| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Circulation. 1995;92:465-473.)
© 1995 American Heart Association, Inc.
Articles |
Transient Outward Currents in Subendocardial Purkinje Myocytes Surviving in the Infarcted Heart
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 |
|---|
 Top Abstract IntroductionMethods Results Discussion References |
|---|
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 |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
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 3
.30 of
Reference
28).
|
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 |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
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
. 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), 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 |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
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 1
. A low
concentration of 4-AP blocks both a sustained and a transient component
in Purkinje myocytes (Fig 1D). 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 1E).
|
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 2;
Table). In addition, the time constant of
decay of Ito1 at Vt=+55 mV in SPCs
(
1=9.2±0.6 ms,
2=78.8±10.8 ms) was no
different from that in NZPCs (1=8.3±0.9 ms,
2=73.1±11.9 ms). Thus, for further comparisons,
we
pooled data obtained from these control cells (NZPCS).
|
|
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 3A
;
Table). 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 3B), the average
densities
were not different in the two cell groups (Table).
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
(1=8.7±0.5 ms,
2=76±7.89 ms, where
A2/Atotal=37%) (Fig 4).
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
(1=6.57±0.38 ms,
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
(1=12.09±1.16 ms) (Fig 4).
|
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
5). 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 5C). 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 5C).
|
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 6). For
14 NZPCS (average peak Ito1=18.3 pA/pF), Ito1
recovery was best described by a biexponential function
(1=109±8.6 ms,
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
(1=238±40,
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 (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 6A). In an
additional
3 IZPC48s, Ito1 was too small to accurately
quantify the recovery process.
|
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
7. 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 7). 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 7).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
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
1).
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 1).
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
7). 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 7).
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
7).
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 |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
1. Furukawa T, Myerburg RJ, Furukawa N, Bassett AL, Kimura S. Differences in transient outward currents of feline endocardial and epicardial myocytes. Circ Res. 1990;67:1287-1291.
2. Antzelevitch C, Litovsky SH, Lukas A. Epicardium versus endocardium: electrophysiology and pharmacology. In: Zipes DP, Jalife J, eds. Cardiac Electrophysiology: From Cell to Bedside. Philadelphia, Pa: WB Saunders; 1990.
3.
Fedida D, Giles WR. Regional variations in
action potentials and transient outward current in myocytes isolated
from rabbit left ventricle. J Physiol (Lond). 1991;442:191-209.
4.
Nakayama T, Fozzard HA. Adrenergic modulation
of the transient outward current in isolated canine Purkinje
cells. Circ Res. 1988;62:162-172.
5. Coraboeuf E, Carmeliet E. Existence of two transient outward currents in sheep Purkinje fibers. Pflugers Arch. 1982;392:352-359. [Medline] [Order article via Infotrieve]
6.
Shibata EF, Drury T, Refsum H, Aldrete V, Giles W.
Contributions of a transient outward current to repolarization
in human atrium. Am J Physiol. 1989;257:H1773-H1781.
7. Escande D, Loisance D, Planche C, Coraboeuf E. Age-related changes of action potential plateau shape in isolated human atrial fibers. Am J Physiol. 1985;249:H843-H850.
8.
Nabauer M, Beuckelmann DJ, Erdmann E.
Characteristics of transient outward current in human
ventricular myocytes from patients with terminal heart failure.
Circ Res. 1993;73:386-394.
9. Konarzewska H, Peeters G, Karwande K, Sanguinetti M. Comparison of K currents from human epicardial and endocardial myocytes. Biophys J. 1994;66:A329. Abstract.
10.
Kilborn MJ, Fedida D. A study of the
developmental changes in outward currents of rat ventricular
myocytes. J Physiol (Lond). 1990;430:37-60.
11.
Jeck CD, Boyden PA. Age related appearance of
outward currents may contribute to developmental differences in
ventricular repolarization. Circ Res. 1992;71:1390-1403.
12.
Beuckelmann DJ, Nabauer M, Erdmann E.
Alterations of K+ currents in isolated human
ventricular myocytes from patients with terminal heart failure.
