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(Circulation. 1995;92:164-174.)
© 1995 American Heart Association, Inc.

Articles

Characterization of Inwardly Rectifying K⁺ Channel in Human Cardiac Myocytes

Alterations in Channel Behavior in Myocytes Isolated From Patients With Idiopathic Dilated Cardiomyopathy

Shin-ichi Koumi, MD, PhD; Carl L. Backer, MD; Carl E. Arentzen, MD

From the Division of Cardiology (S.-i.K.), Department of Medicine, and the Feinberg Cardiovascular Research Institute; and the Department of Surgery (C.L.B., C.E.A.), Northwestern University School of Medicine, Chicago, Ill.

Correspondence to Shin-ichi Koumi, MD, PhD, Department of Medicine, Hazaki Saiseikai Hospital, 8968 Hazaki, Kashimagun, Ibaragi 314-04, Japan.

	Abstract

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Background Little is known about the characteristics of the inwardly rectifying K⁺ channel (I_K1) and the influence of preexisting heart disease on the channel properties in the human heart.

Methods and Results We studied the characteristics of cardiac I_K1 in freshly isolated adult human atrial and ventricular myocytes by using the patch-clamp technique. Specimens were obtained from the atria and ventricles of 48 patients undergoing cardiac surgery or transplantation and from four explanted donor hearts. The action potential in ventricular myocytes exhibited a longer duration (391.4±30.2 milliseconds at 90% repolarization, n=10) than in atrium (289.4±23.0 milliseconds, n=18, P<.001) and had a fast late repolarization phase (phase 3). The final phase of repolarization in ventricle was frequency independent. Whole-cell I_K1 in ventricle exhibited greater slope conductance (84.0±7.9 nS at the reversal potential, E_K; n=27) than in atrium (9.7±1.2 nS at E_K; n=8, P<.001). The steady-state current-voltage (I-V) relation in ventricular I_K1 demonstrated inward rectification with a region of negative slope. This negative slope region was not prominent in atrial I_K1. The macroscopic currents were blocked by Ba²⁺ and Cs⁺. The channel characteristics in ventricular myocytes from patients with congestive heart failure after idiopathic dilated cardiomyopathy (DCM) exhibited distinct properties compared with those from patients with ischemic cardiomyopathy (ICM). The action potential in ventricular myocytes from patients with DCM had a longer duration (490.8±24.5 milliseconds, n=11) compared with that for ICM (420.6±29.6 milliseconds, n=11, P<.01) and had a slow repolarization phase (phase 3) with a low resting membrane potential. The whole-cell current slope conductance for DCM was smaller (41.2±9.0 nS at E_K, n=7) than that for ICM (80.7±17.0 nS, n=6, P<.05). In single-channel recordings from cell-attached patches, ventricular I_K1 channels had characteristics similar to those of atrial I_K1; channel openings occurred in long-lasting bursts with similar conductance and gating kinetics. In contrast, the percent of patches in which I_K1 channels were found was 34.7% (25 of 72) of patches in atrium and 88.6% (31 of 35) of patches in ventricle. Single I_K1 channel activity for DCM exhibited frequent long-lasting bursts separated by brief interburst intervals at every holding voltage with the open probability displaying little voltage sensitivity (0.6). Channel activity was observed in 56.2% (18 of 32) of patches for DCM and 77.4% (24 of 31) of patches for ICM. Similar results were obtained from atrial I_K1 channels for DCM. In addition, channel characteristics were not significantly different between ICM and explanted donor hearts (donors). I_K1 channels in cat and guinea pig had characteristics virtually similar to those of humans, with the exception of lower open probability than that in humans.

Conclusions These results suggest that the electrophysiological characteristics of human atrial and ventricular I_K1 channels were similar to those of other mammalian hearts, with the possible exception that the channel open probability in humans may be higher, that the whole-cell I_K1 density is higher in human ventricle than in atrium, and that I_K1 channels in patients with DCM exhibited electrophysiological properties distinct from I_K1 channels found in patients with ICM and in donors.

Key Words: potassium • myocytes • cardiomyopathy

	Introduction

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Cardiac inwardly rectifying K⁺ channel current (I_K1) has been studied by using several voltage-clamp methods.^{1 2 3} These early studies confirmed that I_K1 plays a critical role in maintenance of the resting membrane potential and the rapid repolarization process of cardiac action potentials. More recently, the use of patch-clamp techniques in single-cell preparations has allowed the direct observation of I_K1 and demonstrated the time- and voltage-dependent kinetics of macroscopic and unitary I_K1 channels in guinea pig,^{4 5} rabbit,⁶ rat,⁷ and cat⁸ ventricular myocytes. Although there are several differences in the macroscopic and single-channel I_K1 currents in the ventricular myocytes of these mammalian species, their basic characteristics are similar. These^{4 5 6 7 8} and other studies^{9 10 11} have defined the fundamental characteristics of mammalian cardiac I_K1 channels.

We demonstrated that the characteristics of the sodium current (I_Na) in isolated human atrial and ventricular myocytes are similar to those of other mammalian species and that I_Na kinetics are identical in several different disease states.^{12 13} Another study from our laboratory involving 137 isolated myocytes from 77 patients indicated that I_Na kinetics in atrium and ventricle were essentially identical.¹⁴ Calcium current (I_Ca) in human atrial and ventricular myocytes also appears to be similar to that in other species.^{15 16} In contrast, much less is known about I_K1 channel characteristics and kinetic properties in the human heart.^{17 18} In the present study, we addressed several fundamental questions concerning the human cardiac I_K1 channel: Does the human ventricular I_K1 channel display characteristics similar to those of other mammalian species? Do different preexisting heart diseases modify I_K1 channel properties? If so, how do the disease-modified channels behave? To answer these questions, we characterized the action potential, whole-cell, and unitary currents through I_K1 channels in freshly isolated adult human atrial and ventricular myocytes; compared the behaviors of I_K1 channels in different preexisting heart diseases; and compared I_K1 channel characteristics in human ventricle with those in cat and guinea pig ventricle.

