IKs protects from ventricular arrhythmia during cardiac ischemia and reperfusion in rabbits by preserving the repolarization reserve

Abstract

Introduction: The function of the repolarization reserve in the prevention of ventricular arrhythmias during cardiac ischemia/reperfusion and the impact of ischemia on slowly activated delayed rectifier potassium current (I(Ks)) channel subunit expression are not well understood.

Methods and results: The responses of monophasic action potential duration (MAPD) prolongation and triangulation were investigated following an L-768,673-induced blockade of I(Ks) with or without ischemia/reperfusion in a rabbit model of left circumflex coronary artery occlusion/reperfusion. Ischemia/reperfusion and I(Ks) blockade were found to significantly induce MAPD90 prolongation and increase triangulation at the epicardial zone at 45 min, 60 min, and 75 min after reperfusion, accompanied with an increase in premature ventricular beats (PVBs) during the same period. Additionally, I(Ks) channel subunit expression was examined following transient ischemia or permanent infarction and changes in monophasic action potential (MAP) waveforms challenged by β-adrenergic stimulation were evaluated using a rabbit model of transient or chronic cardiac ischemia. The epicardial MAP in the peri-infarct zone of hearts subjected to infarction for 2 days exhibited increased triangulation under adrenergic stimulation. KCNQ1 protein, the α subunit of the I(Ks) channel, was downregulated in the same group. Both findings were consistent with an increased incidence of PVBs.

Conclusion: Blockade of I(Ks) caused MAP triangulation, which precipitated ventricular arrhythmias. Chronic ischemia increased the incidence of ventricular arrhythmias under adrenergic stimulation and was associated with increased MAP triangulation of the peri-infarct zone. Downregulation of KCNQ1 protein may be the underlying cause of these changes.

Figure 1. Comparison of epicardial monophasic action potential changes between groups.

(A) Epicardial monophasic action potential durations (MAPDs) and triangulation (MAPD90–MAPD30) recorded from the ischemia/reperfusion zone (apical) in the Sham+vehicle group and the ischemia/reperfusion (IR)+vehicle group. *P<0.05 vs. Sham+vehicle. (B) Epicardial MAPDs and triangulation recorded from the ischemia/reperfusion zone in the Sham+L-768,673 group and the IR+L-768,673 group. *P<0.05 vs. Sham+L-768,673.

Figure 2. MAPDs, triangulations and MAP waveforms comparison between the IR+L-768,673 and IR+vehicle groups.

(A) Epicardial MAPDs and triangulation recorded from the ischemia/reperfusion zone (apical) in the IR+L-768,673 group and the IR+vehicle group. Triangulations of the IR+L-768,673 group were increased compared with those of the IR+vehicle group by 31.1%, 26.5%, and 19.3% at R45, R60, and R75 respectively. Results are mean ± standard deviation (STD). * P<0.05 vs. IR+vehicle. (B) Comparison of monophasic action potential (MAP) waveforms between the IR+L-768,673 and IR+vehicle groups at R45, R60, and R75.

Figure 3. Epicardial MAPD90/60/30 and triangulation (MAPD90 - MAPD30) recorded from the remote zone (basal).

Results were means ± STD. There were no significant differences between groups.

Figure 4. Comparison of waveforms between the Sham (2-d) and Infarct (2-d) groups after bolus injection of epinephrine.

The only morphological differences in peri-infarct MAP were identified in the Infarct (2-d) group. (A) ECG Lead II waveforms showing that both groups had a comparable degree of heart rate increase. (B) MAP recorded at the peri-infarct epicardial zone showing that after the adrenergic challenge, the MAP of the Infarct (2-d) group had a dramatic shortening of MAPD30, whereas the MAPD90 was only minimally changed. (C) Direct overlapping of MAP showing that the MAP of the Infarct (2-d) group demonstrates more prominent triangulation.

Figure 5. Comparison of ventricular premature beats and KCNQ1 expression between groups.

(A) Comparison of the total premature ventricular beats (PVBs) between the groups within 10 min after bolus injection of epinephrine. Results are presented in a box plot format (n = 6–8) where boxes indicate the 25–75% interval along with the median of the data. * P<0.05 vs. the other groups. (B) Reverse transcription-polymerase chain reaction (RT-PCR) of KCNQ1 mRNA levels. Top: Examples of KCNQ1 mRNA with samples harvested from the peri-infarct zone and remote zone of the Healing (2-d; n = 6), Infarct (2-d; n = 8), Sham (2-d; n = 7), Healing (5-d; n = 7), Infarct (5-d; n = 7), and Sham (5-d; n = 8) groups of rabbit hearts. Bottom: mean KCNQ1 mRNA band intensities. (C) Western blot analysis of membrane-associated KCNQ1 protein levels. Top: Representative immunoblot results showing membrane KCNQ1 protein (∼75 kDa) with samples harvested from the peri-infarct zone and remote zone of the six groups of rabbit hearts. Bottom: mean membrane KCNQ1 protein band intensities. * P<0.05 vs. Sham (2-d) and Sham (5-d) respectively; # P<0.05 vs. Healing (2-d) and Healing (5-d) respectively; $ P<0.05 vs. Infarct (5-d).

Figure 6. Study protocol 1 of experiments.

Monophasic action potentials were recorded after the equilibration at both the middle of the infarct zone and the unaffected zone of the epicardium. For L-768,673, The infusion rate of the initial dose was 0.5 µg/kg/h for 30 min, and the maintenance rate was 0.25 µg/kg/h for two hours. MAP duration data were expressed as MAPD90/60/30_{Baseline, Initial Dose, I5, I10, I15, I20, R5, R10, R15, R20, R25, R30, R45, R60, R75, R90, R105, R120}, which stood for MAPD90/60/30 recorded at baseline, after initial dose, at ischemia for 5 min, 10 min, 15 min, 20 min and at reperfusion for 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, 120 min, respectively.

Figure 7. Study protocol 2 of experiments.

Monophasic action potentials were recorded after the equilibration at both the peri-infarct zone and the unaffected zone of the epicardium during each open chest operation and at the time point of peak adrenergic excitation. MAP duration data were expressed as MAPD90/60/30_{pre-op, post-op, epi i.v.}, which stood for MAPD90/60/30 recorded at first operation, at second operation, and after the bolus intravenous injection of epinephrine, respectively.