Circ Res. 1993;73:379-385.
13.
Wettwer E, Amos GJ, Posival H, Ravens U.
Transient outward current in human ventricular myocytes of
subepicardial and subendocardial origin. Circ
Res. 1994;75:473-482.
14.
Mansourati J, LeGrand B. Transient outward
current in young and adult diseased human atria. Am J
Physiol. 1993;265:H1466-H1470.
15. Coulombe A, Momtaz A, Richer P, Swynghedauw B, Coraboeuf E. Reduction of calcium independent outward potassium current density in DOCA salt hypertrophied rat ventricular myocytes. Pflugers Arch. 1994;427:47-55. [Medline] [Order article via Infotrieve]
16.
Tomita F, Bassett AL, Myerburg RJ, Kimura S.
Diminished transient outward currents in rat hypertrophied
ventricular myocytes. Circ Res. 1994;75:296-303.
17.
Xu X, Best PM. Decreased transient outward K
current in ventricular myocytes from acromegalic rats. Am
J Physiol. 1991;260:H935-H942.
18. Jourdon P, Feuvray D. Diabetes induced decreases in outward potassium currents in rat ventricular myocytes. J Mol Cell Cardiol. 1992;24:SVS178. Abstract.
19.
Shimoni Y, Firek L, Severson D, Giles W.
Short-term diabetes alters K+ currents in rat
ventricular myocytes. Circ Res. 1994;74:620-628.
20. Magyar J, Rusznak Z, Szentesi P, Szucs G, Kovacs L. Action potentials and potassium currents in rat ventricular muscle during experimental diabetes. J Mol Cell Cardiol. 1992;24:841-853. [Medline] [Order article via Infotrieve]
21. TenEick RE, Zhang K, Harvey RD, Bassett AL. Enhanced functional expression of transient outward current in hypertrophied feline myocytes. Cardiovasc Drugs Ther. 1993;7:611-619.
22.
Lue W-M, Boyden PA. Abnormal electrical
properties of myocytes from chronically infarcted canine heart:
alterations in Vmax and the transient outward
current. Circulation. 1992;85:1175-1188.
23.
Dillon S, Allessie MA, Ursell PC, Wit AL.
Influences of anisotropic tissue structure on reentrant circuits
in the epicardial border zone of subacute canine infarcts.
Circ Res. 1988;63:182-206.
24.
El-Sherif N, Smith RA, Evans K. Canine
ventricular arrhythmias in the late myocardial infarction period, 8:
epicardial mapping of reentrant circuits. Circ
Res. 1981;49:255-265.
25.
Friedman PL, Stewart JR, Fenoglio JJ Jr, Wit AL.
Survival of subendocardial Purkinje fibers after extensive
myocardial infarction in dogs. Circ Res. 1973;33:597-611.
26. Fenoglio JJ Jr, Karagueuzian HS, Friedman PL, Albala A, Wit AL. Time course of infarct growth toward the endocardium after coronary occlusion. Am J Physiol. 1979;236:H356-H370.
27.
Friedman PL, Fenoglio JJ Jr, Wit AL. Time course
for reversal of electrophysiological and ultrastructural abnormalities
in subendocardial Purkinje fibers surviving extensive myocardial
infarction in dogs. Circ Res. 1975;36:127-144.
28. Wit AL, Janse MJ. The Ventricular Arrhythmias of Ischemia and Infarction: Electrophysiological Mechanisms. Mount Kisco, NY: Futura Publishing Co, Inc; 1993.
29.
Boyden PA, Albala A, Dresdner K.
Electrophysiology and ultrastructure of canine subendocardial
Purkinje cells isolated from control and 24 hour infarcted
hearts. Circ Res. 1989;65:955-970.
30.
Boyden PA, Pinto JMB. Reduced calcium currents
in subendocardial Purkinje myocytes that survive in the 24- and 48-hour
infarcted heart. Circulation. 1994;89:2747-2759.
31. Boyden PA, Dresdner K. The electrogenic Na-K pump in Purkinje myocytes from control noninfarcted and the infarcted myocardium. Am J Physiol. 1989;258:H766-H772.
