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Circulation. 1995;92:465-473

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(Circulation. 1995;92:465-473.)
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Articles

Transient Outward Currents in Subendocardial Purkinje Myocytes Surviving in the Infarcted Heart

Cynthia Jeck, PhD; Judith Pinto, PhD; Penelope Boyden, PhD

From the Department of Pharmacology, Columbia College of Physicians and Surgeons, New York, NY.

Correspondence to Dr Penelope A. Boyden, Department of Pharmacology, Columbia College of Physicians and Surgeons, 630 W 168th St, New York, NY 10032.


*    Abstract
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 *Abstract
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Background Altered electrical activity of subendocardial Purkinje fibers contributes to arrhythmias in the 48-hour infarcted canine heart. Changes in the transmembrane action potentials of these fibers include marked action potential prolongation. The ionic basis for these changes is unknown.

Methods and Results We used whole-cell voltage-clamp techniques to study 4-aminopyridine (4-AP)–sensitive voltage-dependent transient outward currents (Ito1) in Purkinje myocytes isolated from LV subendocardial (n=14) and free-running (n=15) bundles of the normal canine heart. Ito1 in these two groups of control cells (normal-zone Purkinje cells [NZPCS]) did not differ. NZPCS Ito1 was then compared with Ito1 of Purkinje myocytes dispersed from subendocardium of infarcted hearts 48 hours after total coronary artery occlusion (IZPC48, n=14). Ito1 amplitude and current density were significantly reduced (P<.01) in IZPC48s (1650±389 pA, 9±2 pA/pF) compared with NZPCS (2917±267 pA, 20.2±2 pA/pF) at Vt=+55 mV, Vh=-60 mV, where Vt is test potential and Vh is holding potential. Decay of Ito1 was biexponential in all NZPCS but monoexponential in 71% of IZPC48s. Both NZPCS and IZPC48s have a sustained 4-AP–sensitive component (at 250 ms, Vt=+55 mV: 4±1 pA/pF, 3±1 pA/pF, respectively). Ito1 voltage dependence of inactivation did not differ between groups. In IZPC48s, recovery of Ito1 from inactivation was slowed significantly. Furthermore, significantly more Ito1 was seen with rapid pacing in NZPCS (cycle length [CL] 5000 ms=100%, CL 1300 ms=73%, CL 330 ms=46%) than in IZPC48s (CL 5000 ms=100%, CL 1300 ms=58%, CL 330 ms=31%). In three IZPC48s, no Ito1 was seen at CL 330 ms.

Conclusions Ito1 plays a major role in normal Purkinje myocyte electrophysiology, contributing both a large transient and a sustained component that are 4-AP–sensitive. In subendocardial Purkinje myocytes that survive in the 48-hour infarcted heart, density of Ito1 is markedly reduced and the remaining Ito1 showed specific changes in kinetics. The alterations observed in both Ito1 density and function could contribute to abnormally long transmembrane action potentials of these arrhythmogenic Purkinje myocytes of the infarcted heart.


Key Words: potassium channels • ions • Purkinje • myocytes • myocardial infarction


*    Introduction
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 up arrowAbstract
 *Introduction
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down arrowDiscussion
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A voltage-gated transient outward potassium current (Ito1) has been identified in several cell types, including cardiac cells. Studies on myocytes dispersed from normal hearts have shown that Ito1 is a major determinant of action potential duration and thus refractoriness. For example, in adult canine, rabbit, and feline epicardial cells, Ito1 is prominent, whereas it is less so in endocardial myocytes.1 2 3 Ito1 has also been found in Purkinje,4 5 human atrial, and ventricular myocytes.6 7 8 9 The density and kinetics of the channel that underlies whole-cell Ito1 currents appear to be intricately linked to the age of the animal10 11 and to the underlying disease process. In this latter category, it has now been established that the density and kinetics of the Ito1 channel are decreased in human ventricular myocytes dispersed from failing hearts,12 13 in cells dispersed from dilated human atria (secondary to disease),14 in hypertrophied rat myocytes,15 16 and in myocytes from animals with acromegaly17 or with experimentally induced diabetes18 19 20 and increased in hypertrophied feline myocytes.21 Finally, data from our laboratory have shown that a reduction in the density of the 4-aminopyridine (4-AP)–sensitive Ito1 underlies the loss in rapid phase 1 and notch in the transmembrane action potential of the epicardial myocyte dispersed from the infarcted heart 5 days after total coronary artery occlusion.22 These myocytes lie within the epicardial border zone of the canine heart, the site at which serious reentrant ventricular arrhythmias have been shown to occur.23 24

