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72
Action Potential Transfer in Cell
Pairs Isolated From Adult Rat and
Guinea Pig Ventricles
Robert Weingart and Peter Maurer
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An enzymatic procedure was used to obtain ventricular cells from adult rat and guinea pig
hearts. Isolated pairs of cells were selected to study the action potential transfer from cell to cell
and determine the resistance of the nexal membrane, rn. For this purpose, each cell of a cell pair
was connected to a patch pipette so as to enable whole-cell, tight-seal recording. Normal impulse
transmission was observed when rn ranged from 5-265 Mil. In these cases, the action potential
in both cells occurred virtually simultaneously. An occasional failure in action potential transfer
was seen in cell pairs whose rn had increased to 155-375 Mil. In these cases, the impulse
transfer across the nexal membrane occurred with considerable delay. Impulse transfer was
completely blocked once rn was larger than 780 Mil. Assuming a single connexon conductance
of 100 pS, this would mean that more than 13 connexons are necessary to allow impulse transfer
from cell to cell. Two single myocytes, gently pushed together, neither showed electrotonic
interaction nor impulse transfer, thus rendering unlikely the possibility of an ephaptic signal
transmission. (Circulation Research 1988;63:72-80)
I
n cardiac tissue, impulse propagation is determined by two sets of parameters: source factors and sink factors. The source factors
involve excitability of the sarcolemmal membrane
brought about via voltage- and time-dependent
inward currents; the sink factors include passive
electrical properties and the topology of cell arrangements (e.g., see Fozzard1)- A change in each of
these parameters is expected to modify the conduction velocity in the heart. In fact, the influence of
membrane excitability on conduction velocity has
long been recognized, while the importance of intercellular connections was realized more recently.
Interest in intercellular connections increased after
it was established that the functional cell-to-cell
coupling does not remain constant but can be modified under certain conditions (for review, see De
Mello2; see also Spray and Bennett3).
In the past, studies focusing on impulse propagation were carried out on intact hearts,4 preparations
dissected from different regions of the heart,3-9 or
synthetic strands of cardiac cells. 10 " More recently,
the introduction of enzymatic procedures for disasFrom the Department of Physiology, University of Beme,
Berne, Switzerland.
Supported by the Swiss National Science Foundation grants
3.360-0.82 and 3.253-0.85.
Address for correspondence: R. Weingart, Department of
Physiology, University of Berne, BuhJplatz 5, CH-3012 Berne,
Switzerland.
Received August 27, 1987; accepted January 28, 1988.
sociating cardiac tissue (e.g., see Trube12) enabled a
novel approach. Besides single cells, these methods
also yield functionally intact cell pairs (e.g., see
Kameyama13 and Metzger and Weingart14). Such
cell pairs enable impulse transmission to be investigated at the cellular level for the first time.
This article describes experiments performed on
pairs of cells isolated from adult rat and guinea pig
ventricles. The experimental approach adopted
involved current- and voltage-clamp measurements. It allowed simultaneous study of action
potential transfer from cell to cell and the nexal
membrane resistance (rj limiting the intercellular
current flow. Preliminary data on this topic have
been published before.1516
Materials and Methods
Cell Preparation
Ventricular cell pairs were obtained by means of
enzymatic dispersion of adult rat or guinea pig
hearts. The isolation procedures employed have
been described before in detail. For rat hearts, it
involved the use of collagenase (type I, code CLS
4194, Worthington, Freehold, New Jersey14); for
guinea pig hearts, a combination of collagenase
(type 1, code C0130, Sigma Chemical, St. Louis,
Missouri) and hyaluronidase (type I-S, code H3506,
Sigma).17 All experiments were carried out at room
temperature (22° C) in the presence of the following
bathing solution (mM): NaCl 150, KC1 4, CaCl2 5,
Weingari and Maurer
Cell Pairs: Action Potential Transfer
73
MgCl2 1, HEPES 5 (pH 7.4), glucose 5, and pyruvate 2.
