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(Circulation Research. 1996;78:689-696.)
© 1996 American Heart Association, Inc.

Articles

Evidence for Two Components of Delayed Rectifier K⁺ Current in Human Ventricular Myocytes

Gui-Rong Li, Jianlin Feng, Lixia Yue, Michel Carrier, Stanley Nattel

From the Department of Medicine, Montreal Heart Institute (G.-R.L., J.F., S.N.) and University of Montreal (G.-R.L., S.N.), the Department of Surgery, Montreal Heart Institute and University of Montreal (M.C.), and the Department of Pharmacology and Therapeutics, McGill University (L.Y., S.N.), Montreal, Canada.

Correspondence to Dr Stanley Nattel, Research Center, Montreal Heart Institute, 5000 Belanger St E, Montreal, Quebec, Canada H1T 1C8.

	Abstract

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Abstract Previous voltage-clamp studies have suggested that the delayed rectifier current (I_K) is small or absent in the human ventricle and, when present, consists only of the rapid component (I_Kr); however, molecular studies suggest the presence of functionally important I_K in the human heart, specific I_Kr blockers are known to delay ventricular repolarization and cause the long QT syndrome in humans, and we have shown that the expression of I_K is strongly influenced by cell isolation techniques. The present experiments were designed to assess the expression of I_K in myocytes obtained by arterial perfusion of right ventricular tissue from explanted human hearts. Of 35 cells from three hearts, 33 (94%) showed time-dependent currents typical of I_K. The envelope-of-tails test was not satisfied under control conditions but became satisfied in the presence of the benzenesulfonamide E-4031 (5 µmol/L). E-4031 suppressed a portion of I_K in 32 of 33 cells, with properties of the drug-sensitive and -resistant components consistent with previous descriptions of I_Kr and the slow component (I_Ks), respectively. Action potential duration to 95% repolarization at 1 Hz was prolonged by E-4031 from 336±16 (mean±SEM) to 421±19 ms (n=5, P<.01), indicating a functional role for I_K. Indapamide, a diuretic agent previously shown to inhibit I_Ks selectively, suppressed E-4031–resistant current. The presence of a third type of delayed rectifier, the ultrarapid delayed rectifier current (I_Kur), was evaluated with the use of depolarizing prepulses and low concentrations (50 µmol/L) of 4-aminopyridine. Although these techniques revealed clear I_Kur in five of five human atrial cells, no corresponding component was observed in any of five human ventricular myocytes. We conclude that a functionally significant I_K, with components corresponding to I_Kr and I_Ks, is present in human ventricular cells, whereas I_Kur appears to be absent. These findings are important for understanding the molecular, physiological, and pharmacological determinants of human ventricular repolarization and arrhythmias.

Key Words: repolarization • cardiac arrhythmias • ion channels • antiarrhythmic agents • class III drugs

	Introduction

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The delayed rectifier K⁺ current (I_K) was first described in sheep cardiac Purkinje fibers by Noble and Tsien¹ and has since been identified in a wide variety of species and tissue types.^{2 3 4 5 6 7 8 9 10 11 12 13} Noble and Tsien defined two components of I_K that differed in kinetic properties, voltage dependence, and rectification,¹ and similar components were subsequently observed in cardiac cells from the guinea pig,^{4 5} dog,^{9 10 11} and chick¹³ heart. Sanguinetti and Jurkiewicz^{4 5} demonstrated that certain class III antiarrhythmic drugs, notably E-4031 and sotalol, selectively block I_Kr and that the drug-sensitive (I_Kr) and drug-resistant (I_Ks) components of I_K differ in terms of voltage dependence, kinetics, rectification properties, and pharmacological sensitivity. These differing properties may have important implications for understanding the physiological function of I_K and the effects of I_K blockers and agonists in specific tissues.¹⁴

