271

Electrical Uncoupling and Increase of

Extracellular Resistance After Induction of
Ischemia in Isolated, Arterially Perfused Rabbit
Papillary Muscle
Andre" G. K16ber, Christoph B. Riegger, and Michiel J. Janse

Extracellular and intracellular longitudinal resistances (ro and r,), transmembrane potentials, and
conduction velocity were determined in arterially blood-perfused rabbit papillary muscles. Cable
analysis was made possible by placing the muscle in a H,O-saturated gaseous environment, which acted
as an electrical insulator. Ischemia was produced by exchanging the O2 in the atmosphere by N2 (94%
N2-6% CO2) in addition to arresting coronary flow. The first 10-15 minutes of ischemia were
characterized by an increase in r0, while r, remained constant. The early part of the increase in r.
coincided with the drop in perfusion pressure and was probably due to the diminution of intravascular
volume. Rapid electrical uncoupling, reflected by an increase in r, (threefold within 5 minutes),
occurred thereafter. The dissociation between the early increase in r0 and the delayed increase in r,
produced an initial increase in the ratio ro:r,, which subsequently decreased. The decrease in
conduction velocity was less than observed in intact hearts with ischemia. This difference is explained
by the relatively small changes in resting membrane potential and action potential amplitude in the
preparation used. Our results suggest that in the early, reversible phase of ischemia, the increase in
r. contributes to a small but significant extent to the slowing of conduction. After 15-20 minutes, the
rapid cellular uncoupling, which was most likely coincident with breakdown of cellular homeostasis,
may contribute to the occurrence of arrhythmias during this phase of ischemia. Moreover, the early
change in the ratio ro:r, will influence the amplitude of the extracellular electrograms following
coronary occlusion (TQ-segment and ST-segment shifts). (Circulation Research 1987;61:271-279)
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T
he existence of low resistance or diffusion
pathways between cardiac cells' 2 is essential
for normal cardiac function since it ensures the
rapid propagation of the impulse (e.g., see Crane-
field3). Disruption of cells in myocardial necrosis was
already noted in the last century by Engelmann.4 The
association of electrical uncoupling in heart with
mechanical injury was shown by DeMeze,5 with intra-
cellular calcium overload was shown by De Mello,6
with acidification was shown by Reber and Weingart,7
and with application of a cardiac steroid was shown by
Weingart.8
In ischemia, precise knowledge of the time course
and the magnitude of electrical uncoupling is important
to explain the decrease of conduction velocity and the
formation of conduction blocks. Both are major causes
of the malignant ventricular arrhythmias (e.g., see
Janse and K16ber9) that follow the interruption of
coronary flow. Several indirect observations indicate
that uncoupling develops relatively early in the course
From the Department of Physiology, University of Berne, Berne,
Switzerland, the Department of Cardiology and Experimental
Cardiology, University of Amsterdam (M.J.J.), and the Interuni-
versity Cardiology Institute. The Netherlands.
Supported by the Swiss Science Foundation (Nr.3.903-0.85), the
Swiss Heart Foundation, the Roche Foundation, and the Wijnand
M. Pon Foundation.
Address for correspondence. Andr6 G. Kl^ber, Department of
Physiology, University of Berne, Bilhlplatz 5, CH-3012, Berne,
Switzerland.
Received November 17, 1986; accepted March 18, 1987.
of myocardial ischemia: 1) Currents of injury, flowing
across the boundaries between normal and ischemic
tissue, decrease relatively rapidly after the first 15-30
minutes of ischemia.10 This suggests an increase of
intercellular and/or extracellular resistive elements in
the pathway of the injury current during the develop-
ment of irreversible cellular damage. 2) In myocardial
hypoxia, Wojtczak" found a moderate (77%) increase
of intracellular longitudinal resistance (assuming a
constant extracellular resistance) after 30 minutes of
oxygen withdrawal. 3) The first morphologic sign of
uncoupling, consisting of a separation of adjacent cell
membranes at gap junctions, were reported to occur as
early as 20 minutes after perfusional arrest.12
Myocardial ischemia is characterized by, in addition
to oxygen withdrawal, a lack of extracellular washout
with a rapid extracellular accumulation of metabolic
products and ions, e.g., potassium,13 and an increase in
extracellular osmolality.14 An experiment arrangement
to assess the changes in both extracellular and intra-
cellular passive electrical properties of ischemic ven-
tricle must be designed to fulfill two conditions. First,
the myocardial tissue must be arterially perfused in its
normoxic state, and second, the geometric arrangement
of the cells must permit the application of linear cable
theory." We have shown recently that these method-
ologic requirements are met in a blood-perfused rabbit
