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1 Cardiac Rhythm Management Lab and Department of Biomedical Engineering, University of Alabama-Birmingham, Birmingham, Alabama 35294; 2 Department of Biomedical Engineering, Tulane University, New Orleans, Louisiana 70125; 3 Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah School of Medicine, Salt Lake City, Utah 84112; and 4 Department of Physiology and Biophysics, University of Calgary School of Medicine, Calgary, Alberta, Canada T2N 4N1
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ABSTRACT |
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 Top
 Abstract
 Introduction
Materials and methods
Results
Discussion
References
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Purkinje-ventricular junctions (PVJs) have been
implicated as potential sites of arrhythmogenesis, in part because of
the dispersion of action potential duration (APD) between Purkinje (P)
and ventricular (V) myocytes. To characterize electrotonic modulation
of APD as a function of junctional resistance
(Rj), we
coupled single isolated rabbit P and V myocytes with an electronic circuit. In seven of eight PV myocyte pairs, both APDs shortened on
coupling at Rj = 50 M
. This was in contrast to modulation of APD in paired
ventricular myocytes, which demonstrated APD shortening of the
intrinsically longer action potential and APD prolongation of the
intrinsically shorter action potential. Companion computer simulations,
performed to suggest possible mechanisms for the paradoxical shortening
of the V action potential in paired P and V myocytes, showed that the
difference in intrinsic peak plateau potentials
(Vpp) of the P
and V myocytes determined whether the V action potential shortened or
prolonged on coupling. This difference in
Vpp caused a
large, repolarizing coupling current to flow to the V myocyte,
contributing to early inactivation of the L-type calcium current and
early activation of the inward rectifier current. These results suggest
that intrinsic differences in phase 1 repolarization could yield
differing patterns of APD shortening or prolongation in the network of
subendocardial PVJs, leaving some PVJs vulnerable to conduction of
premature stimuli while other PVJs remain refractory.
coupling clamp circuit; membrane models; transient outward current
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INTRODUCTION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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REGIONAL DIFFERENCES in action potential duration (APD) are arrhythmogenic (10, 20). Such differences exist at the Purkinje-ventricular junctions (PVJs), discrete sites of Purkinje (P)-ventricular (V) interaction distributed throughout the subendocardium (26-28). Action potentials from free-running Purkinje strands are typically longer than ventricular action potentials (5). At the PVJs, however, the junctional action potentials are electrotonically modulated, such that there is a distribution of APD across the PVJs (22, 27, 42). To understand how this distribution might contribute to or prevent arrhythmogenesis, it is important to characterize electrotonic modulation of APD as a function of cell-to-cell junctional resistance (Rj). Within the intact myocardium, however, neither a description of intrinsic membrane properties before coupling nor a method for selectively varying Rj is available.
Computer simulations provide a qualitative description of intrinsic action potential characteristics before and after coupling as well as the ability to adjust Rj to any desired value. Theoretical considerations indicate that Rj is an important determinant of spatial heterogeneities in APD and refractory period in multicellular preparations that have heterogeneities in intrinsic (uncoupled) APD. For example, Lesh et al. (21) modeled spatial dispersion of intrinsic APD within a sheet of myocardium and found that increasing Rj unmasked spatial inhomogeneities in APD. Similarly, Joyner et al. (15) observed that coupling two regions with intrinsic differences in APD produced APD shortening throughout the region with intrinsically longer APD and APD prolongation in the region with intrinsically shorter APD.
In the present study, we used a two-cell experimental system (12, 38) to characterize modulation of APD between coupled P and V myocytes. We hypothesized that coupling a single rabbit P myocyte to a single rabbit V myocyte would result in action potentials of durations intermediate to their intrinsic values. Surprisingly, we found that both action potentials shortened on coupling. Companion computer simulations, performed to elucidate the mechanisms underlying this modulation, suggested that the intrinsic P plateau level determined whether the V action potential shortened or prolonged on coupling. These results may be important in understanding how intrinsic differences in action potential configuration and electrotonic current flow at the PVJ contribute to differential responses to premature stimuli and the development of reentrant activity (8, 31).
