Slow delayed rectifier current and repolarization in canine cardiac Purkinje cells

¹ Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec H3G 1Y6; and ² Research Center and Department of Medicine, Montreal Heart Institute, Montreal, Quebec H1T 1C8 and University of Montreal, Montreal, Quebec, Canada H3C 3J7

	ABSTRACT

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Although cardiac Purkinje cells (PCs) are believed to be the source of early afterdepolarizations generating ventricular tachyarrhythmias in long Q-T syndromes (LQTS), the ionic determinants of PC repolarization are incompletely known. To evaluate the role of the slow delayed rectifier current (I_Ks) in PC repolarization, we studied PCs from canine ventricular false tendons with whole cell patch clamp (37°C). Typical I_Ks voltage- and time-dependent properties were noted. Isoproterenol enhanced I_Ks in a concentration-dependent fashion (EC₅₀ ~ 30 nM), negatively shifted I_Ks activation voltage dependence, and accelerated I_Ks activation. Block of I_Ks with 293B did not alter PC action potential duration (APD) in the absence of isoproterenol; however, in the presence of isoproterenol, 293B significantly prolonged APD. We conclude that, without -adrenergic stimulation, I_Ks contributes little to PC repolarization; however, -adrenergic stimulation increases the contribution of I_Ks by increasing current amplitude, accelerating I_Ks activation, and shifting activation voltage toward the PC plateau voltage range. I_Ks may therefore provide an important "braking" function to limit PC APD prolongation in the presence of -adrenergic stimulation.

ventricular arrhythmias; action potential; long Q-T syndrome

	INTRODUCTION

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ADRENERGIC STIMULATION MODULATES Purkinje fiber delayed rectifier K⁺ current (I_K) (4, 5, 11), selectively activating the slow component (I_Ks) in ventricular myocytes (21). Deficiency of either subunit of I_Ks, KvLQT1 or minK, causes congenital long Q-T syndrome (LQTS) (3, 19). KvLQT1 abnormalities cause LQTS1 (1), in which the occurrence of torsades de pointes (TdP) ventricular tachyarrhythmia is particularly adrenergically dependent (2, 28). Shimizu and Antzelevitch (25) showed that -adrenergic stimulation causes a substrate for TdP in the presence of I_Ks inhibition with chromanol 293B. Repolarization abnormalities in the Purkinje system are believed to be important in TdP initiation (10, 16). It is conceivable that I_Ks deficiency in Purkinje cells (PCs) might lead to repolarization abnormalities that play an important role in TdP. Despite the evidence in multicellular Purkinje fiber preparations (5, 17), recent publications have suggested that I_K may be small or absent in single cardiac PCs (9, 18). The present study was designed to evaluate I_Ks in PCs from free-running false tendons in the dog heart, determine the response of PC I_Ks to -adrenergic stimulation, and establish the potential role of I_Ks in PC repolarization.

	MATERIALS AND METHODS

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Cell isolation. Mongrel dogs (20-30 kg) were anesthetized (pentobarbital sodium, 30 mg/kg iv), and their hearts were removed and immersed in Tyrode solution. False tendons were excised into modified MEM (GIBCO-BRL; pH 6.8, HEPES-NaOH) containing collagenase (800-900 U/ml, type II; Worthington) and 1% BSA. The fibers were agitated with 100% O₂ in a 37°C shaker bath (50-100 min). After the endothelial sheath had been digested, the fibers were washed twice with high-K⁺ storage solution and incubated for 10 min at 37°C. Individual PCs were dispersed by trituration, harvested by centrifugation for 1 min, and kept in high-K⁺ storage solution.

Solutions. The solutions contained (mM) 136 NaCl, 5.4 KCl, 1.0 MgCl₂, 1.0 CaCl₂, 0.33 NaH₂PO₄, 5.0 HEPES, and 10 dextrose, with pH adjusted to 7.4 with NaOH (Tyrode solution); 20 KCl, 10 KH₂PO₄, 10 dextrose, 70 glutamic acid, 10 -hydroxybutyric acid, 10 taurine, 10 EGTA, and 0.1% albumin, with pH adjusted to 7.4 with KOH (storage solution); and 110 potassium aspartate, 20 KCl, 1 MgCl₂, 5 Mg₂ATP, 10 HEPES, 5 phosphocreatine, 0.1 GTP, and 5 EGTA (current recording) or 0.05 EGTA [action potential (AP) recording], with pH adjusted to 7.2 with KOH (pipette solution). All solutions were equilibrated with 100% O₂.

