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(Circulation. 1995;92:1179-1187.)
© 1995 American Heart Association, Inc.

Articles

Repolarizing K⁺ Currents in Nonfailing Human Hearts

Similarities Between Right Septal Subendocardial and Left Subepicardial Ventricular Myocytes

Hanna Konarzewska, MD; George A. Peeters, MD; Michael C. Sanguinetti, PhD

From the Division of Cardiology and Program in Human Molecular Biology and Genetics (M.C.S.), Eccles Institute of Human Genetics, University of Utah, Salt Lake City.

Correspondence to Dr Michael C. Sanguinetti, Program in Human Molecular Biology and Genetics, Eccles Institute of Human Genetics, Bldg 533, Room 4220, University of Utah, Salt Lake City, UT 84112.

	Abstract

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Background Studies of cardiac K⁺ channels from animal models have documented tissue-dependent and species-dependent diversity in the types and properties of K⁺ channels responsible for the repolarization of cardiac action potentials. Recent reports of human ventricular K⁺ currents emphasized differences in transient outward K⁺ current (I_to1) recorded from left ventricular (LV) subendocardial and subepicardial myocytes. These myocytes are usually isolated only from the LV free wall. The surface of the interventricular septum is continuous with the endocardium of both ventricular chambers. However, the septum contracts in unison with the left ventricle and therefore might have electric properties consonant with this function. In this study, we compare the characteristics of human cardiac K⁺ currents (I_to1 and inward rectifier K⁺ current [I_K1]) of myocytes isolated from either the LV subepicardium or subendocardium of the right ventricular (RV) septum.

Methods and Results Subendocardial tissues were obtained during routine biopsies of the right interventricular septum of seven heart transplant recipients. Subepicardial tissues were obtained from five patients with normal LV function during open heart surgery. I_K1 amplitude was the same in myocytes isolated from both regions. Delayed rectifier K⁺ currents were small or absent in these cells. I_to1 was only slightly larger in LV subepicardial versus RV septal subendocardial myocytes. For example, at +60 mV, I_to1 was 7.2±0.4 pA/pF (n=33) in subepicardial cells compared with 6.0±0.5 pA/pF (n=36) in subendocardial cells. All characteristics of I_to1 examined, including the voltage dependence of activation and inactivation, rate of inactivation, and percent decline of peak current during repetitive pulsing, were similar in myocytes isolated from both regions. These findings are in contrast to previous studies that demonstrated that I_to1 of subendocardial myocytes isolated from the LV free wall of human hearts was smaller and that recovery from inactivation of this current was much slower compared with that observed in subepicardial myocytes.

Conclusions We conclude that the major repolarizing K⁺ currents in normal human ventricular myocytes are I_K1 and I_to1 and that the properties of I_to1 of subendocardial cells isolated from the right interventricular septum are more similar to subepicardial cells than to subendocardial cells of the LV free wall. The similar electric properties shared by myocytes from these two regions may be functionally important inasmuch as the right side of the interventricular septum functions as an extension of the subepicardium of the left ventricle during the contractile cycle.

Key Words: electrophysiology • myocardium • potassium

	Introduction

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Repolarization of cardiac action potentials depends on activation of several distinct types of voltage-dependent K⁺ channels. The properties of these channels have been studied extensively with voltage clamp techniques in cardiac myocytes isolated from several mammalian species. These studies documented considerable species and tissue differences with regard to the types and magnitudes of ventricular K⁺ currents and raised doubts as to whether myocytes isolated from any one species or anatomic region can serve as an adequate model of human cardiac muscle.

Because of the relative unavailability of normal human cardiac tissue for research purposes, initial studies characterized currents in myocytes dissociated from diseased human hearts.^{1 2 3} However, the properties of currents recorded from cells isolated from diseased tissue may differ from those found in normal tissue. For example, the delayed rectifier K⁺ current (I_K) was reported to be smaller and the inward rectifier K⁺ current (I_K1) was reported to be larger in feline ventricular myocytes dissociated from hypertrophied tissue compared with normal tissue.⁴ In humans, the magnitude of I_K1 and the transient outward K⁺ current (I_to1) were reported to be reduced in myocytes obtained from hearts of patients with terminal heart failure.¹ These differences may reflect adaptations to different loading or activation conditions rather than primary pathological changes. The extent to which transmembrane currents adapt to pathophysiological conditions has not been explored adequately in human myocytes. Thus, there is a need to characterize and compare repolarizing K⁺ currents from a larger number of nonfailing human cardiac myocytes obtained from functionally and anatomically diverse sites. Such information could serve as reference data with which to compare similar voltage clamp measurements with myocytes obtained from diseased hearts.