During the acute phase of arrhythmias in this latter animal model of myocardial infarction, the subendocardial Purkinje fibers that survive 24 to 48 hours after occlusion are the source of multiform ventricular arrhythmias.25 26 27 In particular, the surviving fibers have abnormally long action potential durations. Interestingly, there is still a small component of the rapid phase 1 of repolarization of the action potentials if fibers are driven at slow drive rates. However, although pacing at fast rates causes little or no change in phase 1 of repolarization in normal Purkinje fibers, it has a dramatic effect on the time course of repolarization of subendocardial Purkinje myocytes surviving in the infarcted heart. In some cases, with an increase in drive rate, the rapid phase 1 of repolarization of action potentials in these fibers disappears (see Fig 3Down.30 of Reference 28).



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  		Figure 3. 4-Aminopyridine–sensitive current density–voltage relations are shown for both peak Ito1 (A) and Isus (B) from normal-zone Purkinje cells (n=29) (open circles) and subendocardial Purkinje cells from the infarcted heart 48 hours after coronary artery occlusion (IZPC48s, n=14) (solid circles). The density of peak Ito1 differed significantly between the control and infarcted cells for all Vt >-10 mV. There was no significant difference in Isus density between control and infarcted cells.

We previously described a preparation of single subendocardial Purkinje myocytes isolated from the 24- and 48-hour infarcted myocardium.29 30 Using fine-tipped microelectrodes, we showed that both the total time course of repolarization and the process of action potential restitution are abnormal in these myocytes.29 Further, we determined that altered function of the Na-K pump does not underlie these action potential abnormalities.31 On the other hand, we showed that both types of Ca2+ currents are reduced in density in the Purkinje myocytes dispersed from the 48-hour infarcted heart.30

We hypothesize that the density of the transient outward current in these Purkinje myocytes that survive in the infarcted heart may also be decreased. Furthermore, on the basis of action potential recordings, we speculate that the well-known rate dependence and kinetics of restitution of Ito1 are altered in these cells. Therefore, in the present study, we recorded and compared whole-cell Ito1 from control single Purkinje cells (SPCs) dispersed from free-running fiber bundles, from the subendocardium of the left ventricle (LV) (normal-zone Purkinje cells [NZPCs]) of noninfarcted hearts, and from subendocardium of the LV 48 hours after total coronary artery occlusion (infarcted-zone Purkinje cells at 48 hours [IZPC48]). In this way, we determined the function of Ito1 in arrhythmogenic Purkinje cells that survived in the infarcted heart.


*    Methods
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 *Methods
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Cell Preparation
Healthy male mongrel dogs (12 to 15 kg, 1 to 2 years old) were used in these studies. Surgical production of myocardial infarction was done according to the Harris procedure32 under pentobarbital anesthesia (30 mg/kg IV). With this method, the left anterior descending branch of the coronary artery was isolated at a site approximately 0.5 to 1 cm from the tip of the appendage and then occluded permanently by the two-stage ligation technique. At the time of the second ligature, lidocaine (2 mg/kg IV) was given if multiple ventricular beats occurred. The chest was closed, and no further antiarrhythmic agents were administered. Seven dogs that survived this procedure were used for cell study 48 hours after the coronary artery occlusion (IZPC48).

In addition, 19 dogs that had not undergone any previous surgery served as controls. This larger group was split into two smaller groups. Some hearts were used to prepare single cells from free-running Purkinje fiber bundles (SPCs, n=12), and those remaining were used to prepare subendocardial Purkinje cells from noninfarcted LV myocardium (NZPCs, n=7).

The enzymatic technique used to disaggregate Purkinje myocytes from control and infarcted hearts has been described.29 31 33 In brief, the technique used to disaggregate the subendocardial Purkinje cells from control noninfarcted hearts and from infarcted hearts is the same except for the initial dissection. In both cases, small strips (4x2x2 mm) of LV endocardium containing longitudinally oriented Purkinje fiber bundles were carefully dissected from larger preparations removed from specific regions in the control heart and in the infarcted heart. The specific endocardial regions were identified carefully with a dissection microscope. All small strips prepared from the infarcted hearts were taken from the subendocardium overlying the infarcted portion of the LV. These sections were identified grossly by their pale color.25 After 8 to 10 of these ministrips were prepared, the fiber bundles were subjected to our general method for enzymatic disaggregation of Purkinje fibers.33 No tissue from these infarcted hearts was taken from the noninfarcted regions of the LV. Marked cellular electrical changes are absent in these tissues.27

