Introduction

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Inward and outward ionic currents play critical roles in regulating action potentials and effective refractory period in cardiac tissues. In human heart cells, the major inward current responsible for shaping the action potential plateau is the L-type Ca⁺⁺ current (Grand et al., 1990). The presence of T-type Ca⁺⁺ current (Grand et al., 1990) has not been established. In contrast, multiple K⁺ currents in the plateau region were reported. The most often described is the transient outward current, I_to (Amos et al., 1996, Escande et al., 1987, Shibata et al., 1989). This current is inactivated by positive holding potentials and is Ca⁺⁺ dependent or independent (Escande et al., 1987). Also, there is a rapidly activating, slowly inactivating, sustained outward current, I_so (Amos et al., 1996, Wang et al., 1993) (or I_Kur, I_su). It is blocked by micromolar 4-AP, and is present in human atrial, but not in ventricular cells. In several studies, a delayed outward current, I_K (Beuckelmann et al., 1993, Wang et al., 1993) was described, which can be subdivided into a rapid, I_Kr, and a slow, I_Ks current (Wang et al., 1994, Li et al., 1996). In addition, there is the inward rectifier, I_K1 (Beuckelmann et al., 1993). This current is markedly reduced in the ventricular cells of patients with terminal heart failure.

Blockade of the K⁺ currents, as by class III antiarrhythmic compounds, can effectively prolong the APD and may terminate re-entrant arrhythmia and atrial arrhythmia (Singh and Nademanee, 1985). The projected clinical usefulness of such a mechanism has spurred intense efforts searching for these compounds. Sotalol, which is approved for use in human, is a good example. In animal heart, sotalol, like most class III antiarrhythmic compounds, blocks the delayed rectifier, specifically, the rapidly activating outward potassium current, I_Kr (Sanguinetti and Jurkiewicz, 1990). In human atrial cells, however, sotalol's action is unknown because I_Kr has been difficult to resolve in human heart tissues. Only one laboratory has reported this currents in considerable detail (Li et al., 1996, Wang et al., 1994). Other investigators (Amos et al., 1996, Escande et al., 1987, Shibata et al., 1989), including a recent study of ventricular myocytes in nonfailing human hearts (Konarzewska et al., 1995), have not observed I_Kr. This is not to imply that I_Kr plays no role in APD prolongation because in human papillary muscles, E-4031, a specific I_Kr blocker, clearly prolongs APD (Ohler and Ravens, 1994).

Besides K⁺ channel blockers, inward current activator can also promote class III activity. A typical example is ibutilide currently in clinical use. Ibutilide increases a Na⁺ sensitive inward current in guinea pig ventricular cells (Lee, 1992). This current appears at the plateau potential positive to 20 mV and peaks at 20 mV. It shares many characteristics of the "L"-type Ca⁺⁺ current with the exception that it inactivates more slowly, has a more positive peak-current potential, and is removed upon external Na⁺ removal. In Na⁺ and Ca⁺⁺ containing external solution, ibutilide can increase this inward current at very low concentrations of 10⁹ to 10⁷ M. Removing external Na⁺ abolishes ibutilide's effect. The remaining "L" type Ca⁺⁺ current is relatively insensitive to the drug. At similar low concentrations of 10⁹ to 10⁷ M, ibutilide prolongs APD (Lee, 1992). Thus, the close agreement between ibutilide's effect on the Na⁺ sensitive inward current and the APD prolongation suggests that the inward current is responsible for ibutilide's class III activity. Ibutilide also blocks I_Kr in cultured tumor atrial cells, the AT-1 cells (Yang et al., 1995), and may contribute to APD prolongation.

In human heart cells, there is no mechanistic information on ibutilide. The purposes of this study were: 1) to establish experimental conditions for obtaining single human atrial cells that are suitable for stable recordings of action potentials and ionic currents by the suction pipette method; 2) to characterize ibutilide's ionic mechanism of action on human atrium.

