© 1995 American Heart Association, Inc.
Articles |
Diminished Ca2+ and Ba2+ Currents in Myocytes Surviving in the Epicardial Border Zone of the 5-Day Infarcted Canine 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 West 168th St, New York, NY 10032.
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Abstract Ventricular arrhythmias frequently occur in patients suffering from ischemic heart disease. In a canine model developed to understand the pathoelectrophysiological mechanisms of the ischemia-related arrhythmias, electrical stimulation can initiate and terminate reentrant ventricular tachyarrhythmias, which arise in surviving subepicardial muscle fibers (epicardial border zone [EBZ] fibers) of the left ventricle 5 days after coronary artery occlusion. Both the structural and electrical changes occurring in the EBZ provide the important substrate for generation of reentrant ventricular tachyarrhythmias. In this study, we tested the hypothesis that abnormalities exist in the electrophysiological properties of macroscopic Ca2+ currents in myocytes isolated from the EBZ of the 5-day infarcted canine heart (IZs). We recorded the T-type (ICa,T) and L-type (ICa,L) Ca2+ currents by using the whole-cell voltage-clamp technique with either Ca2+ or Ba2+ (5 mmol/L) as the charge carrier and under experimental conditions (Na+- and K+-free solutions, 10 mmol/L intracellular EGTA) that eliminated contamination by other currents. When Ca2+ served as the charge carrier, the density of peak ICa,T in IZs (0.89±0.5 pA/pF, n=28) was similar to that in myocytes from normal noninfarcted hearts (NZs) (1.1±0.5 pA/pF, n=32). Although no changes existed in the properties of ICa,T, dramatic changes occurred in the density and function of ICa,L in IZs compared with NZs. Density of peak ICa,L at a holding potential of -40 mV (8-second clamp-step interval) was significantly reduced in IZs (4.6±1.5 pA/pF, n=40) compared with NZs (7.2±1.6 pA/pF, n=53). The reduction in peak ICa,L density was not attributable to altered steady state inactivation relations or a delay in recovery of ICa,L from inactivation. The time course of decay of peak ICa,L was described by a biexponential function in both cell types, with the fast and slow time constants (
1 and
2, respectively) of decay being significantly
faster in IZs (1, 12.3±3.6 ms;
2, 55.1±31.1 ms) than in NZs
(1, 16.1±4.1 ms; 2,
85.2±51.7 ms). In addition, rapid clamp stimulation (at 1-s intervals)
of cells produced a larger frequency-dependent decrease of peak
ICa,L density in IZs than NZs, suggesting that at more
physiologically relevant rates, little
ICa,L may be activated. Finally, a significant
reduction and acceleration of decay of the ICa,L persisted
even when Ca2+ was substituted by equimolar
Ba2+ as the charge carrier. These latter findings
suggest that the reduced peak ICa,L density in IZs may be
due to a decrease in the number of functional channels, which also show
an alteration in the voltage-dependent inactivation process. In
summary, we have shown that chronic changes exist in the
electrophysiological properties of
ICa,L in cells that survive in the infarcted heart. Such
changes could contribute to the altered repolarization of action
potentials of myocytes from EBZs of the 5-day infarcted canine heart.
Key Words: L-type Ca2+ current • myocardial infarction • ion channels • ventricular myocytes • T-type Ca2+ current
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After total occlusion of the LAD, several layers of muscle fibers survive on the epicardial surface of the left ventricle of the canine heart. This surviving muscle layer has been termed the EBZ and provides the substrate for ventricular tachycardias in the 5-day infarcted canine heart.1 2 3 4 5 It has been demonstrated that both structural and electrical abnormalities are present in these surviving muscle fibers of the EBZ. Electrically, transmembrane action potentials recorded from myocytes as well as multicellular fiber preparations of the 5-day EBZ have reduced action potential amplitude, action potential duration, and maximal upstroke velocity. Furthermore, the potentials are triangular in shape with loss of the plateau phase.1 6 7 8 The structurally altered cellular arrangement of the surviving muscle fibers enhances nonuniform anisotropy of the EBZ, which also predisposes the EBZ to reentrant excitation and ventricular tachycardias.4
Previous studies have attempted to determine the ionic mechanisms that underlie the electrical abnormalities of transmembrane voltage profiles after myocardial infarction either by studying isolated infarcted muscle excised at various times after coronary artery occlusion1 6 7 or by exposing tissue obtained from normal hearts to conditions that simulate ischemia.9 10 11 12 13 14 Although these studies provide strong indirect evidence that loss of the plateau phase of the action potential and action potential triangularization under conditions of simulated ischemia and after myocardial infarction may be the result of abnormalities in ICa,L, no studies have specifically recorded and measured ICa,L in myocytes surviving in the infarcted canine heart.
Chronic changes in ICa function and density could contribute to the electrical remodeling underlying changes in the repolarization phase of transmembrane action potentials of fibers of the infarcted heart. Therefore, in the present study, we hypothesized that abnormalities in the electrophysiological properties of the inward calcium currents exist in myocytes surviving in the 5-day infarcted canine heart. We used the whole-cell variant of the patch-clamp technique to record and compare the electrical properties of macroscopic ICa,T and ICa,L in myocytes from the normal noninfarcted heart with those dispersed from the EBZ of the 5-day infarcted canine heart.