Most single-cell studies can be faulted in that a process of selection is integral to the experiments, and our experiments are no exception. After enzyme incubation and the dispersion protocol, viable SPCs and NZPCs were routinely obtained. Unlike suspensions of SPCs, cell suspensions of NZPCs included ventricular cells, but most of the ventricular cells do not survive the cell disaggregation procedure and appear as rounded myocytes in a Ca2+-containing solution. The identification of healthy NZPCs was based on our studies of the SPCs.33 Cells possessed fine finger-like processes at either end and did not possess "blebs." Our previous electron microscopic studies of NZPCs confirmed that the healthy cells surviving the disaggregation procedures have structural characteristics of Purkinje cells and characteristics similar to SPCs.29

Cell suspensions from the subendocardium of infarcted myocardium included IZPC48s and ventricular cells. However, often the ventricular cells appear as "ghosts," which is understandable, because histological studies of ventricular muscle in the 48-hour infarct show most ventricular muscle cells to be dead.25 IZPC48s were large, mostly rod-shaped, cells and under the light microscope, cells had slightly corrugated membranes and appeared to be filled with black dots.

Experimental Protocol
For study, cells were allowed to adhere to a polylysine-coated glass coverslip placed at the bottom of a 0.7-mL tissue chamber mounted on the stage of a Nikon inverted microscope and continuously superfused with Tyrode's solution. The Tyrode's solution had the following composition (mmol/L): NaCl 137, NaHCO3 24, dextrose 5.5, NaH2PO4 1.8, MgCl2 0.5, KCl 4, and CaCl2 2. The solution was equilibrated with 5% CO2/95% O2, maintained at pH 7.2, and superfused at a rate of 2 to 3 mL/min (35°C to 36°C).

Whole-cell Ito1 channel currents were recorded by patch-clamp techniques. Pipettes were made of borosilicate glass (Sutter Instruments) and pulled by a two-stage puller (Sutter). All pipettes were heat-polished before use and filled with the following internal solution (mmol/L): KCl 130, HEPES 5, ATP (potassium salt) 5, MgCl2 5, EGTA 10, and creatinine phosphate 5 (pH adjusted to 7.3 with KOH). The tip resistances of filled pipettes ranged between 0.5 and 2 M{Omega}. The amplifier was set to zero before the seal was made and was checked at the completion of the study of the clamped myocyte. If marked changes had occurred, data were not included. After the formation of the gigaohm seal (>5 G{Omega}), pipette capacitance was electronically compensated before membrane rupture. In the whole-cell configuration, series resistance was compensated nearly 100%. No corrections were made for leak currents or junction potentials. Estimated size of the junction potential was 3 to 5 mV for solutions used. No compensation was made for whole-cell capacitance.

After membrane rupture, an interval of 3 to 5 minutes was allowed for internal dialysis to begin, and then the external solution was changed to a Na+- and K+-containing external solution that had the following composition (mmol/L): NaCl 140, KCl 4, dextrose 5.5, MgCl2 0.5, HEPES 10, and CaCl2 1.8 (pH 7.3). This solution also contained CdCl2 (0.5 mmol/L) to block any contaminating inward currents carried by Ca2+ ions. Although Cd2+ ions have been demonstrated to cause a positive shift in the voltage dependence of activation of IK,34 we chose to study both cell types using identical recording conditions in which K+ currents through Ito1 channels were "isolated" from other contaminating membrane currents. Shifts due to the presence of Cd2+ ions have not been compensated.

In preliminary studies, we tested whether a low concentration of 4-AP could be used as a tool to differentiate and quantify the 4-AP–sensitive transient outward current (Ito1) from a 4-AP–sensitive sustained outward current (Isus) in Purkinje myocytes. Recent data obtained from human atrial myocytes suggest that Isus is due to a rapidly activating IK current that is more sensitive to 4-AP (IC50=49 µmol/L) than the transient component.35 Unfortunately, although in canine Purkinje myocytes Isus is sensitive to low concentrations of 4-AP (50 to 100 µmol/L), so is the transient component (Ito1) (see "Results"). Therefore, for comparison of currents in the different cell types, we determined the amplitude of the peak Ito1 and Isus from 4-AP–sensitive currents (2 mmol/L). For peak Ito1, the difference between the amplitude of the peak transient component and the current at the end of the clamp pulse was measured. For Isus, the amplitude of the 4-AP difference current remaining at the end of the test pulse relative to zero current was measured.