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Experimental Setup and Patch Pipettes
The experiments were carried out in a chamber
consisting of a Perspex frame with an attached glass
bottom (volume, 1 ml). The chamber was mounted
on the stage of an inverted microscope equipped
with phase contrast optics (Diaphot-TMD, Nikon;
Nippon Kogaku, Tokyo, Japan). A TV system
(JVC; Victor Company, Tokyo, Japan) facilitated
the visual control of the cells and pipettes. Patch
pipettes were pulled from microhematocrit tubing
(code 1021; Clay Adams, Parsippany, New Jersey)
using a micro-processor controlled puller (model
BB-CH, M6canex, Geneva, Switzerland). Fire polished pipettes (tip size, approximately 2 fim) were
filled with the following solution (mM): NaCl 10,
potassium aspartate 120, KC1 10, CaCl2 1, MgCl2 1,
EGTA 10, and HEPES 5 (pH 7.2). In some experiments, potassium chloride or cesium chloride was
used instead of potassium aspartate.
The equipment employed to manipulate the
pipettes and to measure and record the electrical
signals has been described previously.1418 The settling time of the custom-built amplifiers was approximately 0.1 fjbsec. Control experiments (two patch
pipettes fixed to a single cell) revealed that the
membrane potential was adequately controlled
within 1 msec. Series resistances arising from the
pipette tips (direct current resistances: 1.5-2.5 MH
and 0.8—1.3 Mft, for aspartate and Cl" containing
solutions, respectively) were compensated for previous to an experiment. No corrections were made
for access resistances arising from membrane fragments inside the pipette tip. Previous studies have
shown that this does not severely affect the results.18
Membrane potentials were corrected for liquid junction potentials between the pipette filling solution
and the bath solution (Cl" containing solutions, - 2
mV; aspartate containing solution, - 10 mV).
Signal Recording and Data Analysis
During an experiment, each cell of a cell pair (cell
1, cell 2) was attached to a patch pipette so as to
enable whole-cell, tight-seal recording.19 The pipettes
were in turn connected to separate amplifiers that
allowed operation in the current- or voltage-clamp
mode (see Figure 1 A). The current-clamp mode was
used to inject rectangular current pulses and record
the subsequent action potentials. The voltageclamp mode was used to determine the nexal membrane resistance, rn.18 In essence, this approach
enabled the potential of the sarcolemmal membrane
of each cell to be controlled (V m ,, Vm-2) and thereby
the voltage gradient across the nexal membrane
(VJ to be set, while monitoring the associated
currents flowing through each pipette (I,, y . Thus,
when a voltage pulse was delivered to cell 1 (V,),
while cell 2 was kept at the common holding potential (VH), V, reflected the Vn and I2 corresponded to
B
FIGURE 1. Schematics of the experimental arrangement. Panel A: Isolated ventricular cell pair with two
attached patch pipettes, operated in the tight-seal, wholecell recording mode. The pipettes enable the measurement of imposed voltage changes (V,, V2) and the associated current increments (I,, I2). Panel B: Equivalent
circuit used to analyze the resistive elements involved.
rmJ and rmJ2, resistances of the nonfunctional membranes
of cell 1 and cell 2; rn, resistance of the nexal membrane
located between the cells.
the current flowing across the nexal membrane (see
Figure IB). From these two quantities, that is, V,
and I2, rn was determined.
Results
The experiments were performed on two-cell
preparations in the side-to-side configuration. As a
standard procedure, each cell pair was subjected
sequentially to either of two protocols. In the one
case, action potentials were elicited and recorded
through use of the current-clamp mode. This
involves application of depolarizing current pulses
to one of the cells and recording of the elicited
action potentials from the stimulated cell and the
follower cell. In the other case, transmembrane
currents were determined using the voltage-clamp
mode. This includes establishment of desired transnexal voltage gradients and measurement of the
associated junctional membrane currents. Alternate
application of the two protocols allowed exploration of the relation between intercellular coupling
and impulse propagation.
Normally Coupled Cell Pairs
The electrical behavior of an isolated cell pair
under control conditions is documented in Figure 2.
