INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

IN MOST MAMMALS (including humans), a rapidly activating component of delayed rectifier current (I_Kr) is postulated to play a prominent role in defining ventricular repolarization (8, 15, 22, 25). Indeed, most class III antiarrhythmic drugs (as well as some antihistamines and antibiotics with adverse cardiac effects) prolong the action potential duration and ventricular refractoriness, and they may promote torsade de pointes arrhythmias by blocking I_Kr. Despite the relatively rapid activation kinetics of I_Kr, its contribution during the action potential plateau is limited by inward rectification. Rectification of I_Kr has been attributed to rapid inactivation, which reduces outward current at depolarized potentials and contributes to the prolonged plateau phase of the ventricular action potentials (27, 28, 30). Studies of I_Kr in sinus node cells revealed that rapid recovery from inactivation (along with channel deactivation) occurs during repolarizing square-clamp pulses (26). The role that each process may play in defining I_Kr during a ventricular action potential is uncertain, because ventricular repolarization is slower than repolarizing square pulses and the gating characteristics of I_Kr vary between species.

Recent studies have highlighted differences in the densities of various potassium currents across the ventricular wall with implications regarding arrhythmogenesis and antiarrhythmic drug therapy. Heterogeneity of repolarization across the canine ventricular wall has been linked to regional variations in the density of the transient outward current (16) and the slowly activating component of delayed rectifier current (7, 17). Although pharmacological studies suggest that I_Kr plays a dominant role in defining the ventricular action potential duration, significant differences in ventricular I_Kr density have not been reported. In Purkinje fibers, direct measurements of I_Kr have proven difficult (4, 21), despite the fact that block of I_Kr causes dramatic prolongation of the action potential duration in these tissues (10). Detecting differences in I_Kr density or characteristics is difficult, because I_Kr is typically one of the smaller of the potassium repolarizing currents, and its characteristics may not be fully realized using traditional repolarizing square-clamp pulses.

To further characterize and evaluate the involvement of native I_Kr in ventricular repolarization, voltage-clamp protocols simulating the plateau and repolarization phases of the action potential were applied to isolated canine ventricular myocytes and Purkinje fibers. These results were also contrasted with those from atrial myocytes, allowing for a comparison of I_Kr characteristics from electrophysiologically distinct preparations and different sets of ionic currents. Results demonstrate that I_Kr provides a transient "pulse" of outward current later during the ventricular action potential plateau, consistent with its rapid reactivation over the time course of ventricular repolarization. The peak density of the I_Kr transient is unaffected by repolarization rates encountered physiologically and does not "accumulate" during rapid stimulation if the preceding plateau is sufficiently long to ensure I_Kr activation. Whereas the peak density of the I_Kr transient in ventricular myocytes is relatively constant, the voltage characterizing the peak I_Kr transient varies significantly. This variability provides a novel means for modulating the contribution of I_Kr during repolarization to affect electrical heterogeneity. In contrast, variability of inward rectifier current (I_K1) density is the predominant means by which this current varies between ventricular myocytes. Action potential-clamp techniques (in which the recorded action potential serves as the command waveform) demonstrate the defining role of I_Kr in the transition to phase 3 (terminal) ventricular repolarization and the activation of I_K1 to complete repolarization. Preliminary results have been reported in abstract form (9).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Isolation of Ventricular and Purkinje Myocytes

To isolate Purkinje cells, we removed larger free-running Purkinje fiber bundles from the heart after collagenase perfusion but before removal of the endocardium. Purkinje fibers were dissected free of endocardial tissues to prevent potential contamination by ventricular myocytes. Fibers were sliced, minced, and manually triturated for 5 min in modified Tyrode solution to free the Purkinje cells from the surrounding collagenase matrix. The distinct morphology of canine Purkinje myocytes (greater length than ventricular myocytes, minimal step-like projections along sides, and "crinkle-cut" surface undulations) confirmed their identity during electrophysiological studies. In a few instances slow, spontaneous, uniform contractions of some cells were observed when cells were placed in a HEPES-buffered solution [containing (in mM) 132 NaCl, 20 HEPES, 1.2 MgSO₄, 11.1 glucose, 4 KCl, and 2 CaCl₂, adjusted to pH 7.4 with HCl at 37°C], consistent with spontaneous phase 4 depolarization.

