Action potential changes associated with a slowed inactivation of cardiac voltage-gated sodium channels by KB130015

Abstract

1. We have studied the acute cardiac electrophysiological effects of KB130015 (KB), a drug structurally related to amiodarone. Membrane currents and action potentials were measured at room temperature or at 37 degrees C during whole-cell patch-clamp recording in ventricular myocytes. Action potentials were also measured at 37 degrees C in multicellular ventricular preparations. 2. The effects of KB were compared with those of anemone toxin II (ATX-II). Both KB and ATX-II slowed the inactivation of the voltage-gated Na(+) current (I(Na)). While KB shifted the steady-state voltage-dependent inactivation to more negative potentials, ATX-II shifted it to more positive potentials. In addition, while inactivation proceeded to completion with KB, a noninactivating current was induced by ATX-II. 3. KB had no effect on I(K1) but decreased I(Ca-L) The drug also did not change I(to) in mouse myocytes. 4. The action potential duration (APD) in pig myocytes or multicellular preparations was not prolonged but often shortened by KB, while marked APD prolongation was obtained with ATX-II. Short APDs in mouse were markedly prolonged by KB, which frequently induced early afterdepolarizations. 5. A computer simulation confirmed that long action potentials with high plateau are relatively less sensitive to a mere slowing of I(Na) inactivation, not associated with a persisting, noninactivating current. In contrast, simulated short action potentials with marked phase-1 repolarization were markedly modified by slowing I(Na) inactivation. 6 It is suggested that a prolongation of short action potentials by drugs or mutations that only slow I(Na) inactivation does not necessarily imply identical changes in other species or in different myocardial regions.

Figure 1

Comparison of the effects of KB and ATX-II on voltage-dependent Na⁺ current (I_Na). (a,b) I_Na traces elicited by depolarizations to various potentials before (upper panels) and following 10-min exposure (lower panels) to 10 μM KB (a) or 200 nM ATX-II (b). Inset in (a): voltage pulse protocol. Holding potential (V_H): −80 mV. Test potentials between −120 and 0 mV (a) or +20 mV (b). (c,d) Average normalized I_Na traces at −30 mV in the presence of 10 μM KB (c; n=5) or 200 nM ATX (d; n=4). I_Na in the presence of drug in each cell was normalized relative to the I_Na peak amplitude before exposure to the drug in the same cell. Inset in (d) superimposed traces from (c) and (d) shown at different current gain. The horizontal dashed lines indicate zero current level. Pig ventricular myocytes. [Na]_o=[Na]_i=10 mM; K⁺-free, Cs⁺-containing external and internal solutions; room temperature.

Figure 2

Comparison of the effects of KB and ATX-II on voltage-dependent inactivation of I_Na. (a,b) Superimposed I_Na traces, elicited by depolarizations to −30 mV following 1-s prepulses to various levels (upper panels), and inactivation curves (lower panels) in the absence (unfilled circles) and in the presence (filled circles) of either 10 μM KB (a) or 200 nM ATX-II (b). Inset in (a): voltage pulse protocol. Holding potential (V_H): −80 mV. Horizontal dashed lines indicate zero current level. Fitting of the inactivation curves was made using Boltzmann distribution functions. Average parameters of the Boltzmann functions for ATX: V_0.5=−83±2.85 mV, slope=5.7±0.3, in control; V_0.5=−72±3.7 mV, slope=7.5±0.4, in the presence of the toxin (n=4; P<0.05 vs control). For average KB data, see Macianskiene et al. (2003). (c,d) Pooled data on peak amplitude of I_Na at −30 mV using V_H of either −120 or −80 mV in control (unfilled columns) and in the presence (hatched columns) of 10 μM KB (c; n=5–6) or 200 nM ATX-II (d; n=4). Pig ventricular myocytes. [Na]_o=[Na]_i=10 mM; K⁺-free, Cs⁺-containing external and internal solutions; room temperature. ^**P<0.01 for drug vs control (paired t-test).

Figure 3

Comparison of KB and ATX-II effects on I_Na and action potentials in pig cardiac myocytes. (a,b) Superimposed I_Na traces induced at −30 mV in control (unfilled circles) and in the presence (filled circles) of either 10 μM KB (a) or 200 nM ATX-II (b). (c,d) Superimposed action potential recorded under current-clamp conditions in the same cells, in control (unfilled circles) and in the presence (filled circles) of either 10 μM KB (c) or 200 nM ATX-II (d). In (d) action potential labeled with unfilled square was obtained after 10-min washout of ATX-II. Horizontal dashed lines indicate 0 mV level. Stimulation via the patch pipette at 1 Hz. [Na]_o=150 mM, [Na]_i=10 mM; K⁺-containing external and internal solutions; ruptured-patch recording; room temperature.