A potential molecular equivalent of I_Ks was cloned several years ago,^{15 16 17} and electrophysiological and immunolocalization data suggesting that the corresponding protein (called minK or IsK) underlies I_Ks in the guinea pig heart have been presented.¹⁸ Recently, a gene (human ether-a-go-go–related gene, or HERG) coding for channels carrying currents resembling I_Kr has been identified in the human heart.^{19 20} Despite the demonstrated presence of genes for both IsK^{16 17} and HERG²⁰ in the human heart and the presence of high concentrations of HERG mRNA in human cardiac tissue,²⁰ the presence of significant currents corresponding to I_Kr and I_Ks has been difficult to demonstrate in human ventricular tissue. Beuckelmann et al²¹ found small delayed rectifier currents in 41% of cells from failing human left ventricles and in none of six cells from normal hearts. Although a detailed electrophysiological characterization of I_K was not performed, the authors concluded that the only component present was I_Kr, that minimal or no I_K was present in most human ventricular cells, and that the major current responsible for repolarization in human ventricle appeared to be I_to. We have recently shown that the expression of I_K in cells isolated from the canine right atrium is very dependent on isolation techniques.⁹ We adapted the techniques we previously used to isolate canine atrial cells with robust I_K⁹ in order to obtain cells from right ventricles of three patients receiving heart transplants for severe left ventricular failure without significant right ventricular pathology. The purpose of the present study was to determine the prevalence of I_K in human ventricular cells isolated with these methods and to establish whether components corresponding to I_Kr and I_Ks are present.

	Materials and Methods

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Myocyte Isolation
Explanted hearts were obtained at the time of heart transplantation from three patients, aged 31, 53, and 54 years. The underlying heart disease was congestive cardiomyopathy in two patients and heart failure due to aortic valve disease in one patient. Subsequent examination of the right ventricle by a cardiac pathologist revealed it to be macroscopically normal in all patients. Microscopic examination of the right ventricular myocardium was normal in all hearts, with minor extramyocardial abnormalities consisting of fatty infiltration in one heart and subendocardial fibrosis in another.

All hearts were initially placed in cold (4°C) oxygenated Krebs' solution and then transferred to cardioplegic solution for dissection and coronary artery cannulation. A portion of the free wall of the right ventricle (2x4 to 2x5 cm) was removed along with the coronary artery branch irrigating it, with dissection and arterial cannulation completed within 20 minutes of excision of the heart. The free wall was perfused with oxygenated nominally Ca²⁺-free Tyrode's solution for 20 to 30 minutes, and the solution was then changed to one containing 200 to 300 U/mL collagenase (CLS II, Worthington Biochemical) for 60 to 100 minutes. The digested tissue was cut into small (1.5- to 2-mm³) pieces, placed in a high-K⁺ storage solution (see below), and gently triturated with a Pasteur pipette. Isolated myocytes were kept in the medium at least 1 hour before use. Atrial cells were isolated from right atrial samples of three additional patients without atrial disease by the use of techniques previously described in detail.^{22 23}

A small aliquot of the solution containing the isolated cells was placed in an open perfusion chamber (1 mL) mounted on the stage of an inverted microscope. Myocytes were allowed to adhere to the bottom of the chamber for 5 to 10 minutes and were then superfused at 2 to 3 mL/min with Tyrode's solution. Experiments studying classic I_K were conducted at 36°C, with the temperature controlled by a Peltier-effect device. In studies of I_Kur, cells were evaluated at room temperature in order to resolve the very rapid kinetics of the current.²³ Only quiescent rod-shaped cells showing clear cross striations were used.

Solutions
The Tyrode's solution contained (mmol/L) NaCl 126, KCl 5.4, MgCl₂ 1.0, CaCl₂ 1.0, NaH₂PO₄ 0.33, glucose 10, and HEPES 10 (pH adjusted to 7.4 with NaOH). For voltage-clamp studies of I_K, external Na⁺ was replaced by equimolar (126 mmol/L) choline to suppress I_Na, 4-AP (5 mmol/L, Sigma Chemical Co) was used to block I_to, 0.5 mmol/L BaCl₂ (Sigma) was used to inhibit I_K1, and 0.2 mmol/L CdCl₂ was used to suppress I_Ca. I_Kur was studied with the same external solution, except that 4-AP was omitted under control conditions. The high-K⁺ storage medium contained (mmol/L) KCl 20, KH₂PO₄ 10, glucose 10, potassium glutamate 70, ß-hydroxybutyric acid 10, taurine 10, EGTA 5.0, and mannitol 10, along with 0.1% albumin (pH adjusted to 7.3 with KOH). The pipette solution contained (mmol/L) KCl 20, potassium aspartate 110, MgCl₂ 1.0, HEPES 10, EGTA 5.0, GTP 0.1, Na₂ phosphocreatine 5.0, and Mg₂ATP 5.0 (pH adjusted to 7.2 with KOH). E-4031 was provided as a kind gift by Eisai Ltd (Ibaraki, Japan) and prepared as a 5 mmol/L stock solution in distilled water.

