Abstract Because the number and characteristics of delayed rectifier K+ current (IK) components vary between species, the role of each component in the action potential and modulation by class III agents is uncertain. To address these issues, IK was assessed in adult isolated canine ventricular and atrial myocytes by using whole-cell and perforated-patch techniques. IK components were characterized by using two complementary approaches: a kinetic approach (based on biexponential fits to deactivating tail currents) and a pharmacological approach (using the methanesulfonanilide compound E-4031). In ventricular myocytes, two exponential tail current components were distinguished; these components differed in the voltage and time dependence of activation and the effect of lower [K+]o. Both kinetic components contributed equally to peak tail current amplitude (measured at −35 mV) after a single 300-ms pulse to 5 mV, simulating an action potential. By use of E-4031, rapidly and slowly activating components of IK (IKr and IKs, respectively) that were analogous to tail components described kinetically were identified. The activation kinetics and rectification properties of canine IKr and IKs are qualitatively similar to those described previously for guinea pigs. In contrast, canine IKr and IKs deactivation kinetics differed markedly from those found in guinea pigs, with canine IKr deactivating slowly (time constant τ, 2 to 3 s near −35 mV) and IKs deactivating rapidly (τ, 150 ms near −35 mV and decreasing to 30 ms near −85 mV). E-4031 elicited reverse rate-dependent effects (greater drug-induced prolongation of the action potential at slower stimulation rates); this effect is inconsistent with the hypothesis attributing reverse rate dependence to incomplete IKs deactivation during rapid stimulation (due to rapid deactivation of canine IKs). Two IK components with characteristics comparable to those found in ventricular myocytes were also observed in atrial myocytes. In conclusion, (1) IKr- and IKs-like components of IK are present in canine atrial and ventricular myocytes, with deactivation kinetics strikingly different from those found in guinea pigs, and (2) the rapid deactivation kinetics of canine IKs do not support its role in reverse rate dependence with class III agents in this species.
It is generally accepted that IK provides outward current during the cardiac action potential to facilitate repolarization.1 Recent pharmacological studies have identified two IK components in guinea pig atrial and ventricular myocytes based on the effects of the prototypic class III antiarrhythmic agent E-4031.2 3 4 These two components have been termed IKr (described as a rapidly activating and deactivating component blocked by E-4031 that displays inward rectification) and IKs (an E-4031–resistant component that activates and deactivates slowly and displays minimal rectification). The number of IK components appears to differ when different species are compared. In contrast to findings in guinea pigs, pharmacological evidence suggests that one IK component is present in rabbit5 and feline6 ventricles; IK is reportedly present7 or absent8 9 in adult rat ventricles. In canine ventricles, two IK components have recently been reported.10 11 The characteristics of IK also appear to differ across species. For example, IKr deactivates rapidly in guinea pig myocytes2 3 4 but more slowly in rabbit myocytes.5 Although two IK components have also been reported in atrium from guinea pigs, a description of atrial IK in other animal models has not been reported.
Most class III antiarrhythmic agents cause greater prolongation of the APD at slow versus rapid rates of stimulation. This effect, which has been termed reverse use dependence12 or reverse rate dependence,13 has been implicated in the bradycardia-dependent proarrhythmic effects of various class III agents. Reverse rate dependence has been demonstrated with various IKr blocking agents, including the structurally related methanesulfonanilides E-4031, dofetilide, sotalol, and WAY-123,398.9 14 15 In guinea pigs, reverse rate dependence has been attributed to the “accumulation” of IKs during rapid stimulation (resulting from the incomplete deactivation of this current), which minimizes the effects of IKr block at faster stimulation rates.13
Because of the importance of IK in modulating repolarization and its possible role in defining the heterogeneity of electrical activity across the ventricular wall1 16 17 and because of its use as a potential focus for modulation by class III agents with antiarrhythmic as well as arrhythmogenic potential,12 18 the characteristics of IK were studied by using isolated myocytes derived from the canine left ventricular free wall and atrium. An earlier study identified two components of canine ventricular IK.10 In this study, both components were characterized kinetically and compared with those identified pharmacologically by using E-4031 to avoid possible confounding effects of voltage- and time-dependent block by this agent. With either approach, two IK components were identified, with each component contributing comparable repolarizing current after 300-ms clamp pulses to the plateau range of potentials. Two IK components were also identified in canine atrial myocytes, with characteristics similar to those found in the ventricle. Although the activation kinetics of canine IKr and IKs were found to be comparable to those found in guinea pigs, their deactivation kinetics were distinctly different, with canine IKr deactivating slowly and IKs deactivating more rapidly. Despite the rapid deactivation of canine IKs, block of IKr by E-4031 caused reverse rate-dependent effects on the canine ventricular action potential, an effect inconsistent with the postulated role of IKs in reverse rate dependence. These results suggest that the mechanisms responsible for reverse rate dependence by antiarrhythmic agents require reevaluation in this widely used animal model for electrophysiological studies as well as in other species.
