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Am J Physiol Heart Circ Physiol 279: H466-H474, 2000;
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Vol. 279, Issue 2, H466-H474, August 2000

A comparison of transient outward currents in canine cardiac Purkinje cells and ventricular myocytes

Wei Han1,3, Zhiguo Wang1,2, and Stanley Nattel1,2,3

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although abnormalities in Purkinje cell (PC) repolarization are important causes of cardiac arrhythmias, the detailed properties of repolarizing currents in PCs are incompletely understood. We compared transient outward K+ current (Ito) in single PCs from canine false tendons with midmyocardial ventricular myocytes (VMs). Ito reactivation was biexponential, with a similar rapid-phase time constant (30 ± 5 and 35 ± 4 ms for VM and PC, respectively) but a large, slow component in PCs with a much greater time constant than VM (1,427 ± 70 vs. 181 ± 24 ms, P < 0.001). Tetraethylammonium had no effect on VM Ito but reversibly inhibited PC Ito (IC50 = 2.4 ± 0.4 mM). PC Ito was also more sensitive to 4-aminopyridine (IC50 = 50 ± 7 vs. 526 ± 49 µM in VM, P < 0.0001). H2O2 slowed Ito inactivation in PCs but did not affect VM Ito. We conclude that PC Ito shows significant differences from VM Ito, with some features, such as tetraethylammonium sensitivity, that have been reported in neither cardiac Ito of atrial or ventricular myocytes nor cloned K+ channel subunits (Kv1.4, Kv4.2, or Kv4.3) known to participate in cardiac Ito.

potassium channels; molecular biology of cardiac ion channels; cardiac arrhythmias; action potentials; potassium channel blockers; antiarrhythmic drugs


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PURKINJE FIBERS are responsible for the rapid propagation of the cardiac impulse to the ventricles, provide life-saving ventricular escape rhythms if atrioventricular block occurs, and generate the arrhythmogenic early afterdepolarizations that cause the potentially lethal ventricular arrhythmias associated with the long Q-T syndrome (25, 31). The acquired long Q-T syndrome is the most serious complication limiting treatment with action potential (AP)-prolonging drugs (class IA and III) that are otherwise quite effective for reentrant cardiac arrhythmias. In contrast to atrial and ventricular myocytes, where the ion channel electrophysiology has been well characterized with patch-clamp methods, much less work has been done to study repolarizing currents in single Purkinje cells. Although many voltage-clamp studies of cardiac Purkinje fibers were performed with classical two-electrode voltage-clamp methods, the inherent limitations of this methodology leave important questions about the ionic determinants of repolarization in Purkinje fibers unanswered. The present study was designed to characterize Ca2+-independent transient outward K+ current (Ito) in single canine Purkinje cells and compare it with Ito of canine ventricular myocytes. Because of the known variations in transient outward current of cells from different regions within the ventricular wall (23), midmyocardial cells were used, since these cells exhibit some properties intermediate between those of myocardial and Purkinje cells (34).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell preparation. Adult mongrel dogs (20-30 kg) were anesthetized with pentobarbital sodium (30 mg/kg iv), and their hearts were removed and immersed in Tyrode solution equilibrated with 100% O2. Purkinje fiber false tendons were excised from both ventricles into modified MEM (GIBCO-BRL) with 100 µM CaCl2-containing collagenase (1,000 U/ml, Worthington type II) and 1% BSA (Sigma Chemical). The fibers were bubbled with 100% O2 in a 37°C shaker bath for 50-100 min. After the endothelial sheath had been digested, revealing columns of Purkinje cells under light microscopy, the fibers were washed twice with a high-K+ salt solution and incubated for an additional 10 min. Individual cells were dispersed by hand pipetting, concentrated by centrifugation for 1 min, and kept in a high-K+ storage solution. Ventricular myocytes were isolated from the left ventricular free wall as previously described in detail (22) and kept in a high-K+ storage solution.