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Experimental Model of Myocardial Infarction
Adult mongrel dogs weighing 12 to 15 kg and 1 to 2 years in age were used in these studies. While under isoflurane anesthesia and sterile surgical conditions, dogs underwent complete permanent occlusion of the LAD by a two-stage ligation procedure.15 Dogs were closely monitored for infection, treated with antibiotics, and allowed to recover for a period of 5 or 6 days before acute myocyte experiments. At that time, each dog was reanesthetized with sodium pentobarbital (30 mg/kg IV), and a cardiectomy was performed. The infarcted region of the left ventricle was identified on gross examination as a pale white mottled area on the epicardial surface of the anterior region of the left ventricle adjacent to the LAD. A scalpel blade was used to quickly remove a thin slice of epicardial muscle (EBZ), measuring 1 cmx2 cmx2 to 3 mm, for the preparation of myocytes.
Preparation of Single Myocytes
Myocytes were enzymatically dissociated from the slice of
infarcted epicardial tissue (IZs), as previously
described.8 Myocytes for control experiments (NZs) were
dispersed from the epicardial muscle tissue obtained from noninfarcted
hearts of control animals in a similar fashion. Only NZs that were rod
shaped with staircase ends, clear cross striations, and surface
membranes free of blebs were used for study. The living cell yield was
30% to 40%, although some preparations gave slightly better or
worse results. IZs had a ruffled appearance, appeared less rodlike, and
had various shapes, with cells having somewhat irregular cross
striations. Additionally, as has been previously
described,8 small dark droplets were apparent in IZs and
were used to identify myocytes that had survived infarction. The recent
finding that IZs possessing these specific morphological
characteristics exhibit abnormal transmembrane action potentials
similar to those of the multicellular preparation of the
EBZ1 8 was used as the basis for selection of IZs in this
particular study. Since size of the EBZ could vary in the infarcted
heart, the yield of IZs (10% to 30%) varied.
Electrophysiological Studies
For electrophysiological studies,
aliquots of freshly isolated cells were transferred onto a
poly-L-lysine–coated glass coverslip that had been
placed on the bottom of a Lucite recording chamber (volume, 0.5
mL) mounted onto the stage of an inverted microscope (Nikon Diaphot).
Bath temperature was maintained at 36°C to 36.5°C throughout the
experiments. Myocytes were initially superfused (2 to 3 mL/min) with
normal Tyrode's solution (mmol/L): NaCl 137, NaHCO3 24,
NaH2PO4 1.8, MgCl2 0.5,
CaCl2 2.0, KCl 4.0, and dextrose 5.5 (pH 7.4). Patch
pipettes were fabricated from borosilicate glass (Sutter Instrument Co;
outer diameter, 1.5 mm; inner diameter, 0.86 mm) by using a
Flaming/Brown-type horizontal puller (model P-87, Sutter
Instruments Co). These pipettes had resistances of 1 to 2 M
after
heat-polishing and when filled with an internal solution of the
following composition (mmol/L): CsOH 125, aspartic acid 125,
tetraethylammonium chloride 20, HEPES 10,
Mg-ATP 5, EGTA 10, and phosphocreatine 3.6, pH 7.3 with CsOH. After
gigaohm seal formation and cell membrane rupture, a period of 5 to 10
minutes was allowed for intracellular dialysis before
superfusate was switched to a Na+- and
K+-free solution having the following composition (mmol/L):
CaCl2 5, tetraethylammonium
chloride 140, MgCl2 0.5, dextrose 10, and HEPES 10, pH 7.3
with CsOH.
External and internal solutions were designed to minimize the contamination by other overlapping currents when recording ICa,T and ICa,L.16 In all experiments, 2 mmol/L 4-aminopyridine was added to the external solution to block current flow through the voltage-dependent transient outward K+ channel.17 Thus, these recording conditions allowed for accurate quantification of the chronic changes in properties of ICa,L and ICa,T in both cell types under similar conditions.
Single epicardial myocytes were voltage-clamped by using the
continuous-clamp method and an Axopatch-1D patch-clamp
amplifier (Axon Instruments) as previously described.18
Membrane currents were filtered by the amplifier at 2 kHz, digitized at
a sampling interval of 0.2 ms for whole-cell currents and 0.02 ms
for capacitative transients, and stored on the computer for
off-line analysis. To enable comparison of
ICa,L and ICa,T amplitudes (in pA) between
cells of different sizes, ICa,L and ICa,T
magnitudes were normalized by each cell's membrane capacitance (in pF)
and expressed as current density (in pA/pF). Cell membrane capacitance
was determined in Cs+-containing solutions by integrating
the area under a capacitative transient induced by a 15-mV
hyperpolarizing pulse and dividing this area by the voltage step. Cell
capacitance averaged 140±36 pF in NZs (n=53) and 184±52 pF in IZs
(n=40) (P<.05). The stray capacitance was compensated by
the capacitance compensation circuit of the patch-clamp amplifier.
The residual series resistance was estimated by dividing the time
constant of the capacitative transient by the calculated cell membrane
capacitance. The capacitative transient decayed with an average time
constant of 0.42±0.11 ms in NZs (n=53) and 0.46±0.13 ms in IZs
(n=40). The average residual series resistance was 2.9±0.8 M for
NZs and 2.3±0.7 M for IZs (P>.05). Therefore, the
voltage error introduced by series resistance at a current amplitude of
1 nA was 3 mV. Voltages were not corrected for the liquid junction
potential between the bath and pipette solutions. The currents
displayed are original raw recordings, with no corrections made
for linear leakage currents or whole-cell capacitance.
When myocytes are dialyzed during whole-cell recordings, there is the decrease or "rundown" of peak ICa,L with time.19 20 In contrast, ICa,T is not subject to rundown.21 22 In the present study, the rates of rundown of ICa,L in cells from the two groups were determined to establish a time frame during which specific quantitative measurements of the current could be made. The rate of ICa,L rundown was determined by applying voltage-clamp steps from a VH of -40 mV to various depolarized Vts every 8 s for 30 to 40 minutes after membrane rupture. The relation was assumed to be linear, and the slope of the line was taken as the rate of ICa,L rundown for each cell.