	Methods

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Human Cardiac Specimens
Atrial and ventricular specimens were obtained from 14 transplanted adult human hearts (4 donors and 10 recipients) and from 38 additional patients (mean age, 49±17 years) undergoing cardiac surgery. The National Institutes of Health guidelines for experimentation using human tissues and the institutional guidelines for human subject research were followed in obtaining surgical specimens. All patients gave written, informed consent before the operation. Table 1 describes the characteristics of the patient population. No cardioactive drugs were given during the 48 hours before surgery. All heart transplant recipients had terminal heart failure (New York Heart Association functional class IV) after idiopathic dilated cardiomyopathy ([DCM] mean age, 48±14 years) or ischemic cardiomyopathy ([ICM] mean age, 55±12 years) due to coronary artery disease; cardiac index was 2.0±0.5 L · min^-1 · m^-2 (n=5) for DCM and 2.1±0.4 L · min^-1 · m^-2 (n=5) for ICM, and ejection fraction was 20±6% (n=5) for DCM and 22±6% (n=5) for ICM. These values were not statistically different (P=NS) between DCM and ICM. Three patients with valvular heart disease (VHD) and four patients with ischemic heart disease (IHD) displayed no significant ventricular dysfunction (nonfailing); cardiac index was 3.8±0.4 L · min^-1 · m^-2 (n=7), and ejection fraction was 64±5% (n=7).

View this table: [in this window] [in a new window]   Table 1. Patient Population Characteristics

Immediately after specimens were removed from the patients, they were placed in a chilled solution and transported to the laboratory; the cell isolation procedure was begun within 1 hour.

Cell Preparation
Human atrial myocytes were isolated by an enzymatic dissociation method identical to that we described previously.¹² Briefly, specimens were minced using a fine razor and washed three times, 7 minutes each time, in oxygenated Ca²⁺-free Tyrode's solution. The minced tissue was then incubated in oxygenated Ca²⁺-free Tyrode's solution containing 300 to 350 U/mL collagenase (Type V, Sigma Chemical Co), 0.5 U/mL protease (Type XXIV, Sigma), and 1 mg/mL of bovine serum albumin (Sigma) at 37°C and then gently stirred with a magnetic stirring bar to release isolated myocytes from the minced tissue (40 minutes). The minced tissue was then strained through a 200-µm nylon mesh to remove undigested tissue and to harvest the individual myocytes. Myocytes were stored at room temperature in a modified KB medium.¹⁹ The residual nondigested tissue was then reincubated in enzyme-containing solution for an additional 10 minutes, and isolated myocytes were again harvested in a similar manner. This process was repeated until viable myocytes could no longer be obtained.

Human ventricular myocytes were isolated by an enzymatic dissociation method. The isolation was accomplished with a Langendorff-type apparatus for coronary artery perfusion. If the available coronary artery was not suitable for cannulation, the tissue was perfused with a method we have described previously in which a hypodermic needle is used to infiltrate the tissues.²⁰ Ventricular specimens were trimmed to 1 cm³ using fine scissors and then infiltrated with Ca²⁺-free Tyrode's solution with a 25-gauge surgical needle. Perfusion with 20 mL Ca²⁺-free Tyrode's solution (1 mL/min) was followed by perfusion with Ca²⁺-free Tyrode's solution containing 125 U/mL collagenase (Sigma, Type V) and 1 mg/mL bovine serum albumin (Sigma) for 20 to 30 minutes (1 mL/min). The perfusate was bubbled with 100% O₂ and warmed to 37°C. The specimen was then minced with fine scissors in the same enzyme solution. When ventricular specimens were very small, the isolation method similar to that for atrium was used. Isolated cells were separated from the minced tissue by gravity filtration through 200-µm nylon mesh and were stored in a modified KB solution at room temperature. Only Ca²⁺-tolerant, clearly striated, rod-shaped cells without any blebs were studied. I_K1 channel characteristics did not differ regardless of whether the coronary artery perfusion or the needle infiltration procedure was used.

Cat ventricular myocytes were isolated using a previously described modification of the method of Silver et al.²¹ Adult cats of either sex were anesthetized with pentobarbitone (24 mg/kg IV). The heart was excised, and the coronary arteries were retrogradely perfused after cannulation of the aorta. After 2 to 3 minutes of perfusion with a Ca²⁺-free Krebs-Henseleit buffer solution (KHB), the heart was perfused with KHB containing 0.15% collagenase (Type II, Worthington). After 30 to 40 minutes, perfusion was stopped. Ventricular tissue was then minced and incubated in a shaker bath for 5 to 10 minutes in collagenase-containing solution. The remaining tissue pieces were removed by filtering, and cells were washed free of collagenase and stored in KHB containing 1% albumin and 1 mmol/L Ca²⁺.

Guinea pig ventricular myocytes were isolated by an enzymatic dissociation method similar to that described previously by Mitra and Morad.²² Guinea pigs of either sex weighing 150 to 200 g were anesthetized with sodium pentobarbital (60 mg IP), and the heart was quickly excised and placed in a petri dish filled with normal Tyrode's solution. The aorta was then cannulated, and the heart was mounted in a Langendorff-type apparatus. Retrograde aortic perfusion of the coronary bed was initiated with Ca²⁺-free Tyrode's solution for 5 minutes under a hydrostatic pressure of approximately 100 cm H₂O. The heart was then perfused with Ca²⁺-free Tyrode's solution containing 1.5 mg/mL collagenase (Type I, Sigma), 1 mg/mL bovine serum albumin (Sigma), and 1 mg/mL protease (Type XIV, Sigma) for 3 to 4 minutes, after which the collagenase was washed out by perfusion with KB solution (50 mL). All perfusates were bubbled with 100% O₂ and warmed to 37°C. After the collagenase had been washed out, the heart was gently agitated in KB solution. Next, the heart was minced with fine scissors, and isolated cells were harvested through a nylon mesh (200 µm).

