500
Purkinje and Ventricular Activation Sequences of
Canine Papillary Muscle
Effects of Quinidine and Calcium on the Purkinje-Ventricular
Conduction Delay
Richard D. Veenstra, Ronald W. Joyner, and David A. Rawling
From the Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
SUMMARY. We have studied in vitro preparations of canine right and left papillary muscles to
determine the excitation sequences of the Purkinje and ventricular cells, using both monopolar
surface electrodes and intracellular microelectrodes. Our results show, for right papillary muscles,
that the Purkinje layer covers the basal part of the muscle, and that activation from the right
bundle branch propagates over all of the Purkinje layer, but directly activates the underlying
ventricular layer only at specific junctional sites. Left papillary muscles have attachments to both
apical and basal Purkinje strands and the Purkinje layer covers the entire muscle, but, as for right
papillary muscles, activation from the Purkinje layer to the ventricular layer occurs only at basal
junctional sites. Antidromic conduction in papillary muscles (propagation from the ventricular
layer to the Purkinje layer) can occur at regions other than the specific sites through which the
Purkinje layer activates the ventricular layer. At the identified junctional sites, the Purkinje cell
action potential duration is significantly shorter than in the free-running strand, but it remains
longer than that of the ventricular cells. The time delay at the junctional sites is increased by
quinidine, increased calcium concentration, and increased pacing frequency. (Circ Res 54: 500515, 1984)
THE normal excitation sequence of the heart involves the propagation of action potentials from the
specialized conducting system of Purkinje (P) fibers
into the ventricular (V) cells of the papillary muscles,
interventricular septum, and the ventricular free
wall. Researchers in several laboratories have studied the nature of impulse propagation across the
Purkinje-ventricular junction (PVJ) (Alanis et al.,
1961; Matsuda et al., 1967; Mendez et al., 1969,
1970; Kamiyama and Inoue, 1971; Myerburg et al.,
1972). From these observations, the concept of a
conduction delay occurring across a PVJ (PVJ delay)
was developed. An anatomical study showed that
there are numerous regions of cell-to-cell couplings
among P cells and among V cells, but they noted
that there is a relative lack to cell-to-cell couplings
in the PVJ (transitional) region (Martinez-Palomo et
al., 1970). This anatomical study was preceded by a
series of microelectrode studies in which Alanis and
colleagues (Alanis et al., 1961; Alanis and Benitez,
1970; Benitez and Alanis, 1970) demonstrated that
quinidine, a 50% reduction in external sodium, anoxia, low [Ca++]o, lowered temperatures, and increases in stimulation rate will increase the PVJ
This manuscript from the University of Iowa was sent to Dr. Brian
F. Hoffman, Consulting Editor, for review by expert referees, for
editorial decision, and final disposition.
delay. Their conclusions stress the role of a "functional discontinuity" between the P and V cells and
the possible existence of a population of transitional
cells having a low maximal upstroke velocity (Vm^)
as a means of slowing conduction through the PVJ.
Mendez et al. (1970) models the PVJ as a onedimensional cable-like P fiber input into the much
larger two- or three-dimensional papillary muscle.
Based on the results of their study of the spatial
distribution of action potential duration (APD)
across the PVJ, Mendez et al. (1969) proposed that
the P and V cells are electrically well coupled and
the various PVJ delays observed at different PVJs
are related to the geometry of a given site. The socalled "funnel hypothesis' predicts that the length
over which the tapering from a one-dimensional
system (P strand) to a two-dimensional system (papillary muscle) occurs determines the value of the
PVJ delay, since the papillary muscle represents a
low impedance pathway for current flow.
Whereas some of these early microelectrode studies commonly reported PVJ conduction delays (PVJ
delay) on the order of 5-20 msec (e.g., Alanis et al.,
1961), other reports suggest that the true PVJ delay
is probably less than 5 msec (Matsuda et al., 1967;
Mendez et al., 1970; Kamiyama and Inoue, 1971;
Spach et al., 1971; Myerburg et al., 1972). It has
been proposed that the long PVJ delays reported in
Veenstra et al. /Purkinje-Ventricular Conduction Delay
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
the literature do not reflect the true conduction time
from a P cell into a V cell at a true PVJ (Myerburg
et al., 1972; Hondeghem and Jensen, 1974). Myerburg et al. (1972) stressed that the true PVJ sites are
restricted to certain regions of the endocardial surface, and measured only very short delays between
the "terminal" P fibers and the underlying muscle
fibers. Differences in the activation times for the
superficial P fibers and the deeper V fibers have also
been studied with extracellular and intracellular recordings from the ventricular endocardial surface
(Kamiyama and Inoue, 1971; Spach et al., 1971;
Spach and Barr, 1972).
In this paper, we examined the phenomenon of
PVJ delay by combining extracellular surface mapping with intracellular microelectrode recordings to
explore the activation sequences of canine right and
left papillary muscles in order to definitively identify
a PVJ site. We also explored the changes in action
potential duration associated with the PVJ in greater
detail than has been previously reported (Mendez
et al., 1969), as well as the effects of quinidine and
altered external calcium ion concentration on PVJ
delay. Our overal hypothesis is that: (1) the P and
V layers are electrically coupled to each other with
a coupling resistivity that is significantly higher than
the normal resistivity among P cells or among V
cells, (2) this junctional resistivity between the two
cell layers is spatially inhomogeneous, with sites
near the base of the papillary muscle having a lower
junctional resistivity than sites near the apex of the
muscle, and (3) this spatial inhomogeneity in junctional resistivity produces the discrete pattern of
coupling sites for propagation from the P layers to
the V layer.
Methods
Electrical Measurements
Twenty nine dogs weighing 15-25 kg were anesthetized
with sodium pentobarbital (Nembutal, 30 mg/kg, iv), and
a left lateral thoracotomy was performed to expose the
heart. Their hearts were removed and immersed in a
beaker of ice-cold Tyrode's solution, and then were transferred to a large dissection chamber filled with ice-cold
Tyrode's solution, where we opened the right ventricle by
slicing the pulmonary artery and then down along the
pulmonary outflow tract and into the right ventricle. As
the right ventricle was opened, all free-running Purkinje
fibers were trimmed away from the endocardial surface
and the right anteroseptal papillary muscle (RAP) was
removed and pinned out in the experimental chamber.
On occasion, the left ventricle was also cut open along the
interventricular septum and a left papillary muscle (LPM)
was excised for separate electrophysiological experiments.
The preparation was allowed to recover for 30 minutes
after dissection, before the experiment was begun.
The experimental chamber was perfused with Tyrode's
solution of the following composition (ITIM): NaCl, 125.0;
NaH2PO4, 0.4; NaHCO3, 24.0; KC1, 4.0; MgCl^ 1.0; CaCU,
2.7; and dextrose, 5.5. The solution was gassed with 95%
O2-5% CO2 to give a pH of 7.35 ± 0.05 and warmed to
36-37°C by immersing the inflow line in a hot water bath.
501
The total chamber volume was 16 ml and the flow rate
was set at 5 ml/min. The volume of the tissue in the
chamber was approximately 2 ml, and the perfusion rate
appeared to be adequate, since stable extracellular and
intracellular recordings could be obtained for a period of
up to 8 hours. Each solution change was performed over
a period of 15 minutes, which was adequate for the
preparation to reach a new steady state in the test solution,
as evidenced by superimposing the extracellular waveforms taken 2 minutes apart. When the external calcium
concentration was altered during the course of an experiment, the NaCl concentration was also altered to maintain
the osmolality of the solution. In those experiments in
which quinidine was used, a stock solution of 250 mg/
liter quinidine HC1 (ICN Pharmaceuticals) was prepared
for each experiment and diluted to give a final concentration of 2.5 mg/liter.
Intracellular recordings were obtained using conventional glass microelectrodes that had tip resistances between 15-25 MQ when filled with 2.5 M KC1. The microelectrodes were connected to the probe input of a WPI
model M-707 DC amplifier adjusted to a gain of 10.
Maximum upstroke velocity ( V J measurements were
obtained by means of a differentiator circuit that had been
calibrated with a sawtooth waveform generator. The differentiator circuit was linear in the range from 4 to 1000
V/sec.