	Methods

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Cell Preparations

Atrial tissues procurement procedures were approved by the ethics committee of Bronson Methodist Hospital (Kalamazoo, MI). Segments of atrial appendages removed from patients undergoing heart surgery were immediately immersed in Ca⁺⁺ free-Tyrode's solution at 37°C. The solution contained, in mM: NaCl, 137; KCl 4; MgCl₂, 1.05; glucose, 5; Tris HCl, 3.17; Tris base 0.41; pH at 7.47 and saturated with 100% 0₂ gas. Cell dissociation procedures began 10 to 20 min after tissue removal. The tissue was washed twice in Ca⁺⁺-free Tyrode's solution saturated with 0₂ and contained 30 mM 2,3-butanedionemonoxine (Peeters et al., 1995). This compound, although increased cell yield, reduced cell quality, and its use was discontinued shortly in this study. The cell dissociation procedure was very similar to that for the dissociation of coronary smooth muscle cells (Wilde and Lee, 1989). Briefly, atrial tissue submerged in Ca⁺⁺-free Tyrode's solution was minced by a pair of scissors to about 1-mm pieces and then washed twice with the same solution. The minced tissues were then transferred to the same Ca⁺⁺-free Tyrode's solution that contained 350 U/ml collagenase (type II, Sigma Chemical Co., St. Louis, MO), 34 U/ml elastase (type II, aqueous suspension, Sigma), 200 U/ml soybean trypsin inhibitor, 0.1% taurine and 0.1% bovine serum albumin. In this enzymatic solution, the minced tissues were incubated at 35°C while stirred at 330 r.p.m. with a stir bar. A drop of incubation solution was drawn for examination of single cells every 10 min until cells appeared. Then the solution was decanted for centrifugation at 1380 rpm for 1 min. The pellet was resuspended in a wash solution of 300 µM Ca⁺⁺ Tyrode's solution that contained 1% bovine serum albumin. The remaining minced tissues were placed in fresh enzymatic solution for continual digestion. Normally, cells began to appear within 45 min, peaked in about 60 min. Typical good cell yield was about 30%. For electrophysiological recording, large rod shape cells of approximately 20 µm wide by 120 µm long, with clear striations and shining appearance were selected. Preparations that yielded less cells or cells with small size or unclear striations were discarded. Harvested cells were stored in Tyrode's solution containing 300 µM Ca⁺⁺ and 1% BSA at room temperature for electrophysiological recording in the next 4 to 6 hr.

Electrophysiological Recordings

Membrane currents of single human atrial cells were recorded in whole-cell configuration using the suction pipette method. Pipettes were fabricated from borosilicate glass capillaries (Scientific America, Evanston, IL) with a P.80/PC, Flamming Brown puller (Sutter Instruments, Novato, CA). Pipette resistance was 0.8 to 1 M when filled with experimental solutions. Whole-cell currents were recorded using an AXOPATCH-1D amplifier (Axon Instruments, Inc., Foster City, CA) interfaced with a personal computer. A commercial software "pClamp" (Version 6.0.4, Axon Instruments, Inc.) was used for data acquisition and analysis. Using the suction pipette, spontaneous action potentials were elicited by brief positive current pulses in current-clamp mode. In voltage-clamp, the cell capacitance was calculated from the capacitance transient elicited by a 10 mV pulse. The seal resistance was between 0.5 to 2 G. The series resistance was compensated by about 90%. Currents were filtered at 2 KHz and digitized at 1 to 2 KHz. Cells were held at 40 mV throughout during experiments and dialyzed internally with either K⁺ or Cs⁺ solutions for 10 to 15 min to reach equilibrium before recording. Current amplitudes were not corrected for leak.

Solutions and drugs. For resting and action potentials: A, Pipette solution, in mM: K-glutamate, 115; K-aspartate, 10; KCl, 5; CaCl₂, 1; EGTA, 10; MOPS, 10; glucose, 20; Mg⁺⁺-ATP, 5; creatine phosphate, sodium salt, 5; pH adjusted to 7.2 with KOH . B, External, normal Tyrode's solution, in mM: NaCl, 137; KCl, 4; CaCl₂, 2; MgCl₂ 1.05; N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 10; pH adjusted to 7.5 with NaOH.

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	Results

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In this study, 110 atrial tissues were processed for single cell dissociation. Tissues from 35 patients with various medical conditions and medications yielded successful experiments. Among this group, 12 had hypertension; 5 had coronary artery diseases; 11 had diabetes, including insulin-dependent and noninsulin-dependent diabetes mellitus; 4 had chronic obstructive pulmonary diseases; 2 had myocardial infarction and 1 had atrial fibrillation. They were treated with one or more types of medications such as aspirin, insulin, Ca⁺⁺ channel blockers (diltiazem, nicardipine), heparin, nitroglycerine, glybenclamide and alprazolam. Most were smokers. Due to the small sample for each condition, we were unable to establish a correlation of cell yield or drug response with any of these conditions. The age of the 35 patients ranged from 41 to 82 yr old, with a mean of 64.5 ± 7.2 yr. Tissues from younger patients yielded more and better cells with shiny appearance and clear striations. Other patients produced no viable cells or cells with low resting membrane potential and rapid run-down of inward currents.