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Cell isolation. Single P and V myocytes were isolated from rabbit hearts using previously published techniques (4, 32). Adult male rabbits weighing 2.0-3.0 kg were anesthetized with 1 ml/kg pentobarbital sodium and 0.5 ml heparin. After rapid isolation of the heart, the aorta was cannulated for Langendorff perfusion. The heart was sequentially perfused for 8-10 min with nominally Ca2+-free Tyrode solution, 18-20 min with enzyme solution containing 0.1 mM Ca2+, and 5 min with 0.1 mM Ca2+ Tyrode solution containing no enzymes.
Free-running Purkinje fibers were dissected from both ventricles, put into a small bath containing enzyme solution, and agitated with a stream of 100% O2. The temperature was maintained at 37°C. Single P myocytes were periodically removed from the bath and stored in 0.1 mM Ca2+ solution, and enzyme solution was added to the remaining P fibers in the bath to maintain a 2-ml volume. Cell dissociation required 15-60 min under these conditions. After the P fibers were dissected from both ventricles, the endocardial surface of the left ventricle was minced and gently agitated for 10 min in 0.1 mM Ca2+ Tyrode solution. The isolated cells were stored at room temperature in 1 mM Ca2+ Tyrode solution until use.Solutions. Nominally Ca2+-free Tyrode solution contained (in mM) 126 NaCl, 5.4 KCl, 5.0 MgCl2, 22 glucose, 1.0 NaH2PO4, 20 taurine, 5 creatine, 5 sodium pyruvate, and 24 HEPES, with pH adjusted to 7.4 with NaOH. The enzyme solution had the same composition, except that it also contained 1 mg/ml collagenase (type II, Worthington Biochemical, Freehold, NJ), 0.1 mg/ml protease (type XIV, Sigma Chemical, St. Louis, MO), and 0.1 mM CaCl2.
The normal bathing solution during the experiments contained (in mM) 126 NaCl, 5.4 KCl, 1.0 MgCl2, 1.0 CaCl2, 11 glucose, and 24 HEPES titrated with 13.0 mM NaOH (pH 7.4). In some experiments, we prepared a second bathing solution of the same composition but that additionally contained 2 mM 4-aminopyridine (4-AP). 4-AP reduced phase 1 repolarization by blocking the Ca2+-independent component of the transient outward current (Ito). The pipette filling solution contained (in mM) 15 NaCl, 30 KCl, 1.0 MgCl2, 5.0 HEPES, 10 EGTA, 5.0 K2ATP, and 90 potassium aspartate, pH adjusted to 7.2 with KOH. Transmembrane potentials were corrected for the 10-mV liquid junction potential caused by the potassium aspartate.Electrical recordings.
P and V myocytes were placed in a glass-bottom, temperature-controlled
bath (36°C) and continuously bathed with the normal Tyrode solution
at a rate of 1-2 ml/min. Transmembrane potentials were recorded
with an Axoclamp 2B amplifier system (Axon Instruments, Foster City,
CA). Suction pipettes were made from borosilicate glass (no. 7052, OD
1.65 mm, ID 1.20 mm, A-M Systems, Everett, WA), and when filled with
the pipette filling solution they had resistances of 1-5 M.
Pipette series resistance was carefully compensated before cell
attachment. Pipette capacitance was minimized by maintaining a low
level (1 mm) of solution in the bath. To minimize electrotonic
interactions resulting from sequential activation (12), we
simultaneously stimulated both myocytes with intracellular current
injection. The cycle length was either 1 or 1.5 s but was held constant
in each experiment. The stimulus duration was 3 ms, and the stimulus
magnitude was ~1.1 times the current threshold. The Purkinje
(Vm,p) and
ventricular
(Vm,v)
transmembrane voltages (Vm) were
digitized at 50 kHz for the first 100 ms of the action potential and
then at 4 kHz until the next stimulus was applied. These traces were
digitized with a 12-bit analog-to-digital converter (Digidata 1200A,
Axon Instruments) and recorded with a computer using pCLAMP 6 software
(Axon Instruments) for these coupling experiments.