The extracellular electrodes were made from Tefloncoated silver wire with a diameter of 75 ^m (A-M Systems,
Inc.). We used a modified bipolar electrode to record
monopolarly from the surface of the muscle. Two silver
wires, insulated except at the tip, were used to make each
electrode. One lead was placed on the surface of the
papillary muscle and the second lead was clipped so that
it would be positioned 2-3 mm above the surface of the
muscle. The two signals were recorded differentially with
an AC amplifier (AD521, Analog Devices, Inc.) with a
gain of 100. The purpose of using a second lead as a
reference was to eliminate the large field effect of the
papillary muscle which would be recorded if a bath reference had been used. Our technique yields a low-noise
monopolar recording of the surface extracellular potentials. The output of the AC amplifier is connected to the
input of another amplifier with a variable gain from 1 to
100. The individual channel gains were adjusted to give
us large enough signals (±5V) for digital sampling. The
extracellular signals were sampled digitally (12 bits) at a
rate of 6,666 to 20,000 samples/sec and stored in a VAX
11/780 computer (Digital Equipment Corp.), and the intracellular recordings were generally recorded on a FM
tape recorder (Vetter, model B) for playback and sampling,
but in some cases we did simultaneous digital sampling
of the extracellular and intracellular recordings.
A pacing stimulus of 0.5-1.0 msec in duration and 1.52.0 times the threshold voltage was applied to the preparation through a bipolar stimulating electrode. A digital
pulse generator was used to trigger a Grass S88 stimulator
which drove the stimulus isolation units (Grass model
SIU5A). For studies on RAP's, one stimulating electrode
was placed on the right bundle branch (RBB) of the
Purkinje system where it exits the septal wall and a second
stimulating electrode was placed near the apex of the
papillary muscle. For studies on LPM's, one stimulating
electrode was placed on a basal P strand and another was
placed on the apical P strand (Myerburg et al., 1972).
Circulation Research/Vol. 54, No. 5, May 1984
502
Data Analysis
The digitally recorded waveforms were redisplayed on
a Tektronix 4014 graphics terminal for measurement of
the P and V activation times, using a subroutine of our
sampling program. The activation rimes were selected by
moving a cursor to the peak of the negative deflection, as
illustrated in Figure 1. The solid lines labeled *P TIME' in
Figure 1, A and B, illustrate our definition of the P activation time in our mapping studies. Likewise, the solid
lines labeled 'V TIME" in Figure 1, A and B, represent the
measured V activation times. The time difference between
V ACT and P ACT is defined as the PV delay at a given
recording point. In the case illustrated in Figure 1 A, where
SUM
(1
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
f
IP
P TIME
—
Y
\/v
V TIME
PVJ DELAY
1
1
1
10
MSEC
15
20
B
STIM
-A,—
-
—
•
p TIME
V
I
VP
' V
V TIME
PV DELAY
i
1
10
MSEC
1
15
20
FIGURE 1. Definition of P and V activation times and PV(J) delay.
Part A shows the digitally recorded extracellular waveform from the
PVj of a RAP. The heavy solid lines labeled P ACT, V ACT, and PV}
delay define the time intervals recorded as the P and V activation
times and the PV} delay, respectively. The P and V activation times
are measured from the trailing edge of the stimulus artifact (STIM)
and the PV} delay is defined as V ACT—P ACT. Part B illustrates
the definition of the P and V activation times and the PV delay for
an extracellular recording taken from a nonjunctional site of the same
RAP. The heavy solid lines are again labeled P ACT and V ACT, in
reference to the measured P and V activation times. The time difference of V ACT—P ACT is defined to be the PV delay and is not to be
referred to as the PV} delay.
the recording site has been determined to be a PV junctional site (PVJ, see Figure 2 for a detailed description of
our definition of a PVJ), the PV delay is redefined as the
PVJ delay for that specific junctional site. The peak negative deflection was used as the determinant of P and V
activation because of the previously shown correlation
between this phase of the extracellular waveform and the
local occurance of an action potential (Spach et al., 1972;
Joyner, 1982). Occasional records from sites where the
peak negative deflection was difficult to interpret due to
fractionarion of the recorded wavefronts were not included in the analysis.
The rationale behind using the stimulus artifact as a
reference point is 2-fold. First, the stimulus artifact is fixed
in time, since we used a fixed duration pulse (1 msec)
which followed the computer and oscilloscope external
sync pulse by a fixed duration (5 msec). Second, the P and
V activation times may vary during the course of the
experiment with changes in the stimulus strength. To
prevent large fluctuations in the P and V activation times,
the stimulus voltage was set at 1.5-2 times the threshold
voltage at the beginning of the experiment. To detect any
such changes in the P and V activation times that still
might develop over the course of an experiment, a stationary extracellular electrode was placed on the surface of
the papillary muscle for the duration of the experiment.
During the course of the two extensive mapping studies
reported on in this paper, the P and V activation times at
the reference site did not vary by more than 1 msec. If the
recorded extracellular waveform from the stationary electrode changed its shape during the course of the experiment, an event which is indicative of an alteration in the
activation pattern being recorded, the experiment was
terminated. The contribution of the deeper layers of the
papillary muscle to the activation sequence being measured was assumed to be minimal, since only the outer 12 mm of the papillary muscle have been reported to
remain viable after 2 hours of superfusion (Spach et al.,
1982a). In all of the experiments, the position of the
recording electrodes was recorded using a micrometer
eyepiece. The isochronal lines were sketched onto a scale
drawing of the papillary muscle that had the recording
sites marked by hand.
Action potential durations were measured by a computer program which performed digital sampling (1 kHz)
of the analog tape and then tabulated the peak amplitude
and duration for 90% repolarization (APD90) of each action potential. Graphical displays of the action potential
recordings were copied with a Tektronix 4631 hard copy
unit. All V™,, measurements were made from photographed records taken with a Tektronix C-5A oscilloscope
camera. The changes in PVJ delay, muscle longitudinal
conduction velocity, and V,^ (control vs. test solutions)
were analyzed statistically by Student's paired f-test for
paired observations (Bruning and Kintz, 1977). Statistical
comparisons between the conduction velocity changes and
the changes in the PVJ delay in the various test solutions
were made with the Mest for differences among the means
(Bruning and Kintz, 1977).
Results
Identification of the Purkinje-Ventricular
Junction (PVp
Three criteria were used to define the PVJ of a
canine right anteroseptal papillary muscle (RAP). (1)
Veenstra et al. /Purkinje-Ventricular Conduction Delay
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Upon mapping the surface of the RAP, the site in
question must be the earliest site of V activation
during orthodromic conduction [right bundle branch
(RBB) stimulation]. (2) The site had to have the
shortest PV delay. (3) The recorded extracellular
signal had to have a uniphasic, all-negative V activation waveform.
In nine experiments, we documented that the two
extracellular waveforms recorded at that site were
truly the P and V activation waveforms, and were
not due to asynchronous activation of adjacent fiber
bundles within the P or V layers alone. This was
done by recording the intracellular potentials at the
same location with a microelectrode. One example
of such an experiment is displayed in Figure 2.
Part A of this figure demonstrates the temporal
relationship of activation between the superficial P
cells and the deeper V cells at this site. The surface
recording is shown as a dashed waveform. In this
example, a single microelectrode was advanced from
the surface to make three consecutive cell penetrations labeled P, T (for transitional), and V, respectively. Figure 2B shows the superimposed action
potentials recorded for each penetration. The P cell
had an action potential duration measured at 90%
repolarization (APD90) of 250 msec and a V ^ of
320 V/sec. The T and V cells had identical values
for APD90 of 195 msec, but the T cell had a higher
Vmax (190 and 160 V/sec, respectively). Notice that
the second component of the upstroke of the T cell
action potential coincides exactly with the peak of
the V cell action potential. Conversely, the foot of
the V cell action potential begins to rise with the
activation of the T cell. These results could be interpreted to be the "spike and dome" phenomenon
reported by Matsuda et al. (1967) for junctional P
cells.
503
On the basis of these experiments, we have chosen to define the conduction delay that occurs at the
PVJ as the time difference between the entrance of
activation of the P cell layer into this region and the
time at which V activation has commenced. As
defined, the PVJ delay does not represent the conduction delay that occurs between adjacent junctional cells, but is best described as the time delay
between which activation first enters the region via
the Purkinje system and activation leaves the region
by the ventricular system.