Data Acquisition and Analysis
The tight-seal whole-cell patch-clamp technique was used. Borosilicate glass electrodes (outer diameter, 1.0 mm) were pulled with a Brown-Flaming puller (model P-87) and had tip resistances of 2 to 4 M when filled with pipette solution. Data were acquired with the use of an Axopatch 200A or 1-D amplifier (Axon Instruments). Command pulses were generated by a 12-bit digital-to-analog convertor controlled by pClamp software (Axon Instruments). Recordings were low-pass–filtered at 2 kHz, and data were acquired by analog-to-digital conversion at a maximum rate of 50 kHz (model TM 125, Scientific Solutions) and stored on the hard disk of an IBM-compatible computer. Junction potentials (2 to 10 mV) were compensated before the pipette touched the cell. A tight seal was obtained, and seals with a resistance of <10 G were rejected. The cell membrane was ruptured by gentle suction to establish the whole-cell configuration.

R_s was electrically compensated to minimize the duration of the capacitive transient. R_s was estimated by dividing _cap by the total membrane capacitance obtained during 5-mV hyperpolarizing steps from a holding potential of -60 mV. Before compensation, _cap in ventricular cells averaged 1043±112 µs, and R_s averaged 5.8±0.7 M (cell capacitance, 179±15 pF). After compensation, _cap decreased to 449±21 µs, and R_s decreased to 3.2±0.5 M. In atrial cells, the initial _cap averaged 509±49 µs (cell capacitance, 71±9 pF), and R_s averaged 6.9±0.9 M. Corresponding values after _cap and R_s compensation were 259±30 µs and 4.1±0.8 M, respectively.

Curve fitting was performed with a Marcquardt algorithm and TableCurve software (Jandel Scientific). Results are presented as the mean±SEM. Statistical comparisons between two group means were by t test, and a two-tailed value of P<.05 was taken to indicate statistical significance. Each series of experiments was performed with roughly equal numbers of cells from all hearts, in order to ensure that the results of each analysis were representative of all the hearts studied.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Prevalence and General Properties of I_K
Currents were studied during 3-s depolarizing steps to voltages between -40 and +60 mV from a holding potential of -60 mV, with I_Ktail observed during subsequent repolarization to -30 mV. Thirty-three (94%) of 35 cells showed I_Ktail of at least 50 pA after steps to +60 mV. Fig 1A shows I_Kstep and I_Ktail in a representative cell. The addition of 5 µmol/L E-4031 reduced the amplitude of both I_Kstep and I_Ktail (Fig 1B), and the E-4031–sensitive current obtained by digital subtraction showed rapid activation and relatively large I_Ktails (Fig 1C). Action potentials were recorded at 1 Hz with current-clamp techniques during superfusion with normal Tyrode's solution, and typical results from one cell before and after the addition of E-4031 (5 µmol/L) are shown in Fig 1D. In five cells exposed to 5 µmol/L E-4031, resting potential averaged -83±3 mV, and APD to 95% repolarization at 1 Hz averaged 336±16 ms before and 424±19 ms after (P<.01) exposure to the drug. Of the 33 cells showing I_K, an E-4031–sensitive component was seen in all but one, and an E-4031–resistant component was present in all.

View larger version (20K): [in this window] [in a new window]   Figure 1. IK and action potentials recorded from a human ventricular myocyte. Currents were elicited by 3-s depolarizing pulses (0.1 Hz) from a holding potential of -60 mV to potentials ranging from -40 to +50 mV, followed by a 2-s repolarization to -30 mV to observe IKtail. A, Currents under control conditions. B, Currents recorded after 10-minute exposure to 5 µmol/L E-4031. C, E-4031–sensitive currents obtained by digital subtraction of currents in panel B from those in panel A. The calibration in the inset of panel C applies to panels A and B as well. D, Action potentials recorded under current-clamp conditions at 1 Hz before and after exposure to 5 µmol/L E-4031.