Cell size varied markedly, capacitance ranged from 63 to 207 pF with a mean of 115.5 ± 6.28 pF (n = 36). The cells were rod-shape, many with irregular branches. About 60% of the cells had low resting potential of 60 to 40 mV, and full size action potentials were difficult to elicit (Amos et al., 1996). The other cells had normal resting membrane potentials of 71.4 ± 2.4 mV (n = 16). At 37°C, brief current stimulation of these cells at 1 Hz elicited full size action potentials with overshoot of 36.8 ± 1.8 mV and duration (APD) of 265 ± 89 msec at 90% repolarization. The resting potential, action potential overshoot and duration remained stable in 30 to 60 min recording time. Figure 1 shows three types of representative action potentials that have different plateau heights. Type 1 had an early plateau and a late plateau phase; type 2 had a late plateau, and type 3, had no plateau (Wang et al., 1993). In all cases, the late repolarization phase was slow. Action potentials were recorded from the 16 cells. Type 1 was obtained from three cells, all from a smoking patient with diabetes and treated with glybenclamide and insulin. Type 2 was from five cells obtained in two patients, one with myocardial infarction and coronary artery disease, treated with heparin, diltiazem and the other with hypertension, treated with nitroglycerine. Type 3 was observed in eight cells, all from hypertensive patients on chronic Ca⁺⁺ channel blockers (diltiazem and nifedipine), nitroglycerine. Due to the small patient sample with multiple diseases and drug treatments, it was not possible to relate the action potential shape with any particular patients or treatment. However, the hypertensive patients treated with Ca⁺⁺ channel blockers more often developed type 3 action potentials. For unknown reasons, type 1 and 2 action potentials can convert to type 3 spontaneously.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Three types of human atrial action potentials. Action potentials shown were obtained from three internally dialyzed cells using the suction pipette method and solutions described in "Methods." The spike preceding each action potential is the stimulus artifact in response to a 0.5 to 0.9 nA stimulus lasting 5 to 10 msec. The action potentials shown were elicited by the last pulse in a pulse train with 16 identical stimuli delivered to the cell at 1 Hz frequency. No outward holding current was injected into the cell to artificially hyperpolarize the membrane potential and to generate the repolarization phase (temperature, 37°C).

Ibutilide elevates and prolongs the plateau of human atrial action potentials, in the absence of I_Kr. Type 3 action potential is of special interest, because, according to Wang et al. (1993), it has no I_Kr. If ibutilide were to prolong APD by blocking this current, then it should not affect type 3 action potentials. Nevertheless, figure 2A shows that ibutilide induced a robust and reversible APD prolongation. The effect is specific, by generating a plateau at 15 to 10 mV, and this was observed in all experiments of this kind. The action potential result suggests, although indirectly, that in human atrial cells, ibutilide's class III effect may be independent of I_Kr activity.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   A, Typical effect of ibutilide on a triangular action potential (type 3). Again, suction pipette and solutions for action potentials similar to figure 1 was used. For a steady-state effect, the cell was stimulated by a pulse-train containing 16 pulses delivered to the cell at 1 Hz frequency. The first pulse-train was delivered to the cell in the absence of drug; then after 5 min in drug exposure, a second pulse train was delivered; this was followed by a third pulse train after a 10-min wash in the control solution. Action potentials shown were elicited by the last pulse of each train. Temperature was at 37°C. Note the drug specifically generated a plateau at about 10 mV in these triangular action potentials, but without delaying either the early or late repolarization phase. B, Current traces and I-V relationship (current amplitudes measured at the end of the 2-sec step) obtained in 2 mM 4-AP, 100 nM atropine, 1 µM nifedipine for the isolation of IKr and IKs (Wang et al, 1994). Currents were elicited every 10 sec from holding potential of 50 mV to various step (2 sec long) potentials as indicated. Open symbols, control; solid symbols, in dofetilide (temperature, 36°C).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Separation of Ito from Iso using 4-aminopyridine (4-AP) and depolarization prepulses. A, The cell was held at 40 mV and step depolarized from 20 to 50 mV, at 10 mV increments. The cell was bathed in 0.2 mM Ca2+ Tyrode's solution with 1 µM nifedipine (solution C). The 4-AP sensitive net current traces were obtained by subtracting the current in 0.1 mM 4-AP from the control. Open symbols (, ) are controls and solid symbols (, ) are in 0.1 mM 4-AP; the dotted line on the I-V curve represents net 4-AP sensitive current, Iso. Vertical scale bar = 400 pA; horizontal bar = 100 msec. B, Concentration response curve of Ito () and Iso (). The current amplitude was normalized against the peak current amplitude in control; Imax, peak control current amplitude; I, current amplitude at various 4-AP concentrations as indicated on the abscissa. IC50 was obtained by fitting the data to the equation: (a-d)/(1+[x/c]b)+d where a = asymptotic maximum; b = slope; c = IC50; d = asymptotic minimum; x = drug concentration. C, prepulses lasting 2 sec from 70 to 10 mV, at 10 mV increments, were used to inactivate Ito, with a 5-msec return to 40 mV preceding the 50 mV test pulse. D, Normalized steady-state inactivation curves of Ito () and Iso (). Imax, is the current amplitude at prepulse potential of 70 mV and I, current amplitudes at prepulse potentials positive to 70 mV as indicated on the abscissa. The data of Ito were best-fitted by the Boltzmann function: 1/(1+exp(V-V0.5)/K)+C where V, is prepulse potentials; V0.5, potential at 50% of maximal current; K, the slope factor; and C, the inactivating component. V0.5 = 30 mV and slope = 3.4 at 36°C. For Iso, due to incomplete inactivation, the curve can not be fitted properly.