Activation of the Right Anteroseptal Papillary
Muscle
Figure 3 is a detailed activation pattern of a RAP
during stimulation of the RBB at a basic cycle length
(BCL) of 1 second. The excitation sequences shown
in Figure 3 were constructed from the extracellular
waveforms recorded from 220 sites. A portion of the
base of this RAP was not mapped, since the P
strands obstructed the surface. Separation of the P
and V extracellular waveforms was made on the
basis of observations presented in Figure 2. Additional extracellular recordings from various sites of
this particular RAP are presented in Figure 7.
Part A of Figure 3 is the isochronal activation map
for the Purkinje system, with the labeled lines indicating milliseconds (msec) of delay as measured
from the trailing edge of the stimulus. The dashed
lines refer to activation times for the RBB and the
free-running Purkinje strands, whereas the dotted
lines represent activation times for the superficial
Purkinje cell layer of the RAP. Two observations
can be made in regards to this map. First, it is evident
that the P cell layer is restricted primarily to the
lower half of the RAP. In this instance, however,
there was a small, finger-like projection of P cells
B
MV
MV
-80
-80-
SO
TIME (MSEC)
1OO
150
200
250
300
TIME (MSEC)
FIGURE 2. Identification of cell type at a PV]. Part A shows the digital display of the recorded extracellular waveform (dashed line) and the
superimposed traces of the intracellular recordings (solid lines) from the P, T, and V cells of the PV] for the RAP illustrated in Figure I. Notice
the all-negative V extracellular activation waveform and the short delay between the peak negative deflections corresponding to P and V cell
activation We have defined this delay as the PV] delay. Part B shows the superimposed digitized signals for the P, T, and V cell action potentials
recorded at BCL of 1 second.
Circulation Research/Vol. 54, No. 5, May 1984
504
CANINE RIGHT ANTEROSEPTAL PAPILLARY MUSCLE
PV DELAY
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
FIGURE 3. Activation patterns of canine RAP. The RAP (same RAP as in Fig. 2) was stimulated on the RBB at a BCl of 1 second. Part A shows
the delay in msec from the end of the stimulus to the activation of the P cells. The dashed lines refer to the isochrones for the free-running P
strands, and the dotted lines refer to the delay for the surface P cells which are activated via P fibers originating from the terminal portion of the
RBB. Part B is a similar plot for the V cell activation times, showing that activation of the V cells occurs within localized regions (PVJs) near the
base of the RAP. The PV] is indicated by the star on the surface of the RAP. Part C shows the time delay (PV delay) between activation of the
surface P cells and the underlying V cells. There is a short PV delay associated with the PVJ (PV] delay), whereas there is a longer PV delay at
all of the other recording sites. The PV delay at the other sites does not refer to conduction time from the P to the V cells, but is a function of the
independent activation pathways for the P and V cells.
running toward the apex of the RAP. Second, activation of this P cell layer follows activation of the
RBB, and is not activated by the free-running P
strands which reflect back into the papillary muscle.
Figure 3B is the activation sequence for the V cell
layer of this particular RAP. Only the 11-, 12-, 14-,
17-, 20-, 22-, 25-, 27-, 29-, and 31-msec isochrones
were plotted for the purpose of clarity. There are
two foci for activation of the V cells by the P cell
layer, both located near the base of the RAP. Toward
the apex of the RAP, the isochrones are nearly
parallel and the conduction velocity can be obtained
by measuring the distance between the isochrones
(muscle longitudinal conduction velocity = 0.4 M/
sec). This value for muscle longitudinal conduction
velocity is in agreement with other measurements
for the canine papillary muscle longitudinal conduction velocity (Spach et al., 1982a). From the V activation pattern, it is evident that propagation occurs
from the P cells to the V cells only at localized
regions near the base of the papillary muscle, and
that activation of 1-4 mm2 area of V cells occurs in
a nearly synchronous manner. Figure 3C is a map
of the time difference between P and V activation
times (PV delay). Only the 3-, 5-, 7-, 9-, 12-, and
14-msec isochrones have been plotted for clarity.
The star that appears in every part of Figure 3
identifies the site with the shortest PV delay. This
plot exemplifies the observation that functional coupling of P to V occurs only in localized regions near
the base of the RAP. Our use of the term 'functional
coupling' implies that we believe that the intercellular resistance between the P and V cells at the PVJ
is low enough to allow the current necessary for V
cell activation to flow from the P cell layer to the
adjacent V cell layer through a population of low
resistance gap junctions. This observation does not
imply that we assume the PV junctional resistance
to be the same as the intercellular resistance that
exists in the homogeneous case between P cells or
V cells. This topic will be discussed further in the
discussion section. From this observation, it follows
that most of the PV interface of canine RAP is
functionally uncoupled (i.e., has an increased intercellular resistance relative to the PVJ and to the
homogeneous case) in the orthodromic direction,
and that the measured PV delay in most regions
does not directly relate to conduction from the P
cells into the V cells. Conduction patterns in the
antidromic direction will be discussed later.
Figure 4 shows similar activation sequences for
another RAP. Over 300 recording sites were used to
construct the isochrones for this papillary muscle.
In Part 4A, it again is evident that the superficial P
cell layer is restricted to the base of the RAP, and
that it is activated by fibers from the RBB. Part 4B
demonstrates the complex activation patterns that
can exist due to multiple sites of activation. The
12-, 13-, 15-, 17-, 19-, 21-, 23-, and 25-msec isochrones have been plotted, in this case. The other
sites of activation are not as evident in this plot as
they were in Figure 3B, but these multiple activation
sites are more observable in Figure 4C where there
are four 3-msec isochrones present in the PV delay
map. Uniphasic, all-negative V signals indicative of
the activation initiation site were recorded from
points within the boundaries of each 3-msec isochrone. The star identifies the earliest site of V
Veenstra et al./Purkinje-Ventricular
Conduction Delay
505
CANINE RIGHT ANTEROSEPTAL PAPILLARY MUSCLE
PV DELAY
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
FIGURE 4. Activation patterns of canine RAP. This map is similar to that shown in Figure 3 for a second RAP. Part A refers to the P cell
activation times in msec from the end of the stimulus for the free-running P strands (dashed lines) and the surface P cells (dotted lines). Activation
of the surface P cells begins in the region near the termination of the RBB and spreads out over the base of the RAP and toward the apex to the
point of termination of the P cell layer. Part B illustrates the V cell activation pattern for this RAP. Activation patterns near the base are
complicated by numerous sites of activation (PVJs), with the earliest site being indicated by the star. Propagation of V cell activation proceeds
toward the apex with a relatively uniform conduction velocity. Part C is a plot of the PV delays for this RAP. The four regions with short (3mstc) PV delays were all indicated to the sites of coupling between the P and V cells from the uniphasic, all-negative extracellular V activation
waveform.
activation in this RAP, and this region lies within
the only 12-msec isochrone of Figure 4B. As the V
activation approaches the apex, the isochrones again
become nearly parallel with an average conduction
velocity of 0.7 M/sec.
The same general conclusions can be made concerning activation of the P and V cells for both
papillary muscles. These conclusions are: (1) the
superficial P cell layer is activated by fibers of the
RBB, (2) this P cell layer is restricted to the basal
portion of the RAP, (3) only localized regions of the
P cell layer are functionally coupled to the V cells
of the RAP, (4) V activation within this region of
coupling is nearly synchronous, and (5) the spread
of V activation propagates towards the apex of the
RAP with a nearly constant conduction velocity.
In a total of 18 RAP preparations, we located the
earliest site of ventricular activation following RBB
stimulation. In all cases, we found the site to be in
the basal region of the muscle, generally more basal
than the point of attachment of the RBB to the RAP.
Three examples are shown in Figure 5, in which
parts A, B, and C each represent a scale drawing of
a RAP preparation with the location of the PVJ and
the attachment of the RBB indicated.
Activation of Left Ventricular Papillary Muscle
The left bundle branch (LBB) forms two columns
of Purkinje fiberes which branch and interconnect
to form a complex network of P fibers. The papillary
muscles of the left ventricle receive P fiber input
near the apex of the muscle and, also, have basal P
strands. Unlike the RAP, left ventricular papillary
muscles (LPM) have a P cell layer over the entire
surface of the muscle (Myerburg et al., 1972). Figure 6 illustrates the activation of a LPM during
stimulation of the P strands near the base (part A)
or near the apex (part B) of the LPM. The upper
APEX
FIGURE 5. Location of the PV] sites.