Figure 2A shows the standard signals obtained in
the current-clamp mode. Both cells exhibited the
same resting potential, that is, - 8 5 mV, indicative
of electrical coupling via intact nexal membrane. In
such preparations, it is anticipated that differences
in intrinsic membrane potential are masked by
compensatory currents flowing from cell to cell.
Depolarizing current pulses, constant in duration
(20 msec) and frequency (0.5 Hz), but variable in
amplitude, were applied to cell 1. As indicated,
74
Circulation Research
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Vol 63, No 1, July 1988
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|20mV
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20 mV
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J
0.2 s
2. Electrical properties of a cell pair exhibiting
normal intercellular coupling. Panel A: Records obtained
in the current-clamp mode. Current injection into cell 1
(Ii) evoked an action potential in cell 1 (Vj) and cell 2
(V2). Vm, -85 mV. Panel B: Signals recorded in the
double voltage-clamp mode. Establishment of a transnexal voltage gradient (V,) gave rise to nexal current flow
(I2). Holding potential (VH), -52 mV; /•„, 41 Mil. Guinea
pig cell pair P44-g.
FIGURE
current pulses of just threshold amplitude elicited
an action potential in cell 1 (V,) and cell 2 (V2). It is
tempting to assume that the action potential was
transferred from cell 1 to cell 2 via current flow
across the nexal membrane.
Figure 2B illustrates the standard signals recorded
after switching to the voltage-clamp mode. Initially,
the membrane potential of both cells was clamped
to a VH of - 5 2 mV. Thereafter, a depolarizing
clamp pulse (amplitude, +41 mV; duration, 200
msec; frequency, 0.5 Hz) was applied to cell 1,
while the membrane potential of cell 2 was maintained. This gave rise to the current signals I( and I2.
1, reflects the sum of two current components, I m ,
and In. Im 1 flows across the nonjunctional membrane of cell 1 and corresponds to the spontaneously decaying Ca2+ inward current, I,) (e.g., see
Irisawa and Kokubun20), whereas In flows through
the nexal membrane located between cell 1 and cell
2. The capacitative spikes in I,, inwardly and outwardly oriented, were caused by the rapid "on"
and "off" of the voltage pulse, respectively. I2
corresponds to In. The ratio V,/I2, defining rn, turned
out to be 41 Mft.
Figure 3 repeats selected records from Figure 2A.
To emphasize the temporal relation, the signals
were superimposed and displayed on an expanded
time scale. Trace I, reproduces the current pulse
injected into cell 1, while V, and V2 correspond to
the voltages measured from cell 1 and cell 2, respec-
10 ms
FIGURE 3. Oscilloscope traces of transfer of an action
potential in a normally coupled cell pair. Current injection into cell 1 (It) gave rise to an electrotonic response
followed by an action potential in cell 1 (V,) and cell 2
(V2). As judged from the fast rising phase of the action
potentials, there was no measurable delay between the
two voltage signals. Position of I, marks the zero voltage
level for V, and V2. Its polarity has been turned upside
down. rn, 41 Mil. Guinea pig cell pair P44-g.
tively. Current injection gave rise to electrotonic
responses that were similar in time course and
magnitude. The depolarizations eventually reached
threshold so that each cell fired an action potential.
Close inspection of the fast-rising phase reveals no
measurable delay between the two action potentials. This suggests that the two events occurred
virtually simultaneously.
Immediately after the onset of electrical measurements this cell pair exhibited an rn of 9 Mft. The
records presented in Figures 2 and 3 were obtained
12 minutes later, when rn had increased spontaneously to 41 Mft. The increase in rD presumably was
caused by elevation in [Ca2+], (e.g., see Maurer and
Weingart17) and/or elution of cytosolic compounds
involved in connexon regulation. Of importance is
that up to this stage, there was no indication of a
delay in action potential transfer from cell to cell.