Ibutilide does not inhibit human atrial I_to, I_so, I_K1 and I_Ks-like currents at moderate concentrations. To examine if the APD prolongation by ibutilide is caused by K⁺ current blockade, we separated I_to from I_so by using a 2-sec, 10 mV prepulse to inactivate I_to (fig. 3C). Also, in a separate set of experiments, we isolated the I_Ks-like current using the 2 mM 4-AP, 100 nM atropine and 1 µM nifedipine protocol (fig. 2B) (Wang et al., 1994). We then chose the concentration of 10⁷ M that caused a robust APD prolongation (fig. 2A). In control at 20 mV, for example, I_to, I_so and I_Ks measured 312 ± 80, 219 ± 60 and 122.6 ± 23 pA, respectively; in 10⁷ M ibutilide, the corresponding values became 395 ± 75 (n = 8, P > .05), 214 ± 75 (n = 8, P > .05) and 116 ± 19 pA (n = 4, P > .05). Figure 4A shows total outward current (upper panels), I_so (middle panels) and I_to (lower panels) before (left) and after (right) ibutilide application. Figure 4B are I-V plots of I_to and I_so before and after drug application. Figure 4C further illustrates experiments on the I_Ks-like current. In the physiological temperature and potential range tested, ibutilide did not inhibit these currents. Finally, we examined ibutilide's effect (10⁷ M) on the inward rectifier, I_K1. Again, the result was negative. For example, at 100 mV, I_K1 measured 214 ± 20 pA at 2 sec into the depolarization step; in 10⁷ M ibutilide, it became 188 ± 18 pA (n = 8, P > .05). These results demonstrate that ibutilide, at a concentration that prolongs APD (fig. 2A), produces no apparent changes on these K⁺ currents. At higher concentration of 10⁶ M and above, ibutilide blocked I_to dose dependently (data not shown). However, the high concentration effect can not explain ibuitlide's APD prolongation seen at 10⁷ M. This point will become more apparent later in this report.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Effect of ibutilide on outward K+ currents. A, Superimposed current traces on the left were controls and at right, 5 min in 10 M ibutilide. The cell was held at 40 mV, without (Aa, top panels) and with a 2-sec prepulse to 0 mV (Ab, middle panels). Net Ito current (Ac, bottom) was obtained by subtracting Ab from Aa. Currents lasting 150 msec were elicited from 50 to 60 mV, at 10 mV increments, and repolarized to 20 mV for 50 msec for detection of tail currents. Aa, total current; Ab, Iso; Ac, Ito. Open symbols are controls and solid symbols, in ibutilide. Vertical bar = 400 pA. B, Left, Ito, measured at 10 and 150 ms into the step before (, ) and 5 min after (, ) 10 M ibutilide application. Right, Iso, before (, ) and after (, ) 10 M ibutilide application. C, Ibuitlide has no effect on the IKs- like current. External solution contained 2 mM 4-AP, 100 nM atropine, 1 µM nifedipine (Wang et al, 1994) and step depolarized from a holding potential of 50 mV to various step potentials as indicated for 2 sec long, at 10 mV increments. Ibutilide (10 M) was applied for 5 to 10 min before data were taken. I-V are current amplitudes measured at the end of the 2- second step vs. voltage (temperature, 37°C).

