowed progressively with loss of excitability, CV slowing was accompanied by Cx43 reduction, action potential duration was shortened, Ca(i) transient duration was prolonged, and spontaneous reentry occurred in five of 11 monolayers arising from the border zone. CV slowing indicates the inhibited Na + inflow; Ca(i) transient duration being prolonged indicates the inhibited Ca 2+ inflow; the shortened action potential duration and the prolonged Ca(i) transient duration indicate it is the enhanced K + outflow that causes the shortened action potential duration, but not the enhanced Ca 2+ inflow (the enhanced Ca 2+ inflow also shortens action potential duration).…”