The two different types of Ca2+ currents (ICa,T and ICa,L) were separated by voltage clamping the cell at different VH levels and recording inward currents.16 23 24 25 Peak ICa,L at various Vts was measured as the difference between the maximal inward peak and the current level at the end of a 250-ms voltage-clamp step. Amplitude of peak IBa was measured as the difference between the inward peak current and the holding current level. The time course of ICa and IBa decay was characterized by fitting the current change between the inward peak and the current level 125 ms (for ICa,T) and 250 ms (for ICa,L and IBa) after depolarization (CLAMPFIT, PCLAMP 5.0). As described by others,16 24 25 26 27 28 the time course of ICa,L decay was best fit by using a biexponential function, whereas decay of ICa,T was best fit by using a monoexponential function. Current tracings were judged to be best fit if attempts to fit the current decays with additional exponential components either reduced the quality of the fits or prevented convergence altogether.
The steady state activation variable of ICa,L was estimated for some cells of each group by using the peak chord conductance according to the method of Isenberg and Klöckner.29 Values for V1/2 and the slope factor k, describing the steepness of the activation curve, were obtained for each cell studied. These values were averaged for cells in the same group and then compared. Steady state inactivation variables of peak ICa,L were determined by using a double-pulse protocol.27 For each cell, the peak current elicited at each test pulse was expressed as a fraction of the current obtained with the most negative conditioning prepulse potential (-70 mV; duration, 1000 ms), and normalized values were plotted to obtain V1/2 and slope factor k for each cell. These values were used to determine and compare the average values of V1/2 and k for all NZs and IZs.
The time course of recovery from inactivation of ICa,L was examined because an altered time course of recovery could underlie changes in ICa,L between NZs and IZs. Recovery of ICa,L was studied by using a double-pulse protocol (delivered every 8 s) consisting of a 350-ms prepulse from a VH of -40 mV to a Vt of +20 mV, followed by a similar test pulse (duration, 250 ms) delivered at a progressively increasing IPI ranging from 10 to 2000 ms. The degree of recovery at each IPI was determined by dividing the peak current amplitude at each IPI by the peak current amplitude at the IPI of 2000 ms. The time course of recovery was estimated by fitting the data points to a single or biexponential function by using a simplex algorithm. The time constant of recovery of ICa,L from inactivation was determined for each NZ and IZ, and average values were compared.
For all comparisons, data were expressed as mean±SD or as mean±SEM where indicated. A value of P<.05 was considered statistically significant. The two-sample t test was used to make comparisons of a single parameter between two independent experimental groups, NZs and IZs (for instance, for comparing the mean values of the peak ICa density, rate of current decay, steady state activation and inactivation variables, and time constants of recovery). A paired t test was used to determine whether Ba2+ ion substitution exerted significant changes on the control current in a particular cell. In situations in which multiple comparisons were made, an ANOVA and subsequent F test were performed to determine whether the sample means between groups were significantly different from each other. If the F statistic indicated that significant differences existed between the groups, a modified t statistic with Bonferroni's correction for multiple comparisons was used to compare the mean values. This type of analysis was used to compare the voltage dependence of the time constants of current decay during the control condition.
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For these studies, it was necessary to determine the rate of current rundown in order to decide on the proper time frame during which measurements of peak ICa,L could be made and compared in the two cell populations. The rate of ICa,L rundown in NZs (n=5) and IZs (n=5) was determined to be similar between the two cell types (0.09±0.08 and 0.08±0.06 pA/pF per min, respectively) during a period of 10 and 40 to 50 minutes after cell membrane rupture (Fig 1
). Therefore, we used measurements of peak current
made 15 to 25 minutes after whole-cell membrane rupture for making
comparisons of ICa,T and ICa,L magnitude
between NZs and IZs.
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) and five IZs
(
). Peak ICa,L densities for depolarizations from
VH of -40 mV to Vt of +20 mV are plotted
as a function of time after cell membrane rupture, in the presence of 5
mmol/L Ca2+. Membrane rupture occurred at time zero.
This relation was assumed to be linear, with the slope taken as a
measure of the rate of ICa,L rundown.
Two Types of Ca2+ Currents
Fig 2A, left, shows current tracings obtained in an
NZ when the cell was held at a VH of -70 mV (left
tracings) or a VH of -40 mV (middle tracings) and
stepped to various Vts (as indicated) for 250-ms
clamp-step duration every 8 s; Fig 2A, right, shows current
tracings obtained in a similar manner from an IZ. In both cell types,
at a VH of -70 mV, a small inward transient was
present at Vts of <-10 mV, whereas at a
VH of -40 mV, no inward transients were seen at
Vts of -30 and -20 mV, with only a small
transient present at Vts of -10 mV. Peak
ICa,T was obtained by digitally subtracting the currents at
the two VH levels and is shown in the right tracings for
both the NZs and IZs.
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) and -40 mV (
) for NZ and IZ of panel A. Peak
inward ICa density plotted as a function of
Vt. Note that two inward maxima are present at
VH of -70 mV, whereas only one inward maximum is
present at VH of -40 mV. Peak current
density–voltage relation is shown for the DIFF currents in NZs
(
Typical peak current density–voltage relations for the difference
currents of the NZ and IZ are shown in Fig 2B
.