Aliquots of isolated myocytes were pipetted onto a heated (36-37°C) chamber on a Nikon inverted scope as described previously (7). Myocytes were superfused with the HEPES-buffered solution. Only rod-like, relaxed myocytes (free from contraction bands, "bends," and membrane "blebs") with resting potentials more negative than 75 mV were studied.

Voltage-Clamp Studies

"Traditional" square-clamp pulses and repolarizing ramp clamps. For voltage-clamp studies using traditional square-clamp pulses and repolarizing ramp clamps, whole cell patch-clamp techniques were applied to myocytes using an Axopatch 200A amplifier (Axon Instruments). Myocytes were accessed with the use of an intracellular solution containing (in mM) 125 K-aspartate, 20 KCl, 10 EGTA, 5 ATP (Mg salt), 1 MgCl₂, and 5 HEPES (free acid), adjusted to pH 7.3 with 5 N KOH. Extracellular solution contained nisoldipine or nimodipine (1 µM) to block L-type calcium current; contamination by Na⁺ current (I_Na) during potassium current recordings was prevented by holding the membrane potential at 40 mV before depolarizations to the plateau range of potentials. Series resistance compensation was typically adjusted to values between 60 and 70%, and recorded potentials were offset negatively by 10 mV for junction potential as described previously (7).

Action potential clamps. For voltage-clamp studies using action potential waveforms ("action potential clamps," see Ref. 5), perforated-patch techniques were used to minimize alterations of the intercellular milieu (11). Pipette solutions contained (in mM) 130 K-aspartate, 15 KCl, 5 HEPES (free acid), 10 NaCl, and 0.5 CaCl₂; 0.18 mg/ml amphotericin B was used as the pore-forming agent. To avoid problems inherent in recording action potential with standard patch-clamp head stages that result from input current in the current-clamp mode (19), an Axoclamp 2B amplifier was used to faithfully record transmembrane potentials (current-clamp mode); voltage clamp was performed as discontinuous single-electrode voltage clamp (or "switch" clamp; typical switching range 4-5 kHz). Action potential waveforms were applied to the same ventricular myocyte from which they were obtained.

Data Interpretation and Analysis

I_K1 was defined as current blocked by 20 mM CsCl (12). To minimize contamination from I_Kr when I_K1 was being evaluated, we pretreated myocytes with E-4031 (to block I_Kr); contamination from slowly activating delayed rectifier K⁺ current (I_Ks) was minimized by using short (250 ms), moderately depolarizing (to 25 mV) square conditioning pulses to reduce activation of I_Ks.

Lines were fit by linear regression, and curves were fit by nonlinear least-squares regression analysis (Origin, version 5.0, Microcal Software). t-Tests were used for unpaired samples to evaluate differences between two groups; comparisons between multiple groups were performed using ANOVA with repeated measures followed by appropriate post hoc tests (Systat). Unless otherwise stated, results are presented as means ± SE.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

An IKr Transient Is Present in Ventricular Myocytes

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Rapidly activating delayed rectifier K+ current (IKr) provides a transient late repolarizing current during ventricular repolarization. A: clamp protocol consisting of a 50-ms prepulse to 35 mV [to inactivate sodium current (INa)] and a 250-ms step pulse to activate potassium current, immediately followed by a repolarizing ramp (1.21 V/s). B: membrane currents recorded from a ventricular myocyte in absence (control) and presence of IKr-blocking agent E-4031 (5 µM). E-4031 causes a greater reduction of outward current during repolarization compared with step depolarization. Each trace represents an average of 2 recordings. C: E-4031-sensitive current (obtained by digital subtraction of traces in B). A drug-sensitive outward current transient is evident during the repolarizing ramp, representing delayed contribution of IKr during repolarization. All records aligned on time axis. Note different current calibrations in B and C.