Parts A, B, and C are the outlines of
three different RAP preparations drawn
to scale. The location of the PVJ site is
indicated by the star, in each case. The
PV] sites were located during stimulation of the RBB at a BCL of 1 second.
BASE
Circulation Research/Vol. 54, No. 5, May 1984
506
1.5 MM
LEFT PAPILLARY MUSCLE
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
BASAL STMULUS
APICAL STIMULUS
FIGURE 6. Activation sequences of a LPM. The upper panel is a scale drawing of a LPM and the positions of the seven extracellular recording
sites. Part A shows the recorded signals during stimulation of the basal P strand (STIM B) at a BCL of 1 second. Site 1 is the PV] for this
preparation. Part B illustrates the activation sequence during stimulation of the apical P strand (STIM A). V activation does not commence until
the P activation front reaches the PV] (site 1), again illustrating the functional uncoupling of the apical P and V cells in the orthodromic direction.
panel of Figure 6 is an illustration of the left papillary
muscle used for this experiment, and the positions
of the recording sites 1-7 have been marked accordingly. During basal stimulation (Fig. 6A), at a BCL
of 1 second, both the P and V activation waveforms
begin near the base and propagate toward the apex
of the muscle with different conduction velocities.
V activation is shown to begin at site 1 during basal
stimulation. Our most important observation was
that during stimulation of the apical P strand (Fig.
6B), activation of the V cells did not begin until P
activation reached site 1. V activation then proceeded back toward the apex of the LPM. We have
made three major observations concerning activation of the LPM. They are that there is a superficial
P cell layer covering the entire surface of the LPM,
that activation of the V cell layer by the P cell layer
occurs only at a site near the base of the LPM, and
that much of the P cell layer cannot support propagation of activation into the V cells. We have
located the PVJ sites during apical P strand stimulation in a total of 13 LPM preparations, and the
results present similar findings to those illustrated
in Figure 6.
Antidromic (V to P) Conduction Pathways of the
RAP
Apical stimulation of a RAP produces direct activation of the V cells since the apex of the RAP is
devoid of P cells (see Fig. 3 and 4). Figure 7 is an
example of propagation of activation in both the
orthodromic and antidromic directions in a RAP.
Figure 7 A describes the positions of the recording
electrodes for this experiment. Part B in Figure 7
depicts the activation pathway from base to apex
during stimulation of the RBB at a BCL of 1 second.
Site 1 has been defined as the PVJ, as identified
from the extracellular waveform (see Fig. 2), and
also by mapping (Fig. 3). Propagation through the
P and V layers proceed independently, in the apical
direction. Part 7C illustrates the antidromic conduction pathway in the same experiment. In this example, V activation proceeds all the way to the PVJ
(site 1) before entering the P cell layer. The P activation wave then proceeds back toward the apex.
This result again indicates the extent of electrical
uncoupling that exists between P and V cells of the
RAP.
Figure 8 shows a different set of results obtained
from another RAP (see Fig. 4). As in Figure 7, Figure
8A illustrates the position of the recording electrodes, and Figure 8B demonstrates the activation
pattern during orthodromic conduction. Site 1 was
again verified to be the PVJ by mapping studies (see
Fig. 4) and by the combined intracellular and extracellular recording techniques. However, coupling
507
Veemtra et al./Purkinje-Ventricular Conduction Delay
B
RB8 STIMULATION
CANINE RIGHT ANTEROSEPTAL
PAPILLARY MUSCLE
MUSCLE STIMULATION
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
MM
FIGURE 7. Orthodromic and antidromic conduction pathways in a RAP. Part A is a scale drawing of the RAP (same RAP used in Fig. 3) marking
the locations of the extracellular recording electrodes. Part B illustrates the activation sequence of the P and V cells during stimulation of the
RBB. Site 1 is the PVf Part C shows the antidromic conduction pathway for this RAP. Direct stimulation of the V cells can be obtained by
stimulating the RAP near the apex (V STIM). In this RAP, V to P propagation does not occur until the V activation wave reaches the PVf.
between V and P cells during antidromic conduction
(Fig. 8C) occurred at a site located apical to site 1.
Activation of the P cell layer precedes activation of
the V cell layer at site 1, since the P conduction
velocity is greater than the V conduction velocity.
The area near site 2 represents a region of potential
unidirectional block, since impulse propagation between P and V may proceed in the antidromic
direction (V to P), whereas orthodromic conduction
(P to V) does not appear to occur at site 2. Second,
these results illustrate the fact that antidromic conduction may take a different pathway from orthodromic conduction. Although the actual number of
occurrences was not recorded, in several experiments in which we located the PVJ site during RBB
stimulation and then stimulated the muscle near the
apex of the RAP, we observed that the P signal
preceded the V signal at the PVJ site, as indicated in
Figure 8C.
APD Changes Associated with the PVJ
If P and V cells are well coupled, electrically,
through low resistance gap junctions, then there
should be no detectable difference in the APD of
the P cells and V cells of the PVJ. We investigated
this hypothesis by microelectrode recordings from P
and V cells at various points along the activation
pathway during stimulation of the RBB at a BCL of
1 second. Figure 9 is a scale drawing of the RAP
used for this experiment (same RAP as in Fig. 2),
and the positions of the impalement sites have been
marked accordingly. The results of this experiment
are listed in Table 1. The larger P strands had an
APD90 in the range of 310-340 msec. On the surface
of the RAP, the P cell APD90 declines from 290 to
250 msec as the PVJ is approached. The mean APD90
values among the three groups are significantly different (P < 0.05) as determined by Student's nonpaired f-test. There was no statistically significant
difference in the P cell V^* values for the three
groups. The V cell APD90 remains rather stable from
base to apex, with values ranging from approximately 190 msec near the base to 200 msec at the
apex for a BCL of 1 second (P < 0.05). As with the
P cells, there was no difference in the Vma* values
of the V cell grops. A similar experiment on another
RAP gave us quantitatively similar results. The only
real difference, from the evidence presented in
Table 1, is that the V cell APD90 ranged from 210
msec near the apex to 220 msec near the base of the
RAP, while the P cell APD90 declined from 340 to
250 msec as the PVJ was approached. Together,
Circulation Research/Vol. 54, No. 5, May 1984
508
B
R88 STIMULATION
CANINE RIGHT ANTEROSEPTAL
PAPILLARY MUSCLE
MUSCLE STIMULATION
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
10
15
10
T I E (MSEC)
FIGURE 8. Orthodnmic and antidromic conduction pathways in a RAP. Part A is a scale drawing of the RAP (same RAP used in Fig. 4) marking
the location of the extracellular recording electrodes. Site 1 is the PV] for this RAP. Part B illustrates the activation sequence of the P and V cells
of the four sites during orthodnmic conduction. Part C shows the antidromic activation sequence during apical stimulation (V STIM). In this
RAP, V toP propagation occurs at a site (site 2) apical to the PV] (site 1), identifying site 2 as a site of naturally occurring unidirectional ulock.
these two RAP experiments show that there is a
gradual decline in P cell APD90 of about 90 msec as
the PV] is approached, whereas the APD90 values
for the V cells and the V^x values of the P and V
cells remain relatively stable throughout the preparation.
In six experiments in which the APD90 differences
between P and V cells at the PVJ were recorded at
a BCL of 1 second, the mean (±SD) difference in
APD90 was 83 ± 52 msec. Also, the data from nine
experiments recording from P cells and V cells from
the endocardial surface of the left ventricular free
wall gave us a mean differeence (±SD) of 56 ± 48
msec at a BCL of 1 second. These observations
indicate that the P and RV cell layers are not very
well coupled to each other, even at documented
sites of P to V conduction, suggesting the existence
of a 'resistive barrier' between the two cell layers.