Although both ICa,T and ICa,L were elicited
when the cell was depolarized from a VH of -70 mV,
only ICa,L was elicited when stepped from a VH
of -40 mV. Averaged peak ICa,L density–voltage
relations for all NZs (n=53) and IZs (n=40) are shown in Fig 3. The average current density was significantly reduced
at Vts of -10 to +55 mV in IZs compared with NZs. The
data used to construct these current density–voltage plots were
recorded at similar times after membrane rupture (19.55±3.5
minutes in NZs and 21.91±6.3 minutes in IZs). Although the peak
ICa,L density at various voltages is reduced in IZs, the
current-voltage relations for NZs and IZs have similar shapes (Fig 3B). For IZs, the average ICa,L density–voltage
relation of IZs depicted in Fig 3A was scaled by a factor of 1.71 and
normalized, and the resulting relation could be reasonably superimposed
on the normalized average ICa,L density–voltage
relation of NZs (Fig 3B).
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Table 1 summarizes the properties of ICa,T
in NZs and IZs. As previously reported,16 23
ICa,T is completely absent in some myocytes, occurring only
in about two thirds of the canine myocytes studied.16
Similarly, in the present study, ICa,T was not
present in all myocytes (for both NZs and IZs), yet it occurred as
frequently in NZs as in IZs (
2 test,
P=NS). Furthermore, average values of peak ICa,T
amplitude, density, and current kinetics did not differ between groups.
The average ratio of peak ICa,T amplitude to peak
ICa,L amplitude was similar between the cells in the two
groups. ICa,T decays monoexponentially in
myocytes.16 24 Thus, it appears that by 5 days after total
coronary artery occlusion, there is no change in the frequency
or magnitude of ICa,T in myocytes that survive in the
infarcted heart. Table 2 summarizes the properties of
ICa,L for all NZs and IZs studied. Cell membrane
capacitance was significantly greater in the IZs (by an average of
31%). The amplitude of peak ICa,L was reduced in the IZs
(by an average of 15%) at VH values of -40 and
-70 mV, but this reduction was not statistically different from
the control level. However, when current amplitudes were normalized for
cell membrane capacitance and compared, the maximal peak
ICa,L density was significantly reduced in the IZs (an
average of 36%) compared with NZs at VH values of
-70 and -40 mV.
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Since it is known that Ca2+ entry through ICa,L can contribute both a transient and sustained inward current,28 30 experiments were performed in a subset of cells to determine whether the observed significant reduction in peak ICa,L density in IZs was accompanied by a change in the density of inward current at the end of the clamp step (ICa,L at 250 ms). To complete these studies, experiments were conducted with the dihydropyridine Ca2+ channel antagonist nisoldipine, which blocks ICa,L through L-type calcium channels.31 Superfusion with 3 µmol/L nisoldipine completely blocked the large inward ICa,L at all test potentials in both NZs and IZs (data not shown). The peak nisoldipine-sensitive current was significantly different between NZs (6.7±1.3 pA/pF, n=6) and IZs (4.2±0.7 pA/pF, n=6) (P=.001). In contrast, the density of the nisoldipine-sensitive ICa,L at 250 ms was not different between NZs (1.0±0.3 pA/pF, n=6) and IZs (0.9±0.6 pA/pF, n=6) (P=.6). Thus, although the peak ICa,L was significantly reduced in IZs, the density of the steady inward currents remaining at 250 ms did not differ between the two cell types.
Time Course of ICa,L Decay in Internally Dialyzed
Cells
The time course of decay of the peak ICa,L has been
best described as the sum of two exponential components with time
constants separated by approximately an order of magnitude in
canine,26 bovine,29 and rat32
ventricular myocytes. Quantitative analyses of
ICa,L revealed that the decay phases of the peak
ICa,L currents were best fit by biexponential functions in
both NZs and IZs at Vts between -10 and +40 mV.
However, the decay of the transient ICa,L was significantly
accelerated in IZs compared with NZs at Vts of >+10 mV
(Fig 4). Fig 4A shows
representative fits of the time course of decay of the
maximal peak ICa,L in the two cell types. To determine
whether acceleration of peak ICa,L decay in IZs was
secondary to the time frame (15 to 25 minutes) during which the
currents were recorded, kinetics of peak ICa,L decay at
several different times (ranging from 10 to 40 minutes) after cell
membrane rupture were examined in several NZs and IZs. No
time-dependent change in the time course of ICa,L decay
occurred (Fig 4). Therefore, even after prolonged intracellular
dialysis, the time course of ICa,L decay remained
accelerated in IZs, significantly different from NZs.
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To assess whether the increased rate of peak ICa,L decay
was related to remaining contaminating outward currents, the effect of
2 µmol/L ryanodine, which blocks release of Ca2+
from the sarcoplasmic reticulum33 and thereby inhibits any
intracellular Ca2+–activated outward
current, on ICa,L decay was tested. Application of
ryanodine exerted little change in the 1 values of decay
in an NZ and IZ. The 1 values in the absence and
presence of ryanodine were 17.3 and 16.9 ms, respectively, in one NZ
(peak ICa,L, 6.2 pA/pF) and 14.1 and 13.6 ms in one
IZ (peak ICa,L, 4.9 pA/pF), respectively. Thus,
acceleration of peak ICa,L decay in the IZ cannot be
ascribed to residual contamination from overlapping outward
currents.