Vm,v and
Vm,v Vm,p). That
output was sent to voltage-to-current convertors with fixed gain to
simultaneously supply equal and opposite coupling current
(Ic) of
(Vm,p Vm,v)/Rj
to the V myocyte and
(Vm,v Vm,p)/Rj
to the P myocyte.
Rj was determined
by the gains of the convertors and amplifiers and could be varied from 0 to 2,000 M in our system. We defined
Ic as positive
when it flowed from the ventricular to the Purkinje myocyte.
Our procedure for studying modulation of repolarization in coupled P
and V myocytes was to first establish pipette attachments in both
myocytes. We simultaneously recorded five intrinsic (uncoupled) action
potentials and then coupled the myocytes at
Rj = 1,000 M
and immediately recorded the next five action potentials from both
myocytes. We repeated this procedure for resistances of 500 and 50 M. We characterized modulation of repolarization through changes in
APD and the peak plateau potential
(Vpp), where
APD was the time of 90% repolarization and
Vpp was defined
as Vm at the end
of phase 1. Because the transition from phase 1 to phase 2 was not
always easy to identify, we mathematically defined
Vpp as the local
zero-crossing of
d2Vm/dt2,
which represented the point where phase 1 repolarization was slowest.
Data analysis. Because the action potential configuration recorded from a single myocyte demonstrates beat-to-beat variability (35), we averaged Vpp and APD for the five uncoupled and the five coupled action potentials at each resistance for every myocyte pair. Thus the summary statistics reflect means ± SD of all recorded traces. Statistical analysis included one-way analysis of variance (Minitab 10x, State College, PA). A value of P < 0.05 was considered statistically significant.
Computer simulations. As previously described (12), we used the DiFrancesco-Noble (DN) membrane equations (2) to describe the ionic currents for a single Purkinje cell and the Luo-Rudy (LRd) membrane equations (24, 47) to describe the ionic currents for a single ventricular cell. Action potentials were calculated by numerically solving the following equations
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(1) |
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(2) |
Parameter scaling for
Ito.
In some simulations, we studied the influence of the P plateau
potential on modulation of the ventricular APD. To adjust
Vpp of the model
P cell, we modified the magnitude of the transient outward current
(Ito) in the DN
membrane equations. Figure 1 shows the
uncoupled P action potentials (A)
resulting from variation in
Ito
(B). A peak
Ito of 51.9 µA/cm2 generated action
potentials similar to those recorded in the experiments, with APD = 312 ms and Vpp = 33 mV. Nearly complete block of
Ito increased
Vpp to 17 mV and
shortened APD to 260 ms. Graded reduction of
Ito yielded
graded changes in
Vpp and APD. These modifications were used in all simulations involving analyses of
the contributions of high
(Vpp = 17 mV) and
low (Vpp = 33 mV) P plateau to modulation of APD between P and V cells.
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RESULTS |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Modulation of repolarization in PV and VV' myocyte pairs.
Action potentials from PV and VV' myocyte pairs demonstrated
different patterns of modulation in response to electrical coupling. VV' myocyte pairs demonstrated shortening of the longer action potential and prolongation of the shorter action potential, whereas PV
myocyte pairs demonstrated shortening of both action potentials. Figure
2 shows uncoupled and coupled action
potentials from a PV myocyte pair
(A) and a VV' myocyte pair
(B). In Fig.