Materials and Methods
Isolation of Canine Ventricular Myocytes
Ventricular myocytes from adult male mongrel dogs were isolated by using techniques described previously19 20 and as approved by the institutional animal care and use committee. Briefly, hearts were removed from anesthetized heparin-pretreated dogs and placed in cold nominally Ca2+-free Tyrode’s solution containing (mmol/L) NaCl 118.5, KCl 2.8, NaHCO3 14.5, KH2PO4 1.2, MgSO4 2.7, and glucose 11.1 and aerated with 95% O2/5% CO2. Wedges of left ventricular free wall supplied by the left anterior descending coronary artery were excised, cannulated, and subsequently flushed with 40 mL warm Tyrode’s solution supplemented with 0.1% BSA (fraction V, protease-free, Sigma Chemical Co). The cannulated wedge was then mounted in a warmed jacketed Langendorff perfusion system and perfused with nominally Ca2+-free Tyrode’s solution supplemented with 0.04% collagenase (type IV, Worthington Biochemical Co), 1% BSA, and 1.5 mmol/L MgSO4 (37°C). After <20 minutes of perfusion, the wedge was removed, and epicardial and endocardial layers (minimum, 1 mm thick) were removed by shaving with a dermatome. Chunks of remaining midmyocardial tissue were then minced and immersed in HEPES-buffered “base” solution containing (mmol/L) NaCl 132, HEPES 20, MgSO4 1.2, glucose 11.1, and KCl 4.0, supplemented with 1.5% BSA, 0.04% collagenase, 2.0 mmol/L MgSO4, and 0.3 mmol/L CaCl2 (pH 7.2, termed “digest solution”) and aerated with 100% O2 in a shaker bath (37°C). At 10-minute intervals, digests were filtered through nylon mesh, centrifuged, and resuspended in base solution supplemented with 0.5 mmol/L CaCl2, 2.0 mmol/L MgSO4, and 3% BSA (termed “storage” solution). Larger tissue fragments were returned to fresh digest solution for further dissociation, typically for 20 to 30 additional minutes. Cells resuspended in the storage solution (at room temperature) were used within 12 hours; some cells were “washed” by passage through a Percoll gradient to remove excess debris.
Isolation of Canine Atrial Myocytes
Hearts were removed, placed in a Langendorff apparatus, and perfused (37°C) with a solution containing (mmol/L) KCl 80, KH2PO4 30, MgSO4 4, HEPES 20, glucose 10, taurine 20, creatine 5, succinate 5, and EDTA 1, along with 0.1% BSA (pH 7.2). After a 10-minute rinse, perfusion was switched to a fresh solution supplemented with collagenase (type II, 125 U/mL, Worthington) and recirculated for an additional 30 minutes. A portion of the left atrium was removed and triturated, and myocytes were placed in the recording chamber.
Experimental Apparatus and Solutions
Myocytes were allowed to settle on the bottom of a Peltier-based temperature-controlled perfusion bath mounted on an inverted microscope. Cells were superfused (0.8 mL/min) with HEPES-buffered Tyrode’s solution containing (mmol/L) NaCl 132, MgSO4 1.2, HEPES 20, glucose 11.1, KCl 4, and CaCl2 2 (pH 7.4 with HCl). Bath temperature was monitored with a small thermistor probe (0.35-mm diameter, NBD Enterprises) located on the bath bottom and positioned within 2 mm of the myocyte under study. Whole-cell experiments were conducted at 36°C to 37°C; bath solutions were exchanged within ≈30 s. Only rodlike quiescent (when not stimulated) myocytes with uniform sarcomeric appearance and nonrounded or contracted edges were chosen for study. Most myocytes studied were isolated from the midventricular free wall to minimize differences in Ito density.20
E-4031 was prepared fresh daily as a 5 mmol/L aqueous stock solution. A final bath concentration of 5 μmol/L was chosen on the basis of studies showing that this concentration totally blocked IKr in guinea pig myocytes.2 Nisoldipine was prepared fresh daily in 30% ethyl alcohol and used in a darkened room. The possible effects of ethyl alcohol were not studied.
Membrane currents were obtained by using the whole-cell configuration of the patch-clamp technique.21 Borosilicate glass patch electrodes were coated with either Sylgard or dental wax and heat-polished immediately before filling with a standard high-K+ solution containing (mmol/L) potassium aspartate 125, KCl 20, EGTA 10, ATP (magnesium salt) 5, MgCl2 1, and HEPES-free acid 5, adjusted to 7.3 with 5N KOH; electrode resistances typically ranged from 1 to 2.5 MΩ. The potential was adjusted for a 10-mV junction potential just before sealing. After formation of a gigaseal and compensation of electrode capacitance, the patch was ruptured either electronically or by negative pressure. Analog series resistance compensation was then adjusted to values typically 60% to 70%. An additional initial check of ventricular cell viability was routinely provided by assessing the N-shaped steady state current-voltage characteristics recorded with a 100-mV depolarizing ramp pulse.
Where indicated, perforated-patch recordings of whole-cell ventricular IK were obtained by using techniques adopted from Horn and Marty.22 These experiments examined IK with minimal alterations of the intracellular milieu (which would be expected if whole-cell patch techniques were used). The tip of a freshly coated heat-polished pipette was filled with a solution containing (mmol/L) potassium aspartate 125, KCl 20, Na2-ATP 5, MgCl2 1, and HEPES 5. Subsequently, 20 μL of freshly prepared amphotericin solution (Sigma A-4888, 6 mg/100 μL dimethyl sulfoxide) was added to 5 mL of filling solution and used to backfill the pipette. After formation of a gigaseal, the diffusion of amphotericin to the pipette tip and its partitioning into the cell membrane established electrical continuity between the pipette and the cell interior through newly formed pores. Typical access resistances of 10 MΩ were obtained within 20 minutes of establishing a gigaseal.
For studies of reverse rate dependence with E-4031, changes in the ventricular APD were assessed by using standard microelectrode techniques (as in Reference 19).