The Effect of [Ca++lo and Quinidine on PVJ
Delay
In nine experiments, we altered the [Ca++L, from
its control value of 2.7 mM to 1.35 and 5.4 mM. The
results of these experiments are presented in Figures
10 and 11 A. Figure 10 is a plot of the extracellular
recordings from the PVJ of one RAP (same RAP as
in Fig. 1) during RBB stimulation at a BCL of
1 second. The top set of tracings represents the
superimposed recordings of three controls taken at
the beginning of the experiment, after the washout
of the 5.4 mM [Ca++]o solution, and after the washout
of the 1.35 mM [Ca++]o solution (the end of the
experiment). There is some variability in the control
recordings, but the measured PVJ delay varied only
slightly from 4.0 to 4.1 msec throughout the course
of the experiment. The middle trace in Figure 10 is
the extracellular recording taken in the 5.4 mM
[Ca++]o solution. The P signal has shifted outward
in time only slightly, whereas the V signal has
shifted considerably, thereby increasing the measured PVJ delay to 4.6 msed. The bottom tracing in
Figure 10 is the extracellular recording obtained in
the 1.35 mM [Ca++]o solution. Although there has
been no measurable shift in the P activation time at
the PVJ, in comparison to the control recordings, the
V activation time has become earlier than in the
control. The measured PVJ delay in the low calcium
solution is 3.7 msec. The results from all nine experiments are graphically displayed in Figure 11 A. In
Figure 11 A, each set of symbols connected by lines
represent the results from one experiment. In 2.7
[Ca++]o, the PVJ delay varied from 2.0 to 4.4
Veenstra et al. /Purkinje-Ventricular Conduction Delay
509
CANINE RIGHT ANTEROSEPTAL
PAPILLARY MUSCLE
CONTROL(S)
HIGH CALCIUM
LOW CALCIUM
I
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
MSEC
1 MM
FIGURE 9. Location of the intracellular and extracellular recording
sites for determining the APD and Vmv, distribution of P and V cells
m relation to the PV]. The numbers mark the sites of extracellular
and multiple intracellular recordings during this experiment. The
and Vmm values are shown in Table 1.
TABLE 1
Vou, and APD« of Purkinje and Ventricular Cells of Canine
RAP
P cell
Region
studied
Junchonal
surface
Nonjunctional
stTand
Nonjunctional
surface
APDw
(msec)
1
253
2
3
4
15
FIGURE 10. Effect of [Ca+*]o on PV} delay. The upper trace is the
superimposed extracellular recordings to three control records taken
at the beginning following the washout of the 5.4 mM [Ca**]o
solution, and at the end of the experiment. The PV] is the same PV]
site as that illustrated in Figure 1A. The middle trace is the record
taken after the 15-minute solution change to 5.4 m/slCa**],, showing
the increase in the PV] delay that occurs with elevated [Co**]* The
bottom trace is the same PVJ site recorded after the solution change
to 1.35 mu [Ca**]^ Note that the PV] delay has decreased in the
presence of 1.35 mM[Ca^]o due to the earlier V activation time. The
V waveform remained uniphasic and negative in all three of the
[Ca+*]o solutions.
Vcell
(V/sec)
APD«
(msec)
(V/sec)
345
190
170
248
250
310
335
187
187
188
115
180
115
5
340
290
6
7
8
312
335
318
325
315
325
9
314
325
195
105
10
11
12
13
14
314
290
280
325
185
225
197
190
200
203
203
160
110
110
90
155
Site
10
VfflU
msec at a BCL of 1 second with a mean (±SD) of 3.4
± 0.8 msec. Increasing [Ca++]o to 5.4 mM increased
the PVJ delay in all nine experiments. The PVJ delay
values ranged from 2.6 to 4.9 msec at a BCL of 1
second with a mean of 3.9 ± 0.8 msec. This represents an 18 ± 8% increase in PVJ delay for all nine
experiments and a level of significance of P <
0.0005, by paired /-test (Bruning and Kintz, 1977):
We also measured the longitudinal and transverse
conduction velocities of the papillary muscle, using
the apical region of the RAP where no P layer is
present. The longitudinal conduction velocity decreased by 7 ± 7% and the transverse conduction
velocity decreased by 12 ± 4% with the increase in
[Ca++]o (« = 5). The effect of 5.4 mM [Ca++]o on the
PVJ delay was statistically significant in comparison
to the changes in the longitudinal conduction velocity (P < 0.05), but not in comparison to the transverse conduction velocity changes, as determined
by /-test for differences among the means (Bruning
and Kintz, 1977). Conversely, lowering [Ca++]o from
2.7 to 1.35 mM decreased PVJ delay by 14 ± 5%,
whereas the longitudinal and transverse conduction
velocities increased by only 4 ± 5% and 5 ± 3%,
respectively. The effect of 1.35 mM [Ca++]o on PVJ
delay was statistically significant in comparison to
the changes in the longitudinal and transverse conduction velocities. The PVJ delay values ranged from
1.8 to 3.8 msec, with a mean of 2.9 ± .7 msec at a
BCL of 1 second. The decrease in PVJ delay was
significant to P < 0.0005, by paired /-test.
Five experiments were performed to study the
effect of 2.5 mg/liter (3 X 10"6 M) quinidine on PVJ
delay. We also monitored the longitudinal conduction velocity and V ^ of the papillary muscle during
the experiments as an indirect measure of the degree
Circulation Research/Vol. 54, No. 5, May 1984
510
B
1.504-
Normalized
PVJ Delay .
3(mSec)
• *-
1.00
.
2-
PVJ Delay
1-
\
\
\
1.35
2.7
5.4
.50-
200
400
600
Basic Cycle Length
Catdum Concentration (mM
800
1000
(mSec)
D
1.50
1.50
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Normalized
1.00-
1.00
5O
.501
20O
1
4O0
1
600
1
80O
1
1OOO
Bask: Cycle Length (mSac)
200
400
600
Basic Cycls Length
800
1000
(mSec)
FIGURE 11. Effect of [Ca++]o and qumidine on PVJ delay. Part A is a plot of PVJ delay vs. [Ca**]o for nine experiments. The PVJ delay was
measured at a BCL of J second. Each line connecting a set of three points represents the results from one experiment. The changes in PVJ delay
associated with changing [Ca*+]a are statistically significant (P < 0.0005, Student's paired t-test). Part B illustrates the effect of 15 mg/liter
qumidine on PVJ delay in five experiments. The PVJ delay values for each experiment have been normalized to the value measured m control
solution at a BCL of 1 second. The remaining points in control solution (dashed line) represent the mean ( ± s a n = 5) values for PVJ delay at
each BCL tested (500, 400, 300, and 250 msec). The solid line in part B connects the values obtained in the presence of qumidine. Part C illustrates
the frequently-dependent changes in V cell Vmo in control solution (dashed line) and in the presence of quinidine (solid line). The Vma values are
again normalized to the control value at a BCL of 1 second and expressed in terms of the mean (±SQ n => 5) value at each BCL tested. Part D is
a similar plot for the muscle longitudinal conduction velocity changes in four experiments. See text for further details.
of sodium channel blockade. The results are presented in Figures 11, B-D. The data were normalized
to the values of PVJ delay, V™,,, and longitudinal
conduction velocity present in control solution at a
BCL of 1 second. All of the other points in the
graphs represent the mean normalized values (±SD)
for the five experiments. It should be noted that the
results for the longitudinal conduction velocity were
taken from only four of the five experiments, since
we could not obtain the adequate separation necessary between the recording electrodes and the apical
stimulus for an accurate measurement of the muscle
longitudinal conduction velocity in one experiment.
The dashed lines represent the results obtained in
control solution, and the solid lines represent the
results obtained in the presence of quinidine. The
stimulation rate was increased so that we might
study the effects of frequency on PVJ delay, as well
as to utilize a method for varying the degree of
sodium channel blockade, since quinidine is known
to block sodium channels in a frequency-dependent
manner (Hondeghem and Katzung, 1977, 1980;
Grant et al., Weld et al., 1982). In control solution,
the PVJ delay increases slightly with decreasing BCL
[P < 0.05 for BCL < 300 msec (Fig. 11B)], whereas
the V™, decreases with decreasing BCL [P < 0.05
for BCL of 500, 400, 300, and 250 msec (Fig. 11C)].
Small decreases were generally observed in muscle
longitudinal conduction velocity, but these changes
were not statistically significant (Fig. 11D).