Moderately Uncoupled Cell Pairs
Figure 4 illustrates the performance of a cell pair
with impaired impulse propagation. Figure 4A
depicts current-clamp records displayed at compressed time scale. Both cells showed unstable
resting potentials ranging from —71 mV to - 7 7
mV. The spontaneous changes in Vm occurred synchronously in cell 1 and cell 2, suggesting that the
cells are still coupled electrically. Injection of small
current pulses into cell 1 (I,; duration, 200 msec;
frequency, 0.5 Hz) provoked subthreshold depolarizations in both cells. V, and V2 differed considerably in amplitude, signaling the presence of a substantial voltage drop across the nexal membrane.
Application of larger stimuli to cell 1 evoked action
potentials in both cells in most trials. On two
occasions, however, cell 2 failed to respond with an
action potential. This indicates block of impulse
propagation across the junction. In these cases,
Weingart and Maurer
Cell Pairs: Action Potential Transfer
75
I 20 mV
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20 s
I 20 mV
I 1 nA
02s
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FIGURE 4. Electrical properties of a partially uncoupled
cell pair. Panel A: Subthreshold current injections into
cell 1 (I,) produced electrotonic responses of different
amplitudes in cell I (V/) and cell 2 (V2). Suprathreshold
current injections elicited action potentials in cell 1 and,
with two exceptions, in cell 2. Panel B: Successful transfer of an action potential from cell 1 to cell 2, displayed at
faster time resolution. Vm, -72 mV. Panel C: Determination of the nexal membrane resistance /•„ using the double
voltage-clamp approach. A voltage gradient was applied
across the nexal membrane (V,) which gave rise to the
transnexal current flow I2. /•„, 315 Mil. Rat cell pair
P25-e.
current injection evoked an action potential in the
stimulated cell (cell 1) and an electrotonic response
in the follower cell (cell 2).
Figure 4B illustrates a successful action potential
transfer at faster time resolution. Current injection
into cell 1 was accompanied by a large electrotonic
response in cell 1 and a small one in cell 2. The
former eventually reached threshold, the latter
remained subthreshold throughout. Despite this difference, both cells fired an action potential toward
the end of the current pulse. This pattern of phenomena implies that the two action potentials are of
different origin. In cell 1 it is a consequence of
direct stimulation, whereas in cell 2 it reflects
indirect stimulation via nexal current flow. Thus,
this experimental situation unambiguously documents impulse propagation across the junctional
membrane.
Figure 4C illustrates the voltage-clamp records
used to determine the nexal membrane resistance
prevailing under these circumstances. Starting at a
VH of - 6 9 mV, a hyperpolarizing pulse (amplitude,
- 2 0 mV; duration, 200 msec) was applied to cell 1
(V|), while the membrane potential of cell 2 (VJ was
maintained. This pulse protocol elicited the current
signals I, and I2. Again, I, represents the sum of two
current components, 1^, and Io. Under the prevailing conditions, Im-1 corresponds to the K+ inward
1
1
20 ms
FIGURE 5. Oscilloscope traces of action potential transfer in a partially uncoupled cell pair. Current injection
into cell 1 (/;) gave rise to electrotonic responses of
different amplitudes in cell 1 (V,) and cell 2 (V2). In cell 1,
the threshold was reached and thereby an action potential elicited. The upstroke of this action potential provided extra voltage source to further depolarize cell 2.
Within a delay of 24 msec, cell 2 fired an action potential,
too. To emphasize the temporal relation, the voltage
signals were superimposed and offset by 3 mV. Rat cell
pair P25-e.
rectifier current (e.g., see Sakmann and Trube21). I2
represents a single current component, In. Analysis
of the junctional current signal I2 revealed an rn of
315 Mft, a value 60-80 times above control.
Figure 5 shows selected action potentials displayed at high time resolution. The baseline of the
current trace I, marks the reference potential of cell
1. Application of a short stimulus produced a direct
depolarization of cell 1 and, via nexal membrane
current flow, an indirect depolarization of cell 2.