Three additional series of experiments confirmed the identity of the outward transient as I_Kr. In one set of experiments, the effects of the I_Kr-blocking agent dofetilide (200 nM) on outward currents during repolarization were evaluated. In each of four experiments, a dofetilide-sensitive transient was observed analogous to that observed with E-4031. A similar drug-sensitive transient was also apparent when I_Kr was blocked with 100 µM sotalol. In a second set of experiments, the effects of E-4031 were evaluated in the presence of 0 mM [K⁺]_o to eliminate I_Kr (23). Under these conditions, the E-4031-sensitive transient current was absent during repolarizing ramp clamps (n = 2 myocytes). Together, these results identified the drug-sensitive outward current transient during repolarization as I_Kr and not an artifact unique to E-4031. To examine the unlikely possibility that the I_Kr transient was caused by incremental block (and recovery) of I_Kr during repolarization, we compared the configuration of the transient during equilibration with lower (500 nM) or higher (5 µM) concentrations of E-4031 in the same myocytes. If the drug-sensitive transient had been caused by time-dependent block, the peak of the current transient would have been expected to shift toward more positive potentials as the drug concentration approached maximum blocking concentrations. In each of four experiments, the amplitude of the drug-sensitive transient increased with greater drug concentration, whereas the shape and voltage characterizing the peak of the transient remained unaltered (data not shown). The lack of effect of drug concentration on the I_Kr configuration suggests that incremental block of I_Kr during repolarization is not responsible for the I_Kr transient.

To determine the time course for activation of the I_Kr transient, we clamped ventricular myocytes to +25 mV for increasing durations before a constant repolarizing ramp. Clamp protocols were repeated in the absence and presence of E-4031, and I_Kr was measured as peak drug-sensitive current during repolarization for identical clamp sequences. Figure 2A shows the clamp protocol, consisting of a depolarizing step to 35 mV (to inactivate I_Na) and a second step to +25 mV ranging from 5 to 155 ms in duration (25-ms increments), followed by a repolarizing ramp (1.21 V/s). Figure 2B shows E-4031-sensitive currents recorded from a typical myocyte (with currents offset by 75 pA for clarity). It is evident that the amplitude of the I_Kr transient increases with longer depolarizing step pulses. Figure 2C shows averaged results obtained from seven myocytes, with peak transients normalized as current density. After a 5-ms step pulse, a small I_Kr transient is present that increases after progressively longer depolarizing pulses. The growth of the peak transients was fit to an exponential function with a time constant of 43 ms, attaining half-maximal values within 30 ms. This time constant may slightly overestimate the extent of I_Kr activation during the step pulse, because activation likely continues during early portions of the repolarizing ramp. Despite the increasing current that follows longer depolarizations, the voltage of the peak I_Kr transients was unaffected (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Time course of activation of ventricular IKr transient. A: clamp protocol. B: E-4031-sensitive currents (representing IKr) for plateau durations ranging from 5 (top) to 155 ms (bottom; zero current lines overlay each trace). Current traces have been offset by 75 pA for clarity. Amplitude of IKr transient increases with longer depolarizing pulses. C: summary of results from 7 myocytes. Growth of peak IKr transient with progressively longer step pulses was fit to an exponential function with a time constant of 43.18 ms. Pulses were repeated once every 9 s. See text for further discussion.