Atropine (1 µM) was included in the extracellular solution to eliminate 4-aminopyridine (4-AP)-dependent K⁺ currents (24) and nimodipine (1 µM) to block Ca²⁺ current (I_Ca). Na⁺ current (I_Na) was suppressed by using a holding potential of 50 mV. Chromanol 293B (50 µM) was employed to inhibit I_Ks and dofetilide (1 µM) to block the slow component of I_K (I_Kr). Chromanol 293B fails to alter I_Kr, I_K1, I_Ca, or I_Na but inhibits transient outward current (I_to) by ~65% at a concentration of 50 µM (7). 4-AP (1 mM) was added to the bath to suppress I_to for voltage-clamp experiments. Isoproterenol (Iso) was freshly prepared as stock solutions of 100 µM and 1 mM and stabilized with 100 µM ascorbic acid. L-768673 was kindly supplied by Merck Pharmaceuticals.

Data acquisition and analysis. General voltage-clamp techniques were as previously described (13, 30), with voltage-clamp and AP recordings performed at 0.1 Hz and 37°C. I_Ks step current was measured from the onset of activation to the level at the end of a depolarizing pulse and tail current from initial current on repolarization to the level at the end of the repolarizing pulse. Junction potential offsets averaged 10.0 ± 0.4 mV and were corrected only for APs. Small cells were selected to ensure spatial clamp (capacitance 127 ± 7 pF). Compensated series resistances and capacitive time constants averaged 2.5 ± 0.1 M and 290 ± 10 µs. For AP clamp, APs were recorded with current clamp at 0.1 Hz. The acquired AP waveform was then used as a voltage command signal to measure current flow during the AP, before and after Iso and/or 293B, with the difference current indicating the current inhibited by the drug during the AP in a given cell.

Nonlinear least-square curve fitting was used to fit experimental data. ANOVA and Bonferroni-adjusted t-tests were used for multiple group comparisons and t-tests for single comparisons. Values are means ± SE.

	RESULTS

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Effects of -adrenergic stimulation on I_Ks. Figure 1A shows representative I_Ks recordings from a PC in the absence of Iso (control) and in the presence of progressively increasing Iso concentrations. Iso effects were suppressed by propranolol; for example, in five cells, 500 nM Iso increased I_Ks step current at +40 mV from 8.4 ± 1.3 to 18.1 ± 2.2 pA/pF (P < 0.001), and addition of 1 µM propranolol reduced step current in the continued presence of Iso to 9.5 ± 1.1 pA/pF (P < 0.01 vs. Iso alone). Average step and tail current density-voltage relations (n = 7 cells) are shown in Fig. 1, B and C. Both were significantly increased in a concentration-dependent manner. Concentration-response relations are illustrated in Fig. 1D. The EC₅₀ was in the range of 30 nM. To exclude effects mediated by the vehicle for Iso (ascorbic acid in Tyrode solution), we studied I_Ks before and after the highest ascorbic acid concentration used. In five cells, I_Ks step current at +40 mV averaged 7.0 ± 1.5 pA/pF before and 7.2 ± 1.5 pA/pF [P = not significant (NS)] after 15 min of exposure to the vehicle.

View larger version (37K): [in this window] [in a new window]   Fig. 1.   A: superimposed recordings from a Purkinje cell in response to 4-s pulses from 50 to +50 mV and 2-s repolarizations to 30 mV before (Ctl) and after isoproterenol (Iso). B and C: concentration (conc.)-dependent effects of Iso on slow component of K+ current (IKs) step (B) and tail (C) density (all values positive to 10 mV; P < 0.001 vs. control). TP, test potential. D: Iso concentration dependence for IKs stimulation, based on mean data at +50 mV. Curves are best fit to the following equation: I = Imax{1/[1 + (EC50/x)]}, where I is current increase at a concentration x and Imax is maximum I.