After the addition of quinidine, the PVJ delay is
increased over control values at a given BCL [P <
0.05 for all BCLs (Fig. 11B, dashed lines)]. The V ^
and longitudinal muscle conduction velocity values
are decreased from their respective control values [P
< 0.05 for all BCLs, except for the V ^ value at a
BCL of 1 second, (Fig. 11, C and D)]. The major
observations to be obtained from these results are
(1) that the PVJ delay is more sensitive to changes
in [Ca++]o than are the transverse and longitudinal
Veenstra et al. /Purkinje-Ventricular Conduction Delay
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
conduction velocities of the RAP; (2) that, in the
control solution, PVJ delay is more sensitive to
changes in BCL than is the conduction velocity
within the RAP, although the changes in PVJ delay
are small and are only evident below a BCL of 300
msec; (3) that the V,™ of the V cells declines with
decreasing BCL; and (4) that the addition of quinidine reduces the V m i and muscle longitudinal conduction velocity while simultaneously increasing the
PVJ delay in a frequency-dependent manner. A
reduction in the P cell V™, would reduce the amount
of current supplied for activation of the V layer.
Also, the initiation of a propagating ventricular action potential requires the generation of a net inward
current in the ventricular layer. A reduction in the
amount of inward sodium current that could be
generated by the V layer would lead to an increase
in the amount of current required to be passed by
the P layer to the V layer for V activation. The major
conclusion to be obtained from the experimental
observations is that the PVJ delay is more sensitive
to changes in excitability than is conduction velocity
within the RAP.
Discussion
External monopolar recordings provide the investigator with more information about the local activation patterns of a papillary muscle than a single
microelectrode recording. Spach and Barr (1972) and
Spach et al. (1971, 1972, 1973, 1979) have shown
that the recorded extracellular potentials are directly
related to the spatial distribution of the intracellular
potential, can indicate the presence of one or more
excitation waves at a given site, and are more sensitive indicators of changes in propagation (e.g.,
collisions and conduction velocity). Because multiple
intracellular impalements would be required to obtain the same information contained in a single
extracellular monopolar recording, we chose to use
monopolar extracellular recordings to map out the
activation sequences of canine LPM and RAP muscles. With the use of one or more extracellular electrodes, we were able to record from over 220 to 300
sites on the surface of a RAP within a period of 3
hours. Recordings from various sites 3 hours after
the initial recordings did not reveal any significant
changes in the activation sequences displayed in
Figures 3 and 4. The construction of a similar activation map would have required over 400 intracellular microelectrode impalements in a given RAP to
complete the same analysis, because at least two
cells would have to be penetrated at many of the
sites. Such an undertaking would not be feasible
within the same time frame required for surface
mapping with extracellular electrodes. Instead, micreoelectrode impalements were made at selected
sites to confirm the identity of the extracellular
waveforms (Fig. 2). The results emphasize the advantages of using extracellular recordings to locate
a PVJ by surface mapping. Microelectrode impale-
511
ments can readily be made to provide information
about the membrane properties of P and V cells at
the various sites associated with a RAP (see Figs. 2B,
11, B-D; and Table 1).
Our experimental results provide a two-dimensional description of the P-V activation sequences.
The activation in the third dimension (deeper into
the RAP) has been assumed to be negligible, since
only the superficial 1-2 mm remains viable during
superfusion. The overall pattern is consistent with
the earlier studies from a few recording sites by
Myerburg et al. (1972) concerning the activation
patterns of both LPM and RAP preparations where
they concluded: (1) that the RBB is functionally
isolated from the interventricular septum and the
base of the RAP to the point where it fans out into
multiple free-running P strands; (2) that activation
of the RAP and LPM occurs near the base of the
muscle with a PVJ delay of 1-4 msec; (3) that
activation of the LPM proceeds from base to apex
and that stimulation of either of the basal or apical
P strands does not alter the base-to-apex orientation
of LPM activation (Fig. 6); (4) that long time delays
exist between the P and V cells near the apex of the
LPM; and (5) that LPM and RAP differ in their P
strand input. The RAP has only one P fiber input,
the RBB, and the apical portion of the RAP is devoid
of P cells (Mendez et al., 1969), whereas the LPM
has both apical and basal P strands. The two-dimensional studies presented here clearly show that
large regions of the superficial P cell layer are functionally uncoupled from the underlying V cells. In
addition, the PVJ site is not located at the region
where the RBB strands join the muscle. In every
experiment, the PVJ was located out on the surface
of the RAP, and not immediately adjacent to the
RBB (see Figs. 3-5).
Our results reported in Figure 2, A and B, define
the criteria used for identification of the PVJ and
allow us to identify the P, T, and V cells by their
activation sequence at a given site, rather than by
determination of their action potential characteristics. Sequential microelectrode impalements from
the superficial into the deeper cell layers of the RAP
at the extracellular recording site have revealed that
the P cells are always located on the surface and
that the V cells comprise the deeper cell layers of
the RAP. Comparison of the extracellular recordings
with the multiple intracellular recordings at the same
site in six experiments showed that the temporal
relationship of the P and V cells, as identified by
their action potential shape, matched with the activation sequence recorded using the extracellular
electrode. We have used these findings to justify our
use of the extracellular waveforms to identify the P
and V cells on the basis of their activation sequence.
This allows us to compare the Vm< and APD90 values
from free-running P strands, nonjunctional or junctional surface P cells, and basal or apical V cell
recordings (Fig. 9; Table 1) as independent variables,
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
512
without using any of the above action potential
parameters for identification of cell type. Mendez et
al. (1969) partially relied upon the action potential
configuration to identify the cell type after penetration with the microelectrode. Also, they recorded
only from the superficial cells at the various recording sites in the canine RAP preparation, in their
investigation of the spatial distribution of P and V
cell APD values (Mendez et al., 1969), whereas we
have made sequential penetrations at each recording
site, in order to record from each cell type present
at that site, as identified from the extracellular recordings. Despite these differences in methodology,
our results confirm those of Mendez et al. (1969),
who reported that there was a gradual decline in P
cell APD as the PVJ is approached. However, we
find that the P cell APD does not become equal to
the V cell APD at the juncnonal site. We also do not
find any significant difference in the V cell APD
values from the base to the apex of a RAP. Our APD
differences at the PV] are larger than the APD
differences reported by Myerburg et al. (1970) for
recordings from the base of a P strand and the
endocardial surface of the canine right ventricle.
Some of the differences may be attributed to the
different methods used for identification of cell type.
Some of the differences in our results, in comparison
to the results of Myerburg et al. (1970), can be
attributed to the use of different levels of repolarization in measuring the APD of the P and V cells,
and to the use of different BCL's for comparison of
the APD values (1 second in our experiments vs.
500 msec for Myerburg et al.). A more systematic
comparison of the APD]0o values of Purkinje fibers
and papillary muscle fibers was performed by Moore
et al. (1965), who reported that recordings from nine
Purkinje cells and 13 ventricular cells from a canine
RAP revealed that there is a 150-msec difference in
the APD, oo values of the P and V cells at a BCL of
1 second. This difference in APD decreases to 30
msec at a BCL of 200 msec. A similar comparison of
the results presented in Table 1 shows that four freerunning P strand cell recordings had an average
APD90 of 325 msec at a BCL of 1 second, compared
with an average APD90 value of 195 msec for all 10
of the ventricular cell recordings at the same BCL,
which results in a difference of 130 msec at a BCL
of 1 second. This result compares very well with the
reported differences in APD,oo of 150 msec, as reported by Moore et al. (1965). Furthermore, our
unpublished results of eight experiments from the
left ventricular endocardial surface shows that the
mean difference in APD90 of the P and V cells at a
BCL of 1 second is 56 msec, whereas the mean
difference in the APD50 of the same P and V cells at
a BCL of 600 msec is only 30 msec. It can therefore
be concluded that experimental differences in the
BCL and the level of repolarization of the cardiac
action potential can account for a 2-fold variation in
the published APD differences between Purkinje
Circulation Research/Vol. 54, No. 5, May 1984
and ventricular cells. In addition to the APD results,
we have found that the V^* does not change as the
P cell APD changes in relation to its proximity to
the PVJ.
This relationship is predicted by computer simulations (Joyner et al., 1983) of a linear cardiac strand
composed of 50% P cells and 50% V cells connected
end to end with a uniform coupling resistance. In
this model, we examined APD and V™,, distributions. We used the McAllister et al. (1975) model for
reconstructing a P cell action potential and the Beeler
and Reuter (1977) model for a V cell action potential.