In guinea pig myocytes, trains of conditioning depolarizing pulses do not enhance the amplitude of I_Kr assessed during depolarizing ramps (13). To determine whether the I_Kr transient is augmented by rapid electrical activity in canine ventricle, E-4031-sensitive currents were assessed during repolarizing ramps preceded by conditioning pulse trains; drug-sensitive currents were obtained using identical pulse protocols. Preliminary experiments showed that conditioning pulse trains had minimal effects on the I_Kr transient when repolarizing ramps were immediately preceded by a 100-ms step pulse. These results reflect the nearly complete activation of I_Kr during a 100-ms pulse (see Fig. 2). Thus, to examine possible modulation of the I_Kr transient with rapid electrical activity, the duration of the activating pulse step was set to 12.5 ms. Figure 3A, top, illustrates the test waveform, whereas Fig. 3B, inset, illustrates the conditioning pulse protocol, consisting of trains of 0, 2, or 10 pulses applied before the start of the transient protocol. Trains consisted of 100-ms conditioning square pulses to +15 mV with 20-ms interpulse intervals; 13 ms elapsed between termination of the last conditioning pulse and the start of the I_Kr transient test waveform. To maximize sensitivity when recording these small transients, we repeated conditioning pulse train/test pulse sequences three times (in the absence and presence of E-4031) and averaged the results before digital subtraction was performed. Figure 3A compares typical E-4031-sensitive currents preceded by either no prepulses or a train of 10 conditioning prepulses. The I_Kr transient during repolarization is greater after the conditioning pulse train. Figure 3B summarizes results from 11 myocytes. The peak I_Kr transient is enhanced after a 2-pulse conditioning train and further enhanced after a 10-pulse train. These results show that the amplitude of the I_Kr transient is enhanced by repetitive electrical activity when the test waveform is sufficiently brief to prevent full activation of I_Kr. In each of three additional experiments, no I_Kr transient was observed with or without conditioning pulse trains when myocytes were superfused with 0 mM [K⁺]_o.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Conditioning pulse trains increase amplitude of ventricular IKr transient after brief activating test pulses. A, top: test clamp protocol consisting of a 15-ms step pulse to 35 mV (to inactivate INa), followed by a brief (12.5 ms) test pulse to +15 mV to initiate IKr activation before a repolarizing ramp (1.21 V/s). A, bottom: E-4031-sensitive currents obtained from 1 myocyte in absence of a conditioning pulse train (0 prepulses) and after a 10-pulse train (10 prepulses). Conditioning pulse train increased IKr transient. Pulse sets were repeated once every 12 s. Base of calibration represents 40 pA. B: schematic (inset) of conditioning pulse train consisting of depolarizing square pulses (100-mV amplitude, 100-ms duration, 20-ms interpulse interval) applied from 85 mV holding potential; 13 ms elapsed between last conditioning pulse and start of test pulse. Graph shows that density of peak IKr transient is significantly enhanced after 2 conditioning prepulses, with less enhancement after 10 prepulses. Results represent averages from 11 myocytes. * P < 0.05.

To determine whether repolarization rate affected the I_Kr transient, we compared E-4031-sensitive currents during repolarizing ramps of different slopes. I_Kr transients were characterized on the basis of their peak amplitude and the potential at which the peak amplitude occurred. Experiments employed repolarizing ramps with slopes of 1.21, 0.607, and 0.406 V/s (encompassing rates encountered during ventricular repolarization). Figure 4A shows typical E-4031-sensitive currents obtained from a ventricular myocyte, with each ramp overlaying its respective I_Kr transient. Figure 4B shows the peak density of I_Kr transients obtained from 12 myocytes, along with their average value. Some variability in the density of the peak transient was apparent, with values ranging from 0.25 to 0.6 pA/pF (0.44 ± 0.10 pA/pF, mean ± SD). Altering the rate of repolarization did not affect the amplitude of the peak transient.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Amplitude and potential of peak IKr transient are independent of repolarization rate. A: repolarizing ramps used (a, 1.21 V/sec; b, 0.607 V/s; c, 0.406 V/s) along with superimposed E-4031-sensitive currents obtained from a ventricular myocyte. Repolarizing ramps were preceded by a 2-step pulse protocol (as in Fig. 1), with a 250-ms step pulse to +25 mV immediately preceding repolarizing ramp. Pulses were applied every 10 s. B: peak IKr transient densities for each of 12 myocytes () along with average values (). Although significant variability in peak current density is apparent between myocytes, changing the rate of repolarization had no effect on current density. C: potentials for each peak transient () along with average values (). Rate of repolarization did not affect the potential characterizing peak transient current. Values are expressed as means ± SE. Data in B and C were derived from same myocytes (total of 5 hearts). See text for further discussion.

Figure 4C compares the potentials at which the peak transients occurred during repolarizing ramps. The average potential for the peak transients was slightly more negative for the faster versus slower ramp speeds (53.9 vs. 50.1 mV); this difference did not achieve statistical significance. Significant heterogeneity was apparent in the voltages characterizing the peak transients, with values ranging from 33 to 64.7 mV. Heterogeneity was observed when myocytes from individual hearts as well as myocytes from different hearts were compared, demonstrating that variability in the cell isolation procedure is not responsible for the voltage heterogeneity.