Data Acquisition and Analysis
Voltage-clamp experiments were performed with an Axopatch 1D patch-clamp amplifier (headstage 0.1/100, Axon Instruments). Clamp protocols were generated by a 12-bit digital-to-analog converter (Axon Instruments) controlled by PCLAMP software and a Gateway 2000 computer. All currents elicited were filtered at 2 kHz, digitized at 50- to 100-µs intervals, and stored for later analysis. Membrane capacitance of each cell was measured by integrating the area beneath the capacitive transient and dividing that by the voltage step. Current data could then be expressed as current density (pA/pF) by normalizing each current value by the cell's capacitive value.

The time course of decay of the transient current during depolarization was analyzed by fitting current data with a single or double exponential function (CLAMPFIT). For determination of time course of restitution of peak Ito1, curve fitting was done with a Simplex algorithm.36

Voltage dependence of Ito1 and Isus steady-state inactivation was determined with a double-pulse protocol. In this protocol, a 1000-ms conditioning prepulse from a holding potential (Vh) (-60 mV) to various conditioning potentials (-80 to +40 mV) was followed by a brief return to Vh, then by a 250-ms test pulse to +50 mV. Data were normalized by dividing the test current peak by the maximal current elicited. The normalized inactivation-voltage relation was determined for each cell by fitting the data by the Boltzmann function to obtain V0.5 and k values. All values are represented as mean±SEM unless otherwise noted. To determine the significance of differences, an ANOVA was used followed by Bonferroni's test for multiple comparisons.37 For all data, a difference was significant when P<.05.


*    Results
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*Results
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SPCs and NZPCs
In the first series of experiments, we determined whether a low concentration of 4-AP could be used to separate, and thus quantify, Isus from Ito1 in cells dispersed from noninfarcted hearts (cells from free-running fiber bundles [SPCs] and cells from LV subendocardium [NZPCs]). Therefore, in three cells, 50 to 100 µmol/L 4-AP difference currents were obtained. Results from a typical response in a NZPC are shown in Fig 1Down. A low concentration of 4-AP blocks both a sustained and a transient component in Purkinje myocytes (Fig 1DDown). Importantly, this result is different from data presented for human atrial cells35 but is consistent with the observations previously made during 4-AP superfusion of sheep Purkinje fibers.38 Therefore, for all subsequent results in Purkinje cells dispersed from both noninfarcted and infarcted hearts, we obtained 4-AP–sensitive currents using 2 mmol/L 4-AP (Fig 1EDown).



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  		Figure 1. Current traces from a Purkinje myocyte from a control heart. The cell was held at Vh=-60 mV and clamped in 10-mV increments to various test potentials. Traces were first obtained in control solutions (A), followed by solutions containing 50 µmol/L 4-aminopyridine (4-AP) (B) and 2 mmol/L 4-AP (C). Currents sensitive to 50 µmol/L 4-AP (D) and 2 mmol/L 4-AP (E) were then obtained. As seen in the figure, both transient and sustained components of outward current are revealed with 4-AP. Cell capacitance=136 pF.

In both SPCs and NZPCs, 4-AP difference currents obtained by clamping the cell to various test voltages (Vt) (-35 to +55 mV) from a holding potential of -60 mV had a large transient outward component and a sustained component at the end of clamp pulse (250 ms). Since it has been observed that the density of Ito1 can vary depending on the site of the myocyte within the ventricle, we first compared the characteristics of Ito1 and Isus in Purkinje myocytes dispersed from the two different sites within the noninfarcted control hearts. Fifteen Purkinje myocytes were dispersed from free-running bundles (SPCs) and 14 myocytes from the LV subendocardium of the noninfarcted ventricle (NZPCs). Neither the average amplitude nor density of the 4-AP Ito1 or Isus differed in these two cell groups (Fig 2Down; TableDown). In addition, the time constant of decay of Ito1 at Vt=+55 mV in SPCs ({tau}1=9.2±0.6 ms, {tau}2=78.8±10.8 ms) was no different from that in NZPCs ({tau}1=8.3±0.9 ms, {tau}2=73.1±11.9 ms). Thus, for further comparisons, we pooled data obtained from these control cells (NZPCS).



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  		Figure 2. Plots of 4-aminopyridine–sensitive current density–voltage relations for both peak Ito1 (A) and Isus (B) from the two types of control cells used for this study (see "Methods"). Current density of the single Purkinje cells (SPCs) (n=15) is represented by the solid circles, and density of normal-zone Purkinje cells (NZPCs) (n=14) by the open circles. There is no significant difference in density between the two cell types for either current component. Average cell capacitance: SPCs=159 pF; NZPCs=144 pF.