Figure 2A shows I_Ks at 0.1 Hz before and after two concentrations of Iso, along with best biexponential fits to activation and deactivation data. Iso significantly decreased activation time constants (Fig. 2B) without altering the time course of deactivation. Figure 2C shows activation voltage dependence based on tail currents after steps to various voltages (n = 5 cells). Half-activation voltage was shifted from 23 ± 1 mV (control) to 16 ± 1 mV by 10 nM Iso and to 12 ± 1 mV by 500 nM Iso. Maximum decreases occurred at 50 nM Iso (Fig. 2B).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   A: representative data (data reduction applied so that curve fits can be seen) during 4-s depolarizations to +50 mV and 2-s repolarizations to 30 mV in the absence and presence of 10 and 500 nM Iso. Biexponential fits of activation and deactivation are shown. B: activation time constants (Act ) for 6 cells (means ± SE) before and after exposure to Iso. act,s and act,f, slow and fast activation time constants, respectively. C: current-voltage (I-V) relations of tail currents normalized to current at most positive step potential under control conditions and in the presence of 10 and 500 nM Iso. Tail currents were fit by Boltzmann relations to obtain the half-activation voltage (V50). D: activation V50 in 6 cells studied under control conditions and all Iso concentrations. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control.

Effects of -adrenergic stimulation and I_Ks inhibition on the AP. No clear effect of 293B on the AP is seen in the absence of Iso (Fig. 3A). Iso alone raised the plateau and accelerated phase 3, decreasing AP duration (APD; Fig. 3B) in the cell shown. In the presence of 293B, Iso increased APD (Fig. 3C). Mean APD changes are shown in Fig. 3D. 293B did not significantly alter APD in the absence of Iso; however, APD in the presence of Iso + 293B was significantly greater than with 293B or Iso alone, indicating that, in the absence of I_Ks, -adrenergic stimulation delays PC repolarization. Iso alone increased APD slightly in some cells and decreased it in others, with no significant effect overall.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Influence of 293B, Iso, and 293B + Iso on action potential (AP). Recordings were conducted under current-clamp mode at 37°C. APs were elicited by twice-threshold rectangular pulses at a frequency of 0.1 Hz. A-C: representative Purkinje cell APs in the absence (Ctl) and presence of 293B (A), Iso alone (B), and Iso + 293B (C). D: changes of AP duration (APD) to 90% repolarization (APD90) in 293B, Iso, and 293B + Iso (n = 7 per group). Iso did not significantly change APD vs. control. Each cell was studied under control conditions and after 1 intervention. Control APDs were not significantly different among groups.

The AP-clamp technique was applied to evaluate the mechanisms of the effects of 293B and Iso. Figure 4, A-C, shows APs from one PC under control conditions, in the presence of Iso, and in the presence of Iso + 293B. The control AP waveform was used to voltage clamp the cell before (Fig. 4D) and after (Fig. 4E) Iso. The Iso-sensitive current (Fig. 4F) included inward and outward components, compatible with I_Ca and I_Ks. The AP waveform recorded in the presence of Iso was then used to AP clamp the cell in the presence of Iso before (Fig. 4G) and after (Fig. 4H) 293B. The 293B-sensitive waveform (Fig. 4I) indicates that 293B suppressed early transient and more delayed plateau outward components, compatible with inhibition of I_to and I_Ks (7). These results indicate that 293B prolongation of the AP in the presence of Iso likely resulted from outward current inhibition but that I_to and I_Ks could have been involved. Similar AP-clamp results were obtained in three other cells.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Currents affected by Iso and 293B, as indicated by AP-clamp methods. A-C: APs from 1 cell recorded at 0.1 Hz under control conditions (A) and after the sequential addition of 100 nM Iso (B) and 50 µM 293B + 100 nM Iso (C). The AP recorded under control conditions (A) was used as a voltage-clamp waveform to elicit currents under control conditions (D) and then in the presence of Iso (E). The subtracted current (F) indicates the Iso-sensitive component flowing during the AP. The AP recorded in the presence of 100 nM Iso (B) was used as a voltage command pulse to record currents in the presence of Iso alone (G) and then in the presence of Iso + 293B (H). The subtracted current (I) gives an indication of the 293B-sensitive current during the AP in the presence of Iso. Similar results were obtained in 3 other experiments.