Figure 4 demonstrates a wide voltage range characterizing the peak I_Kr transients. To determine the origin of this variability, we initially compared the voltage describing the peak I_Kr transients with those describing the remaining outward current peak after I_Kr blockade, which occurs at more negative potentials (see Fig. 1). As a working hypothesis, we attributed the residual outward peak to the inward rectifier current (I_K1), which is activated at more negative potentials and is responsible for terminal ventricular repolarization and the ventricular resting membrane potential. We reasoned that the potentials describing both the I_Kr and I_K1 peaks would be shifted in the same direction along the voltage axis if the variability reflected nonspecific changes in individual myocytes. Experimental results were not consistent with this hypothesis. For the 12 myocytes studied in Fig. 4, the potential describing the peak I_Kr transient ranged from 33 to 64.7 mV (52.0 ± 7.17 mV, mean ± SD). In contrast, the peak outward current remaining in the presence of E-4031 spanned a narrower range over more negative potentials (from 67.1 to 74.7 mV; 70.8 ± 2.3 mV, mean ± SD). These results suggest that the voltage heterogeneity characterizing I_Kr transients is not shared with I_K1.

To directly compare dispersion of the voltages describing the peak amplitudes of I_Kr and I_K1, an additional series of experiments was conducted comparing the I_Kr transient with I_K1 in the same ventricular myocytes. The experimental protocol sequentially recorded currents during repolarizing ramps 1) under drug-free (control) conditions, 2) during superfusion with E-4031, and 3) during superfusion with E-4031 and 20 mM CsCl; drug-sensitive currents (obtained by digital subtraction) were used to assess I_Kr (E-4031-sensitive current) and I_K1 (Cs-sensitive current). Currents for each of three ramp slopes (1.21, 0.607, or 0.406 V/s) for each myocyte were averaged and compared. Figure 5A shows E-4031-sensitive current (representing I_Kr) and larger, Cs-sensitive current (representing I_K1) recorded from a myocyte during a repolarizing ramp. Figure 5B displays current densities and corresponding potentials characterizing the peak transients for I_Kr transient and I_K1 from 15 myocytes; individual as well as average values are represented. Results show that the average density of I_Kr is approximately fourfold less than that of I_K1 and occurs 24 mV more positively than I_K1. The variability of the I_Kr density is much less than that of I_K1 (0.38 ± 0.05 vs. 1.46 ± 0.31 pA/pF, respectively, means ± SD). The greater variability is also reflected in the coefficient of variation for I_Kr versus I_K1 (0.132 vs. 0.214, respectively), which considers relative variation normalized to mean values. Surprisingly, the voltage range characterizing the peak outward transients is much wider for I_Kr than I_K1 (51.7 ± 8.05 vs. 77.0 ± 1.44 mV, respectively, means ± SD). Thus, heterogeneity in the characteristics of I_Kr and I_K1 can be ascribed to two different factors, variability in the voltage (but not density) of peak I_Kr transients and variability in the current density (but not voltage range) characterizing I_K1.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Variability of the potential characterizing ventricular IKr transient is greater than that characterizing IK1. A: trailing edge of repolarizing ramp-clamp protocol (top) aligned with drug-sensitive currents (bottom). IKr was obtained as drug-sensitive current recorded after superfusion with E-4031 (5 µM, ). Inward rectifier K+ current (IK1) was obtained as Cs-sensitive current (20 mM, ) after prior IKr blockade with E-4031. Peak IKr current density is smaller and occurs at more positive potentials than that of IK1. Zero current level is indicated by base of calibration. Repolarizing ramps were preceded by a 50-ms depolarizing prepulse to 35 mV followed by a 250-ms step pulse to +15 mV (dashed line segments in protocol). B: summary of characteristics of IKr and IK1 obtained using protocol in A. Individual values for each of 15 myocytes [representing averages from 3 ramp slopes (1.21, 0.607, or 0.406 V/s)] are represented by open symbols; closed symbols represent means ± SD. Greater variability is apparent in potentials characterizing peak IKr transients compared with those characterizing IK1. In contrast, greater variability is apparent in IK1 current density than in that of IKr. Data were obtained from 5 hearts. See text for further discussion.