Data Acquisition and Analysis
Currents were low pass–filtered (500 Hz), digitized (typically 166 Hz), stored, and analyzed on an 80386-based computer by using pclamp software (version 5.5.1, Axon Instruments). Cell capacitance was determined at the resting membrane potential (negative to −80 mV) after cell rupture by methods that account for the series resistance (typically 10 MΩ) and input resistance (typically 15 to 25 M) for canine ventricular myocytes as described previously.10 Values of series resistance (Rs) and cell input resistance (Rin) were calculated from the current response to a 5-mV hyperpolarizing pulse from the zero current potential according to the following equation:
where Cm is cell capacitance and τ is the time constant of the “off transient.” Rin in the range of −40 mV (holding potentials for IK studies) was estimated to be ≥200 MΩ for ventricular myocytes on the basis of the slope of the steady state current-voltage relations (see above).
The kinetics of IK deactivation were determined by using nonlinear least-squares regression analysis applied to tail currents, with two exponential components (IKe1 and IKe2) adequately described the decaying tail currents (see Fig 1⇓; also, Reference 10). In nearly all cases, two exponential components adequately reproduced the tail currents; fits with greater than two exponential components did not significantly enhance descriptions of the time-dependent current, as judged by residual analysis. The amplitude of each tail current component was obtained by extrapolation of fits to the onset of repolarization. Tail currents of 8- to 12-s duration were typically fit to ensure adequate resolution of the slower tail current component; data tracings were truncated in most figures to enhance visual comparisons of the current time course. Because of the typical small amplitude of tail currents, experiments were analyzed and subject to curve fitting only when stable baseline currents were maintained as monitored continuously during experiments and detected from recorded data tracings (see Figs 1⇓, 3⇓, 5⇓, 8⇓, and 10⇓). Depolarizing test pulses were applied once every 20 to 30 s (held constant in any individual experiment) to allow the slower tail component to fully decay.
The two exponential tail current components were termed IKe1 and IKe2 (see Fig 1⇑). Studies of IK in guinea pig myocytes have used the terms IKr and IKs (r referring to rapidly activating and s referring to slowly activating in Reference 2) to describe two IK components based upon pharmacological blockade by E-4031. The IKe1/IKe2 nomenclature was used to describe the present kinetic-based studies, since the IKe1/IKe2 nomenclature follows earlier descriptions of IK in cardiac Purkinje fibers, and kinetic distinctions described are independent of possible voltage- and time-dependent effects of drug block.
IKact was defined as the time-dependent current during depolarizing test pulses measured from the minimum outward current at the onset of depolarization and the maximum outward current immediately before repolarization. The initial time course of current activation is complex and generally does not fit well as two exponential components; this is consistent with a number of native cardiac K+ channels (eg, see Reference 23).
Records characterizing baseline IK were typically obtained within 10 minutes of cell break-in to minimize possible current rundown. Although possible time-dependent changes in whole cell IK were not systematically investigated, biexponential tail currents were observed immediately after clamp “tune-up” (requiring <3 minutes after patch rupture) as well as after 30 minutes of maintained access. The effects of reducing [K+]o and E-4031 were assessed by comparing currents immediately before and after equilibration, typically within 5 minutes. Where appropriate, data are expressed as mean±SEM.
Isolation of Canine IK
The confident evaluation of IK depends on the removal of overlapping contaminating ionic currents, such as inward Na+ and Ca2+ currents and Ito. Holding potentials ranging from −35 to −40 mV were used to inactivate T-type Ca2+ current and fast Na+ current; L-type Ca2+ current was blocked by using nisoldipine (1 μmol/L) under darkened conditions. In contrast to an earlier report,24 nisoldipine had no discernible effect on tail currents at the lower concentration used (n=3; data not shown), in agreement with other studies (see References 2 and 25). These results suggest that IK deactivation is not influenced by transmembrane-dependent Ca2+ influx via L-type Ca2+ channels. Possible contamination from electrogenic Na+-Ca2+ exchange should be minimized by the high EGTA concentrations used in the patch pipettes.
The activation of canine IK likely overlaps the 4-aminopyridine–sensitive Ito, which is minimized (but not fully inactivated) with holding potentials used in this study.20 4-Aminopyridine was not used to block Ito, since preliminary studies showed that it blocked IK in canine myocytes (as has been reported in guinea pig preparations26 ), and block of Ito may be complex.27 As Ito rapidly inactivates, the time course of IK activation for only the first 10 to 20 ms is likely affected. Estimates of the amplitude of IK activation upon depolarization (IKact, above) likely represent a minor underestimation of total IK amplitude because of the rapid kinetics of Ito inactivation compared with slower IK activation at positive test potentials.
Two IK Components Can Be Distinguished Kinetically in Canine Ventricular Myocytes
Fig 1⇑ details typical IK obtained from an isolated canine ventricular myocyte. Panel A illustrates membrane currents during 3-s depolarizing test pulses and tail currents after repolarization to the holding potential of −35 mV. Panel B illustrates the family of tail currents (offset on an expanded scale) labeled according to the preceding test pulse potential. Superimposed on each tail is a biexponential fit. Tail currents were well described as the sum of two exponential components and a constant according to the following equation: I(t)=A1·exp(−t/τ1)+A2·exp(−t/τ2)+Ao, where τ1 and τ2 represent the more rapid and slower exponential components, A1 and A2 represent the amplitude of each respective component, and Ao represents the baseline current. The two components were termed IKe1 and IKe2, with subscripts e1 and e2 referring to the faster and slower exponential tail current components, respectively. A similar approach was previously used to define regional differences in IK components in canine myocytes.10
The biexponential nature of the tail currents is further illustrated in the inset, which plots (on semilogarithmic coordinates) the tail current after a depolarizing pulse to +65 mV. For this tail current, the time constants for IKe1 and IKe2 were 116 and 2297 ms, respectively, with the peak amplitude of IKe1 6.5-fold greater than that of IKe2. Time constants for IKe1 were typically 15 to 20 times faster than those for IKe2 at the −35-mV holding potential. In this myocyte, the time constants for IKe1 and IKe2 ranged from 116 to 140 ms and from 1969 to 2317 ms, respectively. In four myocytes in which perforated patch–clamp technique was used to maintain the intracellular milieu, tail currents were also well described by biexponential fits similar to those used for myocytes by whole-cell patch methods (data not shown). These results demonstrate that the biexponential currents observed by whole-cell recording techniques cannot be attributed to intracellular dialysis.