Solutions
The transport solution for human specimens contained (in mmol/L): NaCl 27, KCl 20, MgCl₂ 1.5, HEPES 5, and glucose 274 (pH 7.0). The control Tyrode's solution contained (in mmol/L): NaCl 140, KCl 5.4, CaCl₂ 1.8, MgCl₂ 0.5, HEPES 5.0, and glucose 5, with pH adjusted to 7.4 with NaOH. Ca²⁺-free Tyrode's solution was made by omitting CaCl₂ from the normal Tyrode's solution. Cl^--free external solution contained (in mmol/L): sodium glutamate 140, potassium glutamate 5.4, CaSO₄ 1.8, MgSO₄ 0.5, HEPES 5.0, and glucose 5 (pH 7.4 with NaOH). The modified KB solution contained (in mmol/L): KCl 25, KH₂PO₄ 10, KOH 116, glutamic acid 80, taurine 10, oxalic acid 14, HEPES 10, and glucose 11 (pH 7.0 with KOH). Ca²⁺-free KHB solution contained (in mmol/L): NaCl 130, KCl 4.8, MgSO₄ 1.2, NaH₂PO₄ 1.2, NaHCO₃ 25.0, and glucose 12.5 (pH 7.4). The internal solution used for whole-cell recording contained (in mmol/L): potassium aspartate 120, KCl 20, KH₂PO₄ 1.0, MgCl₂ 1.0, Na²⁺-ATP 5.0, EGTA 5.0, and HEPES 5.0 (pH 7.2 with KOH). The Cl^--free pipette solution contained (in mmol/L): potassium glutamate 140, KH₂PO₄ 1.0, MgSO₄ 1.0, Na²⁺-ATP 5.0, EGTA 5.0, and HEPES 5.0 (pH 7.2 with KOH). The pipette solution used for single-channel recording contained (in mmol/L): KCl 150 and HEPES 5.0 (pH 7.4 with KOH). Normal Tyrode's solution was used as external solution for both whole-cell and single-channel recording.

Electrical Recordings and Data Analysis
Whole-cell and single-channel currents were recorded using the patch-clamp technique,²³ with an amplifier and head stage designed by M. Yoshii.²⁴ The feedback resistance of the head stage was 100 M (for recording whole-cell currents) and 10 G (for recording single-channel currents). Electrodes fabricated from 1.0-mm-OD glass capillary tubes (Kimax-51, Kimble Products) using a programmable horizontal micropipette puller (model P-87, Flaming/Brown, Sutter Instrument Co) had tip resistances of 1.5 to 2.5 M (for recording whole-cell currents) and 10.0 M (for recording single-channel currents). Seal resistances were 10 to 100 G (for recording single-channel currents).

For whole-cell recording, the series resistance attributed to the pipette tip and the cell interior was compensated by summing a fraction of the converted current signal to the command potential and feeding it to the positive input of the operational amplifier. Series resistance was compensated to minimize the time course of the capacitative surge; the capacitative transient remaining after series resistance compensation was constant throughout the experiments. The cell capacitance (C_m) was calculated from the following equation: C_m=Q/V, where Q is total charge movement determined by integrating the area defined by the capacitative transient in response to +10-mV voltage step (holding potential of -40 mV). The mean cell capacitance was 72.6±12.5 pF (n=26) in human atrial myocytes and 114.8±17.9 pF (n=30) in human ventricular myocytes. The output of the voltage-clamp amplifier was adjusted to give zero current when the tip of the patch pipette (filled with internal solution) was immersed in the bath containing Tyrode's solution. This caused a voltage bias of 7±2 mV positive to what the voltage would have been had the electrode been zeroed in a grounded puddle of internal solution. This bias was not corrected. Voltage-clamp steps of 300-millisecond duration ranging between -120 and +40 mV were applied from a holding potential of -40 mV. The whole-cell membrane currents were filtered at 10 kHz with a two-pole active filter, digitized at a sampling rate of 40 kHz, and stored on the Winchester drive of an LSI 11/73 computer (Digital Equipment Corp) for subsequent analysis. Action potentials were measured by the whole-cell current-clamp mode. Current-voltage (I-V) relations were obtained using ramp voltage-clamp pulses applied from a holding potential of -120 to +40 mV at a rate of 100 mV/s. Current obtained within the first 5 mV after the onset of the ramp was not analyzed because of the contamination of the capacitative transient of the membrane.

Single-channel currents were monitored with a digital oscilloscope (7101A, Kikusui), collected with an AD converter, and stored continuously on videotape using a PCM converter recording system (Unitrade). The recorded signals were reproduced and filtered off-line with a cutoff frequency of 2 to 5 kHz through an eight-pole low-pass Bessel filter (48 dB per octave; model 902-LPF, Frequency Devices, Inc), digitized with 14-bit resolution at a sample rate of 10 kHz, and stored on an LSI 11/73 computer. The data were analyzed using algorithms developed in-house that are based on the half-amplitude threshold analysis method of Colquhoun and Sigworth.²⁵ Channel transitions were calculated using an averaging technique for determining channel amplitude. The measurements derived from the channel transitions were collected into histograms to allow an analysis of the single-channel kinetics. Dwell times were determined from the sum of exponential fits to the distributions of open and closed times recorded from patches with only one channel. Dwell time histograms were constructed from experiments using 150 mmol/L K⁺ internal and external solutions. The external solution was maintained at 37°C using a Peltier thermoelectric device during action potential measurements. All other experiments were performed at room temperature (20° to 22°C).

Statistical Analysis
Results are expressed as mean±SD. Statistical analyses were performed using Student's t test or one-way ANOVA only when the data were suited for parametric tests as judged by normality and equal variance tests. When the data were not suitable for parametric tests, a Mann-Whitney rank sum test (Wilcoxon rank sum test) or a Kruskal-Wallis ANOVA on ranks were used. To consider the interpatient and intrapatient variabilities, each data comparison was also evaluated using a two-way ANOVA. In addition, an ANCOVA was used to determine the influence of age and sex in each comparison, unless otherwise stated. A nonparametric procedure in STATISTICAL ANALYSIS SYSTEM (SAS Institute Inc) on an NeXT computer (NeXT Computer, Inc) was used for these analyses. Results were considered to be significant when P<.05.

	Results

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Action Potentials of Human Atrial and Ventricular Myocytes
Fig 1 shows examples of action potentials in human atrial and ventricular myocytes. The atrial myocyte was isolated from an explanted donor heart (donor), and the ventricular myocyte was isolated from a patient with VHD without ventricular dysfunction. The shape of the action potentials in human cardiac myocytes was similar to those reported previously for other mammalian atrial and ventricular myocytes.^{26 27} Table 2 summarizes the action potential parameters in atrial and ventricular myocytes. The height and duration of the action potential in ventricle were significantly greater than those in atrium. In addition, the action potential in ventricle had a faster late repolarization phase (phase 3) than that in atrium, suggesting that the background I_K1 plays a significant role in ventricle.

View larger version (10K): [in this window] [in a new window]   Figure 1. Tracings of action potentials in human atrial and ventricular myocytes. A, Representative action potential recorded from a human atrial myocyte isolated from donor using the current-clamp mode at 37°C. Action potential duration at 90% repolarization was 268.7 milliseconds. Height of the action potential was 85.7 mV. B, Action potential recorded from a human ventricular myocyte isolated from a patient with valvular heart disease without heart failure under the same conditions as for A. Action potential duration at 90% repolarization was 391.2 milliseconds. Height of the action potential was 101.4 mV.