2A, the uncoupled P action potential
had a lower plateau and longer duration than the uncoupled V action
potential. Vpp
was 37.6 mV and APD measured 437 ms in the uncoupled P action
potential, whereas in the uncoupled V action potential,
Vpp was 28.6 mV
and APD measured 325 ms. On coupling at
Rj = 50 M,
both action potentials shortened dramatically, with concomitant changes
to their plateaus. In the coupled P action potential,
Vpp increased to
15.3 mV and APD measured 119 ms. In the coupled V action
potential, Vpp
decreased to 1.7 mV and APD measured 115 ms.
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, the
values of Vpp and
APD in both action potentials were intermediate to the intrinsic V and
V' values.
We observed this pattern of APD shortening of the longer action
potential and prolongation of the shorter action potential in all eight
VV' myocyte pairs studied. These results are summarized in Fig.
3. The data are organized by coupling
resistance and include measurements taken before coupling and on
coupling at each
Rj. Coupling-induced changes in mean APD were asymmetric (Fig.
3A). Decreasing
Rj progressively
shortened the V action potentials and prolonged the V' action
potentials, with statistically significant changes in APD at all three
values of Rj. At
Rj = 50 M, the
V and V' action potentials had the same mean APD of 242 ms. The shorter V' action potentials had an intrinsically higher
Vpp than the
longer V action potentials (Fig.
3B). However, coupling did not
significantly alter
Vpp in either the
V or V' action potentials.
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, the
P and V action potential durations were not significantly different.
The coupling-induced changes in
Vpp are
illustrated in Fig. 4B. The intrinsic
Vpp of the P action potentials was approximately 20 mV, and the intrinsic Vpp of the V
action potentials was approximately 35 mV.
Vpp increased progressively in the P action potentials with decreased
Rj. In the V
action potentials, statistically significant decreases in Vpp were observed
at Rj = 500 M
and Rj = 50 M.
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Purkinje Vpp and
ventricular APD.
In the one PV myocyte pair that did not demonstrate shortening of both
action potentials on coupling,
Vpp of the
uncoupled P action potential was much higher than the mean
Vpp. Figure
5 shows action potentials from this PV cell
pair before and after coupling. In the uncoupled P action potential,
Vpp was 1.5 mV and APD measured 431 ms. Consistent with coupling-induced changes in
the P action potential observed in the other seven PV myocyte pairs,
Vpp increased and
APD shortened at
Rj = 50 M.
However, the ventricular APD prolonged from 136 to 156 ms when coupled at Rj = 50 M.
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33
mV), both the P and V action potentials shortened on coupling at
Rj = 50 M
(Fig. 6A). This was consistent with changes observed in the experiments (compare with Fig.
2A). When the P plateau was high
(Vpp = 17 mV),
the P action potential shortened by 84 ms, but the V action potential
prolonged by 12 ms on coupling at
Rj = 50 M
(Fig. 6B). This was consistent with
the response shown in Fig. 5.
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. In these simulations (not shown),
Vpp > 20
mV in the uncoupled P action potential resulted in prolongation of the
V action potential on coupling at
Rj = 50 M.
Effect of 4-aminopyridine on coupling-induced changes in APD.
Because the simulations suggested that blocking
Ito would cause
the ventricular APD to prolong rather than shorten on coupling, we
tested this hypothesis experimentally using 4-aminopyridine (4-AP) to
inhibit the Ca2+-independent
component of Ito.
The coupling protocol was first performed in normal bathing solution
and then repeated after equilibration in 2 mM 4-AP (Fig.
7). As described above (Fig. 2), coupling
in normal bathing solution shortened both action potentials, increased Vpp of the P
action potential, and decreased
Vpp of the V
action potential (Fig. 7A). However,
in 2 mM 4-AP (Fig. 7B), the P and V
action potentials came to an intermediate APD. The Purkinje APD
shortened by 107 ms, and the ventricular APD prolonged by 23 ms on
coupling at Rj = 50 M.
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.