Because of the effects of 293B on I_to and I_Ks, we studied the effects of L-768673, reported to be a potent and selective inhibitor of I_Ks (EC₅₀ = 6 nM) in guinea pig ventricular myocytes (23), on PC I_Ks. Unfortunately, L-768673 did not affect PC I_Ks. In five PCs, the density of I_Ks time-dependent activating current at the end of a 4-s pulse to +40 mV averaged 4.9 ± 0.7 and 5.1 ± 0.9 pA/pF (P = NS) before and after L-768673, respectively.

Because we were unable to identify a more selective tool than 293B with which to inhibit PC I_Ks, we used 4-AP to study (in the absence of I_to) the effects of Iso in the absence and presence of 293B. Figure 5 shows AP recordings from one PC under control (A), Iso (B), and Iso + 293B (C) conditions in the continuous presence of 1 mM 4-AP to suppress I_to. Under these conditions, APD at 90% repolarization in eight cells averaged 211 ± 29 and 218 ± 26 ms under control and Iso conditions (P = NS) compared with 281 ± 43 ms (P = 0.02 vs. Iso alone) in the presence of Iso + 293B. In the absence of Iso, APD at 90% repolarization averaged 190 ± 15 ms in six cells before and 193 ± 12 ms after 293B (P = NS). As shown in Fig. 5D, the 293B-sensitive current during AP clamp (current in the presence of Iso alone compared with current in the presence of Iso + 293B) showed a delayed outward current, without any transient component, indicating that the effect of 293B on the AP in the presence of 4-AP is attributable to I_Ks inhibition alone.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effects of Iso and 293B on Purkinje cell APs in the absence of transient outward current (Ito). A cell was exposed to each condition during the continuous presence of 1 mM 4-aminopyridine. APs (at 0.1 Hz) from 1 cell are shown under control conditions (A) and after the addition of Iso (B) and Iso + 293B (C). AP clamp was used to record the 293B-sensitive current in the presence of Iso (D). In the presence of Iso, 293B removed an outward component that activated during the plateau and then deactivated on repolarization, consistent with IKs. Similar results were obtained in 3 other experiments.

	DISCUSSION

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We recorded large I_Ks with typical properties in canine PCs. We were unable to demonstrate an effect of I_Ks inhibition on PC APD in the absence of -adrenergic stimulation; however, in the presence of -adrenergic stimulation, I_Ks appeared to contribute to PC repolarization.

Comparison with previous observations regarding I_K in isolated PCs. In contrast to the very clear I_K recorded from multicellular Purkinje fiber preparations (5, 17), studies in isolated PCs have described very small (22) or absent (9, 18) I_K. In the present study, we were able to record large I_Ks step and tail currents in PCs from canine false tendons, with properties compatible with those reported for I_Ks in multicellular Purkinje preparations (5, 17) and isolated ventricular myocytes (3, 11, 13, 14, 20, 21, 29). The effects of Iso in our study, including increased I_Ks density, negative shift in inactivation voltage dependence, and acceleration of activation, are similar to effects in guinea pig ventricular myocytes (29) and bull frog atrial myocytes (11). The presence of clear I_Ks in our preparations, in contrast to previous studies in PCs, may be due to species differences (rabbit vs. dog) or the site from which cells are isolated (subendocardial tissues vs. false tendons). An additional very important factor may be differences in isolation technique, to which I_K is particularly sensitive (30). Given the physiological significance of I_K and the importance of I_Ks deficiencies in congenital LQTS, the demonstration of clear I_Ks in single PCs is, in itself, a significant contribution.