Components of I_K
The envelope of tails was studied with the use of the protocol illustrated in Fig 2A. Under control conditions, the envelope test was not satisfied, as indicated by the lack of superposition of scaled I_Ktail on I_Kstep. After the addition of E-4031, the envelope test was satisfied (Fig 2B). Mean ratios of I_Ktail to I_Kstep from eight cells are shown in Fig 2C. Under control conditions (open circles), the ratio averaged 2.8±0.3 after a 200-ms activating pulse, and this value gradually decreased to 1.3±0.1, reaching steady state values at a pulse duration of 1000 ms. In the presence of E-4031 (filled circles), time-dependent changes in the ratio of I_Ktail to I_Kstep were eliminated. These data suggest that I_K consists of more than one component under control conditions and that in the presence of E-4031 only one component remains.

View larger version (18K): [in this window] [in a new window]   Figure 2. A, Envelope of tails recorded with protocol at the top delivered at 0.1 Hz under control conditions in a representative myocyte. indicates IKtail scaled onto IKstep. B, Results from same cell as in panel A, after exposure to 5 µmol/L E-4031. C, Ratio of IKtail to IKstep, as measured in eight cells (results are mean±SEM) with the protocol shown at the top, delivered at 0.1 Hz.

Fig 3A shows recordings obtained before and after exposure to 5 µmol/L E-4031 in a different cell from that illustrated in Fig 1. The E-4031–sensitive current (Fig 3B) activated rapidly and had I_Ktails that were relatively large compared with I_Ksteps. At the more positive voltages, E-4031–sensitive I_Kstep density showed a slow time-dependent decay, resulting in strong inward rectification of the end-pulse current as previously shown in human atrial cells.²² Fig 3C shows mean time-dependent I_Kstep density (defined as the current level at the end of the pulse relative to the initial current level, normalized to cell capacitance) as a function of test potential in six cells. The current-voltage relation of total current has a flat portion between +10 and +30 mV, whereas the current-voltage relation for E-4031–resistant current is relatively smooth. E-4031–sensitive current shows strong inward rectification.

View larger version (17K): [in this window] [in a new window]   Figure 3. Voltage dependence of E-4031–sensitive and –resistant components, as determined with the protocol shown in the inset at 0.1 Hz. A, Currents recorded in the absence () and presence () of E-4031 in one cell at the depolarizing pulse voltages indicated. B, E-4031–sensitive currents of cell shown in panel A, obtained by digital subtraction of currents in the presence of the drug from currents in its absence. The dashed horizontal lines correspond to a difference current of 0 pA. C, Mean±SEM time-dependent IKstep (normalized to cell capacitance) as a function of test potential (TP), for six cells studied with protocol illustrated in panels A and B. IKstep is defined as the difference between initial current after relaxation of capacitance (or inactivation of residual Ito or ICa when present) and the current at the end of the pulse.

An analysis of the voltage-dependent activation of E-4031–sensitive and –resistant components is shown in Fig 4. Fig 4A shows mean I_Ktail densities in 11 cells at -30 mV after 3-second depolarizing pulses to the test pulse voltages indicated. At voltages below +10 mV, E-4031 inhibited the majority of the I_Ktail. At more positive potentials, the E-4031–sensitive portion remained constant, whereas the drug-resistant portion continued to increase. Activation voltage dependence (Fig 4B) was determined by normalizing I_Ktail at each test potential in Fig 4A to the current at the most positive test potential. Under control conditions, V_0.5 averaged 0.9±0.3 mV, and the slope factor was 11.2±3.1 mV. In the presence of E-4031, V_0.5 increased to 9.4±2.5 mV (P<.01 versus control), and the slope factor averaged 11.8±2.9 mV. For the E-4031–sensitive component, V_0.5 averaged -14±4 mV (P<.01 versus control), and the slope factor was 7.7±2.7 mV.