4-AP induced a 33.3 ± 17.0 mV increase in
Vpp in the
uncoupled P action potentials. This higher P plateau resulted in an
average prolongation of the ventricular APD of 19 ± 4 ms on
coupling at Rj = 50 M in 4-AP.
Computer simulations suggesting underlying ionic mechanisms.
Because the simulations qualitatively reproduced the experimental
results and suggested that blocking
Ito reversed
coupling-induced shortening of the ventricular APD, we used simulations
to describe how the ionic currents that dominate during repolarization
[L-type calcium current
(ICa), delayed
rectifier current (IK), and inward rectifier
current
(IK1)]
were affected in the model V cell on coupling. Figure
8 shows simulated action potentials
(A),
Ic
(B), and the ionic currents dominant
during repolarization (C-E)
from the model V cell. Three cases are illustrated:
1) the uncoupled V action potential
and currents, 2) the V action
potential and currents when coupled to a model P cell with a high
plateau, and 3) the V action
potential and currents when coupled to a model P cell with a low
plateau. When coupled to the model P cell with a high plateau, the V
action potential had a minimal decrease in
Vpp and a modest
prolongation of APD, relative to the uncoupled V action potential (Fig.
8A). Consistent with the experiments (Fig. 7B), the relatively small
potential gradient established during coupling to the model P cell with
a high plateau yielded a relatively low magnitude, repolarizing
Ic (Fig.
8B) during the plateau. This
coupling current directly caused the reduction in Vpp of the model
V cell, which, in turn, caused a small voltage-dependent increase in
peak ICa (Fig.
8C) and a small
voltage-dependent decrease in
IK (Fig.
8D) throughout the plateau. Once the
P and V action potentials crossed over during late phase 2 and
early phase 3 (see Fig. 6B),
the coupling current became depolarizing, thus prolonging the V action
potential and broadening
IK, relative to
the uncoupled case. Because phase 3 repolarization was slower and later
relative to the uncoupled V action potential,
IK1 was broader
and peaked later for the high P plateau case (Fig.
8E).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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New observations. Results from the present study indicate that direct electrical coupling of a rabbit Purkinje myocyte to a rabbit ventricular myocyte shortened APDs of both cells. This result was surprising initially because electrotonic modulation of repolarization typically is thought to result in shortening of the intrinsically longer action potential and prolongation of the intrinsically shorter action potential (15, 27, 35, 42). In those previous studies, electrotonic interactions during late repolarization induced repolarizing current to flow into cells with longer intrinsic APDs and depolarizing current to flow into cells with shorter intrinsic APDs. This subsequently yielded action potentials of intermediate duration. However, in the present study, the large difference between the P and V plateau potentials induced a significant coupling current during early repolarization. This coupling current accelerated repolarization in both cells.
Current injection during plateau. The accelerated repolarization caused by the coupling current observed in the present study is consistent with previous reports of altered repolarization induced by current injection during the action potential plateau (9, 18, 41, 45). For example, Kass and Tsien (18) showed that small depolarizing pulses applied early in the plateau shortened APD in calf Purkinje fibers by promoting greater activation of IK and accelerating inactivation of ICa. Similarly, small repolarizing pulses prolonged APD. However, a large repolarizing pulse may shorten APD dramatically when "all-or-nothing" repolarization is induced (9, 41, 45). Those early reports described all-or-nothing repolarization as the immediate return of the membrane to its resting potential when a large repolarizing pulse was applied.
In the present study, the coupling current induced by the difference in P and V plateau potentials functioned as a repolarizing pulse during the plateau of the V action potential. In seven of eight PV myocyte pairs, this "pulse" was large enough to accelerate repolarization of the V myocyte. The peak magnitude of this coupling current averaged 0.32 ± 0.11 nA in these seven PV myocyte pairs at Rj = 50 M.