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  		Table 1. Amplitude and Density of 4-Aminopyridine–Sensitive Currents

NZPCS and IZPC48s
In the cells selected for study, there was no difference in the average cell capacitance in the two cell types (NZPCS, 151.3±12.8 pF, n=29; IZPC48, 193±24 pF, n=14). However, the amplitude and density of peak Ito1 elicited with depolarizing clamp steps (Vt) differed (Fig 3AUp; TableUp). In all NZPCS, a prominent 4-AP–sensitive Ito1 was recorded (at +55 mV, 20±2 pA/pF). In Purkinje myocytes dispersed from the infarcted heart, both the average amplitude and density of peak Ito1 were significantly reduced for Vt>-5 mV. Although there was a slight reduction in Isus in IZPC48s (Fig 3BUp), the average densities were not different in the two cell groups (TableUp).

Next we determined whether Ito1 in IZPC48s decayed in a similar fashion during maintained depolarizing steps. For all NZPCS studied (n=28), decay of peak Ito1 (Vt=+55 mV) was best fit by a double exponential function ({tau}1=8.7±0.5 ms, {tau}2=76±7.89 ms, where A2/Atotal=37%) (Fig 4Down). This is in agreement with double exponential decay of Ito1 analyzed in canine Purkinje myocytes4 and other cell types.39 40 For 4 of 14 IZPC48s, peak Ito1 was also best fit by double exponential function ({tau}1=6.57±0.38 ms, {tau}2=50.8±15.5 ms, P>.05, A2/Atotal=30%) and was not different from control. In this subset of IZPC48s, the average density of Ito1 at Vt=+55 mV was 19 pA/pF. However, in 10 other IZPC48s, in which density in each cell was <9 pA/pF (average, 5.2 pA/pF), decay of Ito1 was best described by a monoexponential function ({tau}1=12.09±1.16 ms) (Fig 4Down).



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  		Figure 4. Tracings showing decay of the Ito1 during a maintained depolarizing pulse (Vh=-60 mV). Top, Actual current trace from a normal-zone Purkinje cell (NZPC) (Vt=+55 mV) fit to a double exponential equation (solid line) ({tau}1=10.4 ms, {tau}2=97.2 ms). Subendocardial Purkinje cell from the infarcted heart 48. hours after coronary artery occlusion (IZPC48) trace (Vt=+55 mV) fit well to a single exponential equation (middle) ({tau}1=12.4 ms) and was poorly fit by a double exponential equation (bottom) (fit user terminated). See text for more detail.

Steady-state availability curves were determined for each cell in the two groups to determine whether the decrease in density of Ito1 in IZPC48s was secondary to an altered inactivation process. There was no significant difference in average V0.5 or k for peak Ito1 in NZPCS (V0.5=-19±1.8 mV, k=8.6±0.5, n=20) versus IZPC48s (V0.5=-22.83±2.4 mV, k=7.6±0.6, n=12) (P>.05), yet there was a significant decrease in the maximally available peak Ito1 (at Vc=-80 mV) (NZPCS=14.4±1.1 pA/pF versus IZPC48s=7.1±2.3 pA/pF, P<.05) (Fig 5Down). Note that when 4-AP difference currents were obtained in a cell undergoing this protocol, not only a transient but also a sustained outward current existed that inactivated with conditioning prepulse (Fig 5CDown). The inactivation process of Isus in IZPC48s appeared to be no different from control. Average Boltzmann data for Isus from NZPCS (n=13) were V0.5=-19.5±2.8 mV, k=12.7±1.1 mV and from IZPC48s (n=8) were V0.5=-22.4±5.5 mV, k=10.9±1.8 mV. Moreover, in both NZPCS and IZPC48s, a 4-AP–sensitive noninactivating outward current also existed even after a prepulse step had inactivated Isus and Ito1 (see asterisk in Fig 5CDown).



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  		Figure 5. Actual current traces from a subendocardial Purkinje cell from the infarcted heart 48 hours after coronary artery occlusion (IZPC48, IZ48) illustrating the voltage dependence of inactivation of Ito1 and part of Isus. Tracings of current after several conditioning prepotentials (Vc) are shown in A through C. 4-Aminopyridine (4-AP)–sensitive current traces (C) were obtained by subtracting currents obtained in the presence of 4-AP (B) from control recordings (A). Ito1 and Isus currents are reduced as a function of prepulse voltage. Note also the presence of a 4-AP–sensitive noninactivating current (*). The normalized inactivation-voltage relation was determined for each cell by fitting the data by use of the Boltzmann function. Average Boltzmann data for peak Ito1 from normal-zone Purkinje cells (NZ) (V0.5=-19.4 mV, k=8.6 mV, n=20) and IZPC48s (V0.5=-22.8 mV, k=7.6, n=12) are plotted in D.

