The role of the delayed rectifier component IKs in dog ventricular muscle and Purkinje fibre repolarization

Abstract

1. The relative contributions of the rapid and slow components of the delayed rectifier potassium current (IKr and IKs, respectively) to dog cardiac action potential configuration were compared in ventricular myocytes and in multicellular right ventricular papillary muscle and Purkinje fibre preparations. Whole-cell patch-clamp techniques, conventional microelectrode and in vivo ECG measurements were made at 37C. 2. Action potential duration (APD) was minimally increased (less than 7%) by chromanol 293B (10 microM) and L-735,821 (100 nM), selective blockers of IKs, over a range of pacing cycle lengths (300-5000 ms) in both dog right ventricular papillary muscles and Purkinje fibre strands. D-Sotalol (30 microM) and E-4031 (1 microM), selective blockers of IKr, in the same preparations markedly (20-80%) lengthened APD in a reverse frequency-dependent manner. 3. In vivo ECG recordings in intact anaesthetized dogs indicated no significant chromanol 293B (1 mg kg-1 i.v.) effect on the QTc interval (332.9 +/- 16.1 ms before versus 330.5 +/- 11.2 ms, n = 6, after chromanol 293B), while D-sotalol (1 mg kg-1 i.v.) significantly increased the QTc interval (323.9 +/- 7.3 ms before versus 346.5 +/- 6.4 ms, n = 5, after D-sotalol, P < 0.05). 4. The current density estimated during the normal ventricular muscle action potential (i.e. after a 200 ms square pulse to +30 mV or during a 250 ms long 'action potential-like' test pulse) indicates that substantially more current is conducted through IKr channels than through IKs channels. However, if the duration of the square test pulse or the 'action potential-like' test pulse was lengthened to 500 ms the relative contribution of IKs significantly increased. 5. When APD was pharmacologically prolonged in papillary muscle (1 microM E-4031 and 1 microg ml-1 veratrine), 100 nM L-735,821 and 10 microM chromanol 293B lengthened repolarization substantially by 14.4 +/- 3.4 and 18. 0 +/- 3.4% (n = 8), respectively. 6. We conclude that in this study IKs plays little role in normal dog ventricular muscle and Purkinje fibre action potential repolarization and that IKr is the major source of outward current responsible for initiation of final action potential repolarization. Thus, when APD is abnormally increased, the role of IKs in final repolarization increases to provide an important safety mechanism that reduces arrhythmia risk.

Figure 1. E-4031-sensitive current (I_Kr) in dog ventricular myocytes

A, recording of I_Kr in the absence and presence of 1 μM E-4031. The inset presents the E-4031 (1 μM)-sensitive difference current at +30 mV. B, the peak I_Kr tail current amplitude-voltage relationship in the absence and presence of 1 μM E-4031. Nisoldipine (1 μM) was used to block inward I_Ca and L-735,821 (100 nM) to block I_Ks. Holding potential (V_h) was −40 mV, pulse duration was 1000 ms, and pulse frequency was 0.05 Hz.

Figure 2. E-4031-insensitive current (I_Ks) in dog ventricular myocytes: effect of chromanol 293B

A and B, recordings in the absence and presence, respectively, of 10 μM chromanol 293B. C, the chromanol 293B (10 μM)-sensitive difference current at +50 mV. D, peak I_Ks tail current amplitude- voltage relationship in the absence and presence of 10 μM chromanol 293B. Nisoldipine (1 μM) was used to block inward I_Ca and E-4031 (5 μM) to block I_Kr. V_h was −40 mV, pulse duration was 5000 ms, and pulse frequency was 0.1 Hz.

Figure 3. E-4031-insensitive current (I_Ks) in dog ventricular myocytes: effect of L-735,821

A and B, recordings in the absence and presence, respectively, of 100 nM L-735,821. C, the L-735,821 (100 nM)-sensitive difference current at +50 mV. D, peak I_Ks tail current amplitude-voltage relationship in the absence and presence of 100 nM L-735,821. Nisoldipine (1 μM) was used to block inward I_Ca and E-4031 (5 μM) to block I_Kr. V_h was −40 mV, pulse duration was 5000 ms, and pulse frequency was 0.1 Hz.

Figure 4. Effect of I_Ks block on the action potential in dog ventricular right papillary muscle and Purkinje fibre

Action potential recordings from canine ventricular papillary muscles (A) and Purkinje fibre strands (B) before and after 40 min superfusion with 100 nM L-735,821 (top) or 10 μM chromanol 293B (bottom). Stimulation frequency was 1 Hz.