View this table: [in this window] [in a new window]   Table 2. Action Potential Parameters in Myocytes Isolated From Atrium and Ventricle

Fig 2 shows the effects of cycle length on action potential characteristics in atrial and ventricular myocytes. The relations among preceding cycle length, action potential duration, and the final phase of repolarization were investigated. Action potential duration of both atrial (Fig 2A) and ventricular (Fig 2B) myocytes shortened as cycle length was reduced. Action potential duration measured at 90% repolarization was 289.4±23.0 milliseconds (n=18) at the cycle length of 1000 milliseconds and 214.5±20.7 milliseconds (n=11) at the cycle length of 500 milliseconds (P<.001) in atrial myocytes, and 391.4±30.2 milliseconds (n=10) at the cycle length of 1000 milliseconds and 289.3±27.0 milliseconds (n=8) at the cycle length of 500 milliseconds (P<.001) in ventricular myocytes. In contrast, the final phase of repolarization was frequency independent in both cell types. These results suggest that the final repolarization phase of the action potential appears to be dependent on I_K1 in both atrial and ventricular myocytes.

View larger version (14K): [in this window] [in a new window]   Figure 2. Tracings of interrelationships among cycle length (CL), action potential duration, and characteristics of the final phase of repolarization in human atrial and ventricular myocytes. A, Effects of changing the stimulation rate on action potential characteristics in an isolated atrial myocyte. Myocyte was stimulated at selected CL as shown. B, Effects of changing the stimulation rate on action potential characteristics in an isolated ventricular myocyte. Same pulse protocol as A. Time and voltage calibrations are shown on right.

Characteristics of Whole-Cell I_K1 in Human Atrial and Ventricular Myocytes
Whole-cell atrial and ventricular I_K1 currents were measured with 300-millisecond hyperpolarizing and depolarizing voltage steps from a holding potential of -40 mV. Fig 3 illustrates the characteristics of whole-cell I_K1 currents in human atrial myocytes isolated from a donor and in ventricular myocytes isolated from a patient VHD without ventricular dysfunction. Currents were recorded in the presence of 10 µmol/L tetrodotoxin (TTX) and 5 µmol/L nifedipine in the external solution to block I_Na and I_Ca, respectively. In addition, to eliminate the contamination of the Cl^-current (I_Cl)²⁸ and the fast delayed rectifier current of the 1.5-kV type^{29 30} in atrium, Cl^--free external solution containing 4-aminopyridine ([4-AP] 2 mmol/L) and Cl^--free internal solution were used during recording of atrial I_K1. Atrial I_K1 exhibited small currents that developed in response to hyperpolarization and depolarization (Fig 3A). The average I-V relation was measured at the end of each test pulse (steady state), and data were plotted after normalizing to membrane capacitance (n=10, Fig 3B). Although the I-V relation exhibited weak inward rectification, a negative slope region was not prominent in atrial I_K1. In contrast, ventricular I_K1 exhibited large inward currents at test voltages negative to -80 mV (Fig 3C). The average slope conductance in ventricle was significantly greater (84.0±7.9 nS at the reversal potential, E_K; n=27) than that in atrium (9.7±1.2 nS at E_K; n=8, P<.001). Outward currents were observed at test voltages positive to -70 mV. The averaged I-V plot exhibited inward rectification and displayed a clearly evident negative slope region at potentials between -40 and +20 mV (n=10, Fig 3D). These results suggest that I_K1 in ventricle would provide a greater contribution to the action potential than that in atrium. These differences in whole-cell current properties are consistent with those obtained in action potential measurements; the ventricular action potential exhibited faster late repolarization phase.

View larger version (24K): [in this window] [in a new window]   Figure 3. Whole-cell IK1 in human atrial and ventricular myocytes. A, Representative whole-cell IK1 current traces in isolated atrial myocytes from donor hearts recorded in normal Tyrode's solution. Test pulses (TP) were applied for 300 milliseconds from a holding potential of -40 mV to potentials ranging from -120 mV to +40 mV in 10-mV steps. Recordings were obtained 10 minutes after obtaining intracellular access. Tetrodotoxin (10 µmol/L), nifedipine (5 µmol/L), and 4-aminopyridine (2 mmol/L) were present in the Cl--free external solution, which was used for the pipette solution. B, Plot of average steady-state current-voltage (I-V) relation measured at the end of test pulse and expressed as mean±SD (n=8) after normalization to membrane capacitance. C, Representative whole-cell IK1 current traces in isolated ventricular myocytes from patients with valvular heart disease without ventricular dysfunction, recorded in normal Tyrode's solution. Tetrodotoxin (10 µmol/L) and nifedipine (5 µmol/L) were present in the external solution. D, Plot of average steady-state I-V relation (n=14). Current normalized to membrane capacitance was larger than for atrium at every voltage, and the I-V relation revealed a negative slope region between -40 and +10 mV.

Ba²⁺ and Cs⁺ blockades of I_K1 in atrial and ventricular myocytes were also studied. Fig 4A shows the whole-cell I-V relations obtained during reperfusion with Ba²⁺ (1 mmol/L) and Cs⁺ (10 mmol/L) in atrial myocytes. Voltage-clamp ramps were applied from a holding potential of -120 to +40 mV at a rate of 100 mV/s. TTX (10 µmol/L), nifedipine (5 µmol/L), and 4-AP (2 mmol/L) were added to the Cl^--free external solution. Application of Ba²⁺ (1 mmol/L, top) and Cs⁺ (10 mmol/L, bottom) reverse inhibited atrial I_K1. The insets show examples of whole-cell current families recorded during superfusion with Ba²⁺ (top) and Cs⁺ (bottom). Similar results were obtained in six of six cells. Fig 4B illustrates the effects of Ba²⁺ (1 mmol/L) and Cs⁺ (10 mmol/L) on I_K1 in ventricular myocytes. Similar to atrial I_K1, Ba²⁺ (1 mmol/L, top) and Cs⁺ (10 mmol/L, bottom) reverse inhibited ventricular I_K1.