32. Harris AS. Delayed development of ventricular ectopic rhythms following experimental coronary occlusion. Circulation. 1950;1:1318-1328. [Medline] [Order article via Infotrieve]
33.
Robinson RB, Boyden PA, Hoffman BF, Hewett KW.
The electrical restitution process in dispersed canine cardiac
Purkinje and ventricular cells. Am J Physiol. 1987;253:H1018-H1025.
34.
Follmer CH, Lodge NJ, Cullinan CA, Colatsky TJ.
Modulation of the delayed rectifier IK by cadmium in
cat ventricular myocytes. Am J Physiol. 1992;262:C75-C83.
35.
Wang Z, Fermini B, Nattel S. Sustained
depolarization induced outward current in human atrial myocytes:
evidence for a novel delayed rectifier K current similar to Kv1.5
cloned channel currents. Circ Res. 1993;73:1061-1076.
36. Caceci MS, Cacheris WP. Fitting curves to data: the Simplex algorithm is the answer. BYTE. 1984;9:340-362.
37. Snedecor GW, Cochran WC. Statistical Methods. Ames, Iowa: Iowa State University Press; 1978.
38. Van Bogaert PP, Snyders DJ. Effects of 4-aminopyridine on inward rectifying and pacemaker currents of cardiac Purkinje fibers. Pflugers Arch. 1982;394:230-238. [Medline] [Order article via Infotrieve]
39.
Nakayama T, Irisawa H. Transient outward current
carried by potassium and sodium in quiescent
atrioventricular node cells of rabbits.
Circ Res. 1985;57:65-73.
40.
Hiraoka M, Kawano S. Calcium-sensitive and
insensitive transient outward current in rabbit ventricular
myocytes. J Physiol. 1989;410:187-212.
41.
Fozzard HA, Hiraoka M. The positive dynamic
current and its inactivation properties in cardiac Purkinje
fibers. J Physiol. 1973;234:569-586.
42.
Tseng G-N, Hoffman BF. Two components of
transient outward current in canine ventricular myocytes.
Circ Res. 1989;64:633-647.
43.
Fermini B, Wang Z, Nattel S. Differences in rate
dependence of transient outward current in rabbit and human
atrium. Am J Physiol. 1992;263:H1747-H1754.
44. Po SS, Tamkun MM, Snyders DJ, Bennett PB. Differential modulation of 2 human potassium channels (Kv1.4,Kv1.5) by cAMP and theophylline. Biophys J. 1993;64:A197. Abstract.
45.
Murray KT, Fahrig SA, Deal KK, Hu NN, Snyders DJ,
Tamkun MM, Bennett PB. Modulation of an inactivating human K
channel by protein kinase C. Circ Res. 1994;75:999-1005.
46.
Apkon M, Nerbonne JM. Alpha adrenergic agonists
selectively suppress voltage-dependent K+ currents in rat
ventricular myocytes. Proc Natl Acad Sci U S A. 1988;85:8756-8760.
47.
Fedida D, Shimoni Y, Giles WR. A novel effect of
norepinephrine on cardiac cells is mediated by alpha
adrenoceptors. Am J Physiol. 1989;256:H1500-H1504.
48.
Fedida D, Shimoni Y, Giles WR. 
-Adrenergic
modulation of the transient outward current in rabbit atrial
myocytes. J Physiol. 1990;423:257-277.
49.
Braun AP, Fedida D, Clark RB, Giles WR.
Intracellular mechanisms for alpha adrenergic regulation of the
transient outward current in rabbit atrial myocytes. J
Physiol. 1990;431:689-712.