Fig 2⇓ highlights differences in the voltage-dependent activation of IK over a wide range of test pulse potentials. Panel 2A summarizes differences in the amplitude of each tail component after 3-s test pulses, with peak values of IKe1 and IKe2 normalized to those after a test pulse to +65 mV in the same myocyte. Panel A shows that the amplitude of the IKe1 component continues to increase with more positive test pulse potentials up to +65 mV. (In two additional experiments, IKe1 showed no indication of saturation with a +75-mV test pulse.) In contrast, the peak amplitude of IKe2 is attained with test pulses to 0 mV. When test pulses to +65 mV were used, the density of the IKe1 tail component (1.04 pA/pF) was much greater than that of IKe2 (0.188 pA/pF). The effect of test pulse potential on IK activation (IKact) during depolarizing pulses is illustrated in Panel B. As before, current density was normalized to maximum IKact measured during a +65-mV test pulse. The current-voltage relation for IK was described as an increasing curvilinear function of test pulse potential; the average maximum density of IKact was 2.38 pA/pF (at +65-mV test potential).
To assess the time course of activation of each tail component, the growth of each component was determined from fits following depolarizing pulses of selected durations. Fig 3⇓ shows averaged results obtained from five myocytes by use of test pulses to 60 mV (−40-mV holding potential). When this protocol was used, the amplitude of the slowly decaying exponential component (IKe2) after 50-ms pulses was equivalent to those obtained after 650-ms pulses. This result demonstrates rapid activation of the slowly deactivating tail component. In contrast, the amplitude of the rapidly decaying exponential component (IKe1) continues to increase after longer depolarizing pulses. The inset shows results from a typical experiment; IK during depolarization (IKact) and the IK tail current continue to increase with prolonged depolarizing pulses, consistent with the hypothesis that IKe1 represents the majority of IK activated during strong prolonged depolarizing pulses. Tail currents following pulses shorter than 50 ms were not characterized because of the difficulty in fitting these smaller currents and possible overlap with inactivation of Ito.
The time course of activation of each IK component at other test potentials was not systematically examined. However, tail currents were compared by using 300-ms and 3-s test pulses for select potentials in the same myocyte, providing an indication of the contribution of each IK component during prolonged versus short depolarizations (the later resembling a single action potential). Results from 13 myocytes are summarized in Fig 4⇓. For each test potential, the total tail current density (represented by bar height) is greater after the longer test pulse, largely because of an increase in the more rapid (IKe1) tail component (open bars). A slight increase in the slower tail component (IKe2 [shaded bars]) was noted by comparing 300-ms versus 3-s depolarizing test pulses, which did not achieve statistical significance. This result confirms that IKe2 activation is largely complete within 300 ms for all test potentials examined and does not show evidence of slow inactivation. The figure also confirms that (1) for each test pulse duration, IKe1 increases after stronger depolarizing pulses, and (2) for each test potential, IKe1 increases with longer depolarization. As a consequence, for test potentials ≥25 mV, IKe1 is the predominant tail current component. However, the amplitudes of IKe1 and IKe2 are similar for 300-ms pulses in the range of potentials encountered during ventricular repolarization (<+25 mV), suggesting that both components provide comparable repolarizing current before termination of the plateau of a single action potential.
The two tail components were differentially affected by eliminating [K+]o. Typical results are illustrated in Fig 5⇓, which shows superimposed membrane currents recorded in 4 mmol/L (solid circles) and 0 mmol/L (open circles) potassium Tyrode’s solution. Reducing [K+]o greatly reduces the amplitude of IKe2 while it increases the amplitude of IKe1 (fit parameters indicated in figure). The time constants of either component were unaffected. Similar results were observed in four analogous experiments. Reducing [K+]o to 0 mmol/L also increased the amplitude of IK activated upon depolarization and caused a reduction in net outward current at the holding potentials used (range, −35 to −50 mV).
The voltage dependence of IK deactivation was assessed by using two different protocols. In one series of experiments (summarized in Fig 6⇓), tail currents following 3-s depolarizing pulses were fit with two exponentials. Despite variability of the derived time constants for each component, the components clustered into a faster (IKe1) and slower (IKe2) range through −20 to −60 mV. In most cases, the time constant for deactivation of the faster tail component appeared to decline at more negative potentials.
To further examine the voltage-dependent kinetics of IKe1, two-step clamp protocols were applied in the presence of 0 mmol/L [K+]o. This strategy (1) minimized variability inherent in biexponential fits (by eliminating the slower IKe2 component), (2) increased the amplitude of IKe1, thereby allowing for a wider range of potentials to be examined, and (3) minimized contamination by IK1. The protocol used is illustrated in Fig 7A⇓. To eliminate contamination of time-dependent currents sometimes present during hyperpolarization alone, tail currents without prepulses (Fig 7A⇓, a) were subtracted from those with a depolarizing prepulse (Fig 7A⇓, b). The resultant difference currents (Fig 7B⇓, a−b) were well fit by using a single exponential (shown superimposed on calculated tracings) at all potentials. Fig 7C⇓ summarizes the deactivation kinetics of IKr obtained from five myocytes; the time constant for IKe1 deactivation decreased with hyperpolarization, approaching a value of 30 ms at resting potentials near −85 mV. Similar results were obtained in two experiments in the presence of 4 mmol/L [K+]o with the slower (IKr component; see below) blocked by E-4031 (data not shown). The voltage dependence of deactivation of the E-4031–sensitive current component was not evaluated because of the smaller amplitude of this current.