Solutions. The standard Tyrode solution for cell isolation and patch-clamp studies contained (mM) 136 NaCl, 5.4 KCl, 1.0 MgCl2, 1.0 CaCl2, 0.33 NaH2PO4, 5.0 HEPES, and 10 dextrose; pH was adjusted to 7.4 with NaOH. The high-K+ storage solution contained (mM) 20 KCl, 10 KH2PO4, 10 dextrose, 70 glutamic acid, 10 beta -hydroxybutyric acid, 10 taurine, 10 EGTA, and 0.1% albumin; pH was adjusted to 7.4 with KOH. The pipette solution contained (mM) 110 potassium aspartate, 20 KCl, 1 MgCl2, 5 Mg2ATP, 10 HEPES, 5 phosphocreatine, 0.1 GTP, and 5 EGTA for current recordings or 0.05 EGTA for AP recordings; pH was adjusted to 7.2 with KOH. The high-K+ salt solution contained (mM) 160 potassium glutamate, 5.7 MgCl2, and 5 HEPES; pH was adjusted to 7.0 with KOH. For voltage-clamp studies, atropine (1 µM) was included in the extracellular solution to eliminate basal ACh-dependent K+ current and CdCl2 (200 µM) was included to block Ca2+ current (ICa).

Contamination by Na+ current (INa) was prevented with a holding potential (HP) of -50 mV or by isomolar substitution with choline or Tris when more-negative HPs were necessary.

Data acquisition and analysis. General voltage-clamp techniques were carried out as previously described in detail (22, 41, 42), with recordings performed at 37°C. Ito amplitude was measured from the peak current level to the steady-state level at the end of a depolarizing pulse. Junction potential offsets averaged 10.0 ± 0.4 mV and were corrected for APs only. Smaller Purkinje cells were selected to ensure adequate voltage control. Mean capacitance averaged 125 ± 6 pF (n = 40) for Purkinje cells and 113 ± 6 pF (n = 22) for ventricular myocytes. Compensated series resistance averaged 2.2 ± 0.5 and 2.1 ± 0.6 MOmega , respectively.

A nonlinear least-square curve-fitting program (CLAMPFIT in pCLAMP 6) was used to perform exponential curve fitting. ANOVA with Bonferroni's t-test was used for multiple group comparisons and t-tests for single comparisons. P < 0.05 was considered to indicate statistical significance. Values are means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Purkinje cell APs. Figure 1 shows the behavior of a typical Purkinje cell AP after the onset of stimulation at 2 Hz. For clarity, only the response to the first pulse and the steady-state AP are shown. The first AP shows a clear notch, but as stimulation continues and the AP shortens, the notch is much reduced. The AP morphology and response to a pause are very similar to the well-recognized behavior of Purkinje fibers studied with classical microelectrode techniques (e.g., compared with Fig. 12 in Ref. 24).


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  		Fig. 1.   Purkinje cell action potential (AP) on the onset of stimulation at 2 Hz. First AP after a 60-s pause and steady-state AP after 5 s of stimulation are shown.

Voltage dependence of Ito. Figure 2A shows representative recordings of Ito from a Purkinje cell, and Fig. 2B shows the mean Ito density-voltage relation for 36 cells. Figure 2C shows original recordings illustrating voltage-dependent inactivation in one cell. Mean results for experiments analyzing voltage dependence of inactivation (obtained from data of the type shown in Fig. 2C) and activation (obtained with the type of data shown in Fig. 2B, with correction for driving force by dividing by TP - Erev, where TP is test potential and Erev is the reversal potential of tail currents) are shown in Fig. 2D. The mean values of half-maximal voltage (V1/2) and slope factor for activation (Boltzmann fits) were 8.6 ± 1.4 and 9.8 ± 0.6 mV (n = 7), whereas V1/2 and slope factor for inactivation averaged -29.9 ± 1.9 and -10.7 ± 1.1 mV (n = 13). Corresponding V1/2 and slope factor values for ventricular myocytes (data also shown in Fig. 2D) were 7.1 ± 0.8 and 10.6 ± 0.2 mV for activation (n = 9) and -35.9 ± 1.1 and -4.9 ± 0.5 mV (n = 9) for inactivation. The slope factor for inactivation in Purkinje cells was significantly larger (P < 0.001) than the value for ventricular muscle cells, and the V1/2 was slightly but significantly (P < 0.05) less negative. Activation parameters were not significantly different between ventricular and Purkinje cells.