An IKr Transient Is Present in Atrial Myocytes With Characteristics Similar to Those of Ventricular Cells

View larger version (18K): [in this window] [in a new window]   Fig. 6.   IKr transient is present in atrial myocytes. A: representative experiment with an atrial myocyte, displaying the ramp-clamp protocol (top) and corresponding membrane current recorded in absence (control) and presence of E-4031 (middle). Bottom: E-4031-sensitive current shown on an expanded scale. An IKr transient is apparent during repolarization (ramp slope 0.607 V/s). Note minimal IK1 during repolarization (compare with Fig. 1 showing data for a ventricular myocyte). B: peak IKr transient densities obtained from 10 atrial myocytes () along with average values () using repolarizing ramps of 1.21, 0.607, and 0.406 V/s. C: potentials for each peak transient () along with average values () from myocytes described in B. Rate of repolarization had no effect on peak current density or peak potential of atrial IKr transients. Despite minimal IK1, the heterogeneity characterizing peak potentials in atrial myocytes is similar to that in ventricular myocytes. Values are expressed as means ± SE. Atrial cells were obtained from 4 hearts.

IKr Transient Density Is Greater at More Positive Potentials

View larger version (2121K): [in this window] [in a new window]   Fig. 7.   IKr transient amplitude is greater at positive peak potentials. Mean values for peak IKr densities are plotted against potentials characterizing maximal transients for atrial (A, n = 10) and ventricular (B, n = 12) myocytes. Linear regression results (curves representing 95% confidence limits) are superimposed on data, with equations and correlation coefficients indicated for each myocyte type. For either atrial or ventricular myocytes, peak transient density tends to be larger when transient occurs at more positive potentials. Data were obtained from cells described in Figs. 4 and 6 showing values obtained with repolarizing ramps imposed on each cell. Densities were weighted by error bars (SD) for regression analysis.

Direct Demonstration of IKr in Ventricular Repolarization: Action Potential Clamps

4

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Action potential clamps: a direct demonstration of role of IKr in ventricular repolarization. A: 10 consecutive recordings of a ventricular action potential in absence (control) and presence of 5 µM E-4031 recorded using a conventional voltage follower and perforated-patch techniques. Control action potentials were digitally recorded and applied as voltage-clamp waveform. B: results obtained using action potential-clamp technique. Average action potential-clamp waveform has been superimposed over average membrane current (IKr) required to maintain the shorter action potential in presence of E-4031. IKr appears as an increasing outward current that peaks near transition between phase 2 and phase 3 (terminal) repolarization. Basic cycle length, 2 s. See text for further discussion.

IKr Transient Is Present in Purkinje Myocytes

View larger version (22K): [in this window] [in a new window]   Fig. 9.   IKr transient is also present in canine Purkinje myocytes. A: voltage-clamp protocol (top) and corresponding membrane currents (middle) recorded from a Purkinje myocyte in absence (control) and presence of E-4031. Bottom: E-4031-sensitive current. Note different current waveforms in absence and presence of E-4031 (compared with those for ventricular myocytes in Fig. 1) and different current scales for middle and bottom panels. Ramp slope, 0.406 V/s. Effects of repolarization rate on average peak IKr density (B) and average potential of maximal IKr current (C) in 5 Purkinje myocytes are shown. Repolarization rate had no discernible effect on either parameter. Data represent means ± SE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

IKr Provides a Transient, Late-Repolarizing Current

Shibasaki (26) first suggested that voltage-dependent inactivation was responsible for inward rectification of I_Kr in rabbit sinoatrial nodal cells superfused in high [K⁺]_o. Subsequent studies described I_Kr in guinea pig ventricular myocytes as a rapidly activating outward current that contributes minimal current at plateau potentials because of its inward rectification properties (2). Studies suggest that the human ether-à-go-go-related gene (HERG) encodes a potassium ion-selective channel that displays some electrophysiological and pharmacological characteristics similar to those of I_Kr (24, 29, 32). Studies of HERG expressed in HEK-293 cells or Xenopus oocytes support the role of voltage-gated fast inactivation in the rectification of I_Kr (27, 28). In these expression systems, rectification results from inactivation that proceeds at a rate faster than activation, limiting outward current on depolarization. I_Kr and HERG typically display a "hooked" configuration on step repolarization that has been attributed to rapid recovery from inactivation (relative to deactivation) immediately following a repolarizing square-clamp pulse.