Two IK Components Distinguished Pharmacologically in Canine Ventricular Myocytes
Earlier studies with guinea pig myocytes demonstrated the presence of two IK components on the basis of the effects of a prototypic class III antiarrhythmic agent E-4031.2 In guinea pig myocytes, the E-4031–sensitive component of IK (IKr) rapidly activates and deactivates and displays inward rectification; the E-4031–insensitive current (IKs) activates and deactivates more slowly and shows minimal inward rectification. An initial study had suggested that IKe1 was analogous to IKs, and IKe2 was analogous to IKr.10 In the present study, the effects of E-4031 were further characterized, including its effects on IK activation.
Representative effects of E-4031 on IK are illustrated in Fig 8⇓. Panel A overlays current recordings during and after 3-s depolarizing pulses in the absence and presence of E-4031. In general, E-4031 reduces outward current during moderately depolarized test pulses (−15 to +25 mV) to a greater extent than at strongly depolarized potentials. The time course of IK is parallel in the absence and presence of E-4031 during the later 2.5 s of the pulse. During the first 300 ms of a depolarizing pulse, E-4031 alters the time course of IK activation. This effect is further illustrated in panel B, which shows E-4031–sensitive currents obtained by digital subtraction of currents in the absence versus presence of the compound (ie, drug-free control minus E-4031). With moderately depolarizing pulses (test potentials of +5 to +25 mV), the E-4031–sensitive current is an increasing outward current, which reaches a plateau within 300 ms. Beyond 300 ms, the E-4031–sensitive current is time independent, accounting for the parallel currents observed later during depolarizing pulses in panel A. Upon repolarization to −35 mV, the drug-sensitive tail current is larger than that activated upon depolarization, consistent with the inwardly rectifying properties of IKr. The amplitude of the E-4031–sensitive tail component is similar when test pulse potentials >+5 mV are used, consistent with the characteristics of voltage-dependent activation of the kinetically defined IKe2 component. During strongly depolarized test pulses, E-4031–sensitive current is harder to resolve, consistent with the rectification properties of this current and the small amplitude of this current relative to the larger E-4031–insensitive (IKs-like) current.
Because of the small amplitude of E-4031–sensitive current relative to total IK during depolarization and concerns that small nonspecific changes in membrane current could be incorrectly interpreted as drug effects, an additional series of five experiments was performed by comparing the effects of E-4031 on IK during moderate versus strongly depolarizing pulses. For these experiments, a sequence of two depolarizing pulses (to 5 and 45 mV; 3-s duration; holding potential, −35 mV) was sequentially applied before and during equilibration with E-4031, and drug-sensitive currents were measured. Time-dependent E-4031–sensitive current was greater at the 5-mV test potential in one experiment, greater at 45 mV in two experiments, and equal in the remaining two experiments. It could not be determined whether the approach to the plateau of the E-4031–sensitive current was any more rapid at either test potential. However, in each of the five experiments, E-4031–sensitive current upon repolarization was greater than during depolarization, consistent with the rectification apparent in Fig 8B⇑. Subsequent experiments (see Table⇓) demonstrate no significant effect of E-4031 on IK activation at a test potential of 55 mV. Thus, the E-4031–sensitive (IKr) component of IK activation in canines displays rapid activation and inward rectification, similar to guinea pigs.
The voltage-dependent activation of canine IKr and IKs was assessed from the amplitude of individual tail current components in the absence and presence of E-4031. Fig 8C⇑ illustrates typical effects over a wide range of test pulse potentials. E-4031 consistently blocked the slower (IKe2) tail component (squares), while minimally affecting the more rapid (IKe1) component (circles). These results confirm that IKr (defined as E-4031–sensitive current) is analogous to the slowly deactivating tail component (IKe2) defined kinetically and that IKs (defined as E-4031–resistant current) is analogous to the rapidly deactivating tail component (IKe1). Results obtained from seven similar experiments are summarized in the Table⇑. E-4031 consistently blocked IKe2 without affecting the amplitude or kinetics of IKe1. Thus, despite similar IKr activation between canines versus guinea pigs, the kinetics of deactivation are strikingly different.
E-4031 consistently reduced net outward current at holding potentials ranging from −35 to −45 mV (see Table⇑ and Figs 8⇑ and 10⇓) by values approaching 50% of the amplitude of the fully activated IKr tail component. A number of observations suggest that this effect is not due to block of sustained IKr. First, earlier kinetic-based results (Fig 2⇑; also author’s unpublished data, 1995) demonstrated a threshold for IKr activation near −35 mV, which is positive to the holding potentials used. Second, E-4031 affected the holding current when myocytes were bathed in 0 mmol/L [K+]o (which reduced IKr and electrogenic Na+-K+ pump current). Under these experimental conditions, holding current was reduced in four of five myocytes coincident with drug equilibration; no change in holding current was noted in the fifth experiment. If one assumes that IKr activation occurs negative to −35 mV, reduced net outward current with E-4031 could reflect an incremental block of IKr resulting from depolarizing clamp pulses used to monitor drug equilibration. This interpretation is incorrect, because holding current shifted inward when the membrane potential was maintained at −40 mV and depolarizing pulses were withheld during drug equilibration (three experiments).