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  		Fig. 2.   A: representative recordings of transient outward K+ current (Ito) in a Purkinje cell obtained during 100-ms voltage steps at 0.1 Hz from a holding potential (HP) of -70 mV. B: Ito density-voltage relations for 36 Purkinje cells. TP, test potential. Values are means ± SE. C: recordings from an experiment assessing the voltage dependence of Purkinje Ito inactivation. A 200-ms test pulse to +50 mV was preceded by 1-s conditioning pulses to voltages between -120 and +40 mV (HP -80 mV, protocol applied at 0.1 Hz). For clarity, only recordings obtained with prepulse potentials of -120, -70, -30, -20 and -10 mV are shown. D: data (means ± SE) from experiments assessing the voltage dependence of Purkinje cell (PC) and ventricular myocyte (VM) inactivation (inact) and activation (act). Inactivation was studied as illustrated in C, and activation was assessed from the current-voltage relation with a correction for the driving force (TP - Erev, where Erev is the reversal potential determined from the reversal of Ito tail currents, which averaged -75 mV for Purkinje cells and -72 mV for ventricular myocytes). Curves are best-fit Boltzmann relations.

Time-dependent inactivation and recovery. Examples of curve fits to Ito decay in illustrative Purkinje and ventricular myocytes are shown in Fig. 3A. Whereas current inactivation was biexponential for both, the time constants were larger in Purkinje cells. This is demonstrated by the mean data shown in Fig. 3B, which indicate that time constants were significantly greater in Purkinje cells than in ventricular myocytes.


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  		Fig. 3.   Time-dependent inactivation of Ito in Purkinje cells and ventricular myocytes. A: representative data (points prepared with data reduction so curve fits can be seen) during 100-ms depolarizations to +40 mV, with best-fit biexponentials shown. tau , Time constant. B: voltage dependence of inactivation time constants. Values are means ± SE for 12 Purkinje cells and 10 ventricular myocytes. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. ventricular myocytes.

Figure 4 illustrates the time dependence of recovery from inactivation. Original recordings of currents elicited by depolarizing pulses with varying coupling intervals at an HP of -80 mV are shown for a ventricular myocyte in Fig. 4A and for a Purkinje cell in Fig. 4B. Whereas recovery was almost complete within 100 ms in the ventricular myocyte, it required >4 s in the Purkinje cell. Mean recovery data for ventricular and Purkinje cells are provided in Fig. 4, C and D, respectively. At -80 mV, Purkinje Ito recovered with time constants of 35 ± 4 and 1,427 ± 70 ms (n = 7) compared with 30 ± 5 and 181 ± 24 ms, respectively, for ventricular muscle cells [n = 8, P = not significant (NS) for fast phase, P < 0.0001 vs. Purkinje cells for slow-phase time constant]. The slower kinetic component comprised 62 ± 4% of Purkinje Ito reactivation vs. 34 ± 3% of ventricular muscle reactivation (P < 0.001). At -60 mV in the same cells, reactivation time constants were 56 ± 5 and 1,900 ± 138 ms for Purkinje cells compared with 29 ± 6 and 307 ± 43 ms for muscle cells (P < 0.01 and P < 0.0001, respectively). The slower component accounted for 62 ± 2% of reactivation at -60 mV in Purkinje cells vs. 51 ± 8% in muscle cells (P = NS).