View larger version (17K): [in this window] [in a new window]   Figure 4. Voltage and time dependence of components of IK. A, Current-voltage relations for IKtail of control, E-4031–resistant, and E-4031–sensitive currents. IKtails were recorded during a 2-s pulse to -30 mV after a 3-s depolarization (0.1 Hz) from -60 mV to the test potentials (TPs) shown and normalized to cell capacitance. B, Isochronal activation curve of control, E-4031–resistant, and E-4031–sensitive currents based on an analysis of IKtail in panel A. IKtails at each potential were fitted to a Boltzmann distribution function of the following form: A=1/{1+exp[(V0.5-Vm)/s]}, where A is the activation variable (IKtail at a TP [Vm] divided by IKtail at +50 mV), V0.5 is the 50% activation voltage, and s is the slope factor. C, Time dependence of IK activation as determined from capacitance-normalized IKtail at -30 mV after a pulse to +50 mV of the duration indicated. Curves are nonlinear fits to an equation of the following form: I=If[1-exp(-t/f)]+Is[1-exp(-t/s)] for control and E-4031–resistant currents, and I=Imax[1-exp(-t/act)][exp(-t/inact)] for E-4031–sensitive currents, where I is IKtail after an activating pulse of duration t, and If, Is, f, s, Imax, act, and inact are constants. Results are mean±SEM for 11 cells (panels A and B) and 10 cells (panel C).

An analysis of the kinetics of I_K activation at +50 mV based on I_Ktail densities upon subsequent repolarization to -30 mV (same protocol as in Fig 2) is shown (mean±SEM for 10 cells) in Fig 4C. Under control conditions, I_K was well fitted by a biexponential function, with a ₁ of 161±32 ms and a ₂ of 533±86 ms. The E-4031 resistant component was also biexponential, with a ₁ of 360±87 ms and a ₂ of 8.5±0.3 s. The E-4031–sensitive current had an activation time constant of 192±53 ms (not significantly different from the fast phase time constant under control conditions) and an inactivation time constant of 10.6±0.6 s.

The reversal potential of I_K was closely related to [K⁺]_o. In six cells exposed to [K⁺]_o of 5.4, 10.8, and 21.6 mmol/L, the potential for reversal of I_Ktail was linearly related to log([K⁺]_o), with a slope of 54.8 mV per decade and a correlation coefficient of .999. Fig 5A illustrates the effect of E-4031 on the reversal potential in a representative cell. The amplitude of I_Ktail was determined at various potentials after a 3-second pulse to +40 mV. Under control conditions, the reversal potential was between -70 and -80 mV. After exposure to E-4031, I_Ktail became smaller, and the reversal potential became less negative (between -60 and -70 mV). Fig 5B shows mean I_Ktail densities at various test potentials as measured under control conditions in four cells and in the presence of E-4031 in four cells. The average reversal potential was -75 mV under control conditions and -69 mV in the presence of E-4031. Mean E-4031–sensitive tail current densities, measured in two cells studied under stable conditions before and after E-4031, are shown by the open inverted triangles in Fig 5B. The reversal potential of mean E-4031–sensitive current was -86 mV, very close to the estimated K⁺ equilibrium potential.

View larger version (21K): [in this window] [in a new window]   Figure 5. A, IKtail recorded upon repolarization to the voltages indicated after a 3-s pulse to +40 mV (0.1 Hz). Results are shown for a representative cell studied in the absence (top) and presence (bottom) of E-4031. Note that in the presence of E-4031, IKtail reverses between -60 and -70 mV, whereas IKtail reverses between -70 and -80 mV in its absence. B, Relation between IKtail density and voltage at which IKtail was measured with the use of the protocol illustrated in panel A. TP indicates test potential. Results are mean±SEM for four cells each in the absence and presence of E-4031. E-4031–sensitive current data are mean±SEM for two cells that remained stable for the entire protocol in the absence and presence of E-4031.