Inward current increased by ibutilide. We then examined the inward currents at the plateau potential region, hereafter referred to as the inward current. The current was recorded by bathing the cell in K⁺-free solutions containing K⁺ channel blockers (solutions E and E1, see "Methods"). In addition, holding potential was at 40 mV throughout to inactivate the fast inward Na⁺ current and the T-type Ca⁺⁺ current. Because stable inward currents were critical for this study, we carefully selected cells that had less than 5% "run-down" in the first 10 min of recording time. In a typical good experiment, the inward currents remained stable for 30 min or longer. Figure 5A shows the inward current elicited by a ramp depolarization. Application of 10⁷ M ibutilide caused the peak current to increase significantly over the plateau potential range. Figure 5B is the time course of current increase in response to ibutilide application. In most cells, the onset of drug effect was slow, requiring 10 to 15 min, and took 15 to 20 min to washout. Figure 5C illustrates inward currents elicited by depolarization steps in the absence and presence of ibutilide. At 20 mV depolarization, for example, the peak inward current was increased from 1478 ± 103 to 2347 ± 75 pA (n = 12, P < .05). The sustained inward current, measured at 50 msec into the step, was increased from 296 ± 56 to 425 ± 87 pA (n = 12, P < .05). At the physiological temperature, ibutilide increased both the peak and the sustained inward current. The sustained effect was most evident at 10 mV (fig. 5C). In addition, ibutilide slowed the time course of the inward current inactivation. Double exponential fit to the inactivation time course showed that the average fast time constant at 10 mV was increased from a control of 3.2 ± 0.5 msec to 4.7 ± 0.3, 5.2 ± 0.24 and 4.9 ± 0.2 msec in 10⁹, 10⁸ and 10⁷ M ibutilide, respectively; the average slow time constant was also increased from a control of 26 ± 5 msec to 30 ± 8, 44 ± 4 and 41 ± 3 msec, respectively (n = 12). Qualitatively, the human atrial cell result is similar to the guinea pig ventricular cells at room temperature (Lee, 1992).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Inward currents and their response to ibutilide. K+-free solutions with TEA and 4-AP were used to remove the K+ currents (see "Methods"). Both Na+ and Ca++ were present in the external solution. Cell was held at 40 mV and depolarized to potentials as indicated. A. Ramp depolarization at 1.2 mV/msec was delivered to the cell at potentials as indicated. Upper current trace is the control and lower trace is in ibutilide. Note: at negative potentials, the fast inward Na+ current failed to appear probably due to slow recovery from inactivation. This was consistent in human atrial cell experiments. B. The time course of current increase at 10 mV in response to 10 M ibutilide application. Open symbols are control current amplitudes at the peak () and at 15 msec into the step (); solid symbols are in ibutilide. Dotted lines indicate control current level. C. Another cell, inward current elicited by step depolarization. Steps to potentials as indicated were delivered to the cell at 0.5 Hz. Two to four I-V controls were taken to make sure the current was stable, then ibutilide was applied to the cell bath under gravity while the cell was continuously stimulated to a fixed potential of 10 mV at 0.5 Hz. After 2 to 5 min, the same I-V run was performed. Currents were not corrected for leak. Holding potential, 40 mV and temperature, 37°C.

Ibutilide concentration dependently increases the inward current, effective at 10¹⁰ M. Figure 6 illustrates a continuous response of the inward current of a cell to sequential ibutilide application from 10¹⁰ up to 10⁶ M, then back to 10⁸ M before washing out the drug. Figure 6A shows peak inward current increase with time in response to cumulative drug application. The experiment lasted for about 75 min until it was terminated after a long washout. The increase reached a peak at 10⁶ M. However, subsequent lowering of the drug concentration to 10⁸ M increased the current further. The drug was difficult to washout, requiring 15 to 20 min. Most cells did not last that long. Figure 6B are superimposed I-V curves of inward current obtained from the same cell. Both the peak inward current (left) and the late inward current (right) measured at 50 msec into the step are shown. In the presence of external Na⁺, the inward current, especially the late component, consistently has a more positive I-V relationship than the "L" type Ca⁺⁺ current recorded in Na⁺-free external solution. The positive shift is also obvious at slower rate of depolarization as illustrated by the ramp experiment (fig. 5A).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Continuous exposure of the cell to various ibutilide concentrations as indicated by dashed vertical lines. A, Continuous response of peak inward current amplitude at 0, 10 and 20 mV to changes in ibutilide concentration. Vertical dotted-lines mark approximate time when a new concentration was applied. Each current-time point represents data extracted from an I-V run performed at that time (abscissa). B, Same cell, illustrating peak inward (left) and late inward current at 50 msec into the step (right) over a broad voltage range and drug concentrations. Ibutilide was applied cumulatively in sequence from low to high. Holding potential, 40 mV; temperature, 37°C.