I_to1 is the most prominent outward current in canine and human atrial⁵ and ventricular^{3 6} cardiac myocytes. Antzelevitch and colleagues^{7 8 9} showed that the marked regional variation in the configuration of canine ventricular action potentials can be attributed primarily to a variable magnitude of I_to1. The initial repolarization phase of action potentials recorded from subepicardial and midmyocardial myocytes is characterized by a prominent notch. This notch is virtually absent in subendocardial myocytes because of a much reduced I_to1. I_to1 is also much larger in epicardial than in endocardial ventricular tissue of the cat¹⁰ and rat.¹¹ Lukas and Antzelevitch¹² proposed that such transmural heterogeneity in channel density and gating properties may be important in normal dispersion of refractoriness, arrhythmogenesis, and responsiveness to drugs and ischemia. Recently, Wettwer et al⁶ reported that a marked transmural gradient in the magnitude of I_to1 also exists across the left ventricular (LV) free wall in nonfailing and failing human myocardium. I_to1 was found to be about two times larger in subepicardial relative to subendocardial myocytes dissociated from the LV free wall. The gating of I_to1 channels also exhibited regional variability. The rate of I_to1 recovery from inactivation was much slower in subendocardial than in subepicardial myocytes.⁶

In this study, we have characterized the major K⁺ currents in human ventricular myocytes isolated from biopsy samples taken from the right interventricular septum of heart transplant recipients and the LV subepicardium of patients undergoing coronary artery bypass graft surgery. The tissue obtained by biopsies of these patients is composed of relatively normal myocytes, at least compared with those obtained from explanted failing hearts. We found that the average magnitude and recovery kinetics of I_to1 were not markedly different between these two regions of the human heart in contrast to the transmural gradient reported for LV free wall. This similarity may reflect the functional similarity of tissues that form the outer surface of the LV chamber.

	Methods

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Tissue Procurement
LV biopsies were obtained from five patients undergoing coronary artery bypass graft surgery. These patients had no previous myocardial infarction and had normal global LV function as assessed by angiography. Specimens of approximately 1x1x5 mm were taken during cardioplegia from a region of the LV epicardium that appeared normal on gross inspection. The tissue specimen was excised parallel to the myocardial fiber orientation, and the surgical site was repaired with a single suture. This procedure did not prolong surgery significantly and has been reported to be safe.¹³ Subendomyocardial biopsies were obtained from seven healthy heart transplant recipients during their routine surveillance cardiac biopsies. A single additional specimen of approximately 1.5 mm³ was sampled from the right septal wall with a Stanford biotome by use of standard techniques.¹⁴ The performance of both biopsy procedures was approved by the University of Utah Human Studies IRB Committee, and informed written consent was obtained from each patient. Most of the patients were on multiple medications at the time of biopsy. The Table lists these drugs.

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

Myocyte Isolation
The method used to enzymatically dissociate human ventricular biopsy samples was described previously in detail.¹⁵ Biopsy specimens were immediately transported to the laboratory in ice-cold, oxygenated HEPES-buffered/2,3 butanedione monoxime (BDM) saline (composition described below). The addition of BDM to this solution minimizes cutting and reoxygenation injury of isolated myocytes.¹⁶ After a 30-minute equilibration at room temperature in this transport solution, the tissue sample was placed in Ca²⁺-free HEPES-buffered/BDM saline and sliced into 400-µm pieces with a mechanical vibratome. The slices were then incubated at 37°C with protease and collagenase while the solution was constantly bubbled with 100% oxygen. Slices were transferred to a solution containing fresh enzyme every 10 to 15 minutes for a total of 90 minutes. The enzyme solution containing dissociated cells was diluted with an equal volume of recovery medium and centrifuged at 100g for 5 minutes. The resulting pellets were then resuspended in recovery media and kept at room temperature.

Voltage Clamp Data Acquisition and Analysis
The suction microelectrode voltage clamp technique described by Giles and Shibata¹⁷ was used to record currents in single ventricular myocytes. Pipettes were fabricated from 1-mm-OD square-bore borosilicate glass (Frederick & Dimmock) and had resistances of 3 to 6 M when filled with an intracellular solution that consisted of 0.5 mol/L potassium gluconate and 25 mmol/L KCl (pH 7.3). Series resistance was compensated by at least 80%. The limitations (high series resistance, loading of cells with K⁺ during prolonged recordings) and advantages (no immediate alteration of intracellular contents, ease of seal formation) of using these electrodes and filling solutions were described previously.^{18 19} Command voltage pulses were generated by use of PCLAMP software, a Digidata 1200 interface (Axon Instruments) connected to a 486DX, 50-MHz desktop computer, and an Axopatch 200 amplifier (Axon Instruments). Currents were low-pass–filtered with a cutoff frequency (-3 dB) of 1 or 2 kHz. Currents are usually expressed relative to total cell capacitance to facilitate comparison between cells of various sizes.