Adding 4-AP to the bathing solution in two experiments reduced the
potential gradient between the P and V myocytes, thereby reducing peak
Ic to 0.09 ± 0.07 nA. Similarly, peak
Ic generated during VV' interactions, which prolonged the shorter action
potential and shortened the longer action potential, was only 0.06 ± 0.04 nA. Note that
Ic was only a
repolarizing current to the V myocyte while
Vm,v remained
above Vm,p. Once
Vm,v repolarized
below Vm,p, Ic became a small
depolarizing current to the V myocyte. This depolarizing
Ic did not
prolong the ventricular action potential, however, because final
repolarization of the V action potential had already begun.
Membrane resistance and electrical load.
Asymmetries in the coupling-induced changes to the P and V action
potentials (e.g., larger increase in Purkinje
Vpp than decrease in ventricular
Vpp) were
likely caused by the mismatch in P and V membrane resistances. In the
present study, we measured diastolic membrane resistance by applying
small hyperpolarizing currents from the resting potential. The membrane
resistance of the P cells (Rm,p) averaged
85 ± 21 M, and the membrane resistance of the V cells
(Rm,v) averaged
24 ± 14 M. These values were consistent with previously reported
values (4, 12). Because the coupling current applied to each cell of a
PV cell pair was of equal and opposite magnitude, the larger
coupling-induced change in
Vm,p reflected
the larger Rm,p.
Note that the membrane resistance changes over the course of the action
potential, so diastolic membrane resistance is a measure only of how
the membrane potential will change with a current applied during phase
4. Although we know that
Rm progressively
increases and comes to a peak during phase 3 of the action potential
(45), there have been no systematic comparisons of the differences in
the time courses of
Rm,p and Rm,v to date.
Ito and plateau potential. Our studies show that the repolarizing current applied to the V myocyte was a direct result of the large potential gradient formed by the low P plateau and the high V plateau. This difference in plateau potentials is directly related to the difference in Ito density in these myocytes. Specifically, in rabbit P myocytes, the 4-AP-sensitive component of Ito has a peak current density of 14 pA/pF (4), and in rabbit papillary muscle myocytes, peak Ito density is 3.69 pA/pF (6). Because the difference in Vpp of the uncoupled myocytes is largely caused by intrinsic differences in Ito density, it follows that the difference in Vpp can be reduced by interventions that reduce the difference in Ito density. The Ca2+-independent component of Ito was selectively inhibited by 2 mM 4-AP, and in the present study we showed that by raising the P plateau, 4-AP prevented coupling-modulated shortening of the V action potential.
A change in cycle length will also differentially modulate the P and V action potentials (37), largely because of the intrinsic differences in Ito (4). Because recovery of Ito from inactivation is slow, the magnitude of Ito is smaller at faster rates (7). Although we did not alter the cycle length in the present study, on the basis of our findings, we expect that an increase in the rate of stimulation would reduce peak Ito, subsequently raising the P plateau and decreasing the driving force for coupling current. However, it is unlikely that a moderate increase in the stimulation rate would prevent coupling-modulated shortening of the V action potential, because only a 20% decrease in Ito would be expected with a change in cycle length from 1 to 0.5 s (4).Coupling-induced changes to ICa, IK, and IK1. Although the only experimental modification to the ionic currents considered in the present study was inhibition of Ito by 4-AP, our simulations suggested specific coupling-induced changes to the time course and magnitude of currents dominant during phases 2 and 3. Coupling-induced changes to the magnitude of ICa observed in the present study were consistent with changes generally associated with slow conduction in VV' myocyte pairs (16, 19, 36). In those studies, early partial repolarization of the stimulated leader cell caused by electrical loading by the nonstimulated follower cell induced a large ICa only in the leader cell during conduction (19). In the present study, although the P and V myocytes were simultaneously stimulated to eliminate electrotonic interactions during conduction, the P myocyte imposed an electrical load on the V myocyte because of the large difference in Vpp between the action potentials. This load induced a much larger ICa when the P plateau was low than when the cells were uncoupled or when the P plateau was high (Fig. 8). Studies in isolated rat and rabbit ventricular myocytes also showed an increase in peak ICa when repolarization was accelerated by action potential voltage clamps (1, 46). In the present study, coupling additionally caused a voltage-dependent reduction in IK. Although the combination of increased ICa and decreased IK is generally expected to prolong ventricular APD, the coupling current induced accelerated repolarization by forcing Vm,v into the voltage range for activation of IK1, initiating final repolarization and thereby shortening the ventricular APD.