Again, in cell 1, the depolarization was large enough
to reach threshold, whereas in cell 2 it was too
small. As a consequence, an action potential was
elicited in cell 1. The upstroke of this action potential provided an extra voltage gradient across the
nexal membrane. The subsequent intercellular current flow depolarized cell 2 toward the threshold
and, after considerable delay, released an action
potential. Conversely, the change in membrane
potential of cell 2 also influenced the membrane
potential of cell 1. The early plateau phase of V!
indicates that cell 1 received a repolarizing current
before cell 2 fired an action potential. Thereafter,
cell 1 obtained a depolarizing current, which gave
rise to the notch in V,. These phenomena further
illustrate the electrical interaction between two functionally coupled cells. Comparison of the action
potential peaks reveals a temporal separation by as
much as 24 msec.
The cell pair described above initially revealed an
rn of 3.9 MH. Subsequently, rn increased spontaneously as a function of time (for an explanation, see
above). The functional state depicted in Figures 4
and 5 was reached after 22 minutes.
Severely Uncoupled Cell Pairs
Figure 6 illustrates an experiment in which impulse
transfer from cell to cell was blocked completely.
76
Circulation Research
Vol 63, No 1, July 1988
TABLE 1. Impulse Transmission and Nexal Membrane Properties
Number of
Nexal resistance available Action potential
Functional state
(MO)
connexons delay (msec)
Successful
transmission
(i = 30)
Occasional block
(n = 5)
Complete block
I 20 mV
I 0.2 nA
20s
02s
0.2 s
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FIGURE 6. Electrical properties of a strongly uncoupled
cell pair. Panel A: Injection of depolarizing current pulses
into cell 1 (Ij) elicited action potentials in cell 1 (V,) but
only small electrotonic responses in cell 2 (V2). Panel B:
Section of the records illustrated in Panel A, displayed at
faster time resolution. Panel C: Signals recorded to
determine the nexal membrane resistance by means of
the double voltage-clamp method. rn=l,250 MCI. Rat cell
pair P24-a.
As shown in Figure 6A, cell 1 and cell 2 no longer
experienced a common resting potential. Not only
did the absolute value of Vm deviate from cell to cell
(cell 1, - 6 5 m V t o -70mV;cell2, - 7 3 m V t o - 8 1
mV), but the spontaneous fluctuations also showed
different patterns. Furthermore, Figure 6A also
indicates that electrical shocks, when applied to cell
1, evoked action potentials in this cell, but only
small electrotonic responses of variable amplitude
and no action potentials in the other cell. Figure 6B
repeats some signals at a faster time resolution. The
current pulse Ii gave rise to a large depolarization in
cell 1 and a small, barely visible one in cell 2. This
led to an action potential in cell 1, which in turn
further depolarized cell 2. However, this extra
depolarization still fell short of the threshold. Injection of current pulses into cell 2 gave rise to an
action potential in cell 2 and a small electrotonic
signal in cell 1 (results not shown). This suggested
that the conduction block does not reflect a mere
loss of excitability in cell 2. Figure 6C documents
the experimental run performed to determine rn. VH
was chosen as —42 mV, and the pulse applied to
cell 2 had an amplitude of +41 mV. In this case, the
analysis revealed an rD of 1,250 Mft.
Correlation Between Action Potential Transfer
and rn
Table 1 summarizes the collected data from 30
different cell pairs. It documents the correlation
between the state of impulse transfer (first column)
and the rn (second column) or the putative number
of connexons involved (third column). The single
connexon conductance was assumed to be 100 pS
(see "Discussion"). In cell pairs exhibiting rns of up
£265
£38
0
155-375
25-65
£24
>780
The collected data were grouped into three classes utilizing
nexal transmission as a criterion (first column). Second column:
Experimentally determined values of rD prevailing in the different
situations. Third column: Figures for the putative number of
operational connexons as calculated from rn, assuming a single
connexon conductance of 100 pS (for references, see "Discussion"). Fourth column: Observed delays in action potential
transfer from cell to cell. n = number of cell pairs investigated.
to 265 Mft, no irregularities were observed with
regard to action potential transfer. The critical
range of sporadic failures in action potential propagation was associated with rn values from 155 to
375 Mft. Complete block of impulse propagation
was found with rn values beyond 780 Mft. Table 1
shows that the measurements from different preparations varied substantially. Each type of impulse
propagation covers a large range of rn values. Part
of it could be caused by variations in relative cell
size and/or excitability of the sarcolemma. In this
study, we have made no attempts to explore these
possibilities.
