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 10.6 +/- 3.6 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 1.0 +/- 0.8 minutes, but CV remained depressed for 9.0 +/- 3.0 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.

Fig. 1. Effects of regional ischemia on action potentials and Ca_i transients in NRVM monolayers and typical activation patterns during ischemia

A. Photograph of an NRVM monolayer (22×22 mm) with the 18 mm coverslip in place. B. Snapshot of Ca_i fluorescence (F_Ca, above) and the isochronal activation map (below) during pacing from the upper middle region, at baseline and after 8 and 10 min of coverslip ischemia. The AP (F_V, black) and Ca_i transient (F_Ca, red) traces at sites 1 and 2 on the isochronal map are shown to the right of each map. Propagation was uniform at baseline, but by 8 min had slowed progressively only in the IZ, which became inexcitable by 10 min, with macroreentry circulating around it.

Fig. 2. Summary of electrophysiological changes during regional coverslip IR in NRVM monolayers

Average changes in action potential amplitude ratio (A) and APD (B), Ca_i transient duration (C), conduction velocity (CV, D), and wavelength (APD × CV, E) in the IZ (solid circles) versus the NIZ (open circles) at various time points during coverslip IR. NoE refers to the time point at which the IZ became inexcitable (average 10.6 ± 3.6 min). Values are mean ± 2 SD.

Fig. 3. Cx43 fluorescence during coverslip IR

A. Snapshot of Ca_i fluorescence (F_Ca) after 6 min of coverslip ischemia, showing multiple wave macroreentry around the inexcitable IZ (left panel) and during early reperfusion (right panel), showing reentry in the BZ and fibrillatory conduction block in the IZ. Dashed white circles demarcate the IZ and NIZ. Lower panels show dephosphorylated Cx43 immunofluorescence (green) in a magnified image across the BZ region, demonstrating increased fluorescence in the IZ compared to NIZ. Blue fluorescence indicates nuclei staining with DAPI. B. Average ratio of Cx43 immunofluorescence in the IZ to NIZ, for dephosphorylated Cx43 (solid bars) and total Cx43 (open bars) at various times during coverslip IR. The dephosphorylated Cx43 fluorescence ratio increased during ischemia and recovered partly by 10 min of reperfusion, whereas the total Cx43 ratio did not change. Values are mean ± 2 SD for the number of monolayers indicated.

Fig. 4. Spontaneous reentry during ischemia

Right and left panels show a snapshot of Ca_i fluorescence (F_Ca) and the isochromal activation map, respectively, in a monolayer after 6 min of coverslip ischemia. A rotor anchored in the BZ near 4 o’clock (see isochrone map) generated wavefronts circulating around the NIZ (b1-b4). Representative optical AP (F_V) and Ca_i transient (F_Ca) traces below show reentry at site 1, and Ca_i transient alternans (but not resolvable APD altenans) at site 2 near the BZ, with 2:1 conduction block at site 3 near the center of the IZ.

Fig. 5. Initiation of reentry by an extrasystole during coverslip ischemia

A. Isochronal activation maps of a paced beat (upper panel, beat 1) followed by an ectopic beat arising from the BZ (beat 2) in a monolayer after 8 min of ischemia. Note that the central IZ is inexcitable (black). Beat 1 at the pacing site (site 4) propagated counterclockwise (CCW) to site 5 (dashed arrow) and clockwise (CW) to sites 3 to1 (solid arrow). Then the ectopic beat 2 originating from site 1 propagated in the counterclockwise (CCW) direction to sites 2 and 3, but blocked (dashed arrow). Meanwhile, the same beat propagated clockwise (solid arrow) to reach sites 5 and 4 for the opposite direction, and then continued (through the site of previous block of the counterclockwise impulse) to activate sites sites 3 and 2 as reentrant beat 3. B. Optical AP (F_V) and Ca_i transient (F_Ca) traces at sites 1-5, illustrating the earliest activation of the paced beat at site 5, and the origin for the extrasystole from site 1. Note that the short APD and prolonged Ca_i transient at site 1. Arrows indicate propagation sequence. C. Ca snapshot immediately prior to ectopic beat 2 (corresponding to red arrow in B), demonstrating the persistently elevated Ca_i in the area where the ectopic beat is about to emerge. Note that beat 1 is still propagating clockwise through the NIZ (left side) at this time point D. Diagram showing the site of origin of ectopic beats during ischemia in different monolayers, demonstrating a predilection for the IZ at 6 min and for the BZ at 8 min.

Fig. 6. Spontaneous reentry initiated by a paced beat during coverslip ischemia

A. Isochronal activation map of a paced beat initiating reentry during ischemia. The paced beat at site 1 blocked in the IZ, but conducted clockwise partway around the NIZ, invading the IZ between site 3 and 4 to initiate reentry. B. Optical AP traces (F_V) at sites 1-5 at the initiation of reentry. Arrow indicates the activation sequence.

Fig. 7. Reperfusion arrhythmias

A. Snapshots of Ca fluorescence (F_Ca, upper panels), isochronal maps (middle panels) and representative AP (F_V) and Ca_i transient (F_Ca) traces recorded from sites 1 (reperfused IZ) and site 2 (NIZ) at 1, 5 and 10 min after reperfusion. At 1 minute, a figure-eight rotor anchored at the BZ (6 o’clock) propagated normally through the NIZ but with slow conduction, short wavelength and fibrillatory conduction block in the IZ, resembling mother rotor fibrillation. At 5 min, a single rotor was present in the reperfused IZ, and propagated everywhere without conduction block, resembling ventricular tachycardia. At 10 min, reentry had spontaneously terminated, and propagation was uniform from the pacing site.