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  		Fig. 4.   Time-dependent reactivation of Ito, as determined with paired 100-ms pulses (P1 and P2) delivered at 0.1 Hz from -80 to +50 mV with various P1-P2 intervals. A and B: recordings at P1-P2 intervals from a representative ventricular myocyte and a Purkinje cell, respectively. C and D: ratios of current during P2 (I2) to current during P1 (I1) as a function of P1-P2 interval at HPs of -80 and -60 mV. Values are means ± SE for 8 ventricular myocytes (C) and 7 Purkinje cells (D). Best-fit biexponential functions are shown. E: frequency dependence of Ito as determined by the ratio of the current during the 15th pulse to the current during the 1st pulse of a train of 100-ms pulses from -80 to +50 mV. Trains were separated by 60 s at the HP. Values are means ± SE for 8 Purkinje cells and 6 ventricular myocytes. ** P < 0.01 vs. ventricular myocytes.

The frequency dependence of Ito in Purkinje cells and ventricular myocytes is compared in Fig. 4E, with steady-state Ito at each frequency plotted as a function of Ito of the first pulse after 60 s at the HP. Purkinje Ito showed substantially greater frequency dependence. For example, at 2 Hz, steady-state Ito in Purkinje cells was 55 ± 4% of the value during the first pulse, whereas in ventricular myocytes Ito at 2 Hz was 96 ± 3% of the first-pulse value (P < 0.001).

Pharmacological properties of Purkinje Ito. 4-Aminopyridine (4-AP) is commonly used to block Ito in mammalian cardiac cells (33). Figure 5 illustrates the 4-AP sensitivity of ventricular and Purkinje cell Ito. Concentration-response curves were obtained for the inhibition of Ito elicited by pulses to +50 mV at 0.1 Hz, as illustrated by the original recordings from a ventricular myocyte (Fig. 5A) and a Purkinje cell (Fig. 5B). Whereas 500 µM 4-AP produced <50% Ito inhibition in the ventricular cell, 100 µM 4-AP reduced Purkinje Ito by >50%. Figure 5C shows mean concentration-response data, along with best-fit curves according to the following equation: B = 100/[1 + (IC50/D)n], where B is the percentage of maximal current block at a concentration D, IC50 is the 50% blocking concentration, and n is the Hill coefficient. The 4-AP IC50 in Purkinje cells averaged 50 ± 7 µM, significantly less than the IC50 (526 ± 49 µM) for ventricular myocytes (P < 0.0001). The Hill coefficients averaged 1.0 ± 0.1 for Purkinje cells and 1.3 ± 0.1 for ventricular myocytes, suggesting 1:1 stoichiometry for the 4-AP-Ito interaction in both cases.


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  		Fig. 5.   Inhibition of Purkinje cell and ventricular myocyte Ito by 4-aminopyridine (4-AP). A and B: original recordings of ventricular myocyte and Purkinje cell Ito, respectively, obtained during 100-ms pulses from -70 to +50 mV before and after exposure to 4-AP. C: concentration-response data (means ± SE) for 4-AP inhibition of Ito in 6 ventricular myocytes and 9 Purkinje cells. Curves represent best fit to the equation in RESULTS.

Tetraethylammonium (TEA) is a classical K+ channel blocker that does not affect canine atrial Ito (41). Figure 6A shows original recordings of Purkinje Ito before and after exposure to a series of TEA concentrations (TEA was substituted for choline in the extracellular solution for >1 mM TEA to prevent changes in osmolarity). Inhibition appeared rapidly, was clearly observed at 0.2 mM, increased with increasing concentration, and reversed substantially after drug washout. The Ito density-voltage relation is shown for different TEA concentrations in Fig. 6B. Significant inhibition was seen at all voltages. Maximum inhibition at 100 mM was on the order of 82%. Figure 6, inset, shows the concentration-response relation for TEA inhibition, which had an IC50 of 2.4 ± 0.4 mM. Figure 6, C and D, shows Ito from a ventricular myocyte before and after exposure to 10 mM TEA. In this and six other cells studied in the same way, 10 mM TEA had no effect on ventricular muscle Ito.