The time-dependent recovery of the 4-AP–sensitive transient current has been well described for normal Purkinje myocytes.4 Our inability to record large transient currents in IZPC48s may be due to an alteration in this mechanism of recovery from inactivation. Therefore, the time course of recovery of Ito1 was studied in cells from each of the two groups with two types of protocols. In the first, a typical double-pulse protocol was used. Two depolarizing clamp pulses (Vh=-65 mV to Vt=+60 mV) with varying interpulse intervals (IPIs) were applied every 8 seconds. IPIs ranged from 5 to 5000 ms. The peak Ito1 amplitude was obtained at each IPI and then normalized to peak Ito1 (at IPI=5000 ms) (Fig 6Down). For 14 NZPCS (average peak Ito1=18.3 pA/pF), Ito1 recovery was best described by a biexponential function ({tau}1=109±8.6 ms, {tau}2=1232±95 ms, A2/Atotal=63%). The time course of recovery from inactivation of Ito1 was determined in a total of 12 IZPC48s (average peak Ito1=9.9 pA/pF). In 7 IZPC48s, Ito1 recovery was also best described by a biexponential function ({tau}1=238±40, {tau}2=1853±198 ms), but both time constants of recovery were significantly greater than control (P<.005). In 2 other IZPC48s (peak Ito1=6 and 8 pA/pF), recovery from inactivation was best described by a monoexponential function ({tau}1=1681 and 870 ms, respectively) after a delay (delay, 179 and 135 ms). In these latter 2 cells, it appears that the fast component of recovery was lost (eg, see Fig 6ADown). In an additional 3 IZPC48s, Ito1 was too small to accurately quantify the recovery process.



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  		Figure 6. A, Superimposed current traces from normal-zone Purkinje cells (NZPC) (top trace) and subendocardial Purkinje cells from the infarcted heart 48 hours after coronary artery occlusion (IZPC48) (bottom trace) of second depolarizing clamp step (Vh=-65 to Vt=+60 mV) at selected interpulse intervals during recovery of Ito1 from inactivation protocols. In both cases, first depolarizing step was Vh=-65 to Vt=+60 mV, 500 ms. For the NZPC shown (20 pA/pF), time course of recovery was best described by a biexponential function ({tau}1=109 ms, {tau}2=1208 ms). For the IZPC48 shown (peak Ito1=6 pA/pF), time course of recovery was described by a single exponential function after a delay ({tau}1=870 ms). Note that for this IZPC48, for the two interpulse intervals illustrated (25 and 50 ms), no recovery of Ito1 had occurred. Horizontal calibration line represents 24 ms, and vertical line represents 1000 pA (top) and 200 pA (bottom). B, Average values obtained at each interpulse interval (IPI) from cells from NZPCS (solid circles) and IZPC48s (inverted triangles). Inset, Average relation for the two groups for IPI 5 to 1000 ms. See text for more detail.

In a second series of experiments, the interval dependence of peak Ito1 in IZPC48s was compared with that in NZPCS. For these experiments, clamp steps (Vh=-60 to +50 mV) were given repeatedly at three different cycle lengths (CL) (5000, 1300, and 330 ms). The effects of pacing CL on the 4-AP–sensitive currents in a cell from each of the two groups are illustrated in Fig 7Down. In all control Purkinje myocytes (n=22), a decrease in CL resulted in reduction in peak Ito1 (to 73±0.02% of current at CL=5000 ms for CL=1300 ms and to 46±0.03% for CL=330 ms). Thus, even with a rapid clamp-pacing rate, significant Ito1 remained in control cells. This is consistent with the persistence of the rapid phase 1 of repolarization in normal Purkinje myocytes paced at rapid rates.29 In IZPC48s (n=13), the change in pacing CL produced a more significant effect on the peak Ito1. There was an average of 58±0.05% reduction of the peak with CL=1300, and with pacing at 330 ms, peak currents were reduced to 31±0.06% of control (n=8). Importantly, no peak Ito1 could be recorded in 5 IZPC48s (at Vt=+55 mV, average peak Ito1=5.4 pA/pF) paced at the most rapid rate (for example, see Fig 7Down). Interestingly, the 4-AP–sensitive noninactivating current component observed in both cell types was not rate dependent and remained even after a period of rapid pacing (see asterisks in Fig 7Down).