View larger version (29K): [in this window] [in a new window]   Figure 4. Effects of Ba2+ and Cs+ on human atrial and ventricular IK1. A, Effect of Ba2+ (1 mmol/L, top) and Cs+ (10 mmol/L, bottom) on the current-voltage (I-V) relations measured by ramp voltage-clamp in isolated atrial myocytes. Cl--free internal and external solutions were used. Tetrodotoxin (10 µmol/L), nifedipine (5 µmol/L), and 4-aminopyridine (2 mmol/L) were included in the external solution. I-V relations for the membrane currents were measured during the control period (circle), exposure to Ba2+ (top, square), and Cs+ (bottom, square) and after washout (triangle). Family of whole-cell currents obtained during superfusion with Ba2+ and Cs+ is shown in inset of each panel. Test pulses (300-millisecond duration) were delivered to potentials ranging from -120 to +40 mV in 10-mV steps from a holding potential of -40 mV. B, Effect of Ba2+ (1 mmol/L, top) and Cs+ (10 mmol/L, bottom) on I-V relations measured by ramp voltage-clamp in isolated ventricular myocytes. Same protocol as in A.

Alterations in I_K1 Channel Characteristics in Myocytes From Patients With IDC
To address the question of whether preexisting heart disease can affect I_K1 channel properties, we characterized the I_K1 channel in myocytes isolated from patients with congestive heart failure after DCM and compared their characteristics with those in myocytes from patients with congestive heart failure after ICM.

Fig 5 shows action potentials and whole-cell currents in ventricular myocytes isolated from patients with ICM or DCM. The resting membrane potential was depolarized for DCM (-66.9±4.0 mV, n=12) compared with that for ICM (-72.1±4.7 mV, n=12, P<.05). The resting membrane potential for ICM was also more depolarized than that for VHD (-74.0±4.2 mV, n=14), but they did not differ statistically (P=NS). Action potential duration at 90% repolarization was 420.6±29.6 milliseconds (n=11) for ICM and 490.8±24.5 milliseconds (n=11) for DCM. Although the action potential duration for ICM appeared to be prolonged compared with that for VHD without ventricular dysfunction (391.4±30.2, n=9), the value did not achieve statistical significance (P=NS). In contrast, the action potential duration for DCM was significantly longer than that for ICM and VHD (P<.01). The late repolarization phase (phase 3) for DCM was slower than that for ICM (Fig 5A). Figure 5B shows examples of whole-cell I_K1 in ventricular myocytes isolated from patients with ICM or DCM recorded under conditions similar to those of Fig 3. The whole-cell current slope conductance at E_K for DCM was smaller (41.2±9.0 nS, n=7) than that for ICM (80.7±17.0 nS, n=6, P<.05). In contrast, the whole-cell conductance did not differ significantly between ICM and VHD (P=NS, not shown).

View larger version (19K): [in this window] [in a new window]   Figure 5. Characteristics of the action potential and whole-cell IK1 in ventricular myocytes isolated from patients with ischemic cardiomyopathy (ICM) and idiopathic dilated cardiomyopathy (DCM). A, Representative action potentials recorded in ventricular myocytes from patients with ICM and DCM using the whole-cell current-clamp mode at 37°C. Action potential duration for ICM was slightly prolonged compared with that for valvular heart disease without ventricular dysfunction (see Fig 1B). Action potential duration for DCM was prolonged and the late repolarization was slowed compared with ICM. B, Representative whole-cell current recorded in ventricular myocytes from patients with ICM and DCM. Test pulse was applied for 300 milliseconds from a holding potential of -40 mV to the test potential of -120 mV. Current magnitude for DCM was smaller than that for ICM. C, Averaged current-voltage (I-V) relations in ventricular myocytes frompatients with ICM (circles, n=6) and DCM (squares, n=7).

Characteristics of Single I_K1 Channel in Human Atrial and Ventricular Myocytes
To gain further insights into the differences of the action potential and the whole-cell I_K1 between atrium and ventricle, we studied the characteristics of single I_K1 channels in cell-attached patches. Fig 6 gives examples of individual channel activities in atrial (Fig 6A) and ventricular (Fig 6B) myocytes. Each patch contained only one channel. Channel openings occurred in long-lasting bursts separated by variable interburst intervals at all voltages. By applying more negative holding potentials, we increased unitary amplitude, whereas the duration of individual open events decreased, causing an increase in the fluctuation rate between the open and closed states. Current traces became flat at around resting membrane potential of +80 mV, which was the estimated E_K (see "Discussion"). The I-V relations of atrial and ventricular I_K1 were almost linear in the voltage range between resting membrane potential of -40 mV and +40 mV. No outward currents could be detected at potentials positive to E_K through either atrial and ventricular I_K1 channels, indicating inward rectification (Fig 6C). The slope conductances of the channels were 27±2 pS (n=20) for atrial I_K1 channels and 28±2 pS (n=15) for ventricular I_K1 channels (P=NS).

View larger version (24K): [in this window] [in a new window]   Figure 6. Conductance characteristics of single IK1 channels in human atrial and ventricular myocytes in cell-attached patches. A, Cell-attached patch recordings showing IK1 channel activity at different holding potentials in an isolated atrial myocyte from donor heart. Holding potential is expressed as the voltage deviation from the resting membrane potential and is indicated to the left of each current trace. Current was low pass filtered at 2 kHz. Dotted line indicates the baseline level (zero current, closed channel). B, Example of IK1 channel activity in an isolated ventricular myocyte from patients with valvular heart disease without ventricular dysfunction recorded under the same conditions as in A. No outward currents were detected from either atrial or ventricular IK1 channels. C, Current-voltage (I-V) relations obtained from the traces in A and B. Slope conductance was 28 pS for the atrial IK1 (squares) and 29 pS for the ventricular IK1 (circles) channel. Strong inward rectification was displayed in the voltage range positive to resting membrane potential of +60 mV in both atrial and ventricular IK1 channels.

Fig 7 illustrates open time histograms from atrial and ventricular I_K1 channels. The distributions of open times in atrial and ventricular I_K1 channels were well described by a single exponential function. The mean open lifetime was 30.8±4.2 milliseconds (n=20) for atrial I_K1 and 31.6±4.5 milliseconds (n=15) for ventricular I_K1 (P=NS). There results indicate that channel characteristics of I_K1 are similar for atrial and ventricle. In contrast, the percent of patches in which I_K1 channels were found (incidence) was 34.7% (25 of 72) of patches in atrium and 88.6% (31 of 35) of patches in ventricle.