Clearly, ventricular repolarization depended not only on the intrinsic ionic currents flowing through the V cell membrane but also on the coupling current that resulted from potential differences between the P and V cells. Neglecting the contributions of other ionic currents (such as the sodium-calcium exchange current, the sodium-potassium pump current, etc.), we can write the total membrane current (Im) flowing through the V cell as Im = ICa + IK + IK1 + Ic. By convention, Vm,v increases (depolarizes) when Im is negative and decreases (repolarizes) when Im is positive. From Fig. 8, the ventricular APD prolonged on coupling to the P cell with a high plateau because 1) the inward current (ICa) approximately balanced the outward currents (IK and Ic) during the plateau, keeping the plateau relatively high so that 2) IK1 did not activate early, and reversal of Ic delayed final repolarization. Conversely, the ventricular APD shortened on coupling to the P cell with a low plateau because 1) the outward currents (IK, but particularly Ic) were larger than the inward current (ICa), resulting in a rapid decline of Vm,v during the plateau, so that 2) IK1 activated early, accelerating repolarization despite the reversal of Ic during phase 3.Limitations. Our results must be considered within certain limitations. For example, we represented the PVJ simply, as one electrical connection between a single P myocyte and a single V myocyte. Within the syncytium, however, there is a network of PVJs with multidimensional interactions. This complex structure, in concert with intrinsic differences in P and V membrane properties, determines the distribution of APD across the PVJ. However, we wanted to eliminate the structural complexities, so that we could focus on how the intrinsic differences in action potential configuration contribute to dispersion of APD across the high resistance barrier at the PVJ (30, 39). Thus we represented the PVJ in this most basic of structures so that we could record the intrinsic, uncoupled action potentials and the changes to the action potentials induced by electrical coupling. With this preparation, we were also able to vary Rj between the myocytes, which cannot be accomplished within a syncytial preparation without affecting other membrane properties.
Nearly all previous syncytial studies on P-V interactions have used false tendon and Purkinje fiber-papillary muscle preparations from the canine heart (25-27, 42). In these studies, investigators observed prolongation of the ventricular APD at the junction, whereas we observed shortening of the ventricular APD. A possible explanation for this difference is the species dependence of the P action potential configuration. Phase 1 repolarization is less pronounced in canine P fibers, because the plateau potential is typically above or near 0 mV (11, 33). As demonstrated in Figs. 5-7, a higher plateau results in modulation of the action potentials to durations intermediate to the intrinsic APDs. In our isolated P myocytes, the mean plateau potential was approximately20 mV (Fig.
4B). This low
Vpp was not a
consequence of the cell isolation procedure because similar action
potential configurations have been recorded from intact rabbit P
fibers. Colatsky and Tsien (3) documented a mean plateau potential of
22 mV in 15 intact Purkinje fibers from rabbit hearts,
indicating that the low P plateau in rabbits is a result of the balance
between the ionic currents dominant during phase 1. Because no studies
on modulation of repolarization have used the rabbit PVJ to date, it is
unclear what the distribution of APD is in the syncytium of this preparation.
Limitations of the computational studies include the mathematical
models used to represent the P and V membrane properties. These models
were not developed from rabbit heart cell data. Although there is a
rabbit atrial cell model (23), there are no P or V models developed
solely from rabbit heart cell data. The DN model incorporates data from
several species, whereas the LRd model was developed from guinea pig
ventricular myocytes. A specific limitation of the DN model that is
pertinent to the present study is the description of
Ito. There are
two components of
Ito in rabbit P
myocytes, a 4-AP-sensitive component and a
Ca2+-dependent component (7).