The time course of recovery from inactivation of ICa,L was
examined to determine whether a delay in the recovery of L-type calcium
channel function could account for the reduced peak ICa,L
density observed in IZs. In normal myocytes, ICa,L recovery
has been described by a monoexponential
function.16 26 Fig 5A illustrates that
recovery of ICa,L was dependent on the IPI (increasing with
longer IPI) in a typical NZ and IZ. In the present study, recovery
of ICa,L was found to be best described by a
monoexponential function in some NZs and IZs and by a
biexponential function in other NZs and IZs (Fig 5B). In these cells,
the peak ICa,L density was significantly reduced in IZs
(4.2±1.5 pA/pF, n=16) compared with NZs (5.7±1.7 pA/pF, n=28)
(P=.004). A monoexponential function best
described the time course of recovery in 75% of NZs (21 of 28 cells)
and in 50% of IZs (8 of 16); data from the other 7 NZs and 8 IZs were
best described by a biexponential function. Whether cells were best fit
by a mono- or biexponential function did not depend on the density of
peak ICa,L in the two cell groups. Although the fast time
course of recovery of ICa,L was found to be slower in IZs
(n=8), it was not significantly different from NZs (n=21)
(P=.06). For data from cells best described by a
biexponential function, the average values for the time constants and
relative amplitudes were also found to be similar (Fig 5). Thus, there
may be a slight slowing in recovery from inactivation of
ICa,L in IZs, but it is not significant.
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Although the time constant of recovery of peak ICa,L at
slow pacing rates (8-s BCL) was not significantly different between the
two cell types, we hypothesized that the small differences observed may
become significant at more rapid stimulation rates. Therefore, the
effect of a rapid stimulation rate on peak ICa,L density
was tested. In this protocol, each cell was first stepped from a
VH of -40 mV to a Vt of +20 mV at a BCL
of 8 s for a total of 20 pulses. Then the stimulation rate was
increased such that depolarizing clamp steps occurred at a BCL of 1 s.
Although stimulation at a BCL of 8 s produced little change in peak
ICa,L density, a decrease of peak ICa,L density
occurred at a BCL of 1 s in both cell types (Fig 6A). Overall, peak ICa,L
decreased by 3.0±0.8% in NZs (average ICa,L,
6.0±1.3 pA/pF; n=20) and 0.4±5.5% in IZs (average peak
ICa,L, 4.5±1.2 pA/pF; n=14) after stimulation at a
BCL of 8 s. In contrast, there was a significantly greater decrease in
peak ICa,L in IZs (by 25.4±9.7%, n=14) than in NZs (by
11.8±9.6%, n=20) at a BCL of 1 s. Thus, at rapid rates of
stimulation, the significantly different peak ICa,L
densities of IZs are further reduced, becoming 53% of the average
ICa,L density of NZs at the same rate.
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) and IZ (
).
Voltage Dependence of Activation and Inactivation
On the basis of data obtained from current-voltage relations,
an activation curve of peak ICa,L was determined for cells
in each group.29 Activation relations for IZs
(V1/2, 6.6±5.2 mV; slope factor k, 5.8±0.6 mV;
n=17) were no different from those for NZs (V1/2,
9.3±3.3 mV; k, 5.7±0.5 mV; n=23), yet peak ICa,L density
was significantly reduced in IZs (4.3±1.2 pA/pF, n=17) compared with
NZs (6.8±1.3 pA/pF, n=23). Furthermore, since a reduction of L-type
calcium channel availability could account for the reduced
ICa,L density observed in IZs, steady state availability of
ICa,L was determined. No significant differences were found
in average values of V1/2 (-11.6±5.8 mV in NZs
[n=41] and -12.3±6.3 mV in IZs [n=37]) and slope factor k
(5.5±1.2 in NZs and 5.5±0.8 mV in IZs) between the two groups of
cells, yet the maximally available ICa,L density (at a
conditioning prepulse potential of -70 mV) was significantly
reduced in IZs (4.6±1.4 pA/pF, n=37) compared with NZs (6.7±1.8
pA/pF, n=41) (P<.05).
In summary, the peak ICa,L density is significantly reduced and the time course of peak ICa,L decay is significantly accelerated in IZs compared with NZs. However, the reduced peak ICa,L in IZs is not due to differences in steady state availability nor in the time course of recovery from inactivation of L-type calcium channels, yet reductions in peak ICa,L are exaggerated at more rapid rates in IZs. Finally, these data show that the occurrence and magnitude of peak ICa,T do not differ between NZs and IZs.
Effects of Equimolar Ba2+
Substitution on ICa,L
Ca2+ channels are known to
inactivate by both voltage-dependent as well as
intracellular Ca2+–dependent
mechanisms.34 35 Therefore, to determine whether the
reduced peak ICa,L density in IZs was due to changes in
voltage-dependent, but not intracellular
Ca2+–dependent, inactivation properties of L-type
channels, experiments were completed in which extracellular
Ca2+ was replaced with Ba2+ ions.
With Ba2+ ions as the primary charge carrier,
intracellular Ca2+–dependent inactivation is
minimized such that ICa,L inactivation is due to a
voltage-dependent process.34 36
In a subset of NZs and IZs, clamp protocols were completed first with 5
mmol/L Ca2+ as the charge carrier and then repeated
in the presence of 5 mmol/L Ba2+–containing
solutions. Data were included in this analysis only if the same
cell was exposed to both solutions. Similar to results obtained for
normal myocytes,16 23 Ba2+ caused an
increase in the current amplitude (Fig 7)
as well as a slowing of the decay of the peak L-type current in the NZ.