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  		Fig. 6.   Response of Purkinje cell and ventricular myocyte Ito to tetraethylammonium (TEA). A: currents recorded from a Purkinje cell in response to a 100-ms step from -70 to +50 mV under control conditions, in the presence of TEA, and after TEA washout. B: concentration-dependent effects of TEA on the current-voltage relation of Purkinje cell Ito. Values are means ± SE of 5 cells. * P < 0.01 vs. control. Inset: TEA concentration dependence for Ito inhibition on the basis of mean data at +50 mV from 5 cells. Values are means ± SE, but error bars fall within symbols. C and D: Ito recordings from a ventricular myocyte obtained before and after 10 mM TEA, respectively.

The antiarrhythmic drug flecainide has been used to obtain information about the potential molecular basis of Ito, because the cloned K+ channel subunit Kv4 is more sensitive to the drug than the cloned K+ channel subunit Kv1.4 (40). Figure 7 shows the concentration-dependent effects of flecainide on ventricular cell and Purkinje Ito. As shown by the results of representative cells in Fig. 7, A and B, flecainide produced concentration-dependent inhibition in both cell types. The IC50 averaged 29 ± 13 µM (n = 6) in ventricular myocytes and 42 ± 13 µM in Purkinje cells (n = 5, P = NS). Flecainide accelerated current decay, as previously reported in human atrial myocytes (38), in ventricular and Purkinje cells, decreasing the rapid-phase time constant to a significant and similar extent in each (Fig. 7, C and D).


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  		Fig. 7.   A and B: original recordings of Ito in the presence of flecainide during 100-ms depolarizations from an HP of -50 mV to +50 mV at 0.1 Hz in a ventricular myocyte and a Purkinje cell, respectively. C and D: inactivation time constants before and after exposure to 10 µM flecainide in 10 ventricular myocytes and 6 Purkinje cells, respectively. ** P < 0.01 vs. control.

Oxidative stress is known to produce significant slowing of Ito carried by the Kv1.4 subunit and to have little effect on Kv4 channels (10). Figure 8A shows the effects of 0.01% H2O2 on Ito in a Purkinje cell and a ventricular myocyte. H2O2 did not alter Ito in the ventricular myocyte but clearly slowed Ito inactivation in the Purkinje cell. Mean inactivation time constants in seven Purkinje cells and eight ventricular myocytes before and after H2O2 are shown in Fig. 8B. A significant increase in the slow-phase time constant was seen in Purkinje cells, with no changes in ventricular myocytes. In addition to slowing the slow-phase time constant, H2O2 increased the proportion of inactivation comprised by the slow phase from 41 ± 4 to 54 ± 6% (P < 0.05).


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  		Fig. 8.   Effects of H2O2 on Ito inactivation in Purkinje cells and ventricular myocytes. A: original recordings of Ito during 100-ms depolarizations from an HP of -50 mV to voltages between -30 and + 50 mV in 10-mV increments before and after 0.01% H2O2. B: inactivation time constants before and after exposure to 0.01% H2O2 in 7 Purkinje cells and 8 ventricular myocytes. * P < 0.01 vs. control (Ctl).

Finally, we evaluated the effects of two toxins known to affect cloned K+ channels. Blood-depressing substance (BDS) is a sea anemone toxin that specifically and potently (IC50 ~ 50 nM) inhibits Ito carried by the expression of Kv3.4 channels (9). Figure 9A shows the lack of effect of 100 nM BDS on Purkinje cell Ito, as seen in this and three other cells studied at the same concentration. At the highest concentration evaluated, i.e., 1 µM, Purkinje cell Ito density before and after BDS averaged 16 ± 2 and 15 ± 3 pA/pF (P = NS) on stepping from -50 to +50 mV in four cells.