View larger version (17K): [in this window] [in a new window]   Figure 7. Dwell time histograms in human atrial and ventricular IK1 channels. A, Histogram of open times in cell-attached patch recordings at holding potential of -60 mV in a human atrial myocyte isolated from donor heart filtered at 2 kHz. Measurements were made from a patch containing only one channel. Lifetimes of openings were distributed according to a single exponential function with a time constant (o) of 27.9 milliseconds. B, Histogram of open times in a human ventricular myocyte isolated from patients with valvular heart disease without ventricular dysfunction determined under the same recording conditions as that for the atrial myocyte in A. As with the atrial IK1 channel, the open lifetimes were distributed according to a single exponential function with o of 28.6 milliseconds.

To compare characteristics in more detail, we studied I_K1 channels in myocytes isolated from the atrium and ventricle of the same patient. This approach allowed us to compare channel characteristics directly without involvement of possible additional complicating factors such as age, sex, and disease state. A total of 18 atrial myocytes and 23 ventricular myocytes were studied from pairs of atrial and ventricular tissue from four patients with IHD and two patients with VHD. Table 3 summarizes the results; I_K1 channel characteristics did not differ between atrium and ventricle. These results indicate that the slope conductances and gating parameters of atrial and ventricular I_K1 were not significantly different and that the difference in whole-cell I_K1 conductance may be caused by a difference in the number of functional channels between atrium and ventricle.

View this table: [in this window] [in a new window]   Table 3. Electrophysiological Characteristics of Single Ik1 Channels From Human Atrium and Ventricle

Fig 8 describes the effects of specific blockers for I_K1 on human atrial and ventricular I_K1 channel activity. Ba²⁺ (20 µmol/L) in the pipette solution shortened the individual bursts with a concomitant prolongation of the interburst interval, resulting in a decrease of channel open probability (P_o) at every holding voltage tested in both atrial and ventricular myocytes. As summarized in Table 4, P_o was reduced by Ba²⁺ in atrial and ventricular I_K1. Unitary amplitude was unchanged by Ba²⁺. Addition of Cs⁺ (50 µmol/L) to the pipette solution caused flickering to occur during individual bursts of open events at different voltages in atrial and ventricular myocytes. However, the frequency of bursting was not affected at this concentration, nor was unitary amplitude altered by Cs⁺. Although P_o of both atrial and ventricular I_K1 decreased with Cs⁺ (50 µmol/L), it was not of statistical significance. These results indicate that both human atrial and ventricular I_K1 channels are sensitive to these blockers, which is similar to many other mammalian species.^{5 6}

View larger version (28K): [in this window] [in a new window]   Figure 8. Tracings of effects of Ba2+ and Cs+ on human atrial and ventricular IK1 channel. A, Effect of 20 µmol/L Ba2+ or 50 µmol/L Cs+ in the pipette in atrial IK1 channel. Single IK1 channel current traces were recorded with 20 µmol/L BaCl2 or 50 µmol/L CsCl in the pipette in cell-attached patch configuration between holding potential equals resting membrane potential and resting membrane potential of -40 mV as indicated to the left of each current trace. In Ba2+ experiments, currents were low pass filtered at 2 kHz. Individual burst length shortened with prolongation of the interburst interval. Probability of open events decreased at all test voltages. In Cs+ experiments, currents were low pass filtered at 5 kHz. B, Effect of 20 µmol/L Ba2+ or 50 µmol/L Cs+ in the pipette in ventricular IK1 channel. Experiments were performed under the same conditions as A. Ventricular IK1 channel activity was blocked by Ba2+ and Cs+ in a similar manner to the atrial IK1 channel.

View this table: [in this window] [in a new window]   Table 4. Inhibition of Human Atrial and Ventricular IK1 by Ba2+ or Cs+

Characteristics of Single I_K1 Channel in Myocytes Isolated From Heart of Patient With DCM
To assess the alteration in I_K1 channel characteristics in myocytes isolated from DCM, we examined single I_K1 channel behavior for DCM. Fig 9 illustrates a typical example of single I_K1 channel activity in ventricular myocytes from patients with ICM or DCM. Channel activity was observed in 77.4% (24 of 31) of patches for ICM and in 56.2% (18 of 32) of patches for DCM. The single-channel activity exhibited bursting behavior in which long-lasting bursts were separated by brief interburst closed periods. Individual bursts were maintained for long periods with many brief closing events occurring at holding voltages between resting membrane potential of -60 mV and of +40 mV (Fig 9A). This type of bursting behavior was seen in every patch from ventricular myocytes obtained from patients with DCM (18 of 18 cells). We never observed such bursting behavior in myocytes from patients with ICM and the other disease states. The I-V relation exhibited a slope conductance of 27±3 pS (n=18), which was not significantly different from that for ICM (28±2 pS, n=8). The open time distributions were best described by a single exponential function with a mean open lifetime of 31.9±3.5 milliseconds (n=14). Slope conductances and mean open times did not differ between myocytes from patients with DCM or ICM. In contrast, P_o was different for the two groups. Table 5 summarizes these results.

View larger version (25K): [in this window] [in a new window]   Figure 9. Characteristics of single IK1 channels in ventricular myocytes isolated from patients with ischemic cardiomyopathy (ICM) and idiopathic dilated cardiomyopathy (DCM). A, Unitary currents from IK1 channels in ventricular myocytes isolated from patients with ICM or DCM recorded from cell-attached patches. Holding potential is expressed as the voltage deviation from the resting membrane potential and is indicated to the left of each current trace. No outward currents were detected positive to resting membrane potential. Currents were low pass filtered at 2 kHz. B, Current-voltage relations of IK1 channels derived from A. Slope conductance was 29 pS for both ICM and DCM. C, Histograms of open times of IK1 channels in ventricular myocytes isolated from patients with ICM or DCM recorded at holding potential of -60 mV, filtered at 2 kHz. Measurements were made from a patch containing only one channel. Lifetimes of openings were distributed according to a single exponential function with mean open lifetime of 31.4 milliseconds for ICM and 29.9 milliseconds for DCM.