To further study the steady state effects of E-4031 over a wider range of membrane potentials, 5-s depolarizing ramps were applied to approximate steady state current-voltage relations (holding potential, −85 mV; peak potential, +15 mV; slope, 20 mV/s). Currents obtained using 5-s and 3-s ramps were superimposable, demonstrating that longer ramps were of sufficient duration to produce a “quasi–steady state” current-voltage relation. E-4031 reduced outward currents during these depolarizing ramps only at more positive potentials. E-4031–sensitive current was first observed at “threshold” potentials between −27 and −15 mV (−21.4±4.4 mV [mean±SD], n=5) and was apparent up to the ramp peak (+15 mV) but absent at more negative potentials. This observation is consistent with the threshold potential for IKr activation determined using square clamp pulses but inconsistent with decreased outward current when the membrane potential is maintained at −40 mV for prolonged periods. This discrepancy suggests that maintained depolarization near −40 mV is necessary to elicit this steady state component of E-4031–sensitive current. Further studies are necessary to resolve the basis for this drug-sensitive component.
Possible use-dependent block of IKr by E-4031 was assessed by using trains of 200-ms depolarizing pulses applied at frequencies of 2.5, 1.0, and 0.25 Hz (21, 21, and 6 pulses per train, respectively; holding potential, −40 mV; pulse potential, +20 mV). Pulse trains were terminated with a standard 300-ms depolarizing test pulse, and the amplitude of fast and slow tail current components was subsequently evaluated. In each of five experiments, 5 μmol/L E-4031 abolished IKr irrespective of the pulse train frequency. Thus, block of canine IKr by E-4031 is frequency independent under these experimental conditions.
E-4031 Produces Reverse Rate-Dependent Effects on Canine Ventricular Action Potential
In numerous tissues, class III agents including the methanesulfonanilides dofetilide and E-4031 (see “Discussion”) elicit a greater prolongation of the APD at longer versus shorter stimulation rates, an effect that has been termed “reverse use dependence”12 or “reverse rate dependence.”13 In guinea pigs, this effect has been attributed to the accumulation of IKs at rapid stimulation rates (due to the slow deactivation kinetics of IKs in this species), which mitigates the effects of IKr blockade at rapid rates.13 To determine if the rapid deactivation kinetics of canine IKs affected reverse rate dependence, the effects of E-4031 on the APD-rate relation were examined. For these studies, standard microelectrode techniques were used to minimize alterations to the intracellular milieu. Fig 9⇓ illustrates the typical effects of E-4031 on an action potential stimulated at basic cycle lengths of 800 ms (panel A) and 2 s (panel B). E-4031 prolonged the APD by reducing the slope of phase 2 repolarization; neither early nor late repolarization was appreciably affected. E-4031 caused a greater prolongation of the APD at the slower cycle length despite the fact that at either cycle length the diastolic interval was long enough to ensure full deactivation of IKs. Panel C further illustrates reverse rate dependence, with E-4031 observed with three midmyocardial myocytes over a wider range of stimulation rates. Because APD showed variations on a beat-to beat basis, three action potentials were recorded, and durations were averaged for each stimulation rate. E-4031 caused prominent reverse rate dependence in each of the myocytes. Reverse rate dependence was also observed in three additional experiments in which myocytes were accessed by using perforated-patch techniques (data not shown).
Two IK Components Are Present in Canine Atrial Myocytes
The characteristics of IK in canine left atrial myocytes were assessed to determine (1) whether two components of IK were present in atrial myocytes and (2) whether IK deactivation kinetics of atrial myocytes were comparable to ventricular myocytes. Typical results are presented in Fig 10⇓. Panel A displays currents representing IK activation and deactivation that qualitatively resembled those of ventricular myocytes (compare with Fig 1⇑). During 3-s pulses, IK attained steady state values with modest depolarization but continued to increase throughout stronger depolarizing test pulses. Similarly, IK deactivation was well described when two exponential components were used; the inset illustrates a biexponential fit after the depolarizing pulse to +25 mV, which was fit with time constants of 128 and 2088 ms. Two tail components were also observed with shorter (300-ms) depolarizing test pulses; by use of this protocol, the amplitudes of the fast and slow tail components were comparable for test pulses to 5 mV; with more depolarized test pulses, the faster component predominated (data not shown). Panel B illustrates the effects of E-4031 on atrial IK: as with ventricular myocytes, E-4031 (1) reduced the holding current, (2) abolished the slower tail current component, and (3) had minimal effects on IK activation at more positive depolarized potentials. Panel C shows the voltage dependence of activation of each tail current component. As in ventricular myocytes, IKe1 was fully activated after test pulses to −10 mV, whereas the amplitude of IKe2 continued to increase with progressively stronger depolarizations. Thus, the number and characteristics of the atrial IK components resembled IK described in ventricular myocytes.
Two IK Components Are Present in Canine Ventricle and Atrium
The present study concludes on the basis of independent kinetic and pharmacological evidence that two components of IK are present in canine atrial and ventricular myocytes. Two tail current components can be distinguished kinetically on the basis of (1) two exponential components describing IK deactivation, (2) differences in the voltage and time dependence of activation of each tail component, and (3) the differential sensitivity of each component to reduced [K+]o. IK components described kinetically (IKe1 and IKe2) were subsequently compared with components identified pharmacologically by using E-4031. The voltage dependence, activation kinetics, and rectification properties of E-4031–sensitive current (typically defined as IKr) were analogous to IKe2 (defined kinetically), whereas E-4031–insensitive current (IKs) was analogous to the more rapid IKe1 in atrial and ventricular myocytes. These similarities argue against the possibility that pharmacologically defined deactivation of IKr is dependent on the blocking characteristics of E-4031.