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  		Fig. 9.   Effects of blood-depressing substance and dendrotoxin on Purkinje cell Ito. Original recordings of Ito during 100-ms depolarizations from an HP of -50 mV to +50 mV are shown before and after blood-depressing substance (A) and dendrotoxin (B).

Some members of the Shaker subfamily of voltage-gated K+ channel subunit proteins, including Kv1.1, Kv1.2, and Kv1.3, can be inhibited by TEA as well as dendrotoxin (DTX) (15, 35). As shown in Fig. 9B, relatively high concentrations of DTX (100 nM) slightly reduced Ito in Purkinje cells. In eight Purkinje cells exposed to 100 nM DTX, the drug reduced current density by 27 ± 6% (P < 0.001 vs. control). DTX was not found to significantly affect ventricular Ito.

Effects of blocking Ito on the Purkinje cell AP. To obtain an indication of the potential functional role of 4-AP- and TEA-sensitive Ito in the Purkinje cell AP, we exposed Purkinje cells to concentrations of these agents that would be expected to have a selective effect on Ito. To confirm the effects of the drugs on ionic currents, we performed voltage clamp with an HP of -50 mV (to inactivate INa) on each cell from which APs were recorded. Figure 10 shows APs and ionic currents (on depolarization to +50 mV) recorded at 0.1 Hz from representative cells exposed to 50 µM 4-AP (Fig. 10A) and 5 mM TEA (Fig. 10B). The AP changes are consistent with the strong Ito inhibition shown by the voltage-clamp recordings, with a marked slowing in repolarization (particularly the earlier phases to 50% repolarization) and elevation of the plateau. AP duration to 20, 50, 70, and 90% repolarization were increased by 86 ± 44, 119 ± 77, 12 ± 9, and 2 ± 4%, respectively (n = 4), by 4-AP and by 53 ± 7, 84 ± 27, 30 ± 11, and 12 ± 5%, respectively, (n = 6) by TEA.


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  		Fig. 10.   Effects of 50 µM 4-AP (A) and 5 mM TEA (B) on APs of Purkinje cells. Insets: currents in each cell elicited by a 100-ms pulse from -50 to +50 mV. As expected, 4-AP and TEA strongly inhibited Ito and delayed repolarization, particularly the earlier phases.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have studied the properties of Purkinje cell Ito in detail and compared them with those of Ito in ventricular cells in the midmyocardium. We found several important differences between Purkinje cell and muscle cell Ito, notably in terms of the kinetics of inactivation and recovery from inactivation, frequency dependence, and sensitivity to block by 4-AP and TEA.

Comparison with previous single-cell voltage-clamp studies of Ito in cardiac myocytes and putative cloned cardiac Ito subunits in heterologous expression systems. Whereas the properties of canine ventricular Ito that we observed are quite consistent with previous observations regarding cardiac Ito in a variety of tissues and species (1), those we noted for Purkinje cell Ito were quite different in several important respects. Most noteworthy is the sensitivity of Purkinje cell Ito to TEA. Similar to canine ventricular Ito, canine atrial Ito is not affected by TEA at up to 100 mM (41). Similarly, Ito in atrial, ventricular, and nodal cells from a variety of species are insensitive to TEA (1). The molecular species recognized to participate in cardiac Ito include Kv4.2 (1, 12, 17, 37), Kv4.3 (10, 12, 17, 37), and Kv1.4 (37). None of these are inhibited by TEA, even at relatively large concentrations (10, 29, 35). In addition, the 4-AP sensitivity of Purkinje cell Ito is greater than that reported for native atrial (41) or ventricular Ito, as well as for Kv1.4 (27) and Kv4.2 (29).