View this table: [in this window] [in a new window]   Table 5. Comparison of Conductance and Kinetic Parameters in Human Ventricular Myocytes Between Ischemic Cardiomyopathy and Dilated Cardiomyopathy

The voltage dependence of P_o in I_K1 channels of ventricular myocytes from patients with DCM was also different from that from patients with ICM. Because no significant differences were found in P_o between ICM and other disease states, they were grouped as non-DCM. Fig 10 depicts the voltage dependence of P_o for DCM and non-DCM. I_K1 channels for DCM exhibited decreased sensitivity to the holding voltage compared with that of non-DCM. Their values were not significantly different at resting membrane potential of +20 mV. However, the values were different at the holding voltages negative to resting membrane potential. The non-DCM groups exhibited a voltage-dependent decrease in P_o with membrane hyperpolarization, whereas the DCM displayed little sensitivity of P_o to holding voltage. The values of P_o for DCM were significantly different from those for non-DCM at holding potentials negative to resting membrane potential.

View larger version (15K): [in this window] [in a new window]   Figure 10. Plot of voltage dependence of open probabilities (PO) in ventricular IK1 channel from patients with idiopathic dilated cardiomyopathy (DCM) or other different disease states (patients without DCM). Ventricular IK1 channel PO values from patients with DCM (squares) and from patients without DCM (circles) were plotted as a function of holding voltages from cell-attached patch recordings. Po in patients with DCM was less sensitive to holding voltage than was that from patients without DCM. Vertical bars through each point represents SD. Values were not significantly different from each other at holding potential equals resting membrane potential of +20 mV. They were statistically different in the voltage range negative to resting membrane potential. *P<.05, **P<.01, ***P<.001, n=14 in DCM group, n=26 in non-DCM group.

We made similar comparisons between atrial I_K1 channels. The results were similar to those observed for ventricle (Table 6). Again, the channel characteristics for DCM were different from those for ICM and donors, and the channel characteristics for ICM did not differ from those for donors.

View this table: [in this window] [in a new window]   Table 6. Comparison of Conductance and Kinetic Parameters in Human Atrial Myocytes Between Ischemic Cardiomyopathy, Dilated Cardiomyopathy, and Donors

Comparison of Human Ventricular I_K1 Channels With Cat and Guinea Pig
We also compared human ventricular I_K1 single-channel characteristics with those of cat and guinea pig. Fig 11 shows examples of conductance in a single I_K1 channel recorded in isolated cat and guinea pig ventricular myocytes. Fig 11A shows the original traces and the I-V relation in cat ventricle. Open bursts separated by long closed interburst periods were typical. Outward currents were not observed during strong depolarizations. The I-V relation was linear in the voltage range between -40 and +40 mV. The mean value of the slope conductance was 30±3 pS (n=8).

View larger version (26K): [in this window] [in a new window]   Figure 11. Single IK1 channel characteristics in isolated cat and guinea pig ventricular myocytes. A (Top), Cell-attached patch recordings of IK1 channels in freshly isolated cat ventricular myocytes with 150 mmol/L K+ in the pipette and normal Tyrode's solution in the bath. Holding potential was expressed as the voltage deviation from the resting membrane potential and was indicated to the left of each current trace. No outward current was detected indicating inward rectification. Currents were low pass filtered at 2 kHz. Bottom, Current-voltage (I-V) relation plotted from the currents in top panel was linear between resting membrane potential of -40 mV and resting membrane potential of +40 mV with slope conductance of 30 pS. B (Top), Cell-attached patch recordings of IK1 channels in freshly isolated guinea pig ventricular myocytes with 150 mmol/L K+ in the pipette and normal Tyrode's solution in the bath. No outward current was detected. Bottom, I-V relation obtained from the traces in top panel was linear between holding potential equals resting membrane potential of -40 mV and resting membrane potential of +40 mV with a slope conductance of 34 pS.

Fig 11B similarly describes the I_K1 channel characteristics in an isolated guinea pig ventricular myocyte. Individual bursts, separated by long periods with the channel in the closed state, were again observed. The probability of open events decreased with membrane hyperpolarization, and outward currents were not seen during strong depolarizations. The I-V relation was linear in the voltage range between -40 and +40 mV with a slope conductance of 32±3 pS (n=14). Table 7 summarizes the species comparison. Because I_K1 channels from patients with DCM did not behave like those from patients with other disease states, we excluded the data from patients with DCM in this comparison. Although there were some minor differences in each parameter, the overall quantitative characteristics of human I_K1 (non-DCM) were similar to those for both cat and guinea pig. The different isolation method did not affect the channel characteristics (n=5).

View this table: [in this window] [in a new window]   Table 7. Comparison of Human Ventricular Ik1 Channel Characteristics With Those of Cat and Guinea Pig

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The major findings in the present study are as follows. First, the action potential in human ventricular myocytes exhibited a longer duration and a faster late repolarization phase compared with that in atrium. Second, although whole-cell I_K1 in human atrial and ventricular myocytes exhibited inward rectification, only ventricular I_K1 had a prominent negative slope region as determined from the I-V relation and the slope conductance was greater in ventricle than in atrium. Third, ventricular myocytes isolated from patients with DCM exhibited low resting membrane potential, prolonged action potential duration, and a slow late repolarization phase compared with those of patients with ICM. Whole-cell current slope conductance was smaller for DCM than for ICM. Fourth, single I_K1 channels in ventricular myocytes had conductance properties and kinetics similar to those in atrium. However, the percentage of patches in which I_K1 channels were found was greater in ventricle than in atrium, suggesting that smaller whole-cell current magnitude in atrium may be caused by low functional channel density in atrium. Fifth, single-channel P_o for DCM did not show voltage dependence. Sixth, I_K1 channels in human atrial and ventricular myocytes had characteristics that were similar to those in cat and guinea pig heart. However, at potentials negative to resting membrane potential, the ventricular I_K1 channel P_o in human heart was greater than in cat and guinea pig myocytes.

Characteristics of I_K1 in Human Atrium and Ventricle
The action potential in human atrial and ventricular myocytes showed characteristics generally similar to those observed in other mammalian species.^{26 27} A difference in action potential duration in atrium and ventricle was shown in guinea pig²⁶ and rabbit.²⁷ The action potential characteristics in the present study were also similar to those previously reported for human atrium^{31 32} and ventricle.¹⁸ A different density of whole-cell I_K1 current in atrium and ventricle was shown in guinea pig and rabbit.^{26 27} The I-V relation exhibited a negative slope region in the voltage range between -40 to +20 mV in human ventricular I_K1. In contrast, human atrial I_K1 exhibited much lower slope conductance without a prominent negative slope region in the I-V curve.