Though only one lumped
Ito is described in the DN model, we were able to vary the magnitude of this current to
model P action potentials with values of
Vpp that closely
approximated the experimental recordings. Similarly, a specific
limitation of the LRd model pertinent to the present study is the
species dependence of
Ito. Because the
LRd model was formulated from guinea pig heart cell data, no
description of
Ito is provided
in the model. However, the phase 1 repolarization attributed to
Ito in rabbit V
myocytes is small and variable (4, 7) and likely does not contribute to
coupling-induced shortening of the V action potential. Furthermore,
Varro et al. (40) showed that the action potentials and membrane
currents during repolarization were similar in guinea pig and rabbit
ventricular myocytes. Thus, although the simulation results are
necessarily qualitative in nature, the intrinsic differences in plateau
potentials and action potential durations in the DN and LRd models
allowed us to use the simulations to suggest mechanisms for the
seemingly paradoxical shortening of the ventricular APD on coupling.
Specifically, the intrinsic difference in plateau potentials caused by
intrinsic differences in
Ito density
induced a large repolarizing coupling current in the V myocyte and
subsequently led to accelerated repolarization in both myocytes on coupling.
Implications. At the PVJ, the arrangement of cells is complex. A thin layer of Purkinje cells is connected to a much thicker layer of ventricular cells via either strands or strands and sheets of transitional cells (39). Although the transitional cells have been histologically characterized (25, 39), little is known about their ionic membrane properties because there is no documented method for isolating the transitional cells at this time. The transitional cell layer is thought to act largely as a high-resistance barrier between the P and V layers (17, 30), so we modeled this function of the transitional cells as a large Rj between the P and V myocytes. Teleologically, this high-resistance barrier may prevent arrhythmogenesis by isolating the P layer from the ventricular load, thus allowing rapid activation of the subendocardial layer (14, 30). However, under pathological conditions (8, 13, 29, 30, 42, 44), the resistive barrier may promote unidirectional (P-to-V) conduction block (12). Furthermore, differing patterns of APD shortening or prolongation within the network of subendocardial PVJs could promote dispersion of repolarization, leaving some PVJs vulnerable to conduction of premature stimuli while other PVJs remain refractory. In this setting, impulses that block at some PVJs but not others may reenter the Purkinje network retrogradely (V-to-P conduction) at the sites of initial P-to-V block, thereby initiating circus movement reentry (8, 31).
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ACKNOWLEDGEMENTS |
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The authors thank Kate Sreenan for assistance with the statistics, Massimiliano Zaniboni for assistance with the animal preparation, and Adam Cates for helpful comments and suggestions on the manuscript.
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FOOTNOTES |
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This work was supported by the Board of Regents of Louisiana under Education Quality Support Funds GF-15 to D. J. Huelsing; the National Science Foundation under National Young Investigator Award BES-9457212, the Whitaker Foundation Special Opportunities Award to the University of Alabama-Birmingham and the Department of Biomedical Engineering, and the US Public Health Service under Grant R29-HL54024 to A. E. Pollard; National Heart, Lung, and Blood Institute Grants HL-42873, HL-42357, and HL-17682 and awards from the Nora Eccles Treadwell Foundation and the Richard A. and Nora Eccles Harrison Fund for Cardiovascular Research to K. W. Spitzer; and a Research Fellowship from both the Heart and Stroke Foundation of Canada and the Alberta Heritage Foundation for Medical Research to J. M. Cordeiro.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: D. J. Huelsing, Cardiac Rhythm Management Lab, Univ. of Alabama-Birmingham, Volker Hall B140, 1670 University Blvd., Birmingham, AL 35294.
Received 2 April 1998; accepted in final form 19 October 1998.
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REFERENCES |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Statistically significant difference between uncoupled (U)
and coupled (C) action potential characteristics.