The model showed that the APD distribution is
asymmetrical with respect to the PVJ. APD shortening occurs throughout the P cell region (up to
eight resting length constants from the PVJ), with
only a slight prolongation of the V cell APD in the
proximity of the PVJ. This difference presumably is
due to the differences of the plateau phases of the
two models. In the theoretical model, the V ^ values
of the P cell region remained high (Vmax = 310 V/
sec) throughout the P cell region, whereas the V,™,*
of the proximal V cell region increased only slightly
from their normal values (Vn^ =105 V/sec).
Our results, as shown in Figures 7 and 8, indicate
that one cannot always define conduction delays in
both the orthodromic (P to V) and antidromic (V to
P) directions at a given site, as previously reported
by Alanis et al. (1961) and Benitez and Alanis (1970),
since the orthodromic and antidromic pathways may
differ. The most interesting phenomenon presented
in Figure 8 is the possible existence of undirectional
block of recording site 2. In Figure 8B, it appears
that propagation in the orthodromic direction fails
at site 2, as indicated by the biphasic extracellular V
activation waveform, the existence of a longer PV
delay, and a later V activation time for site 2 in
relation to site 1 (PVJ). However, the extracellular P
activation waveform at site 2 during direct muscle
stimulation is contained within the extracellular V
activation waveform, indicating nearly simultaneous
activation of the P and V cell layers at site 2. The
existence of unidirectional block implies that there
is a spatial asymmetry in some parameter between
the P and V cells. Using numerical solutions for
cable equations, Joyner (1981) and Joyner et al. (in
press) have modeled various spatial inhomogeneities
which result in unidirectional block. For a system
with a uniformly low coupling resistance, unidirectional block is more readily produced by an asymmetrical electrical load than by regional changes in
excitability. However, when a localized region of
partial electrical uncoupling is present (a "resistive
barrier*), unidirectional block occurs when either
the excitability or the electrical load of the tissues
on the two sides of the barrier is different. An
example of varying electrical load is the 'funnel
hypothesis" advanced by Mendez et al. (1970), who
proposed that each junction could be modeled as a
funnel-shaped system composed of a cable-like P
Veenstra et al./Purkinje-Ventricular Conduction Delay
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
strand connected to an increasing number of interconnecting muscle fibers. The model predicts that
the delay encountered at a PVJ would depend on
the length of the funnel, with very short taper
lengths producing conduction block due to the
impedance mismatch. This effect has been modeled
theoretically by Joyner et al. (in press) for a cardiac
strand with a constant cell surface-to-volume ratio
and uniform membrane properties [the Beeler and
Reuter ventricular model (1977)]. Propagation failure can be obtained in this theoretical strand by
abruptly increasing the radius in the center of the
strand by a factor of 1.9 or more. However, there
are three drawbacks to the funnel hypothesis as
advanced by Mendez et al. (1970). First, the funnel
hypothesis would not predict conduction delay or
block in the anndromic direction, whereas it is
clearly evident, from Figures 6-8, that antidromic
conduction block may exist at most sites during
direct muscle stimulation. Second, the difference in
P and V cell APD values near the PVJ are much
larger than would be expected if P and V cells were
well coupled, electrically. Finally, our mapping studies reveal that the base of the RAP is covered by a
'sheet' of P cells, and that the PV interface is better
represented by a three-dimensional model of overlying two-dimensional sheets of V and P cells, than
by the one-dimensional system of cable-like terminal P fibers connected to a three-dimensional V cell
layer.
It is possible that differences in electrical coupling
between P and V cells may be responsible for the
PV] delay. Evidence already exists for a relative
paucity of gap junctions between cells of the PVJ
(Martinez-Palomo et al., 1970). Original predictions
regarding the nature of slow conduction in the heart
(e.g., the AV node) assumed that, in order to have
slow conduction, one must have action potentials
with a low Vmax (i.e., slow-response action potentials). There is a new line of evidence suggesting
that one can obtain slow conduction velocities in
cardiac muscle fibers with normal membrane properties (Lieberman et al., 1973; Spach et al., 1981,
1982a, 1982b; Joyner, 1982). Although the AV node
is known to have slow-response action potentials,
measurements of the passive electrical properties of
AV node (De Mello, 1977) and of dye passage
between cells of the AV node (Pollack, 1976) suggest
that increases in the intercellular resistance may be
largely responsible for the slow conduction velocity
of the AV node. The hypothesis that increases in
intercellular resistance are responsible for the PVJ
delay and conduction block seen in canine papillary
muscle preparations is lacking in direct experimental
support, however. Comparative histology of junctional and nonjunctonal sites and measurements of
electrotonic spread between P and V cells would
improve our understanding of the extent of cell-tocell coupling in the PVJ, but such experiments have
yet to be performed. Another approach to under-
513
standing the mechanism responsible for the PVJ
delay would be to attempt to regulate the degree of
cell-to-cell coupling by varying various factors that
have been reported to regulate cell-to-cell coupling
in the heart (see review by De Mello, 1982) such as
pH, (Reber and Weingart, 1982) and [Ca++]i (De
Mello, 1975; Dahl and Isenberg, 1980) while monitoring the PVJ delay.
Our experimental results regarding the action of
[Ca++]o on PVJ delay differ from the results of Benitez and Alanis (1970), who concluded that low
[Ca++]o increased the PVJ delay by reducing the V M I
and resting potential of the P and T cells. However,
they used nominally zero [Ca++]o solutions in their
experiments while we reduced [Ca++]o by only 50%
in our experiments. The differences in our results
probably are due to the deleterious effects of perfusing cardiac tissue with zero [Ca++]o (Weidmann,
1955; Alto and Dhalla, 1979), as manifested by the
reduction in resting potential, action potential amplitude, and Vnum of cardiac Purkinje fibers reported
by Benitez and Alanis (1970) during the course of
their experiments. The only changes that we saw in
the action potentials of nonjunctional P cells during
perfusion with 1.35 M [Ca++]o was a 10% increase in
the APD90 of the P cells, whereas perfusion with 5.4
mM [Ca++]o produced a 10% decrease in the P cell
APD90. These changes probably are due to alterations in the magnitude of the calcium-activated K+conductance of cardiac P fibers (Kass and Tsien,
1976; Isenberg, 1977). Furthermore, long PVJ delays
of 30 msec were reported in control solutions by
Benitez and Alanis (1970). We find that the true PVJ
delay is in the order of 2-4 msec, and that PV delays
of up to 30 msec are associated with nonjunctional
sites of the RAP and LPM. Therefore, changes in
muscle conduction velocity and pathway changes
would also contribute to the measured PV delays of
Alanis and Benitez (1970).
Alanis et al. (1961) offered a similar explanation
for the increase in PV delay associated with the
addition of quinidine to the perfusion solution. Our
results confirm their conclusion of a quinidine-induced increase in the PVJ delay, but we have shown
that quinidine increases this delay in a frequencydependent manner, along with reducing the muscle
longitudinal conduction velocity and the V cell V m>
in a similar fashion. Since Alanis et al. (1961) probably were recording from nonjunctional sites, part
of the increase in PV delay in their experiments can
be associated with the reduction in the muscle conduction velocity that occurs with the addition of
quinidine.
The use of altered [Ca++]0 in our experiments
should not be interpreted as fully supporting the
hypothesis that increases in PVJ delay are due to
changes in the intercellular resistance, since the increased [Ca++]o in our experiments could be increasing the PVJ delay by either increasing the intercellular resistance by raising [Ca++]i or by displacement
Circulation Research/Vol. 54, No. 5, May 1984
514
of the activation threshold (Hille et al., 1975; Brown
and Noble, 1978). Our interpretation of the results
would favor the second alternative, since we have
no direct evidence for any increase in [Ca++], with
increased [Ca++]o, and since our quinidine results
show that decreases in excitability will produce dramatic increases in PVJ delay.
We would like to thank Alfredo Chorro for his technical assistance
with the computer programming.
Supported by Grants HL22562 and HL26021 from the National
Institutes of Health, and Grant 82-G-26 from the Iowa Heart Association issued to Dr. [oyner.