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  		Figure 7. Traces showing effects of altering clamp-pacing cycle length on 4-aminopyridine (4-AP)–sensitive currents in normal-zone Purkinje cells (NZPCS) (left). Tracings obtained during 20 consecutive beats have been superimposed. Note that with the short pacing cycle length (330 ms), a reasonable Ito1 remains after 20 beats. Right, Effects of altering clamp-pacing cycle length on a subendocardial Purkinje cell from the infarcted heart 48 hours after coronary artery occlusion (IZPC48) are shown during control, during 4-AP, and as difference currents. Note the significant decrease in Ito1 with rapid pacing (330 ms). Also note the existence of a non–rate dependent, noninactivating 4-AP–sensitive current (*). See text for more detail. Small vertical bar at asterisk indicates size of 4-AP–sensitive current that is not rate dependent.


*    Discussion
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*Discussion
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The results from these experiments demonstrate that there are significant differences in the density and kinetics of the 4-AP–sensitive transient outward currents in subendocardial Purkinje myocytes that survive 48 hours after total coronary artery occlusion.

4-AP–Sensitive Currents in Purkinje Myocytes From Noninfarcted Hearts
We have shown that in control Purkinje cells with depolarizing clamp steps, both a voltage-dependent transient and a sustained outward current occur and are sensitive to 4-AP. However, unlike the transient and sustained outward currents studied in human atrial myocytes,35 Isus of control Purkinje myocytes could not be distinguished from Ito1 in the same cell on the basis of voltage dependence of inactivation or 4-AP sensitivity. In fact, we found that low concentrations of 4-AP (50 µmol/L) produced partial block of both Isus and Ito1 (Fig 1Up). Therefore, as described in the "Methods," we chose to use 4-AP at a high concentration (2 mmol/L) in this study. This concentration resulted in complete block of currents studied (Fig 1Up).

4-AP–sensitive currents in Purkinje myocytes dispersed from free-running fiber bundles and the subendocardium of the LV do not differ. In both types of control cells, these currents are large and would most likely make a significant contribution to total time course of repolarization, as suggested by other studies. Peak Ito1 and Isus 4-AP–sensitive current-voltage relations of control cells (NZPCS) in this study are similar to those previously reported for normal Purkinje myocytes.4 The decay of peak Ito1 currents in control cells was fit by a double exponential function similar to those reported for Purkinje myocytes4 41 and rabbit cells.39 40 The transient component shows a reasonably rapid decay and a biphasic recovery from inactivation such that significant transient and sustained currents exist even during rapid-pacing protocols (see Fig 7Up). This is consistent with the observation that in the action potentials of Purkinje fibers or myocytes during rapid pacing, a large phase 1 of repolarization remains. This is unlike that described for normal myocytes dispersed from the canine midwall42 and epicardium11 but similar to observations made in human myocytes.8 43

During the clamp steps chosen to be used in this study, a considerable steady noninactivating component of outward current persisted in the normal Purkinje myocyte even after the peak component had inactivated. This current, Isus, was 4-AP sensitive, partially inactivated with prepulse potential, and partially reduced in size with rapid pacing. A sustained depolarization-induced outward current has been observed in both human atrial and ventricular myocytes.12 35

4-AP–Sensitive Currents in Subendocardial Purkinje Myocytes That Survive in the 48-Hour Infarcted Heart
Unlike our study on epicardial myocytes that survive in the infarcted heart 5 days after coronary artery occlusion,22 a 4-AP–sensitive transient outward current was observed in every subendocardial Purkinje myocyte studied from a 48-hour infarcted heart. However, the density and kinetics of the transient component of these 4-AP–sensitive currents in IZPC48s differed dramatically from control.

Our finding of reduced density of peak Ito1 in IZPC48s cannot be accounted for by a shift in the availability of the current, since availability curves did not differ, whereas maximally available Ito1 did. It is unlikely that the disaggregation procedure is the cause of the loss in ion channel function observed, for several reasons. First, 4-AP–sensitive currents observed in both SPCs and NZPCs from control noninfarcted hearts were similar in density and kinetics to previously published transient outward current data from Purkinje myocytes.4 Furthermore, we chose to isolate and compare 4-AP–sensitive currents using patch-clamp techniques with intracellular dialysis. Therefore, all cells as studied were compared under identical whole-cell conditions. This suggests, then, that our findings are due to chronic changes in ion channel density as well as function (kinetics) occurring secondary to disease rather than due to changes in function secondary to acute ischemia.