In some cases of successful impulse transfer, we
explored the possibility of a delay between the
action potentials fired by the individual cells of a
cell pair (e.g., see Figures 3 and 5). These experiments revealed no indication of a discrete impulse
propagation across the nexal membrane for rn values of 5-100 Mft.
Current Flow Between Two Isolated Myocytes
The question arises whether or not two myocytes,
when brought into physical contact, show impulse
transfer from cell to cell. To investigate this possibility, the patch pipettes were connected to two
myocytes clearly separated from each other. Subsequently, gentle agitation of both micromanipulators allowed the cells to be maneuvered close
together and thus to establish an intimate side-toside contact.
Figure 7 illustrates the result of such an experiment. Figure 7A shows the associated currentclamp records. Depolarizing current pulses applied
to cell 1 evoked action potentials in this cell but not
in the other one. Figure 7B shows the records
obtained after switching to the voltage-clamp mode.
Starting from a VH of - 6 5 mV, a strong depolarizing voltage pulse (amplitude, +41 mV; duration,
200 msec) was applied to cell 1. This gave rise to a
large inward current in cell 1, followed by a timedependent decay. In addition, it led to a small nexal
Weingart and Maurer
B
1
20 mV
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500/10 pA
20 s
0.2 s
FIGURE 7. Electrical interaction between two separated
cells. Panel A: Current-clamp signals. Small current
pulses {li; left hand side) produced graded depolarizations in cell 1 only (compare V, and V2). Large current
pulses (right hand side) evoked action potentials in cell 1
and no responses in cell 2. Panel B: Voltage-clamp
signals. Application of a voltage step to cell I (+41 mV,
200 msec; VH, —63 mV) gave rise to a large sodium
inward current in cell 1 (I,) and a minute transnexal
current (I2). The analysis revealed an rn of 4.0 GO. Note
the different amplification in I, and I2- Guinea pig single
cells P58-b.
current signal, which was barely resolvable, detectable with pipette 2. The analysis yielded a resistance located between the cells of 4.0 GCl, equivalent to a conductance of 250 pS. Roughly 2 minutes
elapsed between the establishment of the cell contact and the measurements. This interval appears
too short for a de novo formation of connexons in
adult heart cells.22 An alternative explanation is that
the apparent nexal current signal was caused by a
leak of the amplifiers. Since the equipment was not
designed to study single channel properties, this
possibility cannot be ruled out completely.
Discussion
In the present study, experiments were performed that explored the transfer of the cardiac
impulse at the cellular level. Cell pairs were used
that had been isolated from adult guinea pig ventricles by means of an enzymatic method. As previously shown, such cell pairs exhibit normal
electrical 13141823 and diffusional24 coupling. They
are the simplest preparation appropriate for a study
of impulse transfer from cell to cell.
Impulse Transfer and Nexal Resistance
Regular impulse propagation was found in the
presence of rns ranging from 5 to 265 Mft (see Table
1, second column). If we assume a single connexon
Cell Pairs: Action Potential Transfer
77
conductance of 100 pS (adult guinea pig heart cells,
100 pS [P. Maurer and R. Weingart, unpublished
observations]; neonatal rat heart cells, 33-50 pS25;
embryonic chicken heart cells, 165 pS26; adult rat
lacrimal cells, 90 pS27), this suggests that at least 38
connexons arranged in parallel are required to enable
normal impulse transfer (see Table 1, third column).
The first signs of disturbances in impulse propagation were observed in cases where rn had increased
to 155-375 MO; this would correspond to 25-65
connexons in operation. Complete block of impulse
propagation was observed when rn had reached
values larger than 780 Mft. From this finding it is
inferred that at least 13 connexons are required to
achieve a transfer of the cardiac action potential.
In coupled cell pairs, as in functional syncytia,
the electrotonic voltage response of the follower
cell depends on both the nexal membrane current
generated by the leader cell and the electrical load
on the leader cell. Therefore, the ratio of junctional
resistance to input resistance (rJRin) represents a
useful parameter for expressing the uncoupling data.