In most experiments, cells were bathed in a nominally Ca²⁺-free "extracellular solution" (room temperature) containing 1 mmol/L CoCl₂ to block Ca²⁺ currents and prevent cell contraction. Unless indicated otherwise, currents were elicited with stepped pulses from a holding potential of -60 mV. At -60 mV, a small Na⁺ current (I_Na) remained available for activation on depolarization. Therefore, each test pulse was preceded by a 20-ms pulse to -30 mV, which served to inactivate any remaining I_Na. To determine the current-voltage relation for I_to1, pulses were applied every 8 seconds in 10-mV increments to test potentials ranging from -30 to +70 mV. This pulsing rate was sufficiently slow to allow recovery of I_to1 from inactivation at -60 mV between successive test pulses. The time course of I_to1 inactivation was fit with a single exponential relation: I_to1(t)=A₀+A₁e^(-t/) with a Chebyshev noniterative fitting technique (PCLAMP, Axon Instruments). A nonlinear least-squares fitting routine (KALEIDAGRAPH, Abelback Software) was used to fit normalized data describing the voltage dependence of I_to1 activation and inactivation to a Boltzmann distribution. The voltage dependence of I_to1 activation was determined from the amplitude of tail currents after 20-ms pulses to potentials (V_t) ranging from -40 to +20 mV. Tail currents were normalized to the maximum value (I_to1max) determined by fits of data from an individual experiment. Cumulative data were then averaged and fit with a Boltzmann relation, I_to1/I_to1max=1/{1+exp[(V_1/2-V_t)/k]}, to estimate the average half-point (V_1/2) and slope factor (k) for this relation.

The voltage dependence of I_to1 inactivation was determined by use of 5-second conditioning test pulses. Each conditioning pulse was applied to a potential ranging from -70 to -5 mV and was followed by a pulse to -30 mV (to inactivate any I_Na present) and then to +50 mV to monitor the extent of I_to1 inactivation. The currents were normalized to the largest current for each experiment; then cumulative normalized data were fit in a manner similar to that described for the activation curve.

To determine the current-voltage relation for I_K1, 140-ms pulses were applied every 4 seconds from a holding potential of -60 mV. Test pulses were applied in -10-mV increments to potentials ranging from -40 to -170 mV. The amplitude of I_K1 at each potential was determined by measuring peak current relative to the zero current level.

Solutions
The HEPES-buffered/BDM solution used for transport of tissue specimens contained (in mmol/L) NaCl 120, KCl 5.4, MgSO₄ 0.5, CaCl₂ 0.25, sodium pyruvate 5, glucose 20, taurine 20, BDM 30, and HEPES 10; pH was adjusted to 7.0 with NaOH. The enzyme solution was prepared by adding 0.1 mg/mL protease type XXIV (Sigma Chemical Co) and 1 mg/mL collagenase type 2 (Worthington) to HEPES-buffered/BDM solution in which the CaCl₂ concentration was reduced to 50 µmol/L. The recovery medium was the same as the HEPES-buffered/BDM solution, except that it also contained 1% BSA and the pH was adjusted to 7.2.

The extracellular solution bathing the cells during the voltage clamp experiments contained (in mmol/L) NaCl 132, KCl 4, MgCl₂ 1.2, HEPES 10, glucose 10, and CoCl₂ 1; pH was adjusted to 7.4 with NaOH.

Statistics
All cumulative data are expressed as mean±SEM. Comparisons were performed by a two-way ANOVA where appropriate followed by a multiple-comparisons test (Tukey-Kramer) to test for differences between unpaired data (eg, currents recorded from subepicardial and subendocardial cells at a given test potential). Differences between values were considered statistically significant when P<.05.

	Results

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Cell Capacitance
The average capacitance of the right septal myocytes was 91±1 pF (seven hearts, 36 cells; range, 30 to 189 pF). The average capacitance of the left subepicardial myocytes was 96±1 pF (five hearts, 33 cells; range, 38 to 249 pF). The currents described below are given relative to total cell capacitance determined for each cell. Not all pulse protocols were applied to each cell; therefore, the number of determinations is presented for each experiment.

Inward Rectifier K Current
The magnitude of I_K1 was similar in LV subepicardial and right ventricular (RV) septal subendocardial myocytes throughout the voltage range examined (-60 to -170 mV). At the test potential of -140 mV, I_K1 averaged -18.2±1.4 pA/pF (range, -10.4 to -27.8 pA/pF) for LV subepicardial cells (n=15, two hearts) and -18.7±1.0 pA/pF (range, -9.1 to -33.1 pA/pF) for RV septal subendocardial cells (n=35, six hearts). Outward current recorded at -60 mV was small: 0.076±0.046 pA/pF in subendocardial cells and 0.097±0.042 pA/pF in subepicardial cells. I_K1 exhibited marked time-dependent inactivation at very negative potentials. For example, at -170 mV, current measured at the end of a 140-ms test pulse decayed to about half its initial value (Fig 1). The time course of I_K1 inactivation was best fit with a single exponential relation. The time constants for inactivation were significantly slower in the LV subepicardial than in the RV septal myocytes at all potentials (Fig 1C). The biological significance of this difference is uncertain because of the limited sample size (I_K1 was recorded in myocytes isolated from only two RV septal biopsies).