Figure 4

Variable effects of KB on action potentials in pig preparations. Superimposed action potential traces (leftmost and middle panels; different cells) recorded in control (unfilled circles) and in the presence of 10 μM KB (filled circles), and average durations (columns; rightmost panels) of the APD at 50% (APD₅₀) and 90% repolarization (APD₉₀). Horizontal dotted lines indicate 0 mV level. Pacing frequency: 1 Hz. (a) Pig ventricular myocytes; room temperature; (b) pig ventricular trabecula; 37°C.

Figure 5

Effect of KB on action potentials in mouse myocytes. (a) Action potentials measured in control, at 5 and 10 min after application of 10 μM KB, and following 60-min washout of KB in 0.1% DMSO. Myocyte from the left ventricular free wall; ruptured patch recording. (b,c) Action potential recorded in control and in the presence of 3 or 10 μM KB; perforated patch recordings. Myocytes from the left ventricular free wall (b) and from the septum (c). Horizontal dashed lines indicate zero level; pacing at 1 Hz.

Figure 6

Effect of KB on steady-state currents. (a, b) Traces of currents elicited by 1-s steps to various potentials before (upper panels) and after application (lower panels) of either 10 μM KB (a) or 200 nM ATX-II (b). Horizontal dashed lines indicate zero current level. (c, d) Current–voltage relations in control (unfilled circles) and in the presence (filled circles) of either 10 μM KB (c) or 200 nM ATX-II (d). Currents measured at the end of the 1-s pulse. Notice the change of steady-state currents by ATX-II but not KB at potentials positive to −70 mV. Pig ventricular myocytes; K⁺-containing external and internal solutions; [Na]_o=150 mM; [Na]_i=10 mM; room temperature.

Figure 7

KB effect on the L-type Ca²⁺ current (I_Ca-L). (a) I_Ca-L currents elicited by steps to various potentials in the absence (left) and in the presence (right) of 10 μM KB. The horizontal dotted line indicates zero current level. Inset in left panel shows the voltage protocol: holding potential (V_H) of −80 mV, prestep for 1 s to −40 mV to inactivate I_Na and any T-type Ca²⁺ current. Inset in right panel contrasts the failure of KB to change the time course I_Ca-L (at 0 mV) at a time when it causes slowing of I_Na (during the prestep to −40 mV). (b) Current–voltage relations in control (unfilled circles) and in the presence (filled circles) of 10 μM KB. Pooled data from various cells (n=9). (c) Time constants (obtained using biexponential fitting) of I_Ca-L inactivation measured at 0 mV, in control conditions (unfilled columns) and in 10 μM KB (filled columns). Same cells as in (b) pig myocytes; K⁺-free, Cs⁺-containing external and internal solutions; room temperature. ^*P<0.05 vs control.

Figure 8

Lack of effect of KB on I_to and I_K1 in mouse myocytes. (a) Traces of currents elicited by steps to various potentials before (left panel) and after application (right panel) of 10 μM KB. Horizontal dotted lines indicate zero current level. (b) Current–voltage relations in control (unfilled circles) and in the presence (filled circles) of 10 μM KB. Currents measured at peak level. (c) Pooled data on amplitude of currents at −120 mV (largely I_K1; n=10) and +60 mV (largely I_to; n=3). Notice lack of KB effect. [Na]_o=[Na]_i=0 mM; K⁺-containing external and internal solutions; room temperature.

Figure 9

Simulation of the effect of slowed I_Na inactivation on the action potential. Na⁺ currents (I_Na) and action potentials generated using different channel kinetic parameters. Upper panels: I_Na induced by simulated voltage-clamp from −80 to −30 mV. Middle panels: action potentials generated during simulated pacing at 1 Hz in the absence of the transient outward current (I_to). Lower panels: action potentials in the presence of a large I_to (maximal conductance 4 μS pF⁻¹). (a) Control conditions. Na⁺ channel with standard kinetics and conductance, as given in the Luo–Rudy model formulated from single channel kinetics (Clancy & Rudy, 1999). (b) Na⁺ channel with slowed inactivation kinetics but normal maximal conductance. The default rate constants of transitions from the open to the inactivated state (α₂) and from the closed to the open state (α₁₃) were decreased by a factor of 20 and 3.8, respectively, whereas the rate constant for the recovery from the inactivated to the closed state (α₃) was changed by a factor of 10. α₂=0.459 × e^lV/29.68 m s⁻¹; α₁₃=1/(0.1027 × e^−V/12+0.25 × e^−V/150) m s⁻¹; α₃=3.79 × 10⁻⁸ × e^−V/5.2 m s⁻¹. (c) Na⁺ channel with slowed inactivation kinetics and decreased maximal conductance. The rate constant α₂ and the maximal conductance (G_Na) were decreased. α₂=0.2 × e^V/29.68 m s⁻¹; G_Na=3 instead of 16 μS pF⁻¹.