Single-channel study supported the results of whole-cell measurements. Single I_K1 channels were identified based on the following observations. First, E_K satisfied the theoretical value predicted by the Nernst equation. In cell-attached patches with pipette (150 mmol/L K⁺) and bath solution (5.4 mmol/L K⁺), with intracellular K⁺ concentration assumed to be 140 to 150 mmol/L, E_K was estimated to be +80 mV. The results satisfied these theoretical estimations. Second, outward unitary currents were not observed at potentials positive to E_K, indicating inward rectification typical for I_K1. Third, The slope conductances and mean open times in atrial and ventricular channels in cell-attached patches were similar to those previously reported for guinea pig⁴ and human atrium.¹⁷ Fourth, channel activity was blocked by Ba²⁺ and Cs⁺. Single-channel analysis in the present study demonstrated that the conductance and gating kinetics do not differ between atrial and ventricular I_K1. The fact that the percentage of patches with channel activity is higher in ventricular myocytes than in atrial myocytes is consistent with the results in whole-cell experiments; higher percentage of channel activity of single I_K1 channels in ventricle compared with atrium could underlie the differences in the magnitudes of the whole-cell currents and their conductances in the two tissue types. The facts that whole-cell current conductance of human atrial I_K1 is much smaller than that of the ventricular I_K1 and that the whole-cell atrial I_K1 does not exhibit a prominent negative slope region are consistent with the difference observed in the action potential characteristics in atrium and ventricle.

Alterations in I_K1 Channel Characteristics for Patients With DCM
Previous studies in our and other laboratories using multicellular specimens have demonstrated that human atrium with various disease states exhibited several different electrophysiological characteristics from nondiseased tissues.^{33 34 35 36 37 38} Similar results were obtained in human ventricular myocardium.^{39 40 41 42 43} In isolated single-cell experiments, altered action potential shape and decreased density of whole-cell I_K1 were reported in ventricular myocytes from patients with terminal heart failure.^{18 44} However, electrophysiological alterations in different preexisting disease states were not evaluated in these previous studies. We considered the possible involvement of I_K1 in different preexisting disease states and were interested in comparisons of I_K1 characteristics in myocytes from patients with different heart diseases.

Low resting membrane potential, prolonged action potential duration, and decreased whole-cell I_K1 conductances were found in ventricular myocytes from patients with DCM. It is unlikely that these alterations were caused by heart failure, because electrophysiological characteristics in myocytes from patients with the same functional class heart failure (class IV) after ICM did not exhibit these changes. Although prolonged action potential duration and decreased whole-cell current slope conductance were observed for ICM, they were not significantly different from those for other disease states. Single I_K1 channel properties in atrial and ventricular myocytes from patients with DCM exhibited a distinct burst behavior compared with patients with ICM, persons with other heart diseases, and healthy donors. These results suggest that the alterations in individual single-channel behavior may not parallel the severity of heart failure but may be related to the underlying nature of DCM.

As we have reported previously, we did not detect any alterations in I_Na characteristics in atrial and ventricular myocytes isolated from human hearts.^{12 13 14} In addition, there were no clear-cut differences in I_Na characteristics between DCM and other disease states^{12 13} in atrial and ventricular myocytes isolated from the same patient group that was used the present study, in which alterations in I_K1 channel characteristics were detected. Thus, it is likely that one characteristic of DCM includes alterations of I_K1 but not of I_Na in the human heart.

The exact origin of this alteration in channel behavior for DCM is unclear at present. Although there have not been previously described electrophysiological alterations in different disease states in human heart, biochemical alterations for DCM have been reported; M₂-cholinergic receptor–linked G_i protein subunit G_i is increased for DCM but not for ICM and for nonfailing human hearts.^{45 46} Under conditions of cell-attached patches, we have never observed ATP-sensitive K⁺ channel [I_K(ATP)] activity in atrial and ventricular myocytes from patients with DCM. After formation of excised inside-out patch configurations, we have observed I_K(ATP) activity when perfusing with ATP-free bath solution (data not shown). This finding indicates that although the intracellular metabolic state is maintained, the channel and/or an associated regulatory protein may be involved in DCM.

Comparison With Other Mammalian Species
I_K1 channel characteristics in human atrial and ventricular myocytes from patients without DCM showed many similarities to those from other mammalian species. Although there were several differences in single-channel parameters in human (non-DCM), cat, and guinea pig ventricle, the basic characteristics were not significantly different from one another. One difference was that human ventricular I_K1 channel exhibited a relatively higher P_o value than that for cat or guinea pig despite the fact that channel mean open lifetime in human I_K1 is smaller than that for cat or guinea pig. Our present findings suggest that I_K1 channel behavior is similar between mammalian species but that I_K1 channels for DCM behave in a manner that is distinct from others. In addition, abnormal electrophysiological properties were reported in cardiomyopathic Syrian hamster heart.^{47 48 49} Prolongation of the action potential has been shown in the cardiomyopathic animal models.^{47 50} Thus, information derived from animal studies appears to be applicable to considerations of "normal" and "abnormal" human I_K1 channels.

Study Limitations and Implications
One of the limitations of the present study is the small number of control hearts. In addition, ventricular specimens from persons with normal function were obtained from patients with inhomogeneous disease states (IHD and VHD). The availability of control human hearts without any dysfunction (including donor hearts) is always extremely limited for laboratory investigation. However, electrophysiological properties in myocytes obtained from these control hearts were quite similar to each other. Basic characteristics of I_K1 from ventricles without dysfunction behaved similar to those from donor atria and animal heart. In addition, our statistical treatment using the two-way ANOVA and ANCOVA can effectively eliminate the statistical errors generated by using a different sample size for each group. There were no significant interpatient differences in data comparisons for each group. The possible modulating effects of age and sex also were eliminated by using the ANCOVA in each data comparison.

Finally, the I_K1 channel is likely to be one of the main targets of the involvement of DCM. Depolarized resting membrane potential and altered action potential shape may be caused at least in part by the alteration in I_K1. These changes may increase the vulnerability to malignant arrhythmias for patients with DCM. Because electrophysiological alterations in vitro have been associated with preexisting disease in human heart, further estimation of these properties in vivo remains to be established.

	Acknowledgments

 
The authors wish to thank Drs Ruth L. Martin (University of Chicago), J. Andrew Wasserstrom, Ana-Maria Vites, and Robert E. Ten Eick (Northwestern University School of Medicine) for their thoughtful discussion and comment on this work.

	Footnotes

 
Dr Koumi's present address is The First Department of Internal Medicine, Nippon Medical School, Japan.

Received September 14, 1994; revision received December 28, 1994; accepted January 9, 1995.

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