Unlike I_Kr, for which a variety of highly selective blockers are available, there is no similarly recognized tool for the study of I_Ks. Recently, however, the diuretic agent indapamide has been reported to be a selective I_Ks blocker.²⁴ To study further the pharmacological properties of E-4031–resistant current in human ventricle, we exposed cells to 1 mmol/L indapamide (a concentration reported to fully block I_Ks in guinea pig ventricle²⁴ ). Fig 6A shows an envelope of tails recorded from one myocyte in the presence of 5 µmol/L E-4031. Exposure to indapamide fully suppressed both I_Kstep and I_Ktail (Fig 6B). Partial reversal of current suppression was observed after 15 minutes of indapamide washout (Fig 6C). Similar results were obtained in a total of four cells.

View larger version (20K): [in this window] [in a new window]   Figure 6. Effects of indapamide on E-4031–resistant currents in a representative human myocyte. Currents were elicited by the protocol shown in inset delivered at 0.1 Hz. A, Currents recorded in the presence of 5 µmol/L E-4031. B, Currents recorded after the addition of 1 mmol/L indapamide. C, Currents recorded after 15 minutes of indapamide washout. Similar results were obtained in a total of four cells.

In the final series of experiments, we sought to establish whether the ultrarapid delayed rectifier reported in human atrium is also present in human ventricular myocytes. Fig 7A (top) shows typical recordings of I_Kur, obtained in a human atrial myocyte with the use of a 100-ms prepulse to +40 mV (to inactivate I_to) delivered 10 ms before a 150-ms depolarizing test pulse. As described previously,²³ 50 µmol/L 4-AP substantially inhibited I_Kur (middle). Similar results were obtained in all of five atrial myocytes studied over the course of these experiments. Typical recordings from a ventricular myocyte obtained with the same protocol are shown in Fig 7B (top). The small outwardly rectifying current recorded under these conditions shows no time dependence, and 50 µmol/L 4-AP does not cause any obvious inhibition (middle). In contrast to the clear rapidly activating 50 µmol/L 4-AP–sensitive current observed in atrial cells (Fig 7A, bottom), no corresponding component was noted in this (Fig 7B, bottom) or four other ventricular cells studied in the same fashion. Furthermore, exposure to 4-AP concentrations as high as 10 mmol/L failed to reveal any drug-sensitive component comparable to I_Kur.

View larger version (29K): [in this window] [in a new window]   Figure 7. Currents elicited by voltage protocol (shown in inset) used to study IKur. A, Results from a typical atrial cell. B, Results from a ventricular cell. Under control conditions (top), currents typical of IKur were present in the atrial cell but absent in the ventricular cell. Exposure to 50 µmol/L 4-AP (middle) clearly inhibited atrial cell currents but did not alter those in the ventricular cell. Drug-sensitive currents obtained by digital subtraction (bottom) typical of IKur were seen in the atrial cell, but no significant 4-AP–sensitive current was detected in the ventricular cell. Similar results were obtained respectively from five atrial and five ventricular cells examined with this protocol over the course of these studies. A 100-ms prepulse was used to inactivate Ito and was followed 10 ms later by a 150-ms test pulse to each of the voltages indicated.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we have demonstrated that significant quantities of I_K are present in a large proportion of human ventricular cells and that the current can be dissociated into components corresponding to I_Kr and I_Ks. Before the present study, whole-cell voltage-clamp data in the literature suggested that I_K is small in amplitude and is expressed in a minority of human ventricular cells and that the I_Ks component is absent. We found no evidence for the presence in ventricular cells of an ultrarapid delayed rectifier comparable to I_Kur previously reported in human atrial cells.

Comparison With Previous Studies of I_K
The E-4031–sensitive and –resistant components that we observed share a variety of properties with I_Kr and I_Ks previously described in other systems. As in guinea pig ventricle⁴ and atrium,⁵ canine ventricle,¹⁰ and human atrium,²² the E-4031–sensitive component (I_Kr) activates more rapidly, at a more negative voltage, and with a more steep slope factor than does the E-4031–resistant component (I_Ks) and shows inward rectification. The reversal potential of I_Kr is more negative than that of I_Ks, which is compatible with previous observations in other species,^{1 4 10} and suggests a greater K⁺ selectivity for I_Kr. Like I_Ks in guinea pig ventricle,²⁴ E-4031–resistant current in human ventricle is effectively inhibited by the diuretic agent indapamide. The rapid-phase time constant of I_K activation in human ventricle is similar to the activation time constant of E-4031–sensitive current, suggesting that it is due to the activation of I_Kr. E-4031–resistant current activated in a biexponential fashion, with time constants differing by approximately an order of magnitude. This finding differs from results previously described by Sanguinetti and Jurkiewicz⁵ in guinea pig ventricle and Wang et al²² in human atrium but closely resembles results recently reported in canine ventricular cells.¹⁰ The discrepancies may be due to species- and/or tissue-related differences in I_Ks behavior or to methodological differences among studies. We observed a slow decline of E-4031–sensitive current during sustained depolarization in the present study, similar to previous findings in human atrium.²² This slow decline may be due to I_Kr inactivation, as suggested by previous reports of experiments in rabbit nodal cells⁶ and in AT-1 cells.^{25 26}