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View larger version (13K): [in this window] [in a new window]   Fig. 7.   Averaged peak inward current densities at increasing ibutilide concentrations. A, Concentration-response of peak current density to ibutilide at potentials as indicated at right. Number in parentheses indicates cell number. Current density was calculated based on averaged cell capacitance of 115.5 p. To extract the half-maximal current activation concentration (Kd), the data were fitted by the equation: (a-d)/(1+[x/c]b)+d where a = asymptotic maximum; b = slope; c = Kd; d = asymptotic minimum; x = drug concentration. The Kds are 0.1, 0.26, 0.65, 0.22, 0.69 and 0.9 nM, respectively at 10, 0, 10, 20, 30 and 40 mV. B, Kd at different voltage steps. Each symbol represents a Kd at the respective voltage obtained by the curve-fit equation of above. The Kd data were fitted to the Woodhull equation:Kd(V) = Kd(0)exp(zVF/RT) where z is the effective valence and F, R,T have their usual thermodynamic meaning. Assuming z = 1, the  value is 0.78.

The inward current and APD prolongation have similar concentration response curves. Although ibutilide is highly effective on the inward current at very low concentration, there is no evidence yet that this effect can directly contribute to APD prolongation. We therefore examined ibutilide's effect on APD at similarly low concentrations. Figure 8 (left panels) illustrates that ibutilide, at these low concentrations, elevated the plateau to prolong APD. For example, at 70 and 90% repolarization, the controls were 75 ± 9.8 and 223 ± 53 msec, respectively; in 10⁸ M ibutilide, the corresponding values became 117 ± 19 msec (56% increase) and 263 ± 47 msec (18% increase) (n = 4). Thus, the greatest effect appeared at the plateau phase. Curve-fit analysis of the APD-concentration response curve gave K_ds of 0.7 and 0.23 nM at 70 and 90% repolarization, respectively (fig. 8, right panel).

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Ibutilide prolongs human atrial action potentials at very low concentrations. Left panels: superimposed action potentials before and after ibutilide application at 1010 M (upper left panel) and 109 M (lower left panel) concentrations. Data shown on each panel were collected from different cells. B, Concentration-response curves of action potential prolongation by ibutilide at 70% (upper curve) and 90% repolarization (lower curve). Data were fitted by the equation: (a-d)/(1+[x/c]b)+d where a = asymptotic maximum; b = slope; c = Kd; d = asymptotic minimum; x = drug concentration. The Kds for 70 and 90% repolarization are 7*1010 M and 2.3*1010 M, respectively. Number in parentheses indicates cell number. Temperature, 37°C; stimulation frequency, 1 Hz. Drug response was assayed 5 min after each drug application.

Ibutilide shifts the steady-state inactivation curve to positive potentials and reduces the current decline by repetitive depolarization. Prepulse experiments indicated that ibutilide shifted the steady-state inactivation curve (H-V) of the current to more positive potentials. Curve-fit analysis indicated that the mid-point of the curve (V_0.5) was shifted from 21 to 12.2 mV. Figure 9 shows normalized H-V curves (n = 6, P < .05) superimposed on normalized conductance curves (G-V) (n = 12) before (, ) and after (, ) 10⁷ M ibutilide application. The inset illustrates changes in peak current amplitude at various prepulse potentials. The drug shifted the H-V curve to more positive potentials but without effect on the G-V curve. The shift broadened and elevated the "window" substantially. This is consistent with the increase in peak and persistent inward current in the potential range of 10 to +10 mV.

View larger version (27K): [in this window] [in a new window]   Fig. 9.   Steady-state inactivation (H-V, , ) and conductance curves (G-V, , ) of the inward current. H-V curve was obtained using a 500-msecprepulse from 50 to +20 mV, at 10 mV increment, to be followed by a test pulse to 0 mV. IMax, test pulse current amplitude at 50 mV prepulse potential; I, test pulse current amplitude at prepulse potentials as indicated on abscissa. The inset illustrates a H-V curve obtained from one experiment before () and after () ibutilide application. The G-V curve was obtained by converting the averaged I-V curve, using (I/E-Er) where I is the current amplitude, E, the corresponding voltage step and Er, the assumed reversal potential of +60 mV. Open symbols are controls, and solid symbols are normalized currents in 10 M ibutilide. Holding potential, 40 mV; temperature, 37°C.