View larger version (20K): [in this window] [in a new window]   Figure 1. Graphs showing inward rectifier K+ currents (IK1). A, IK1 recorded in a human left ventricular (LV) subepicardial myocyte in response to 140-ms pulses to test potentials (Vt) ranging from -60 to -170 mV. B, Current-voltage relations for IK1 in isolated LV subepicardial and RV septal subendocardial myocytes. Currents are normalized relative to total cell capacitance. Data represent mean±SEM for 15 cells from 2 hearts (subepicardium) and 35 cells from 6 hearts (subendocardium). C, Time constants for rate of inactivation of IK1. Smooth curves are second-order polynomial fits to the data.

Transient Outward K Current
The current-voltage relation for I_to1 was determined from a holding potential of -60 mV. The small I_Na that remained available for activation was further inactivated during a 20-ms prepulse to -30 mV (Fig 2A). The amplitude of I_to1 was estimated by measurement of the time-dependent component from its peak outward value to its steady-state value at the end of 500-ms test pulses. The sustained current remaining at the end of these test pulses is referred to as I_sus. The transient outward current activated by this protocol is assumed to be the voltage-dependent component (I_to1) because Ca²⁺-dependent transient outward current (I_to2) requires entry of Ca²⁺ through Ca²⁺ channels for its activation.^{5 20} Further evidence that only I_to1 was present under these experimental conditions was the finding that inactivation of the current was usually best fit with a single exponential function. However, because the cell interior was not perfused and the recording pipette did not contain a Ca²⁺ buffering agent such as EGTA, we cannot exclude the presence of a small I_to2 component. The average amplitudes of I_to1 and I_sus were only slightly larger in subepicardial than in subendocardial myocytes at all test potentials (Figs 2 and 3). For example, I_to1 was 1.8±0.1 pA/pF (range, 0.1 to 3.2 pA/pF) at 0 mV and 7.2±0.4 pA/pF (range, 3.2 to 11.3 pA/pF) at +60 mV in subepicardial cells (n=33 cells, five hearts). In RV septal subendocardial myocytes, I_to1 was 1.3±0.1 pA/pF (range, 0.2 to 2.8 pA/pF) at 0 mV and 6.0±0.5 pA/pF (range, 1.3 to 12.9 pA/pF) at +60 mV (n=36 cells, seven hearts). I_to1 was significantly (P<.05) larger in subepicardial than in subendocardial myocytes only at test potentials ranging from -10 to +20 mV (Fig 2B). There was considerable variation in the magnitude of peak I_to1 recorded in myocytes isolated from a single preparation. For example, at the test potential of +60 mV, I_to1 varied from 4.6 to 10.9 pA/pF (n=13) in cells dissociated from one subepicardial specimen and from 1.5 to 12.9 pA/pF (n=12) from one subendocardial specimen. There was no significant correlation (correlation coefficient, .05) between the current densities of I_to1 and I_K1 within a given cell. With the assumption that I_K1 and I_to1 are subject to the same detrimental effects of metabolic or other imbalances, this suggests but does not prove that the variation in the magnitude of I_to1 does not simply reflect the overall condition of a given myocyte.

View larger version (18K): [in this window] [in a new window]   Figure 2. Graphs showing transient outward K+ currents (Ito1). A, Ito1 recorded in a human subepicardial myocyte. Inset shows voltage pulse protocol. B, Current-voltage relations for Ito1 in isolated human subepicardial and subendocardial ventricular myocytes. Currents are normalized relative to total cell capacitance. Data represent mean±SEM for 33 cells from five hearts (subepicardium) and 36 cells from seven hearts (subendocardium). *Ito1 significantly different (P<.05) between subendocardial and subepicardial myocytes. C, Time constants for rate of Ito1 inactivation as a function of test potential. Smooth curves are third-order polynomial fits to the data.

View larger version (16K): [in this window] [in a new window]   Figure 3. Graph showing current-voltage relations for the sustained current present at the end of the 500-ms test pulses after complete inactivation of transient outward K+ currents (Isus) in isolated human left ventricular subepicardial and right ventricular septal subendocardial myocytes. Currents are normalized relative to total cell capacitance. Data represent mean±SEM for 30 cells from five hearts (subepicardium) and 30 cells from seven hearts (subendocardium). *Isus significantly different (P<.05) between subendocardial and subepicardial myocytes.