Interestingly, both NZPCS and IZPC48s exhibited another voltage-dependent, 4-AP–sensitive steady outward current that was noninactivating and not rate dependent (eg, see Fig 7Up). This type of 4-AP–sensitive current is consistent with the reported effects of 4-AP on sustained "plateau" currents in sheep Purkinje fibers.38

In addition to the observed significant decrease in density, Ito1 in IZPC48s showed specific kinetic changes. In most IZPC48s (10 of 14), the slower component of current decay during positive clamp steps was not observed, whereas it was seen in every control Purkinje cell, contributing 37% of total amplitude of peak current. This variability in our finding in IZPCs is not surprising, because of the nature of studying changes in ionic currents in cells from the infarcted heart. What we can say is that the loss of this second component of current decay occurred in IZPC48s in which peak Ito1 was dramatically reduced (<9 pA/pF; average, 5.2 pA/pF).

On the basis of their work with isoproterenol on Ito1 in normal Purkinje myocytes, Nakayama and Fozzard4 suggest that the two components of macroscopic Ito1 current decay may result from two populations of Ito1 channels with different inactivated states. In their scheme, under basal conditions, the slow-to-inactivate channels were phosphorylated, whereas dephosphorylated channels had rapid kinetics.4 A possible corollary to this may be that in IZPC48s, in which kinetics of Ito1 decay are fast and lack a second component, Ito1 channels in cells that have survived in the infarcted heart are dephosphorylated.

On the other hand, although direct evidence is lacking regarding the importance of various protein kinases (eg, PKA, PKC) or phosphatases in alteration of the level of phosphorylation of the Ito1 channel in normal Purkinje myocytes, recent studies using either native or cloned K+ channels have suggested that fast-inactivating K+ channels (presumably those inactivated by N-type inactivation mechanism) respond to both PKC and PKA stimulation.44 45 46 47 48 49 In an even more recent study using Shaker K channels, N-type inactivation appears to be accelerated by direct application of the catalytic subunit of PKA.50

Additionally, in all IZPC48s studied, we found a significant increase in the time course of recovery of Ito1 as well as a change in the rate dependence of Ito1. This slowing of recovery implies that less outward current would be available during action potentials occurring at 60 beats per minute or faster. As we have shown, a reasonable amount of Ito1 remains in control Purkinje cells even with rapid pacing (Fig 7Up). However, in IZPC48s, less transient outward current was observed, especially at the more rapid rate of clamp steps. The effects of changing the level of phosphorylation on the recovery kinetics of Ito1 in normal Purkinje myocytes have not been studied.

The molecular mechanisms that could lead to reduced macroscopic Ito1 currents in IZPC48s remain speculative at this time. The observed reduction in whole-cell current may result from a change in channel function (eg, microscopic activation or inactivation) and/or a reduction in the number of channels. For instance, a smaller whole-cell Ito1 would be consistent with a population of Ito1 channels that are faster in inactivation with or without a slowing in activation. A reduction in the actual number of Ito1 channel proteins is one possibility that may account for a portion of the reduced density of the macroscopic Ito1 current in IZPC48s. Whether this occurs because of a change in the rate of transcription or translation of mRNA that encodes the channel protein subunits needed for a fully functioning K+ channel or by some other mechanism is not known at this time. The regulation of K+ channel expression after ischemia/infarction has not been determined. If the number of Ito1 channels decreased and new channels were not synthesized, then the remaining channels would generate less total whole-cell Ito1 current. However, if only the number of channels were to decrease, then the reduced currents should have kinetics similar to control, as has been suggested for diminished Ito1 found in rat hypertrophied cells.16 In studies presented here, we have identified both a reduction in density and alteration in kinetics of whole-cell Ito1 in IZPC48s. This suggests that the ischemic insult may alter the total number as well as the kinetics of Ito1 channels. Whether these latter Ito1 channels are preischemic channel proteins that have been modified or new channel proteins exhibiting different whole-cell kinetics is not known at this time.

In conclusion, the arrhythmogenic subendocardial Purkinje fibers that survive in the 48-hour infarcted heart have abnormally long transmembrane action potentials. Both a reduction in the density of iCaL30 and Ito1 (this study) could underlie these electrical changes.


*    Acknowledgments
 
This study was supported by grants HL-34477 and HL-07271 from the National Heart, Lung, and Blood Institute, Bethesda, Md.

Received November 1, 1994; revision received January 11, 1995; accepted January 22, 1995.


*    References
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*References
 

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