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View larger version (35K): [in this window] [in a new window]   Fig. 10.   Current decline by repetitive depolarization in the absence and presence of 10 M ibutilide. A pulse train of 16 pulses was delivered to the cell at 2 Hz. A, 4 current traces taken from a 0 mV step pulse train, showing the change in current amplitude at 1, 2 ,5 and 16 pulses before (left) and 5 min after ibutilide application (right). B, Time course of double exponential current decline in response to a pulse train at 0 mV. Curves shown represent amplitudes measured at the peak (left), 50 msec (middle) and 150 msec (right) into the pulse. Controls (); +10 M ibutilide (). C, Fast (left) and slow time constants (middle) before () and 5 min after () 10 M ibutilide application. The slow time constants (middle) at 0 and 10 mV were too large (>500 msec due to very slow decline) to be shown in this plot. The bar graph at right is the % of peak inward currents remaining at the end of the 16th pulse. Open bar are controls and shaded bars, in 10 M ibutilide, at step potentials as indicated on the abscissa; P < .05; holding potential, 40 mV; temperature, 36°C.

Identity of the ibutilide sensitive inward current. In human atrial cells, the ibutilide sensitive inward current resembles the L-type Ca⁺⁺ current. In our earlier study on guinea pig ventricular cells (Lee, 1992), it was suspected to be a new type of inward current because removal of external Na⁺ removes this drug induced inward current. However, subsequent experiments indicate that it is blocked by nifedipine. In human atrial cells, similar observations were made consistently. Figure 11 is a good example, demonstrating, on the same cell, that the drug induced Na⁺ current is through a nifedipine inhibited channel. By using this protocol, figure 11 highlights the importance of Na⁺ for ibutilide's effect. Four other cells showed similar response. By pooling data from different cells, in Na-free solution, the peak inward current amplitude was 1244 ± 65 pA; and in ibutilide (10⁷ M), it remained relatively unchanged at 1302 ± 108 pA (n = 8, P < .05). In full-Na solution, peak inward current amplitude was slightly larger, at 1344 ± 98 pA (n = 17); and in ibutilide (10⁷ M), the current increased to 2173 ± 116 pA (n = 26, P < .05). TTX, of up to 10 µM, did not affect the currents (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 11.   External Na+ removal and nifedipine effect on the inward currents. Upper panels are superimposed current traces obtained under conditions as indicated. Currents were elicited every 15 sec by a 150-msec step to 10 mV from a holding potential of 40 mV. Below are peak inward current amplitudes plotted against time under conditions as indicated. Full-Na is the E1 solution; Na-free is the E2 solution (see "Methods"). In some cells, choline-Cl was used instead, but the results remained similar. The current amplitude of this cell is smaller than the average. Symbols represent current traces as shown. In most cells, external Na+ removal shifts the holding current outward. This shift occurs rapidly and its I-V relationship is linear and time-independent. Hence, although not done here, it can be subtracted from the voltage and time-dependent inward current by using a 10 mV leak current scaled up to the corresponding step potential (temperature, 36°C).

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View larger version (21K): [in this window] [in a new window]   Fig. 12.   Nifedipine reversed ibutilide-induced action potential plateau and prolongation. Strong elevated plateau and APD prolongation appeared in about 5 min after application of 10 M ibutilide to the bath. The effect became so strong that an abortive local response reaching to 0 mV can be seen. The effect remained stable for the next 20 min until 10 M nifedipine was applied on top of ibutilide, and ibutilide's effect was promptly removed. Note, both the early and late repolarization phases were not affected by nifedipine. Each action potential represents an average trace of five action potentials from a given file (insert) collected under that experimental condition. In control, part of the late repolarization phase was missing due to mistake in choosing high sampling rate (temperature, 36°C).