Figure 5. Effect of I_Kr block on the action potential in dog ventricular right papillary muscle and Purkinje fibre

Action potential recordings from canine ventricular papillary muscles (A) and Purkinje fibre strands (B) before and after 40 min superfusion with 1 μM E-4031 (top) or 30 μM D-sotalol (bottom). Stimulation frequency was 1 Hz.

Figure 6. Frequency-dependent effect of I_Kr and I_Ks block on action potential duration

Frequency-dependent effect of I_Kr (by 1 μM E-4031 or 30 μM sotalol) and I_Ks block (by 10 μM chromanol 293B or 100 nM L-735,821) on action potential duration (APD) in canine ventricular papillary muscles (A) and Purkinje fibre strands (B). Pacing cycle length (1/frequency) is plotted on the abscissa and the ordinate indicates percentile changes in APD₉₀. Bars represent s.e.m.

Figure 7. Activation and deactivation kinetics of I_Kr and I_Ks in dog ventricular myocytes

A and C, activation kinetics of I_Kr and I_Ks, respectively, measured as tail currents at −40 mV after test pulses to +30 mV with duration gradually increasing between 10 and 5000 ms. B and D, deactivation kinetics of I_Kr and I_Ks outward tail current, respectively, at −40 mV after a 1000 or 5000 ms, respectively, long test pulse to +30 mV. The inset in D shows I_Ks tail current at higher resolution.

Figure 8. Comparison of the magnitude of I_Kr and I_Ks after short and long voltage pulses

A, recordings of E-4031 (I_Kr, left)- and L-735,821 (I_Ks, right)-sensitive currents after application of a short (150 ms) depolarizing test pulse to +30 mV from a holding potential of −40 mV. B, recordings of E-4031 (I_Kr, left)- and L-735,821 (I_Ks, right)-sensitive currents after a long (500 ms) depolarizing test pulse to +30 mV from a holding potential of −40 mV. C, average I_Kr and I_Ks currents at the end of a short (150 ms, right panel) and a long (500 ms, left panel) depolarizing test pulse to +30 mV, and peak tail current at −40 mV. Bars represent s.e.m.

Figure 9. E-4031 (I_Kr)- and L-735,821 (I_Ks)-sensitive difference currents during short and long ‘action potential-like’ test pulse

A, E-4031-sensitive (1 μM) (I_Kr) and L-735,821-sensitive (100 nM) (I_Ks) difference currents recorded during an ‘action-potential-like’ test pulse in canine ventricular myocytes. The ‘action-potential-like’ test pulse was obtained by recording a normal canine ventricular action potential with a conventional microelectrode in a multicellular papillary muscle preparation and adding a 50 ms prepulse from −80 to −40 mV. B, recordings of E-4031-sensitive (I_Kr) and L-735,821-sensitive (I_Ks) currents when the action-potential-like test pulse duration was increased by a factor of 2 (i.e. to ≈500 ms). Recordings in A and B were obtained in the same myocyte. Similar results to those illustrated were obtained in 4–7 additional myocytes.

Figure 10. Effect of 100 nM L-735,821 on dog ventricular action potentials recorded in the presence of 1 μM E-4031 and 1 μg ml⁻¹ veratrine

A, the time course of a representative experiment. At 0 min 1 μM E-4031 and 1 μg ml⁻¹ veratrine were added and measurements were taken every 5 min until a ‘quasi’ steady state was achieved. Then 100 nM L-735,821 was added to the bath in the continuous presence of E-4031 and veratrine. The relation prior to addition of L-735,821 was fitted by the equation Y = A+Bexp(-X/C) to estimate the time-dependent changes that would have occurred in the absence of the I_Ks blocker (continuous line) so that the magnitude of its effect at 140 min is indicated by the arrow. B, representative action potentials recorded at baseline (0 min), after exposure to E-4031 and veratrine alone (70 min), and following addition of L-735,821 (130 min). C, comparison of the effect of L-735,821 on ‘short’ (open bar) and on ‘long’ (filled bar) dog ventricular action potentials, respectively, recorded in the absence or presence of E-4031 and veratrine. Small asterisks represent significant changes from baseline measurements (i.e. at 0 min). The filled star represents significant changes between the bars (P < 0.01 in both cases). Columns and error bars indicate means and s.e.m.