I_sus exhibited outward rectification at test potentials >0 mV (Fig 3). At test potentials of 0 and +60 mV, I_sus was 0.22±0.03 and 1.50±0.13 pA/pF in LV subepicardial cells (n=30 cells, five hearts) and 0.11±0.02 and 1.31±0.12 pA/pF in RV septal subendocardial cells (n=30 cells, seven hearts), respectively. The channel type underlying I_sus is not known. It is probably not a K⁺ current because it was not blocked by 5 mmol/L 4-aminopyridine or 2 mmol/L BaCl₂ (data not shown).

The time course of I_to1 inactivation was determined by fitting the decay phase of currents recorded during 500-ms pulses to potentials ranging from -10 to +70 mV with either one or two exponential functions. In a few cells, inactivation of I_to1 was best fit with two exponentials at potentials +30 mV. However, the currents at all potentials for the majority of cells were best fit with a single exponential function. The time constants for I_to1 inactivation showed only a slight voltage dependence, ranging from 60 to 80 ms for both subepicardial and subendocardial cells (Fig 2C).

The voltage dependence of I_to1 activation was determined by tail current analysis as described in the "Methods" section (Fig 4). The half-points for activation of I_to1 were -8.9±0.19 and -7.8±0.2 mV for subepicardial (n=15 cells, four hearts) and subendocardial cells (n=20, six hearts), respectively. The slope factors of the activation curves were 6.2±0.2 and 5.7±0.2 mV for subepicardial and subendocardial cells, respectively. Activation curves were also constructed from peak values of outward I_to1 (data from Fig 2B) after correction for changes in driving force for K⁺ and with the assumption of a reversal potential of -80 mV. The V_1/2 and slope factor determined by this method were more positive than that determined by tail current analysis; however, these data were not well fit with a single Boltzmann distribution.

View larger version (13K): [in this window] [in a new window]   Figure 4. Graphs showing voltage-dependence of transient outward K+ currents (Ito1) activation. A, Example of currents recorded from a subepicardial myocyte during 20-ms pulses to test potentials ranging from -25 to +20 mV applied in 5-mV steps. Tail currents were measured at -40 mV. B, Relative tail current amplitudes are plotted as a function of test potential as described in "Methods." Data represent mean±SEM for 15 cells from four hearts (left ventricular subepicardium) and 20 cells from six hearts (right ventricular septal subendocardium). Smooth curves are least-squares fits of the data to Boltzmann distributions. The half-point (V1/2) and slope factor for these curves were -8.9±0.2 and 6.3±0.2 mV (subepicardium) and -7.8±0.2 and 5.7±0.2 mV (subendocardium), respectively.

The voltage dependence of I_to1 inactivation was assessed with 5-second conditioning prepulses (Fig 5). The half-points of this relation were -36.3±0.4 mV for subepicardial myocytes (n=23, four hearts) and -33.0±0.4 mV for subendocardial myocytes (n=25, six hearts). The slope factors for the inactivation curves were -5.6±0.2 and -5.2±0.2 mV for subepicardial and subendocardial myocytes, respectively. There was only a slight overlap between the voltage dependence of activation and inactivation, delineating the range of potentials over which I_to1 could activate but not inactivate. The resulting "window current" would occur between -35 and -5 mV and represent 5% of peak current amplitude at potentials from -20 to -5 mV (Fig 5C).

View larger version (16K): [in this window] [in a new window]   Figure 5. Graphs showing voltage dependence of transient outward K+ current (Ito1) inactivation. A, Currents were recorded from a subendocardial myocyte at a test potential of +50 mV after 5-second pulses to conditioning potentials (Vc) of -70 mV and -50 to -15 mV (in 5-mV steps). A 20-ms pulse to -30 mV preceded each test pulse to inactivate Na+ current, in this example seen after the Vc to -70 mV. Note that currents are displayed on two time scales. B, Relative current amplitudes recorded during test pulse to +50 mV are plotted as a function of the potential of the 5-second conditioning pulse. Data represent mean±SEM for 23 cells from four hearts (left ventricular subepicardium) and 25 cells from six hearts (right ventricular septal subendocardium). Smooth curves are least-squares fits of the data to Boltzmann distributions. The half-point (V1/2) and slope factor for these curves were -36.3±0.4 and 5.6±0.3 mV (subepicardium) and -33.0±0.4 and 5.2±0.2 mV (subendocardium), respectively. C, Overlap of isochronal inactivation curves and activation curves for Ito1. Region of overlap defines voltage range over which Ito1 channels would remain open once activated. Data are the same as those plotted in Figs 4 and 5. Error bars were omitted to enhance clarity. Subendo indicates subendocardium; subepi, subepicardium.