50. Drain P, Dubin AE, Aldrich RW. Regulation of Shaker K channel inactivation gating by the cAMP-dependent protein kinase. Neuron. 1994;12:1097-1109.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  S. Wasson, H. K. Reddy, and M. L. Dohrmann Current Perspectives of Electrical Remodeling and Its Therapeutic Implications Journal of Cardiovascular Pharmacology and Therapeutics, April 1, 2004; 9(2): 129 - 144. [Abstract] [PDF]
|
|
|
|
 |
|
 P. A Boyden, C. Barbhaiya, T. Lee, and H. E.D.J ter Keurs Nonuniform Ca2+ transients in arrhythmogenic Purkinje cells that survive in the infarcted canine heart Cardiovasc Res, March 1, 2003; 57(3): 681 - 693. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. J. Padanilam, T. Lu, T. Hoshi, B. A. Padanilam, E. F. Shibata, and H.-C. Lee Molecular Determinants of Intracellular pH Modulation of Human Kv1.4 N-Type Inactivation Mol. Pharmacol., July 1, 2002; 62(1): 127 - 134. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. R Sipido, P. G.A Volders, M. A Vos, and F. Verdonck Altered Na/Ca exchange activity in cardiac hypertrophy and heart failure: a new target for therapy? Cardiovasc Res, March 1, 2002; 53(4): 782 - 805. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Decher, O. Uyguner, C. R Scherer, B. Karaman, M. Yuksel-Apak, A. E Busch, K. Steinmeyer, and B. Wollnik hKChIP2 is a functional modifier of hKv4.3 potassium channels: Cloning and expression of a short hKChIP2 splice variant Cardiovasc Res, November 1, 2001; 52(2): 255 - 264. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Han, D. Chartier, D. Li, and S. Nattel Ionic Remodeling of Cardiac Purkinje Cells by Congestive Heart Failure Circulation, October 23, 2001; 104(17): 2095 - 2100. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-G. Wang, M. B. Wagner, R. Kumar, W. N. Goolsby, and R. W. Joyner Fast pacing facilitates discontinuous action potential propagation between rabbit atrial cells Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2095 - H2103. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Nattel Acquired delayed rectifier channelopathies: how heart disease and antiarrhythmic drugs mimic potentially-lethal congenital cardiac disorders Cardiovasc Res, November 1, 2000; 48(2): 188 - 190. [Full Text] [PDF]
|
|
|
|
|
|
W. Han, Z. Wang, and S. Nattel A comparison of transient outward currents in canine cardiac Purkinje cells and ventricular myocytes Am J Physiol Heart Circ Physiol, August 1, 2000; 279(2): H466 - H474. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. A. Boyden, J. Pu, J. Pinto, and H. E. D. J. t. Keurs Ca2+ Transients and Ca2+ Waves in Purkinje Cells : Role in Action Potential Initiation Circ. Res., March 3, 2000; 86(4): 448 - 455. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kiyosue and M. Arita Altered expression of cardiac K+ channel genes during sub-acute and healing phases of myocardial infarction Cardiovasc Res, October 1, 1999; 44(1): 13 - 16. [Full Text] [PDF]
|
|
|
|
|
|
J.-A. Yao, M. Jiang, J.-S. Fan, Y.-Y. Zhou, and G.-N. Tseng Heterogeneous changes in K currents in rat ventricles three days after myocardial infarction Cardiovasc Res, October 1, 1999; 44(1): 132 - 145. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M.B Pinto and P. A Boyden Electrical remodeling in ischemia and infarction Cardiovasc Res, May 1, 1999; 42(2): 284 - 297. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Wang, J. Feng, H. Shi, A. Pond, J. M. Nerbonne, and S. Nattel Potential Molecular Basis of Different Physiological Properties of the Transient Outward K+ Current in Rabbit and Human Atrial Myocytes Circ. Res., March 19, 1999; 84(5): 551 - 561. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Rosen, I. S. Cohen, P. Danilo Jr., and S. F. Steinberg The heart remembers Cardiovasc Res, December 1, 1998; 40(3): 469 - 482. [Full Text] [PDF]
|
|
|
|
|
|
D. J. Huelsing, K. W. Spitzer, J. M. Cordeiro, and A. E. Pollard Conduction between isolated rabbit Purkinje and ventricular myocytes coupled by a variable resistance Am J Physiol Heart Circ Physiol, April 1, 1998; 274(4): H1163 - H1173. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||