On the basis of these reports, the configuration of the I_Kr transient during repolarization (in this study) is most easily explained by assuming rapid voltage-dependent recovery from inactivation along with slower deactivation kinetics. Because the amplitude and voltage characterizing the peak I_Kr transient are unaffected by repolarization rate (Figs. 4, 6, and 9), recovery from inactivation must be rapid relative to the physiological repolarization rates tested. The lack of effect of repolarization rate on the I_Kr transient is also consistent with slow channel deactivation relative to repolarization. Prior studies of canine I_Kr demonstrated slow tail-current kinetics after step repolarizations under experimental conditions identical to those in this study [time constant of inactivation () = 2-3 s at 40 mV, Ref. 7]; two time constants have also been reported ( values in range of 250-500 ms and 2-20 s, Ref. 17). Slow deactivation kinetics have also been reported for rabbit (0.8 s, Ref. 3) and cat (0.7 s, from Fig. 7A in Ref. 6) ventricular myocytes and can be gleaned from studies of human ventricular myocytes (  500 ms at 30 mV, derived from Fig. 3 in Ref. 15); faster deactivation kinetics have been reported with guinea pigs (<175 ms near 30 mV, Ref. 25). The rapid recovery from inactivation and slow deactivation kinetics of canine I_Kr provide a voltage-dependent (and functionally time- independent) outward transient during ventricular repolarization, analogous to that provided by I_K1 at more negative potentials during final repolarization.

In contrast to I_K1, greater heterogeneity dispersion is apparent in the potentials characterizing the peak I_Kr transients (Figs. 4-6). The I_Kr transient density is typically greater if it occurs at more positive potentials (Fig. 7). Thus the voltage of the peak transient modulates the contribution of I_Kr during an action potential by two means, namely, 1) by affecting the timing of the transient and 2) by affecting the amplitude of the transient during repolarization. A more detailed kinetics analysis of I_Kr inactivation in ventricular myocytes [using brief (a few ms) step-clamp protocols] is not technically feasible because of their large capacitance (mean value near 180 pF, unpublished observations) affecting temporal clamp fidelity and the presence of larger time-dependent currents present in this preparation. The use of repolarizing ramps enhances the resolution and characterization of native I_Kr and provides an effective means to assess the effects of antiarrhythmic drugs and electrophysiological changes accompanying heart disease.

An outward I_Kr transient is observed during repolarizing ramps (28) and with imposed action potential clamps (32) using heterologous expression systems expressing HERG. In oocytes, expressed peak HERG current increases with progressively slower repolarizing ramps (range 0.1 to 1 V/s) and peaks at more negative potentials with faster repolarization (28). In contrast, native canine I_Kr amplitude is not affected by ramp slopes (range 1.21 to 0.4 V/s, Fig. 4) and peaks at similar potentials as the amplitude grows after longer activating pulses (Fig. 2). It is uncertain whether these disparate results can be attributed to differences in experimental conditions, species, or differences in channel gating. A comparison of results obtained using native I_Kr and heterologously expressed HERG requires caution because of the generally slower kinetics of HERG in heterologous expression systems, which are exaggerated at lower experimental temperatures (32). Computer simulations of guinea pig I_Kr (assuming instantaneous recovery from inactivation) predict an outward I_Kr transient during repolarization (31) but do not predict variability in the voltages describing the peak transients. This suggests that other factors are responsible for modulating native I_Kr in ventricular myocytes.