If we assume an RjD of 50 Mfl,18 r,/Rin is then 0.1-5
in the case of regular impulse transfer, 3-7.5 in the
case of occasional transfer failure, and >15 in the
case of complete block. DeHaan and coworkers28-30
explored the synchronization of action potentials
between embryonic heart cell aggregates exhibiting
different intrinsic rates of automaticity. With this
preparation, synchronous activation was established when rJRin was <13 (rn, 20 MH; Rjn, 1.5 MH).
This value is in good agreement with the limit for
impulse propagation in adult ventricular cell pairs.
It is useful to compare the experimental cell pair
data with the predictions gained from numeric simulations of impulse propagation. It should be noted,
however, that the validity of these computations
depends critically on assumptions about cell geometry and passive and active membrane properties
(e.g., see Joyner31). Several investigators adopted a
segmented model consisting of two cellular domains
with excitable membranes connected by a single
resistance.32-34 Lieberman et aJ,33 using linear cables
as cellular domains, reported a ratio of TJR^ of 6 as
an upper limit for successful impulse transfer.
Employing single cells as cellular domains, Joyner
and van Capelle36 found that the requirement of
successful transmission was met with ratios of r^R;,,
in the range of 1.7-10. Thus, the simulations yield
critical values of r^R;,, that lie between those for
regular impulse transfer and complete block in
cardiac cell pairs. Adopting a one-dimensional multicellular model with distributed intracellular resistance, Tj, Sharp and Joyner37 observed intact conduction up to a 100-fold increase in Tt. This compares
well with the results of our experiments on cell
pairs, which yielded a 150-fold increase in rn as an
upper limit.
It is conceivable that morphologically detectable
connexons differ in number from functional channels. As a matter of fact, data in the literature
78
Circulation Research
Vol 63, No 1, July 1988
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suggest that junctional membranes may contain
connexons of different functional states. For example, experiments exploring the de novo formation of
connexons between embryonic chicken heart cells
revealed an open-state probability of 0.16.26 From
this, it is inferred that 84% of the connexons may be
silent at any given time. A similar conclusion is
reached by comparing morphological and functional
data applicable to cell pairs isolated from adult rat
ventricles. While morphometrical considerations
attribute 50,000 connexons to this preparation
(assuming that disassociation and patching does not
affect the number of connexons; for computation
and references, see Weingart18), electrical measurements furnish 2,500-5,000 (rn, 2-4 Mfl;17-18 single
connexon conductance, 100 pS; see above). This
means that 90-95% of the channels may be closed
on the average. Since regular impulse transfer
requires 38 connexons or more (see Table 1), a cell
pair must possess at least 380-760 intercellular
channels. Provided all connexons have the same
kinetic properties, this implies that impulse transfer
is supported by a safety factor of 65-130.
Our studies demonstrate that the functional state
of impulse transfer is correlated with rn. However,
it should be kept in mind that the quantitative
conclusions subsequently derived must be considered cautiously. This is because distinct functional
differences exist between cell pairs and multicellular preparations. On the one hand, the electric load
imposed on a single cell is different in cell pairs and
multicellular tissues. In the case of a cell pair, the
excitatory current feeds into a large impedance
element, that is, a single follower cell. In case of
intact tissue, the stimulating current provides for a
low impedance element, that is, a large number of
electrically coupled cells. On the other hand, in
multicellular preparations, at a given time there
exists a large number of excited cells that can
deliver current to depolarize cells at rest. Furthermore, the propagating action potential is not confined to the limits of a single cell. Its depolarizing
phase extends over approximately five cell lengths.