The recovery from inactivation of I_to1 is known to depend on voltage.^{6 20} As an indirect measure of this phenomenon, we recorded the decrease in I_to1 magnitude during a train of 10 pulses applied from a variable holding potential to a common test potential of +40 mV. Test pulses of 200-ms duration were applied at a rate of 120 pulses per minute. Cells were held for at least 1 minute at a holding potential of -40, -50, or -60 mV before the train of test pulses was applied. The amplitude of I_to1 during the first pulse was dependent on holding potential (smallest at -40 mV) as expected from the voltage dependence of inactivation described in Fig 5. To facilitate comparison of the data obtained at different holding potentials, the data are plotted relative to the amplitude of I_to1 for the first pulse at a given holding potential. The rate-dependent decrease in I_to1 was the same for both left subepicardial and right septal subendocardial myocytes (Fig 6). This suggests that the recovery from inactivation of the currents was the same for cells isolated from the two regions of the ventricle.

View larger version (17K): [in this window] [in a new window]   Figure 6. Graph showing the effect of holding potential and repetitive pulsing on the magnitude of transient outward K+ currents (Ito1) in left ventricular (LV) subepicardial and right ventricular (RV) septal subendocardial myocytes. The magnitude of Ito1 during a train of 10 pulses applied at 120 pulses per minute from the indicated holding potential was plotted relative to the amplitude of the current recorded for the first pulse. The test pulses were 200 ms in duration to a fixed potential of +40 mV. Number of cells at each holding potential were as follows: LV subepicardium, -60 mV, n=10; -50 mV, n=10; -40 mV, n=7; RV septal subendocardium, -60 mV, n=16; -50 mV, n=6; -40 mV, n=5. The SEM bars are smaller than the symbol for many data points. Smooth curves represent single exponential fits to the data. The time constants were (subepicardium, subendocardium) -60 mV (144 and 144 ms), -50 mV (178 and 146 ms), and -40 mV (194 and 141 ms).

Delayed Rectifier K⁺ Current
Slowly activating delayed rectifier K⁺ current (I_K) was not measurable under the conditions used in this study. It is possible that I_K was present in these cells but that the absence of extracellular Ca²⁺, the presence of 1 mmol/L Co²⁺, and the recording of currents at room temperature reduced its size. Cobalt blocks I_Kr in guinea pig cardiac myocytes.²¹ Therefore, several myocytes were voltage clamped while bathed in a solution containing 1.8 mmol/L CaCl₂ and at 35°C. These same conditions were used previously to record robust I_K currents in isolated guinea pig ventricular myocytes.¹⁸ Even under these conditions, I_K was very small or absent. The average time-dependent I_K during a 3.5-second test pulse to +60 mV in 6 subepicardial myocytes from one heart was 28±23 pA (range, 0 to 128 pA), equivalent to about 0.028 pA/pF.

	Discussion

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Regional Variability in the Magnitude of I_to1
Wettwer et al⁶ and Nabauer and Beuckelmann²² observed a large difference between LV subepicardial and subendocardial myocytes in the amplitude and kinetics of recovery from inactivation of I_to1. We did not find such differences between subendocardial and subepicardial myocytes in the present study. However, our subendocardial cells were dissociated from biopsy samples collected from the right interventricular septum of heart transplant patients, whereas in the previous studies, subendocardial myocytes were dissociated from the inner surface of the LV free wall. Because the interventricular septum contracts in unison with the left ventricle, it is not surprising that the LV subepicardium and the RV septum have similar electric properties. Activation of the interventricular septum is initiated at the endocardial surface of the left ventricle and proceeds to the right side of the septum.²³ Activation of both ventricular chambers proceeds from endocardial to epicardial surface. Thus, in terms of the direction of membrane depolarization, the left-to-right activation sequence of the interventricular septum mirrors the endocardial-to-epicardial direction of normal ventricular activation. Myocytes of the endocardium and septum have a common embryological origin. The primordial cells that form the interventricular septum arise from a coalescing of primitive trabeculae (derived from the endocardium) near the ventricular apex.²⁴ The reported differences in the magnitude of I_to1 between LV subepicardial (and right septal) and LV subendocardial myocytes may simply reflect the level of channel expression between different regions of the heart. However, variation in kinetic properties of I_to1 (ie, the rate of recovery from inactivation) may indicate regional differences in the expression of specific channel subunits that combine to form functional transient outward K⁺ channels. However, because we obtained the RV septal specimens from transplant patients, we cannot rule out the possibility that denervation of the heart may alter the expression or properties of I_to1 channels recorded from these myocytes. Only the transplant patients received prednisone (the Table) before biopsy. Steroid hormones can modulate the expression of K⁺ channel genes. For example, dexamethasone induces expression of mRNA encoding Kv1.5 channels in cultured GH₃ cells^{25 26} and rat ventricular muscle.²⁷ In the rat, this induction was specific for Kv1.5; it had no effect on mRNA levels of Kv1.4 or Kv2.1.²⁷ The effect of long-term prednisone treatment on the expression of human cardiac K⁺ channel genes is unknown. If this steroid induces transcription of the I_to1 channel gene, then the magnitude of I_to1 may be abnormally large in myocytes isolated from heart transplant patients.