	Discussion

Top Abstract Introduction Methods Results Discussion References

The main body of knowledge concerning ionic mechanisms of class III antiarrhythmic compounds is derived from animal heart cells. Few studies (Wang et al., 1995) have systematically examined the action of clinically useful drugs on human heart cells. One reason may be the low accessibility to viable human heart tissues that yield cells suitable for electrophysiological experiments. An alternative approach has been the cloning of channels from human heart libraries that has yielded valuable information (Kiehn et al., 1995). However, the physiological and pharmacological properties of these cloned ion channels have to be carefully validated against native human heart ion channels which are not readily available. In this study, we have examined ibutilide's effect on the K⁺ currents and plateau inward currents in freshly isolated human atrial cells. At low concentrations less than 10⁶ M, ibutilide is ineffective on the K⁺ currents. Instead, it specifically increases a plateau inward current with half maximal concentration (K_d) of about 0.1 nM at 10 mV. The K_d for the inward current matches with the K_d for APD prolongation. In most cells, however, the drug induced plateau appears below or at the inward current threshold of 20 mV, probably due to large I_to. However, in other cells bathed in high concentration of ibutilide, a large plateau near 0 mV could be observed (fig. 12) which was removed by nifedipine. The agreement suggests that a nifedipine sensitive inward current is mainly responsible for mediating ibutilide's class III antiarrhythmic action, although contribution by I_Kr can not be ruled out at present. The other finding of this study is the ability of ibutilide to reduce the amount of inward current inactivation. This could help maintain the drug effect at high heart rates.

Human atrial cells have variable action potentials shapes. Internally dialyzed cells with resting potentials of 70 mV generate full size action potentials in response to brief positive current stimulations. Using our internal solution, the action potential and its duration remained relatively stable. They resemble that of the intact tissues recorded by the microelectrode technique (Gelband et al., 1972). Wang et al. (1993) reported that the shape of human atrial action potentials can vary quite extensively from triangular shape to that with a full plateau. The triangular shape is the majority and may be consequential to the patient's chronic use of Ca⁺⁺ channel blockers. A study of isolated human atrial cells by Grand et al. (1990) has documented that chronic use of nifedipine, nicardipine or diltiazem could triangulate the action potential as a result of Ca⁺⁺ channel blockade. In agreement, about half the cells with triangular action potentials and small inward current we examined were from patients treated with Ca⁺⁺ channel blockers.

Potent effect of ibutilide on a plateau inward current. In contrast to the K⁺ currents, the inward current recorded in Na⁺ and Ca⁺⁺ solutions is sensitive to subnanomolar ibutilide. To rule out that the drug effect is not contaminated by the short "run-up" followed by "run-down" commonly observed in internally dialyzed cells, we applied the drug only after the "run-up" was completed. In fact, most drug application took place during the "run-down" phase. The ability of ibutilide to reduce inward current inactivation is interesting. This may have the net effect for preserving the drug effect on fast pacing, and potentially more arrhythmogenic tissues. By comparison, agents that increase inactivation, such as Bay K-8644 (Sanguinetti et al., 1986) would be less effective on fast pacing cells, but more effective on slower pacing, normal cells. Such agent with "reverse use-dependence" may be inherently more proarrhythmic.

Ionic identity of the Na⁺-sensitive inward current. In guinea pig ventricular cells, we have shown that in the presence of ibutilide, the inward current consists of two components: a "L" type Ca⁺⁺ current and a Na⁺-sensitive component (Lee, 1992). Our data on human resemble that of the guinea pig. However, over the years, we have not been successful in separating it from the L-type Ca⁺⁺ channel because application of nifedipine would removed the drug effect, on both the inward current (fig. 11) and APD prolongation (fig. 12). Furthermore, neither the current-voltage relationship nor the steady-state inactivation differs substantially from that of the L-type channel. The logical interpretation of our result would be that ibutilide promotes Na⁺ permeability through the L-type Ca⁺⁺ channels that are normally impermeable to this ion (Lee and Tsien, 1984). While a drug induced change in Na⁺ permeability through the L-type Ca⁺⁺ channel has not been reported previously, it is well established that such changes can be brought about simply by lowering external Ca⁺⁺ (Hess and Tsien, 1984), thereby reducing occupancy of the high affinity Ca⁺⁺ binding sites by Ca⁺⁺, permitting monovalent cations to bind and permeate the channel. Subsequent site-directed mutagenesis indicates that the L-type Ca⁺⁺ channel selectivity is determined by four conserved glutamate residues in equivalent positions in the pore lining regions of repeats I-IV in the Ca⁺⁺ channel ₁ subunit (Yang et al., 1993). Neutralization of the glutamic acid sites in repeats II and IV by substitution with alanine or glutamine resulted in 10-fold reduction in affinity of the channel for Ca⁺⁺ (Yantani et al., 1994) allowing Na⁺ to permeate the channel. Interestingly, at neutral pH, ibutilide is positively charged. It is possible that the drug may alter the polar fields of the glutamate resides, thereby modifying channel selectivity in due process.