Two dominant exponential components for canine IK deactivation are readily identified during control conditions, and a single dominant exponential component remains after removal of IKr (by either lowering [K+]o or E-4031). Parameters derived from exponential fits are dependent on (1) adequate isolation of the current under study and (2) the accuracy of the dissociation of overlapping components, which may not be easily resolved for components of similar time constants and/or peak amplitudes. Complicated multistate (multiexponential) gating schemes23 28 29 have described IK (or IK components) as a single conductance with multiple closed states, which were not considered in the present study. In addition, concluding that a kinetic component represents an individual conductance relies on the implicit assumption that the component deactivates as a monoexponential function. Despite these limitations, the effects of E-4031 and low [K+]o on canine IK are consistent with two functional IK components, with each component most readily interpreted as a conductance displaying different pharmacological sensitivity and rectification properties.
The presence and characteristics of IKr and IKs in working ventricular myocardium are species dependent; IKr appears to be essentially lacking in adult rat ventricle (References 8 and 9, but see Reference 7), but an IKr-like component is the predominant (sole?) component in feline6 myocytes. In rabbit ventricular myocytes, IKr5 (or perhaps both IKr and IKs30 ) is present. Although direct IK measures suggest that delayed rectifier current is minimal in human ventricular cells,31 indirect evidence suggests that both IKr and IKs are present in human ventricle; E-403132 and dofetilide33 (a blocker of IKr5 ) prolong ventricular repolarization, and mRNA for an IKs-like current is present in human myocardium,34 which expresses an IKs-like current in oocytes.35 In atrial myocytes isolated from guinea pig3 and humans,36 two IK components have been distinguished. Knowledge of the number, characteristics, and amplitude of individual IK components is essential to understanding the role of IK in repolarization and the effects of class III agents.
In canine and guinea pig ventricular myocytes, IKs tail current density is greater than that of IKr. For canine ventricular myocytes, IKs density is approximately fivefold greater than that of IKr (1.04 versus 0.188 pA/pF; see legend, Fig 2⇑) measured from tail currents at −35 mV after 3-s test pulses to +65 mV. This value is about half that reported in guinea pig myocytes (measured at −40 mV after 7.5-s test pulses to +60 mV [Fig 9⇑, Reference 2]) because of the greater IKr density in guinea pigs. The relative proportion of IKr to IKs is likely to remain despite regional variations in IKs density in canine ventricle.10 11 In canine ventricular myocytes, a single depolarizing pulse ≤300 ms in duration (to potentials encountered during the action potential plateau) elicited equal contributions from IKr and IKs measured at −35 mV (potentials typically encountered near termination of the plateau). Thus, the contribution of IKs, the slowly activating component, after a short depolarization is comparable to IKr, the fully activated component, because of the greater IKs density (Fig 4⇑). During strong and prolonged depolarizations, IKs becomes increasingly dominant because of (1) its greater activation at more positive potentials, (2) the greater driving force for K+ ions, and (3) the reduced IKr due to inward rectification. Further experimental studies will be necessary to determine the contribution of each component to repolarization at different rates of activity. Recent computer simulations (based on guinea pig IK kinetics) stressed the importance of the relative densities of IKr and IKs in repolarization and arrhythmogenesis.37
Two exponential components of IKr deactivation have been reported in guinea pig ventricular myocytes2 and AT-1 cells.38 In feline myocytes (which display predominantly IKr5 6 ), two exponential components of IK deactivation are present in an external solution deficient in Na+ and K+.17 In the present study, one exponential component adequately described the deactivation kinetics of canine IKr. If present, two kinetic components of canine IKr would be difficult to resolve because of the small amplitude and slow decay of this drug-sensitive current. In all three species, IKr saturates at potentials in the range of 0 to +40 mV and displays inward rectification. IKr activation in guinea pigs and dogs appears similar, with rapid activation (within 300 ms) at potentials near 0 mV, full activation near 0 mV, and a half-activation voltage ranging between −10 to −20 mV. IKr activates slightly slower in AT-1 cells (400 ms time constant at 0 mV), with a component of slow inactivation apparent at strongly depolarized potentials not present in other tissues.
IKr was originally defined as an E-4031–sensitive current with rapidly activating and deactivating kinetics and IKs as a slowly activating and deactivating current insensitive to E-4031.2 Although IKr activation is qualitatively similar in dogs and guinea pigs (see above), the deactivation kinetics of canine IKr are strikingly slower (with time constants of 2000 to 3000 ms near −40 mV) compared with the slowest deactivation time constant of 630 ms4 and an undetermined time constant slower than 200 ms sometimes present in guinea pig myocytes (mentioned in Reference 2). The slow deactivation kinetics of canine IKr are revealed by using either E-4031 or the antihistamine terfenadine,39 which blocks IKr in feline myocytes.40 In AT-1 cells, the slowest time constant for IKr deactivation (400 ms) occurs near −40 mV; at similar potentials, IKr deactivation in rabbits38 and cats5 appears intermediate compared with either guinea pigs or dogs. Correspondingly, although IKs activation in canine and guinea pig myocytes is qualitatively similar, the deactivation kinetics of canine IKs are much more rapid (τ, 100 ms at −40) than observed in guinea pig myocytes (τ, 775 ms; Reference 4). It is unknown whether the same channels are responsible for IKr and IKs in these preparations or whether species-specific isoforms, differences in levels of expression, modulatory subunits, or other factors account for the kinetic differences. IKr and IKs subcategories (based on distinctions in deactivation kinetics) would be useful when comparing each IK component in native membranes and heterologous expression systems.