These changes with Ba2+ substitution were greatly
attenuated in the IZ. In these subsets of cells, peak ICa,L
density was significantly reduced in the IZs (4.4±1.5 pA/pF, n=10)
compared with NZs (5.9±1.3 pA/pF, n=12) (P<.05). Peak
L-type current density was significantly increased to 16.8±7.0 pA/pF
in NZs and to 7.4±2.9 pA/pF in IZs, with 5 mmol/L
Ba2+ substitution (Fig 8). However, the average peak
IBa density obtained in IZs remained significantly reduced
(by 56%) from that of NZs. Furthermore, the relative increase in
ICa,L with equimolar Ba2+ ion
substitution was significantly less in IZs (1.6±0.4-fold, n=10) than
in NZs (2.8±0.9-fold, n=12) (P=.002).
|
|
P<.05 vs L-type current density in the presence of 5
mmol/L Ba2+. Numbers in parentheses indicate number
of cells. Only cells exposed first to 5 mmol/L Ca2+
and then 5 mmol/L Ba2+ were used, so the bars
represent paired observations from a given cell.Since the diminished effect of Ba2+ ion substitution may be due to reduced availability of ICa,Ls at VH of -40 mV, steady state inactivation relations in the presence of 5 mmol/L Ca2+ and after substitution with 5 mmol/L Ba2+ were completed in both cell types. Substitution of Ca2+ with Ba2+ ions produced a similar hyperpolarizing shift in the average value of V1/2 in NZs (-13±0.3.4 to -19.7±4.9 mV) and IZs (-11.6±3.8 to -17.9±3.9 mV) (data not shown), indicating that the reduced effect of Ba2+ on ICa,L magnitude in IZs was not due to differences in Ca2+ channel availability between the two cell types.
As described for normal cells,16 23 27 30
Ba2+ substitution for Ca2+ causes
a slowing in the rate of L-type current decay, indicating removal of
Ca2+-dependent inactivation processes35
during step depolarizations. In an NZ, when Ca2+
served as the charge carrier, decay of the peak L-type current was
rapid and best fit by using a biexponential function, but when
Ba2+ was the permeant ion through the L-type
Ca2+ channels, there was a slowing in decay of the
maximal peak current (Fig 4A, upper tracings). In contrast, for the IZ
(Fig 4A, lower tracings), both the maximal peak ICa,L and
IBa were rapidly decaying and best fit with biexponential
functions. Maximal peak ICa,L was best fit with
1 of 13.4 ms and 2 of 62.9 ms, and
IBa was best fit with 1 of 28.1 ms and
2 of 104 ms. The average 1 values for
ICa,L and IBa plotted against Vt in
the NZs and IZs studied in both Ca2+ and
Ba2+ solutions are shown in Fig 9. In
these cells, peak ICa,L decay of IZs
(1, 12.8±3.6 ms; 2,
65.0±38.9 ms [relative amplitude of 2,
0.21±0.09]; n=10) was significantly different from NZs
(1, 18.7±2.6 ms; 2,
110.7±55.4 ms [relative amplitude of 2l,
0.18±0.11]; n=11) (P<.05). In NZs (Fig 9A), average
1 values of peak ICa,L and peak
IBa decay exhibited voltage dependence, as assessed by
one-way ANOVA, with minimum values occurring at +20 mV
(1, 18.2±3.1 ms [ICa,L]) and +40
mV (1, 29.7±11.0 ms [IBa]). Decay
of peak L-type current was slowed when Ca2+ was
replaced with Ba2+ as the charge carrier. The
differences in the time constants of current decay were significant at
Vts of -10, 0, and +10 mV (Fig 9A). Similarly, in
IZs, the average 1 of peak ICa,L and peak
IBa decay also exhibited voltage dependence. In contrast,
substitution of Ca2+ by Ba2+ ions
resulted in a significant increase of 1 only at
Vt of -10 mV (Fig 9B). The relations of
1 and Vt for IBa of IZs and NZs
are parallel with the curve for IZs shifted significantly
(P<.05) downward (Fig 9C).
|
Thus, the significant reduction in peak ICa,L persisted even when Ba2+ carried the current in IZs, suggesting that the reduced peak ICa,L density may be secondary to a decrease in the number of functional L-type Ca2+ channels. However, these experiments do not rule out intracellular Ca2+–dependent inactivation of the L-type Ca2+ channel as a mechanism that may also contribute to a further reduction of peak ICa,L density in IZs. Furthermore, when Ba2+ ions carried the current, decay of the peak current flowing through the L-type Ca2+ channel was still accelerated in IZs, suggesting a change in the voltage-dependent inactivation process of ICa,L in IZs. Finally, the reduced relative effect of Ba2+ substitution on peak ICa,L density may indicate a change in the process of ion permeation through the channel pore of functional L-type Ca2+ channel proteins remaining in sarcolemma of IZs.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For the present study, single myocytes were enzymatically dispersed from the epicardial border zone of the 5-day infarcted canine heart to investigate the electrophysiological properties of ICa,T and ICa,L by using whole-cell voltage-clamp techniques. The first major finding of the present study is that both ICa,T and ICa,L are present in myocytes that survive in the infarcted heart. ICa,T did not differ between myocytes isolated from the normal noninfarcted heart (NZs) and myocytes isolated from the 5-day infarcted heart (IZs). Average values for ICa,T parameters for NZs were in a range similar to values of ICa,T reported by others for the sinoatrial node and atrial, ventricular, and Purkinje myocytes from normal hearts.16 24 25 37 38 In contrast to ICa,T, dramatic changes occurred in the function and density of ICa,L in epicardial myocytes that survive in the infarcted canine heart. In IZs, the average density of peak ICa,L was reduced significantly compared with that in NZs, and this reduction of peak ICa,L density was frequency dependent, being exacerbated when the pacing cycle length was decreased from 8 to 1 s. These changes were observed to occur without a statistically significant change in the time course of restitution of ICas with double-pulse protocols. Furthermore, the reduced peak ICa,Ls in IZs persisted when extracellular Ca2+ was replaced with equimolar Ba2+ as the primary charge carrier. Finally, the kinetics of decay of both peak ICa,L and IBa were significantly accelerated in IZs compared with NZs.
The finding in the present study of altered ICa,L
properties in cells that survive in the EBZ of the infarcted heart is
similar to previously published studies of ICa changes that
occur in myocytes from hearts in different pathological states.