Role of I_Ks in PC repolarization and potential importance in LQTS. We were unable to identify a role for I_Ks in PC repolarization in the absence of -adrenergic stimulation. These results are similar to those of Varro et al. (26) and Burashnikov and Antzelevitch (8). The lack of I_Ks involvement in PC repolarization is consistent with PC plateau voltage in the range of 0 mV, at which relatively little I_Ks is activated. We found three mechanisms through which -adrenergic stimulation can increase the contribution of I_Ks to PC repolarization: 1) increased maximal conductance, 2) accelerated activation, and 3) an activation-voltage shift toward the plateau potential. -Adrenergic stimulation is well known to enhance L-type I_Ca (11), an effect that in itself would tend to delay repolarization and promote arrhythmogenic afterdepolarizations (12, 15). An important functional role of I_Ks in PCs might therefore be to act as a "brake" to prevent excessive APD prolongation in the face of -adrenergic enhancement of L-type I_Ca. Varro et al. (26) recently suggested a similar type of I_Ks "braking" function against excessive APD prolongation caused by I_Kr blockade. I_Ks may therefore function more as a physiological safety mechanism protecting against factors prolonging APD than as an important repolarizing current under normal conditions.

Our results point to a possible mechanism whereby Purkinje fibers could participate in producing ventricular tachyarrhythmias in congenital LQTS caused by dysfunctional I_Ks, LQTS1 and LQTS5 (1, 3). If I_Ks activation by -adrenergic stimulation serves as a brake for the APD-prolonging effect of adrenergic enhancement of I_Ca, unopposed I_Ca stimulation in patients with I_Ks dysfunction could provoke excessive APD prolongation, leading to arrhythmogenic afterdepolarizations in PCs. These afterdepolarizations could act as a trigger on the reentrant substrate caused by transmural repolarization abnormalities (25) to produce TdP. This notion is consistent with the preferential occurrence of drug-induced early afterdepolarizations (EADs) in the Purkinje fiber network (16), initiation by subendocardial-triggered activity of reentry in an animal LQTS model (10), and mathematical modeling studies of polymorphic ventricular tachyarrhythmias (6). However, EADs were not recorded in the present study or in the study of Burashnikov and Antzelevitch (8). The in vitro conditions may be suppressing EAD generation, or other mechanisms similar to delayed afterdepolarizations (8) may be provoked by prolonged APs.

Potential limitations. The isolation of PCs is difficult, particularly because of the connective tissue sheath around false tendons. This may account for discrepant results in the literature and increases variability in PC APs. Some of the variability in single-cell APs may also be due to intrinsic differences among cells in ionic current composition (27). Another problem that we encountered was a lack of highly selective I_Ks blockers for pharmacological dissection.

We used AP clamp to evaluate the currents responsible for 293B-induced AP prolongation in the presence of Iso. The AP-clamp technique requires that electrophysiological conditions be stable over the course of measurement before and after an intervention. To avoid potential time-dependent confounding factors, we recorded the AP under control conditions before drug superfusion and then used the recorded control AP to perform AP clamp immediately before and immediately after equilibration with a drug (5 min). Nonetheless, some current rundown may still have occurred, with I_Ca rundown possibly explaining the small early inward component in Fig. 4I. We did not perform a detailed characterization of all repolarizing currents in PCs and, therefore, cannot exclude Iso- or 293B-mediated effects via processes such as the Ca²⁺-dependent Cl current, the Na⁺/Ca²⁺ exchanger, Na⁺-K⁺-ATPase, or intracellular Ca²⁺ handling.

	ACKNOWLEDGEMENTS

The authors thank Chantal St-Cyr for excellent technical assistance and Annie Laprade and France Thériault for secretarial help with the manuscript.

	FOOTNOTES

This work was supported by operating grants from the Medical Research Council of Canada and the Quebec Heart Foundation.

Address for reprint requests and other correspondence: S. Nattel, Montreal Heart Institute, Research Center, 5000 Belanger St. East, Montreal, PQ, Canada H1T 1C8 (E-mail: nattel{at}icm.umontreal.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 25 April 2000; accepted in final form 12 October 2000.

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