Comparison with previous reports regarding cardiac Purkinje cell Ito. The presence of Ito in cardiac Purkinje fibers was recognized in early dual-microelectrode voltage-clamp studies of multicellular preparations (8). The ionic specificity of Purkinje cell Ito was the subject of some controversy until Kenyon and Gibbons (19, 20) showed in 1979 that ~20% of the current was sensitive to Cl- substitution, whereas the vast majority could be suppressed by the K+ channel blockers 4-AP and TEA (19, 20). Formal concentration-response studies were not performed, but 500 µM 4-AP caused full inhibition of the Cl--insensitive component (19). TEA was effective at much higher concentrations (20-40 mM) and required prolonged perfusion (in the range of hours) to act (20). Ito recovery in Purkinje fibers has also been shown to be relatively slow, with a reactivation time constant in the range of 500 ms at -80 mV (13). These dual-microelectrode voltage-clamp studies of multicellular preparations were limited by great technical difficulty, potential problems of voltage control, and issues of slow and uneven distribution of drugs requiring diffusion across superfused multicellular preparations. Furthermore, we were unable to find in the previous literature detailed concentration-response analyses for TEA or 4-AP effects on Purkinje fibers or direct comparisons between Ito properties in Purkinje and muscle tissue with similar methods. Furthermore, we are not aware of previous observations regarding the effects of flecainide, oxidative stress, BDS, or DTX on Purkinje cell Ito. Cordeiro et al. (7) recently performed voltage-clamp studies of rabbit Purkinje cells from free-running false tendons. They noted a substantial Ito, with a current density at +60 mV of ~15 pA/pF, similar to Ito density in canine Purkinje cells in the present study. Rabbit Purkinje cell Ito was strongly inhibited by 2 mM 4-AP, but concentration-response analyses were not performed.

Potential mechanisms and significance. The present study is the first of which we are aware to characterize in detail the pharmacological response of canine Purkinje cell Ito and to compare directly the biophysical and pharmacological properties of Purkinje cell Ito with those of ventricular myocytes from the same species. It is now clear that the Shal (Kv4) family of genes play a prominent role in encoding pore-forming K+ channel subunits of Ito in mammalian atrial and ventricular myocytes (2, 12, 37). Recent evidence suggests that Kv1.4 subunits, the first Ito-like channel subunits to be cloned from cardiac tissue (30, 36), are also likely to contribute to a slowly recovering Ito component in rabbit atrial myocytes (37), rat ventricular septum (39), and ferret ventricular subendocardium (5).

The properties of Purkinje cell Ito do not match those of Ito carried by any single cloned subunit. The response to oxidative stress and the slowly recovering component resemble the behavior of Kv1.4 channels but are not compatible with the rapidly recovering component and the response to flecainide. The latter responses are compatible with Kv4.x channels, but the former are not. Moreover, the TEA sensitivity of Purkinje cell Ito is not compatible with currents carried by any of the subunits (Kv1.4, Kv4.2, or Kv4.3) implicated in atrial and ventricular Ito, which are insensitive to TEA at up to 100 mM (10, 29, 35). Furthermore, a large proportion of Purkinje cell Ito is TEA sensitive (Fig. 6), indicating that a TEA-sensitive subunit must participate in the formation of most Purkinje cell Ito channels. K+ currents resulting from the expression of the Kv3 (Shaw Kv3) family of genes are typically sensitive to TEA, and two Kv3 genes, Kv3.3 and Kv3.4, carry an Ito-like current on heterologous expression (14, 28, 32). Although Kv3 genes have yet to be cloned from cardiac tissue, Brahmajothi et al. (6) showed the presence of Kv3 transcripts with in situ hybridization in the ferret heart, and we have described a TEA-sensitive ultrarapid delayed rectifier in dog atrial cells with functional properties that most closely resemble those of currents carried by Kv3.1 subunit expression (41). On the other hand, the TEA sensitivity of Kv3 channels is about an order of magnitude greater than that of Purkinje cell Ito, and the Kv3.4 blocker BDS had no effect on Purkinje cell Ito. It thus remains to be determined whether the TEA sensitivity of Purkinje cell Ito is due to the presence of a novel K+ channel subunit, to coassembly of different alpha -subunits, or possibly to associated accessory subunits.