Recently, Boyden and Pinto18 reported reductions in peak
ICa,L density and function in arrhythmogenic subendocardial
Purkinje myocytes that have survived for 48 hours after
coronary artery ligation. Pressure overload in experimental
cardiac hypertrophy has also been reported to alter
properties of ICa,L. Although magnitude of the
ICa,L is unchanged in myocytes isolated from hearts with
moderately compensated hypertrophy,39
significant reductions in ICa,L density occur in myocytes
isolated from more chronically hypertrophied feline
hearts.40 In this latter study, however, changes have also
occurred in the time course of ICa,L decay in hypertrophied
myocytes. In contrast, there were no differences in macroscopic
ICa magnitude in ventricular myocytes from
human hearts explanted from patients suffering from terminal heart
failure.41 However, mRNA for 
1C
Ca2+ channel protein is downregulated in ventricles
from patients with end-stage heart failure.42
Experimental Considerations
Several considerations need to be addressed when interpreting the
ICa,L data in the present study. The first
consideration concerns the criteria used for selection of myocytes for
study in the two groups. For NZs, only myocytes having a rod shape,
clear striations, and a membrane free of blebs were studied. As
previously described,8 myocytes dispersed from the 5-day
infarcted heart constituted a heterogeneous population
composed of cells having various morphological features. A few myocytes
were rod shaped and resembled normal myocytes, whereas most had a
ruffled appearance, were irregularly shaped, and appeared to have dark
droplets on the membrane surface. Myocytes with these latter
morphological features were selected for study in the infarct
preparation, since a previous report had shown that these cells
possessed abnormal transmembrane action potential voltage
profiles.8 Although the cell selection procedure for any
myocyte study may be biased, results from studies using this population
of myocytes provide an important starting point for examination of
chronic abnormalities in
electrophysiological properties of myocytes
that survive in the infarcted heart.
The second consideration concerns whether the reduced ICa,L in IZs is related to the cell dispersion process. This is an unlikely possibility, since an identical disaggregation procedure was used to prepare both NZs and IZs. In addition, values for peak ICa,L density in NZs were similar to values reported in normal myocytes.16 18 Also, the observed reduction in ICa,L magnitude recorded in IZs is consistent with the loss of the plateau phase of the action potential configuration recorded from both the myocyte and multicellular preparations of EBZ of the 5-day infarcted heart.1 7 8
The third consideration is that the observed reduction of ICa,L is a consequence of greater time-dependent rundown during our recordings and that under nondialyzed conditions the currents are really not different between the cell types. It could be argued that although the rate of ICa,L rundown for 15 to 40 minutes after membrane rupture were similar in NZs and IZs, rundown during the first 15 minutes occurs at a much faster rate in IZs and accounts for the reduced ICa,L density. However, this is unlikely, because during the first 15 minutes the rate of rundown would have had to have been nearly three times faster in IZs than NZs. Although we were not able to accurately measure rundown of ICa,L during the first 15 minutes because of the time necessary for complete intracellular dialysis,43 our determinations of rundown between 15 and 40 minutes are similar to previously reported values for ICa,L rundown during the first 30 minutes after membrane rupture.19 20
The final consideration concerns the possibility that contaminating outward currents present only in IZs are responsible for the measured reduced peak ICa,L in these cells. Compositions of the internal pipette and external solutions were chosen carefully to isolate ICas from possible overlapping currents. For instance, all solutions were Na+ and K+ free to eliminate Na+ currents, K+ currents, and Na+-K+ and Na+-Ca2+ exchanger currents. Also, the pipette solution included Cs+ to block K+ currents. Maximal concentrations of ATP were included in the internal pipette solution in order to prevent ATP-dependent outward currents from overlapping the ICas. The pipette solution also included a high concentration of EGTA to chelate intracellular Ca2+ levels, since elevated intracellular Ca2+ levels can decrease peak ICa,L44 45 as well as cause activation of intracellular Ca2+–dependent outward currents.46 Furthermore, it is unlikely that the reduction of peak ICa,L in IZs is due to the existence of contaminating intracellular Ca2+–dependent outward currents, because in experiments in which sarcoplasmic Ca2+ release was reduced either by replacing Ca2+ ions with equimolar Ba2+ ions as the charge carrier or by application of ryanodine, peak ICa,L density of IZs remained reduced compared with that of NZs.
Possible Mechanisms for the Reduction of Density of Macroscopic
Whole-Cell ICa,L and IBa
A possible explanation for the observed reduction of peak
ICa,L density in IZs is that an increase in cell surface
membrane area or capacitance without a concomitant increase in L-type
Ca2+ channel number occurs after coronary
artery occlusion. Physical enlargement of the myocyte, an increase in
the amount of surface membrane infolding, and/or an increase in the
dielectric constant of the cell membrane could underlie the slight
increase in cell membrane capacitance in IZs. In order to explain the
reduced ICa,L density, there must also be a decrease in
functional sarcolemmal L-type Ca2+ channel proteins
in IZs. The mechanism responsible for reduced channel proteins could
involve altered protein expression as a result of changes in gene
transcription, translation, or posttranslational modification or,
alternatively, may involve altered incorporation of channel protein
into the cell membrane.
It is now known that the level of steady state L-type Ca2+ channel message expression can be regulated by both catecholamines and intracellular Ca2+.47 48 49 However, although it is thought that during acute myocardial ischemia, levels of catecholamines and intracellular Ca2+ are known to change,50 it remains unknown whether these factors play a role in downregulation of Ca2+ channel protein expression in cells that survive in the infarcted heart.