Beuckelmann et al²¹ observed small I_Kr-like currents in a minority of left ventricular cells from explanted failing human hearts and in none of six cells from normal hearts. They concluded that I_Ks is absent in the human ventricle and that I_K is unlikely to be important in human ventricular repolarization. APD in the failing heart cells studied by Beuckelmann et al was >1 s, compared with an average of 336 ms in the cells in the present study. The latter value is in the same range as the mean APD recorded from normal multicellular human ventricular preparations (300 to 360 ms)^{27 28} and the mean in vivo monophasic APD of 300 ms obtained by Bargheer et al²⁹ during clinical electrophysiological studies in 10 patients. The differences in APD between the cells used by Beuckelmann et al and those used in the present study are consistent with the much smaller amounts of repolarizing I_K recorded in the former study. Veldkamp et al³⁰ reported the presence of single I_Kr channels in human ventricular myocytes in a preliminary communication, but the prevalence and kinetics of these channels were not described. In a recently published study, Konarzewska et al³¹ analyzed the properties of I_to and I_K1 in myocytes obtained from biopsies of normal human ventricle but did not detect I_K. They noted the discrepancy between indirect evidence pointing toward a role for I_K in human ventricular repolarization and the lack of direct recordings of I_K in their study and in the previous literature. The present study, which shows that both components of I_K are demonstrable in the vast majority of normal human right ventricular myocytes, stands to resolve this discrepancy.

We found no evidence in human ventricular myocytes for the presence of currents resembling I_Kur in human atrial cells. Konarzewska et al³¹ were similarly unable to demonstrate a highly 4-AP–sensitive current in human ventricular cells. The molecular component believed to underlie I_Kur, the Kv1.5 channel,^{23 32 33} has recently been detected by immunohistochemical techniques in both human atrium and ventricle.³⁴ The apparent discrepancy between the immunohistochemical and electrophysiological evidence requires explanation. One possibility is that I_Kur is not carried by Kv1.5 channels, but there is substantial evidence pointing to the contrary.^{23 33} A second possibility is that the isolation procedure damages I_Kur in the ventricle and renders it nonfunctional; however, we have observed that I_Kur is more resistant than other K⁺ currents (like I_K and I_to) to damage during isolation of human atrial myocytes. A third potential explanation relates to differences in the pattern of immunohistochemical expression of Kv1.5 channels in human atria and ventricles. Although Kv1.5 protein is found at intercalated disks in both atrium and ventricle, longitudinal staining of the cell membrane is found only in the atria.³⁴ It is possible that only channels in the longitudinal cell membrane carry transmembrane current and that although channels in the intercalated disk are involved in intercellular communication, they do not contribute to ion flux between the intracellular and extracellular spaces. Finally, we examined a limited number of cells from the free wall of the right ventricle, and it is possible that we failed to record Kv1.5-like currents in ventricular cells because of limited sampling and/or regional distribution of the channel. Further work is clearly necessary to determine the mechanisms and functional importance of the cellular localization of cardiac ion channel proteins in general and Kv1.5 in particular.

Potential Significance
Our results shed potentially important light on the K⁺ currents governing cardiac repolarization in humans. These findings are particularly relevant in the context of recent molecular studies, which point to the presence of molecular substrates for I_Kr and I_Ks in the human ventricle.^{15 16 17 18 19 20} The physiological expression of these channels has significant implications, especially in view of the evidence that one form of the congenital long QT syndrome is due to mutations that interfere with the expression of a gene that appears to code for I_Kr.^{19 20} I_Kr blockers are known to be particularly likely to cause the acquired long QT syndrome.^{14 35} Our results demonstrate the electrophysiological substrate, in terms of I_Kr expression, for these important clinical problems.