Address for reprints: Dr. R.W. foyner, Department of Physiology
and Biophysics, The University of Iowa, Iowa City, Iowa 5224Z
Received June 3, 1983; accepted for publication February 9, 1984
References
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Alanis J, Benitez D (1970) Rate of nse of Purkinje and transitional
cells action potential and the propagation across the Purkinjemyocardium )unction. Jpn J Physiol 20: 217-232
Alanis J, Benitez D, Pilar G (1961) A functional discontinuity
between the Purkinje and ventricular muscle cells. Acta Physiol
Lat Am 11: 171-183
Alto LE, Dhalla NS (1979) Myocardial cation contents during
induction of calcium paradox. Am J Physiol 237: H713-H719
Beeler GW, Reuter H (1977) Reconstruction of the action potential
of ventricular myocardial fibres J Physiol (Lond) 268: 177-210
Benitez D, Alanis J (1970) Calcium ions and the propagation of
impulses through the mammalian heart junctional regions. Jpn
J Physiol 20: 233-249
Brown RH, Noble D (1978) Displacement of activation thresholds
in cardiac muscle by protons and calcium ions. J Physiol (Lond)
282: 333-343
Bruning JL, Kintz BL (1977) Computational Handbook of Statistics. Glenview, Illinois, Scott, Foresman and Company
Dahl G, Isenberg G (1980) Decoupling of heart muscle cells:
Correlation with increased cyptoplasmic calcium activity and
with changes of nexus ultrastructure. J Membr Biol 53: 63-75
De Mello WC (1975) Effect of intracellular injection of calcium
and strontium on cell communication in heart. J Physiol (Lond)
250: 231-245
De Mello WC (1977) Passive electrical properties of the AtnoVentricular node. Pfluegers Arch 371: 135-139.
De Mello WC (1982) Cell-to-cell communication in the heart and
other tissues. Prog Biophys Mol Biol 39: 147-182
Grant AO, Trantham JL Brown KK, Strauss HC (1982) pHDependent effects of quinidine on the kinetics of dV/dt,™, in
guinea pig ventricular myocardium. Circ Res 50: 210-217
Hille B, Woodhull AM, Shapiro BI (1975) Negative surface charge
near sodium channels of nerve: Divalent ions, monovalent
ions, and pH. Phil Trans R Soc Lond [Biol] 270: 301-318
Hondeghem LM, Jensen RA (1974) Purkinje-myocardial conduction delay: An alternative explanation. J Mol Cell Cardiol 6:
485-490
Hondeghem LM, Katzung BG (19771) Time- and voltage-dependent interactions of antiarrhythmic drugs with cardiac sodium channels. Biochim Biophys Acta 472: 373-398
Hondeghem LM, Katzung BG (1980) Test of a model of antiarrhythmic drug action. Effects of quinidine and lidocaine on
myocardial conduction. Circulation 61: 1217-1224
Isenberg G (1977) Cardiac Purkinje fibres. [Ca2+], controls the
potassium permeability via the conductance components gk,
and gu. Pfluegers Arch 371: 77-85
Joyner R (1981) Mechanisms of unidirectional block of cardiac
tissues. Biophys J 35: 113-125
Joyner R (1982) Effects of the discrete pattern of electrical coupling on propagation through an electrical syncytium. Circ Res
50: 192-200
Joyner R, Picone J, Veenstra R, Rawling D (1983) Propagation
through electrically coupled cells: Effects of regional changes
in membrane properties Circ Res 53: 526-534
Joyner R, Veenstra R, Rawling D, Chorro A (in press) Propagation
through electrically coupled cells: Effects of a resistive barrier.
Biophys J
Kamiyama A, Inoue F (1971) Conduction delay from Purkinje
fiber to ventricular muscle studied with extracellular microelectrodes. Can J Physiol Pharmacol 49: 678-684
Kass RS, Tsien RW (1976) Control of action potential duration by
calcium ions in cardiac Purkinje fibers. J Gen Physiol 67: 599617
Lieberman M, Kootsey JM, Johnson EA, Sawanobori T (1973)
Slow conduction in cardiac muscle. A biophysical model. Biophys J 13: 37-55
Martinez-Palomo A, Alanis J, Benitez D (1970) Transitional cardiac cells of the conductive system of the dog heart. J Cell Biol
47: 1-17
Matsuda K, Kamiyama A, Hoshi T (1967) Configuration of the
rransmembrane action potential at the Purkinje-ventricular fiber junction and its analysis. In Electrophysiology and Ultrastructure of the Heart, edited by T Sano, V Mizuhira, K Matsuda. New York, Grune & Stratton. pp 117-187
McAllister RE, Noble D, Tsien RW (1975) Reconstruction of the
electrical activity of cardiac Purkinje fibers. J Physiol (Lond)
251: 1-59
Mendez C, Mueller WJ, Merideth J, Moe GK (1969) Interaction
of rransmembrane potentials in canine Purkinje fibers and at
Purkinje fiber-muscle junctions. Circ Res 24: 361-372
Mendez C, Mueller WJ, Urguiaga X (1970) Propagation across the
Purkinje fiber-muscle junctions in the dog heart. Circ Res 26:
135-150
Moore EN, Preston JB, Moe GK (1965) Duration of rransmembrane action potentials and functional refractory periods of
canine false tendon and ventricular myocardium: Comparisons
in single fibers. Circ Res 17: 259-273
Myerburg RJ, Stewart JW, Hoffman BF (1970) Elecrrophysiological properties of the canine peripheral A-V conducting system.
Circ Res 26: 361-378
Myerburg RJ, Nilsson K, Gelband H (1972) Physiology of canine
inrraventricular conduction and endocardia! excitation. Circ Res
30: 217-243
Pollack GH (1976) Intercellular coupling in the atrioventricular
node and other tissues of the rabbit heart. J Physiol (Lond) 255:
275-298
Reber WR, Weingart R (1982) Ungulate cardiac Purkinje fibres:
The influence of intracellular pH on the electrical cell-to-cell
coupling. J Physiol (Lond) 328: 87-104
Spach MS, Barr RC (1972) Intracellular-extracellular action potentials: Considerations for the formation of wavefronts and their
detection on the body surface. In Advancements in Electrocardiography, edited by JW Hurst, DT Mason. New York, Grune
& Stratton, pp 19-26
Spach MS, Barr RC, Serwer GA, Johnson EA, Kootsey JM (1971)
Collision of excitation waves in the dog Purkinje system. Extracellular identification. Circ Res 29: 499-511
Spach MS, Barr RC, Serwer GA, Kootsey JM, Johnson EA (1972)
Extracellular potentials related to intracellular action potentials
in the dog Purkinje system. Circ Res 30: 505-519
Spach MS, Barr RC, Johnson EA, Kootsey JM (1973) Cardiac
extracellular potentials. Analysis of complex wave forms about
the Purkinje networks in dogs. Circ Res 33: 465-473
Spach MS, Miller WT, Miller-Jones E, Warren RB, Barr RC (1979)
Extracellular potentials related to intracellular action potentials
during impulse conduction in anisorropoic canine cardiac muscle. Circ Res 45: 188-204
Spach MS, Miller WT, Geselowitz DB, Barr RC, Kootsey JM,
Johnson EA (1981) The discontinous nature of propagation in
normal canine cardiac muscle. Evidence for recurrent discontinuities of intracellular resistance that affect the membrane currents. Circ Res 48: 39-54
Spach MS, Kootsey JM, Sloan JD (1982a) Active modulation of
electrical coupling between cardiac cells of the dog. A mecha-
Veenslra el al. /Purkinje-Ventricular Conduction Delay
nism for transient and steady state variations in conduction
velocity Circ Res 51: 347-362
Spach MS, Miller WT, Dolber PC, Kootsey JM, Sommer JR,
Mosher CE (1982b) The functional role of structural complexities in the propagation of depolarization in the atrium of the
dog. Cardiac conduction disturbances due to discontinuities of
effective axial resistance. Circ Res 50: 175-191
Weidmann S (1955) Effects of calcium ions and local anaesthetics
515
on electrical properties of Purkinje fibres. J Physiol (Lond) 129:
568-582
Weld FM, CoromilasJ, RottmanJN, Bigger JT (1982) Mechanisms
of quanidine-induced depression of maximum upstroke velocity in ovine cardiac Purkinje fibers. Ore Res 50: 369-376
INDEX TERMS: Purkinje fibers • Ventricle • Action potential
propagation • Quinidine • Calcium • Papillary muscle
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Purkinje and ventricular activation sequences of canine papillary muscle. Effects of
quinidine and calcium on the Purkinje-ventricular conduction delay.
R D Veenstra, R W Joyner and D A Rawling
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Circ Res. 1984;54:500-515
doi: 10.1161/01.RES.54.5.500
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1984 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/54/5/500
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/