Block of IKr by dofetilide in guinea pigs13 and rabbits5 is independent of frequency because of the slow recovery from block, which is further slowed at potentials near the resting membrane potential.5 However, the methanesulfonanilides E-4031 and dofetilide have been shown to have reverse rate-dependent effects on the guinea pig action potential.9 41 42 These observations were resolved by attributing reverse rate dependence to the accumulation of IKs at rapid stimulation rates resulting from the slow deactivation kinetics of guinea pig IKs.13 According to this hypothesis, the increased contribution of IKs during rapid pacing is responsible for mitigating the effect of IKr block, thereby causing reverse rate dependence. Reverse rate dependence with E-4031 in canine ventricular myocytes is inconsistent with this hypothesis, because the rapid deactivation kinetics of canine IKs (τ, 30 ms at −85 mV; Fig 7⇑) predicts that IKs would accumulate only at very rapid rates. However, reverse rate dependence was observed at rates slower than 1 Hz and with diastolic intervals sufficiently long to ensure full deactivation of IKs. Two other reports provide information refuting the role of IKs in reverse rate dependence: (1) In cat ventricular myocytes (a preparation that reportedly lacks IKs), the selective IKr blocking agent WAY 123,398 causes reverse rate dependence.15 (2) Reverse rate-dependence has been observed in canine ventricular myocytes when both IKr and IKs were reduced with the class III agent azimilide43 ; however, the specificity of IK block by azimilide requires further study. It is unlikely that E-4031 block of canine IKr is modulated by stimulation rate, because 5 μmol/L E-4031 consistently blocked all IKrs (as judged from tail currents), and block of canine IKr (assessed with a holding potential of −40 mV) was frequency independent. Similar results have been reported with other species (see above). Thus, the present results suggest that currents other than IKs play a role in reverse rate dependence in canine ventricle. The same arguments apply to canine atrial myocytes; reverse rate dependence with E-4031 has been demonstrated in canine atrium in vivo44 despite the rapid deactivation kinetics of IKs in atrium as in ventricle.
Presumably, increased net outward current with rapid stimulation, resulting from ionic current(s) other than IKs, reduces the effect of IKr block to promote reverse rate dependence. Further studies are necessary to identify these currents. Alternatively, increased net outward current during rapid stimulation might not be required for reverse rate dependence if the membrane conductance during the plateau is sufficiently increased so as to minimize the role of IKr in defining the action potential configuration at faster rates.
In canine myocytes, E-4031 consistently reduced the outward holding current when the holding potential was maintained near −40 mV. This effect appears to require a sustained depolarization, because it is not observed at comparable potentials when slowly depolarizing ramps are used. The ionic basis for this effect in canines is still uncertain. It is presently unknown whether this effect in canines is specific to E-4031 or shared with other class III agents. An effect of E-4031 on holding current was not reported in guinea pig atrial or ventricular myocytes.2 3
In summary, the present study demonstrates that IK in canine ventricular and atrial myocytes can be functionally considered as two distinct components. In ventricular myocytes, both components participate in repolarization by contributing outward current at slow stimulation rates. Although the activation and voltage dependence of IKr and IKs in dogs and guinea pigs are comparable, their deactivation kinetics are distinctly different, with canine IKs deactivating as rapidly as guinea pig IKr. Despite these kinetic differences, reverse rate dependence with E-4031 is apparent in both species, suggesting that the incomplete deactivation of IKs is not necessary for reverse rate dependence by class III agents in canine myocytes. These results indicate that further studies are necessary to elucidate the ionic mechanism responsible for reverse rate dependence, which has been implicated in the potentially life-threatening proarrhythmic effects of class III agents, including excessive prolongation of the QT interval at slow rates and torsade de pointes.
Selected Abbreviations and Acronyms
|APD||=||action potential duration|
|IK||=||delayed rectifier K+ current|
|IK1||=||inward rectifier K+ current|
|IKe1||=||faster exponential tail current component of IK|
|IKe2||=||slower exponential tail current component of IK|
|IKr||=||rapidly activating component of IK|
|IKs||=||slowly activating component of IK|
|Ito||=||transient outward K+ current|
This study was supported in part by a Grant-in-Aid from the American Heart Association, New York State Affiliate, Inc, and by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute (HL-49918 and HL-37396). E-4031 was the generous gift of the EISA Chemical Co. Nisoldipine was provided courtesy of Miles Pharmaceuticals, Inc. Thanks are also extended to Judy Hefferon, Robert Goodrow, Daryl Perraino, and Dr Richard Vander Heide for superb technical assistance and myocyte preparation.
Reprint requests to Dr Gary A. Gintant, Cardiology Division, Department of Internal Medicine, Wayne State University School of Medicine, 1107 Elliman Research Bldg, 421 E Canfield Ave, Detroit, MI 48201.
From the Masonic Medical Research Laboratory, Utica, NY, and the Cardiology Division, Department of Internal Medicine, Wayne State University School of Medicine, Detroit, Mich.
Previously presented as preliminary reports in abstract form (Circulation. 1993:88[suppl 1, pt 2]:I-89; Biophys J. 1994:66[suppl 2, pt 2]:A-210).
- Received August 9, 1994.
- Accepted September 7, 1995.
- © 1996 American Heart Association, Inc.
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