The I_Kr density in canine ventricular myocytes measured during repolarizing ramps (0.44 pA/pF, Fig. 4) is more than twofold greater than that measured during step repolarizing pulses to 40 mV under identical experimental conditions (0.2 pA/pF, Ref. 6). The greater current density with repolarizing ramps is present despite the reduced driving force for potassium ions (peak I_Kr transients typically occurring between 45 and 65 mV, see Figs. 4 and 7). The lesser current density obtained using square repolarizing pulses is consistent with incomplete recovery from inactivation at 40 mV, which is likely reduced at more negative potentials during repolarizing ramps. Despite the greater canine I_Kr density measured with repolarizing ramps, these values remain ~50% less than those used in simulations of guinea pig action potentials on the basis of experiments utilizing square-clamp pulses (1 pA/pF, Ref. 29, based on Ref. 25). The greater current density with guinea pig myocytes may contribute to the shorter action potentials and more rapid heart rates of this species. The mean peak transient current density was statistically greater in atrial versus ventricular myocytes (0.62 vs. 0.44 pA/pF, P = 0.04, Fig. 7). Whether this represents greater functional I_Kr current in all atrial myocytes is uncertain, because the range of I_Kr densities for atrial myocytes overlaps that of ventricular myocytes, and the shorter atrial action potential duration may limit the extent of I_Kr activation. It remains to be determined whether the greater average transient density in atrial myocytes compensates for the shorter atrial action potential duration.

IKr Also Provides a Transient, Late-Repolarizing Current in Purkinje Fibers

Possible Mechanism of Heterogeneity of IKr Inactivation

The voltage range characterizing the I_Kr transients is most easily attributed to differences in the voltage dependence of I_Kr. A recent study has linked a mutation of HERG associated with long-QT syndrome to a shift in the voltage dependence of steady-state inactivation toward more negative potentials (20). In addition to enhancing rectification and reducing channel availability at positive potentials, this mutation would be expected to shift the peak potential of the I_Kr transient to more negative potentials, further delaying repolarization. Studies have reported at least two isoforms of ERG in mouse ventricle (14, 18). The expression of these individual isoforms in Xenopus oocytes results in currents with kinetics either slower or faster than those of native I_Kr, whereas the expression of both isoforms results in intermediate kinetics (comparable to I_Kr) consistent with the coassembly of heteromultimers. The voltage dependence of the I-V relationship of one of the proposed homomultimers (the relatively rare Merg1a') was shifted by +10 mV compared with either of two other homomultimers or the heteromultimer Merg1a/1b. Although results with the mouse homomultimer likely would not account for the shifts in peak transient potentials observed in the present study, they do raise the possibility that some HERG isoforms may display altered voltage dependence. Whether such isoforms exist in canine myocardium is unknown. A recent study of HERG expressed in Xenopus oocytes showed that a small integral membrane protein [termed minK-related peptide 1 (MiRP1)] assembles with HERG and shifts the voltage dependence of activation in a positive direction without appreciably affecting rapid recovery from inactivation (1). Channels associated with MiRP1 also display reduced single-channel current and increased deactivation rate. The effects of MiRP1 on I_Kr during repolarizing ramp clamps were not studied and are difficult to predict when oocyte studies (conducted at room temperature) are extrapolated to native channels and physiological temperatures. Thus, it is possible that HERG isoforms, accessory subunits, and/or metabolic factors may contribute toward the heterogeneous voltage dependence of I_Kr transients; further studies combining cellular and molecular approaches with electrophysiological recordings are necessary to test these hypotheses in native tissues. Because ventricular myocytes in this study were derived from the entire left ventricular midwall, it is unknown whether the variability of I_Kr is related to anatomic origin within the wall or how it may contribute to the electrophysiological distinctions between isolated epicardial, midmyocardial, and endocardial myocytes.

Functional Consequences of Heterogeneity of the IKr Transient

In summary, this study demonstrates that, despite its rapid activation, I_Kr provides a delayed outward current transient during repolarization of ventricular myocardium and Purkinje fibers. This resurgent current plays an increasingly prominent role later during the action potential plateau by promoting the initiation of terminal (phase 3) repolarization and activation of I_K1. Variability in the voltage dependence of the native I_Kr transient and variability in the density of I_K1 provide two distinct mechanisms for modulating the role of these potassium currents during repolarization and contributing toward ventricular electrical heterogeneity.