Because of the risks of proarrhythmia attending currently available class III antiarrhythmic drug therapy,³⁵ there has been interest in defining novel ionic targets for new drug development. I_Ks may contribute to rate-dependent action potential abbreviation,³⁶ and it has been suggested that selective I_Ks blockers may have a more desirable profile of rate-dependent action and safety than currently available compounds. Recent modeling work indicates an important role for I_Ks in repolarizing guinea pig ventricular myocytes.³⁷ Our observation of I_Ks in human ventricular cells is therefore potentially significant for both the understanding of mechanisms of human ventricular repolarization and the development of new antiarrhythmic drugs. The absence of I_Kur in the human ventricle is also of potential importance for new drug development. Since I_Kur plays an important role in human atrial repolarization²³ and is absent in human ventricle, it is a potentially promising target for the development of drugs that prevent reentrant atrial arrhythmias without a risk of ventricular proarrhythmia.

Potential Limitations
We studied right ventricular cells from patients with severe left ventricular failure. We cannot exclude the possibility that our results were influenced by the presence of heart disease. However, expert pathological examination at both macroscopic and microscopic levels did not reveal abnormalities in the right ventricular myocardium of our patients, and action potential characteristics of our cells are similar to previous results for normal human ventricle.^{27 28} There may also be regional differences in the quantity and properties of I_Kr and I_Ks. Since all of our studies were based on tissue from a similar zone in the free wall of the right ventricle, our results are not affected by this possibility and certainly cannot exclude it.

Because of the limited availability of normal human ventricular tissue and the exacting requirements of our isolation technique, we have been able to evaluate the presence of I_K systematically in only the three hearts presented in this article. In fact, initial observations of a similar type were made in cells from another heart and led to the present studies; however, since protocols described in the present article were not applied systematically in that heart, we have based the present article on only the results from the subsequent three hearts that were studied in the same systematic fashion.

Divalent cations are known to have effects on I_K,^{38 39} and since we used Cd²⁺ to block I_Ca, our results must be interpreted in this light. It is important to inhibit I_Ca in studying the components of I_K.⁴⁰ Although divalent cations can modify I_K, organic Ca²⁺ channel blockers are associated with well-known voltage- and use-dependent effects that can also complicate analysis. Finally, the human ventricular cells that we studied do not readily tolerate repeated prolonged (>5-s) depolarizing pulses. This limits precise kinetic calculations for processes occurring during depolarization to those with time constants of <1.5 s. Thus, the time constants for I_Kr decay during a depolarizing pulse and for the slow phase of I_Ks activation should be considered only approximations.

Conclusions
Results presented in this article indicate that I_K is detectable under appropriate conditions in most ventricular myocytes obtained from relatively normal human right ventricle and that rapid (I_Kr) and slow (I_Ks) components with properties similar to those described in other species can be detected. I_Kur, present in human atrium, appears to be absent from human ventricle. These results have important implications for our understanding of the physiological, pharmacological, and molecular control of repolarization in the human heart.

	Selected Abbreviations and Acronyms

 

cap = capacitive time constant 1, 2 = fast and slow time constants 4-AP = 4-aminopyridine APD = action potential duration ICa = Ca2+ current IK = delayed rectifier K+ current IK1 = inward rectifier current IKr = rapid component of IK IKs = slow component of IK IKstep = Ik step current IKtail = Ik tail current IKur = ultrarapid delayed rectifier current INa = Na+ current Ito = transient outward current Rs = series resistance V0.5 = 50% activation voltage

	Acknowledgments

 
This study was supported by grants from the Medical Research Council of Canada, the Quebec Heart Foundation, and the Fonds de Recherche de l'Institut de Cardiologie de Montréal. Dr Li is a research scholar of the Fonds de la Recherche en Santé du Québec. The authors thank Johanne Doucet for technical support and Carolyn Gillis for secretarial help with the manuscript.

Received July 14, 1995; accepted December 14, 1995.

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