The magnitude of I_to1 reported previously from midmyocardial myocytes was much larger than we or others have reported for this current in subepicardial or subendocardial myocytes. Beuckelmann et al¹ reported that peak I_to1 measured at +40 mV averaged 9.2 pA/pF in nonfailing myocytes. The average peak I_to1 at +40 mV in our cells were 4.3 (subendocardium) and 5.4 (subepicardium) pA/pF. The size of our biopsy samples (1.5-mm depth) was small enough to ensure that only subendocardial and subepicardial tissues and not midmyocardial tissue were collected. Further study is needed to confirm whether K⁺ currents are indeed larger in midmyocardial than in subepicardial or subendocardial myocytes.

A large transmural gradient in the magnitude of I_to1 has been documented for the left ventricle of diseased human hearts. In explanted diseased hearts, I_to1 was about 2.5 times larger in LV subepicardial than in subendocardial myocytes when assessed at a test potential of +40 mV.²² In another study, myocytes isolated from failing hearts differed regionally by a factor of 3.4-fold in failing hearts but by only 1.5-fold in nonfailing hearts (6.8 pA/pF, n=16, versus 4.4 pA/pF, n=5, at +60 mV in LV subepicardial and subendocardial myocytes, respectively).⁶ A nonuniform distribution in the magnitude of endocardial I_to1 was also reported to exist in rabbit ventricle. I_to1 of rabbit subepicardial myocytes was reported to be two times larger than in subendocardial cells isolated from papillary muscles but only 1.16-fold larger than that recorded from subendocardial cells isolated from the ventricular free wall.²⁸ We found that I_to1 averaged 7.2 and 6.0 pA/pF at +60 mV in LV subepicardial and RV septal subendocardial myocytes, respectively (a 1.2-fold difference).

A significant transmural difference in the voltage dependence of I_to1 activation was previously reported for myocytes isolated from undiseased human LV free wall. Half-maximal activation of I_to1 was +9.7 mV in subepicardial cells compared with +23.1 mV in subendocardial cells.⁶ We determined the voltage dependence of I_to1 activation by tail current analysis and did not detect any significant regional difference between the LV subepicardial and RV septal subendocardial layers, either in the potential at which I_to1 was half-activated or in the slope of the activation curve. This apparent discrepancy may be related to experimental conditions. We used pipettes with very small tip diameters (to minimize cell dialysis) that were loaded with 0.5 mol/L K gluconate and 25 mmol/L KCl and bathed the cells in a nominally Ca²⁺-free solution containing 1 mmol/L CoCl₂. Differences in pipette (internal) solution and extracellular divalent cations (Cd²⁺ versus Co²⁺ to block Ca²⁺ current; presence or absence of CaCl₂) may explain some of these apparent differences in the voltage dependence of I_to1 activation. The voltage dependence of I_to1 determined by tail current analysis in our study was similar to that reported for rabbit atrial cells (V_1/2, -12 mV; slope factor, 9.6 mV).²⁹ The slope and V_1/2 of the isochronal (5-second) inactivation curve for I_to1 determined in our study were similar to those reported previously when 2-second conditioning pulses were used in normal⁶ or failing ventricular myocytes.²

Our findings confirm the major findings of Wettwer et al,^{3 6} Nabauer et al,² and Beuckelmann et al¹ that the most prominent K⁺ currents of human ventricular myocytes are I_K1 and I_to1. Like these previous investigators, we found that delayed rectifier K⁺ currents were small or absent in these cells. All these studies are complicated by the poor yield and low viability of myocytes isolated from human heart tissue and by the potential, yet unknown, effects of the many medications that patients received before biopsy. Despite these numerous complicating factors, we felt it was reasonable to compare voltage clamp data obtained from myocytes dissociated from both types of biopsy samples because all tissues were handled the same and the methods used to record currents were identical.