Discrete Nature of Impulse Propagation
In a homogeneous medium, the electrical impulse
propagates continuously. In a multicellular tissue,
made of electrically coupled excitable cells, however, conduction is expected to be "step-wise" or
discontinuous.38 Whether or not discretization is
visible may depend on the contribution of rD to the
overall rf (rf = rn + rc; rc, cytoplasmic resistance). For
example, under physiological conditions, parallel to
the cellular axis, the contribution of rn and rc are
comparable.18 Therefore, little or no sign of discrete
propagation is anticipated. In nonphysiological situations, that is, when rn is increased,17 discrete
conduction might become apparent.39
Our studies support this concept. In cell pairs
exhibiting values of rn ranging from 5-100 Mfl, we
did not observe measurable delays between action
potentials. The discrete nature of impulse transfer
became apparent only when rn had increased substantially. Figure 5 illustrates an extreme case in
which the transnexal conduction time was as large
as 24 msec. Other investigators also reported delays
in impulse propagation under conditions of elevated
rn. For example, Clapham et al28 (see also Veenstra
and DeHaan29), studying the synchronization of
action potentials between heart cell aggregates,
observed delays up to 100 msec at the onset of
spontaneous activity. Their data allowed to establish a linear relation between delay and rn. This
suggests that r0 must play a major role in causing
delays in transnexal impulse transfer. Delays have
also been observed in simulation studies (e.g., see
Lieberman et al,35 Sharp and Joyner37).
In intact cardiac tissue, a transmission delay of 24
msec would lead to an extremely low conduction
velocity, for example, 0.5 cm/sec in the longitudinal
direction (intemexal distance, 110 /im14). This is
roughly 1/100 of the conduction velocity observed
under normal conditions.4-9 This dramatic effect
may be explained by the observation that cell-tocell coupling becomes the dominating factor in
controlling conduction velocity as rn increases.35 In
general, slow impulse conduction is regarded as a
necessary requirement for the phenomenon of reentry, a mechanism postulated for the genesis of
cardiac arrhythmias (e.g., see Cranefield40 and
Janse41). Therefore, a decrease in conduction velocity via elevation of rn may well contribute to these
incidents.
Delmar et al42 explored the effects of cell-to-cell
coupling on anisotropic conduction. Exposure to
heptanol caused a gradual uncoupling that was
associated with a stepwise decay of the conduction
velocity. The authors also noticed that transverse
propagation was more sensitive to an increase in r0,
a phenomenon that might set the stage for discrete
changes in impulse conduction. Evidence supporting this view has recently been obtained by K16ber
et al,43 who showed that an increase in r0 is accompanied by discontinuous longitudinal propagation.
As outlined in the introduction, conduction velocity not only depends on passive cable properties,
but is also determined by geometric factors and
active properties of the sarcolemmal membrane
(e.g., see Fozzard and Arnsdorf44). Therefore, modification of membrane excitability represents an
alternative method for influencing transnexal conduction. However, so far we have made no attempts
to explore this aspect with cell pairs.
Ephaptic Impulse Transmission
Based on the experimental data discussed so far,
it has been concluded that transmission of an action
potential from one cell to another one requires at
least 13 connexons conducting simultaneously. From
this it is anticipated that an electrical impulse is not
propagated in the absence of a specialized junctional membrane. As a matter of fact, we were able
Weingart and Maurer Cell Pairs: Action Potential Transfer
to verify this point directly with the following
approach. Two isolated myocytes, when maneuvered into physical contact, failed to show transmission of an action potential and spread of an
electrotonic response (see Figure 7). Analysis of the
voltage-clamp records yielded an "intercellular"
resistance of 4 Gfi. This corresponds to a conductance of 250 pS, which is roughly equivalent to two
connexons in parallel (see above). This finding rules
out the possibility of an ephaptic mechanism of
impulse transmission in cardiac tissue.45 Furthermore, it does not support the concept of electric
field coupling that has been propagated by
Sperelakis.46
Acknowledgments
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We are grateful to Miss M. Herrenschwand for
her expert technical assistance and to Drs. S. Weidmann, A. K16ber, and J. Shiner for critical comments on the manuscript.
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KEY WORDS • ventricular muscle • isolated cell pairs •
intercellular coupling • nexal membrane resistance • action
potential propagation
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Action potential transfer in cell pairs isolated from adult rat and guinea pig ventricles.
R Weingart and P Maurer
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Circ Res. 1988;63:72-80
doi: 10.1161/01.RES.63.1.72
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