Ito clearly plays a significant role in Purkinje fiber repolarization. This role is evidenced by the effect of 4-AP and TEA to raise the plateau and prolong AP duration (Fig. 10). The major limitation to antiarrhythmic drug therapy today is the risk of proarrhythmic responses, of which a prime type is torsade de pointes in association with excess Q-T prolongation. The origin of drug-induced torsade appears to be early afterdepolarizations from the Purkinje fiber network (11, 25). Patients with congestive heart failure have a high incidence of sudden death (3) and are particularly predisposed to drug-induced torsade (21). Experimental congestive heart failure causes downregulation of ventricular myocyte Ito (18) and is associated with ventricular repolarization abnormalities and a high incidence of sudden death (26). Furthermore, coronary artery disease is also a risk factor for drug-induced torsade (21), and Ito is reduced in the subendocardial Purkinje network overlying a myocardial infarction (16). Given the importance of Ito in Purkinje fiber repolarization, changes in Purkinje Ito may be involved in the genesis of spontaneous and drug-induced ventricular tachyarrhythmias caused by arrhythmogenic afterdepolarizations arising from cardiac Purkinje fibers. The present studies indicate that Purkinje cell Ito clearly has important properties that distinguish it from ventricular or atrial myocyte Ito and point to a potentially different molecular basis.

Potential limitations. The isolation of cells from free-running Purkinje fiber false tendons is a difficult technique, requiring enzymatic digestion of the surrounding sheath and with enzyme access to cells occurring via diffusion rather than coronary perfusion as for ventricular myocytes. The method of cell isolation can influence cell properties, although Ito appears to be relatively resistant to damage by isolation (42). The generally high quality of isolated cells is indicated by our ability to record APs of relatively normal appearance (Fig. 1), preserving the characteristic rate-dependent behavior of the Purkinje cell notch (24), which is due to the slow recovery kinetics of Ito (4). On the other hand, there was clear variability in Purkinje cell APs (cf. Figs. 1 and 10), likely because of the greater susceptibility of some currents (e.g., ICa) to damage by the isolation technique, leading to a more-negative plateau and shorter APs in some cells.

Conclusions. Canine Purkinje cell Ito properties show important differences from canine ventricular muscle Ito. Furthermore, some of the properties of Purkinje cell Ito (notably its TEA sensitivity) differ from those reported for ventricular, atrial, and nodal myocyte Ito in a variety of species and from the cloned channel subunits (Kv1.4, Kv4.2, and Kv4.3) presently recognized to underlie cardiac Ito. These findings suggest that Purkinje cell Ito may have a molecular composition different from that of atrial and ventricular muscle. Given the importance of Purkinje fibers in cardiac electrophysiology and, in particular, in the genesis of ventricular tachyarrhythmias associated with delayed repolarization, these findings are of great potential importance.


    ACKNOWLEDGEMENTS

The authors thank Chantal St-Cyr for expert technical assistance, Luce Bégin and Annie Laprade for typing the manuscript, and Sylvie Diochot and Michel Lazdunski for providing BDS toxin.


    FOOTNOTES

This work was supported by operating grants from the Medical Research Council of Canada and the Quebec Heart Foundation and by the Fonds de Recherche de l'Institut de Cardiologie de Montréal. Z. Wang is a Canadian Heart Foundation research scholar.

Address for reprint requests and other correspondence: S. Nattel, Research Center, Montreal Heart Institute, 5000 Belanger St. E., Montreal, QC, 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. §1734 solely to indicate this fact.

Received 5 November 1999; accepted in final form 3 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 279(2):H466-H474
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