Although the reduction in densities of both peak ICa,L and IBa in IZs is consistent with a reduction in the number of functional Ca2+ channel proteins in IZs, the finding of a reduced relative effect of Ba2+ ion substitution on the L-type current magnitude and the acceleration of ICa,L and IBa decay in IZs cannot be adequately explained by such a scheme. For instance, if the total number of channels was decreased in IZs, then, as observed, a reduced macroscopic ICa would exist. However, since we know that decay of ICa,L is both voltage and current dependent,27 a reduced ICa,L (and hence reduced Ca2+ influx) in IZs would then be expected to decay either at the same or at a slower rate than the correspondingly larger-amplitude ICa,L27 present in NZs. On the contrary, the reduced ICa,L in IZs was found to decay more rapidly than did the larger-amplitude ICa,L in NZs, with the acceleration in current decay persisting even when Ba2+ ions served as the charge carrier. Furthermore, based on Ba2+ data, changes in the permeation properties of the channel could also exist. This suggests that other factors besides a change in total channel number may be involved.
One possibility that may account for the observed changes involves a
direct effect of intracellular Ca2+ levels on
ICa,L. In normal myocytes, intracellular dialysis with
increased levels of Ca2+ produces a decrease in
ICa,L amplitude44 51 and an acceleration in
the rate of ICa,L decay.44 In the experiments
in the present study, a high concentration of EGTA (10 mmol/L)
included in the pipette was sufficient to chelate bulk cytoplasmic
Ca2+, as evidenced by the lack of any visible
signs of cellular contraction. However, some have proposed that
intracellular Ca2+ in the subsarcolemmal
"fuzzy" space can contribute to channel closing or inactivation
without there being a generalized elevation of bulk intracellular
Ca2+ concentration.52 Direct binding of
intracellular Ca2+ to a cytoplasmic region of the
1 subunit near the inner mouth of the
Ca2+ channel initiates the inactivation
process.45 53 Whether such a scheme involving
Ca2+ influx–mediated inactivation underlies
changes in ICa,L kinetics in IZs remains to be
explored.
However, since the reduction of L-type current persisted even under
conditions in which intracellular Ca2+–dependent
inactivation of the L-type current was minimized by the replacement of
extracellular Ca2+ (ICa,L) with
Ba2+ (IBa), another possibility must be
considered. This would involve a change in the voltage-dependent
inactivation process of the L-type Ca2+ channel.
This possible lesion could result from changes in 1
subunit structure and/or in its interaction with ß subunits, which
are known to modulate both the magnitude and kinetics of currents
carried by the 1 subunits.54 55 56 The
voltage-dependent inactivation process of the L-type current is
controlled by amino acids within a specific region of the
1 subunit IS6, which interacts with the ß subunit.
This region differs from the III-IV intracellular loop, which may be an
important determinant for the voltage-dependent inactivation
process of Na+ channels.57
Alterations in the lipid environment of the sarcolemma that surrounds the Ca2+ channel protein may provide an alternative explanation for the observed changes in ICa,L in IZs. Two amphiphilic lipid metabolites, long-chain acylcarnitine and lysophosphatidylcholine, are known to accumulate acutely and rapidly in the sarcolemma of ischemic tissue, producing changes in the action potential configuration58 59 60 and modulation of several ionic currents in normal myocytes.61 62 63 Although these agents can incorporate themselves into the sarcolemma and have been implicated as a cause of electrical stages during acute ischemia, the role these agents play in modulating ionic currents in the 5-day infarcted heart is not known. From the present study, it appears unlikely that the reduced ICa,L in IZs can be attributed to these particular metabolites, because prolonged intracellular dialysis and extracellular superfusion would be expected to reverse the inhibitory effect of these agents on ICa,L, such that peak ICa,L density would be restored to similar levels as recorded in NZs. Alternatively, the finding that the cytoskeletal structure of the myocyte can play a role in the biophysical properties of the Ca2+ channel64 65 suggests that alterations in properties of membranes surrounding the channel could underlie the observed changes in IZs.
Physiological Implications of
Findings
Diminished inward ICa,L has important implications in
the infarcted heart. Since ICa,Ls play a crucial role in
excitation-contraction coupling, a reduction of peak
ICa,L density would produce decrease influx of
Ca2+ and could result in altered myocardial
contraction in the 5-day infarcted heart. The recent findings that
myocytes from the infarcted heart have impaired contractile function
that may be the result of altered intracellular Ca2+
transients in these cells66 67 is consistent with
such an idea. Furthermore, the frequency dependent decrease in
ICa,L density demonstrated in the present study is
consistent with a previous report7 and, together
with reported frequency-dependent changes in contraction and levels
of peak intracellular
Ca2+,67 suggests that at
physiologically relevant stimulation rates, the
activation of inward ICa and myocardial contraction may be
dramatically reduced or does not occur at all in IZs.
Finally, the finding of reduced peak ICa,L density may also be important in that it could contribute to the ionic mechanisms responsible for altered repolarization of action potentials in IZs, accounting for the decrease in plateau phase of potentials as well as reduction in amplitude of slow-response action potentials.7 In sum, the findings of the present study, together with the previous observation of reduced density of 4-aminopyridine–sensitive transient outward K+ current,8 firmly establish that chronic changes in ion channel function occur in myocytes of the EBZ as remodeling of the electrophysiological substrate continues during the healing phase of myocardial infarction.
|
Selected Abbreviations and Acronyms |
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|
Acknowledgments |
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This study was supported by grants HL-34477 and HL-30557 from the National Heart, Lung, and Blood Institute, Bethesda, Md, and a Grant-in-Aid from the American Heart Association. Dr Aggarwal was also a Berlex Laboratories predoctoral fellow during a portion of these studies.
Received May 23, 1995; accepted August 28, 1995.
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