The plateau outward current in canine ventricle, sensitive to 4-aminopyridine, is a constitutive contributor to ventricular repolarization

Abstract

Background and purpose: I(Kur) (Ultra-rapid delayed rectifier current) has microM sensitivity to 4-aminopyridine (4-AP) and is an important modulator of the plateau amplitude and action potential duration in canine atria. Kv1.5 encodes I(Kur) and is present in both atria and ventricles in canines and humans. We hypothesized that a similar plateau outward current with microM sensitivity to 4-AP is present in canine ventricle.

Experimental approach: We used established voltage clamp protocols and used 4-AP (50 and 100 microM) to measure a plateau outward current in normal canine myocytes isolated from the left ventricular mid-myocardium.

Key results: Action potential recordings in the presence of 4-AP showed significant prolongation of action potential duration at 50 and 90% repolarization at 0.5 and 1 Hz (P<0.05), while no prolongation occurred at 2 Hz. Voltage clamp experiments revealed a rapidly activating current, similar to current characteristics of canine atrial I(Kur), in approximately 70% of left ventricular myocytes. The IC(50) of 4-AP for this current was 24.2 microM. The concentration of 4-AP used in our experiments resulted in selective blockade of an outward current that was not I(to) or I(Kr). Beta-adrenergic stimulation with isoprenaline significantly increased the 4-AP sensitive outward current density (P<0.05), suggesting a role for this current during increased sympathetic stimulation. In silico incorporation into a canine ventricular cell model revealed selective AP prolongation after current blockade.

Conclusions and implications: Our results support the existence of a canine ventricular plateau outward current sensitive to micromolar 4-AP and its constitutive role in ventricular repolarization.

Figure 1

Blockade of I_Kur using 50 or 100 μM 4-AP causes reverse use-dependent action potential prolongation. Representative action potential recordings are shown at 0.5 Hz (a) and 1 Hz (b). (c) Summary data for APD₅₀ plotted as a function of stimulation frequency. (d) Summary data for APD₉₀ plotted as a function of stimulation frequency. The numbers on each bar indicate the number of cells tested. (^*P<0.05 compared to baseline recordings). 4-AP, 4-aminopyridine; APD, action potential duration; I_Kur, ultra-rapid delayed rectifier current.

Figure 2

4-AP at 100 μM does not block I_to or I_Kr. (a) Representative I_to traces from a ventricular myocyte (cell capacitance—154 pF) recorded at baseline (left), after superfusion with 100 μM 4-AP (middle, voltage protocol is inset) and the averaged I_to I–V curves (right). (b) The peak ramp I_Kr from a ventricular myocyte (cell capacitance—148 pF) recorded at baseline and 100 μM 4-AP using the ramp protocol (inset). Current was measured as the peak outward current during the voltage ramp. For purposes of clarity, only the traces obtained at the last test pulse (plateau duration=80 ms) are shown. The current densities plotted as function of the plateau duration are shown on the right. The dashed lines represent the zero current line. The interpulse interval for I_to and I_Kr was 2 and 7 s respectively. 4-AP, 4-aminopyridine; I_to, transient outward potassium current; I_Kr, rapid delayed rectifier K⁺ current.

Figure 3

4-AP at 100 μM inhibits canine ventricular-sustained outward K⁺ current. (a) The baseline (top), 100 μM 4-AP (middle) and difference currents from a ventricular myocyte (cell capacitance—166 pF) using the voltage clamp protocol shown in the inset. The dashed lines represent the zero current line. (b) The averaged I–V curve for the 4-AP-sensitive sustained outward K⁺ current (from myocytes exhibiting measurable 4-AP-sensitive plateau outward current), with current measured as the average during the last 20 ms of the test pulse. (c) Dose–response relationship for 4-AP inhibition of sustained outward K⁺ current, fitted to the Hill equation ((r²=0.99), n=5–8 myocytes at each concentration). (d) A representative 4-AP-sensitive current trace from a ventricular myocyte using the voltage clamp protocol shown in the inset. Circles on the step current trace indicate the time at which the tail currents were elicited. (e) The mean envelope-of-tails test data where the ratio of tail to step current amplitude is plotted as a function of step pulse duration. 4-AP, 4-aminopyridine.

Figure 4

Canine ventricular plateau outward current is augmented by isoprenaline (Iso). Representative 100 μM 4-AP-sensitive current traces from myocytes recorded at baseline (a; 166 pF) and after exposure to isoprenaline (b; 154 pF). The dashed lines represent the zero current line. Summary I–V curves are shown in (c). (^*P<0.05 vs baseline control values). 4-AP, 4-aminopyridine.

Figure 5

DPO-1, an I_Kur-selective blocker, prolongs APD in canine left ventricular myocytes. (a) Representative action potential trace recorded at 1 Hz before and after superfusion of 1 μM DPO-1. Average data, for APD₅₀ and APD₉₀ measured at 0.5 (b), 1 (c) and 2 Hz (d) at baseline (open bar) or after superfusion with 0.3 μM (grey bar), or 1 μM (black bar) of DPO-1. (^*P<0.05 vs control). APD, action potential duration; DPO-1 (2-isopropyl-5-methylcyclohexyl diphenylphosphine oxide; I_Kur, ultra-rapid delayed rectifier current.

Figure 6

‘I_Kur-like' current blockade prolongs canine ventricular action potential in silico. (a) Fitting of a computer model of the 4-AP-sensitive (‘I_Kur-like') current to current data from patch clamp experiments. For purposes of clarity, current fitting to test steps to −10, 0, 20 and 40 mV are shown. Model parameters after fitting are provided in Table 2. The fitted model was incorporated into the Fox–Gilmour and Hund–Rudy canine action potential models. Steady-state action potential tracings from the Fox–Gilmour model obtained at 2000 (b), 1000 (c) and 500 ms (d) basal cycle lengths. Control action potential traces are shown in black and action potentials in the presence of ‘I_Kur-like' current blockade are shown in grey. 4-AP, 4-aminopyridine; I_Kur, ultra-rapid delayed rectifier current.