Regional Variability in Rate-Dependent Decrease in I_to1
The decrease in I_to1 during a train of pulses applied at 120 pulses per minute was nearly identical for RV septal and LV subepicardial myocytes, regardless of holding potential (Fig 6). The decrease in I_to1 during repetitive pulsing results from a combination of the rate of current inactivation during the test pulse and the rate of recovery from this inactivation. Because the rate of I_to1 inactivation was essentially the same in all myocytes, we can conclude that the rate of recovery from inactivation of I_to1 was also the same at holding potentials of -40, -50, and -60 mV for myocytes isolated from both regions of the heart. Using a similar pulse train protocol applied from a holding potential of -60 mV, Nabauer and Beuckelmann²² reported that I_to1 was decreased by 16.3% in subepicardial cells and 85.4% in subendocardial cells isolated from explanted hearts. We found that I_to1 decreased by about 15% in myocytes from both the LV subepicardium and RV septal subendocardium. This is further evidence that myocytes isolated from the right interventricular septum are more like myocytes of the LV subepicardium than LV subendocardium with respect to properties of I_to1. The pulse-dependent decrease in ventricular I_to1 observed in our study is quite similar to that reported by Fermini et al³⁰ for human atrial myocytes. I_to1 of human atrial cells was reduced by about 5% when paced at 2 Hz from a holding potential of -60 mV. Thus, the previously reported findings that I_to1 of human subendocardial myocytes is characterized by a reduced amplitude, a more positive V_1/2 for voltage-dependent activation, and a comparatively slow recovery from inactivation do not apply to the entire subendocardium.

I_sus and I_K
The sustained outward current remaining after inactivation of I_to1 in isolated human atrial myocytes represents an ultrarapidly activating delayed rectifier K⁺ current (I_Kur) that is half-blocked by 0.05 mol/L 4-aminopyridine (4-AP).^{31 32} The outward current (I_sus) remaining at the end of 500-ms pulses was not blocked by 4-AP or BaCl₂. This is in agreement with an earlier report² that 5 mmol/L 4-AP had no effect on sustained current after complete inactivation of I_to1 in ventricular myocytes isolated from failing human hearts. The lack of effect of 4-AP indicates the absence of I_Kur in these cells. The nonlinear current-voltage relation of I_sus indicates that this current is not merely a leak current. We did not determine the ionic selectivity of this conductance. It is possible that I_sus is an undescribed nonselective cation current, a Cl^- current, or some combination of these and other small currents.

I_K is extremely small or absent in nonfailing human ventricular myocytes. Beuckelmann et al¹ also reported that I_K was absent in cells isolated from nonfailing human hearts. These findings are somewhat puzzling because several drugs known to specifically block one type of I_K (I_Kr) have been reported to lengthen action potential duration of isolated human papillary muscles in vitro³³ and prolong QT in patients, and both types of I_K (I_Kr and I_Ks) have been recorded from human atrial myocytes.³⁴ I_K was observed in human atrial myocytes even in the presence of 2 mmol/L CoCl₂ and 2 mmol/L 4-AP, used to block Ca²⁺ current and I_to2.³¹ However, a significant number of atrial myocytes (28% of total) were found to have a large I_to but no I_K. These myocytes were called type 3 cells to distinguish them from those that had large I_to and I_K (type 1 cells) or large I_K and no I_to1 (type 2 cells). With regard to the major types of repolarizing K⁺ currents, human ventricular myocytes are most like type 3 atrial cells characterized by Wang et al.³¹ As discussed previously,¹⁵ our myocyte isolation technique results in a relatively poor yield compared with the more commonly used retrograde perfusion of entire animal hearts. The most obvious potential explanation for the findings that I_K was not observed in isolated cells and yet drugs such as E-4031 prolong the action potential duration of cells recorded from intact human muscle is that I_K channels were somehow altered during tissue dissociation with protease. However, I_K is still present in guinea pig ventricular myocytes isolated from strips of tissue with the same procedures we used to dissociate cells from human tissue.¹⁵ Another unexplored possibility is that E-4031 and related methanesulfonanilide compounds lengthen cardiac action potentials by affecting a repolarizing current other than I_Kr. This is an unlikely possibility because these drugs were shown to block I_Kr specifically and have no effect on I_to1 or I_K1 in isolated human atrial myocytes.³⁴

Conclusions
We found only small or no differences in the magnitude and properties of I_K1 and I_to1 in LV subepicardial myocytes compared with cells isolated from the right interventricular septum. Previous studies clearly demonstrated that I_to1 of human LV subendocardial myocytes is significantly smaller than and is characterized by gating properties that are distinctly different from LV subepicardial myocytes. Future studies are needed to determine whether the right interventricular septum shares other electric properties with the LV subepicardium and whether such properties are important in the functional coupling between these two anatomic regions.

	Acknowledgments

 
This study was supported by a SCOR in Sudden Cardiac Death, HL-52338-01, from the NIH and grants from the American Heart Association, Utah Affiliate. Special gratitude is expressed to Drs Kay S.V. Karwande, Stephanie Olsen, Dale G. Renlund, Richard Stahl, and David Taylor for providing the cardiac biopsy tissues and to Dr William Barry for insightful discussions.

Received January 9, 1995; revision received March 9, 1995; accepted March 17, 1995.

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