papillary muscle surrounded by a gas mixture, which
acts as an electrical insulator.16 In this preparation, a
modification of the classic linear cable analysis for
 272
myocardial tissue2 can be applied. This approach was
chosen in the present study to determine the time course
and the magnitude of the changes in extracellular and
intracellular longitudinal resistance after the arrest of
coronary flow. Our results demonstrate that electrical
coupling remains unaffected during the first 10-15
minutes of ischemia. Rapid uncoupling thereafter
probably marks the onset of irreversible cellular dam-
age. The extracellular resistance shows a biphasic
increase that has a significant effect on the decrease of
conduction velocity in compact cardiac tissue.
Materials and Methods
Arterially Perfused Rabbit Papillary Muscle
Rabbits weighing 2-3 kg were anesthetized with 50
mg/kg pentobarbital i.v. and killed by a blow on the
head. Heparin (200 U/kg) was administered intrave-
nously, and the heart was rapidly excised and brought
to a dissecting chamber. The basis of the heart
(including both atria and the atrioventricular valves)
and the left ventricular free wall were removed with a
razor blade. Afterwards, the septal artery was cannu-
lated (e.g., Weiss and Shine17), and the nonperfused
parts of the interventricular septum and the right
ventricular free wall were carefully removed. The
average time between excision of the heart and artificial
perfusion was 4 minutes. The final preparation con-
sisted of an arterially perfused part of the rabbit
interventricular septum to which 2-3 papillary muscles
usually were attached. Muscles selected for measure-
Downloaded from http://ahajournals.org by on September 30, 2022
ment had a single insertion of the tendon, a mean
cross-sectional area of 1.12 mm2 (±0.21 mm2, SEM,
n = 8) and showed no marked tapering at the apical end.
Perfusion and Recording Chamber
The perfusate contained a mixture of washed bovine
erythrocytes (hematocrit 25%), albumin (2 g/1), dex-
tran (70,000 MW, 40 g/1), insulin (1 U/l), heparin (400
U/l), and Tyrode's solution of the following compo-
sition (in mM): Na+ 149, K + 4 . 5 , Mg 2 + 0.49, Ca2+ 1.8,
Cl" 133, HCOj 25, H 2 POj 0.4, and glucose 20. The
pH of the perfusion fluid ranged from 7.35-7.40.
A detailed description and illustration of the perfu-
sion apparatus and the recording chamber is given
elsewhere.16 The perfusate was oxygenated in a mem-
brane oxygenator and driven to the recording chamber
by an Ismatec rol ler pump (Switzerland). The perfusion
pressure was set to 40-45 mm Hg by adjustment of the
roller pump. The myocardial blood flow amounted to
approximately 80-100 ml/100 g/min. The Perspex
plate carrying the preparation was placed in the
recording chamber at an angle of 30-40° from the
horizontal plane (see schematic drawing in Figure 1).
The tendon of the papillary muscle was fixed to a
platinum wire that protruded from the hollow core of
a cylindrical Perspex holder. This holder was attached
to a micromanipulator at its base. It served to position
the papillary muscle in a horizontal position, to adjust
its resting length (30% above slack length), and to carry
one of the two electrodes through which subthreshold
or excitatory currents were applied between the apex
Circulation Research Vol 61, No 2, August 1987
Arterial perfusion
FIGURE 1. Schematic presentation of preparation in recording
chamber. Arterially perfused interventricular septum (1) is
mounted on oblique, wax-covered Perspex plate (2) that
contains large AgCI-coated Ag-electrode (3). Papillary muscle
(4) is running freely through recording chamber. Its tendon at
apical end is fixed to platinum wire (5) and connected to constant
current source (6) that delivers subthreshold and excitatory
current pulses. Extracellular potentials are measured between
electrodes E, and E, (tungsten wires, 50-fim diameter). Intra-
cellular potentials are measured between intracellular (float-
ing) microelectrode £, and E2. Dotted area symbolizes artificial
atmosphere of recording chamber (for composition, see text).
and the base of the muscle. In 3 experiments, the
developed tension was measured by a Hottinger Bald-
win transducer (Darmstadt, Germany) fixed to the
platinum wire. In such a way, an arterially perfused
papillary muscle was obtained that was surrounded by
the artificial atmosphere of the recording chamber,
which acted as an electrical insulator. The artificial
atmosphere consisted of an H2O-saturated mixture of
either O2 and CO2 or N2 and CO2 (see below), which
flowed into the chamber through multiple holes drilled
into the anterior and lateral walls. Temperature was
maintained at 35° C by warming the tubing with the
perfusion solution and the gas mixture in a water bath.
Furthermore, the water of the thermostat was pumped
through the partition of the double-walled cover of the
recording chamber. An opening (1 -cm diameter) in this
cover allowed access to the preparation with the
recording electrodes. Normally, a preparation re-
mained mechanically and electrically stable for 6 hours
when perfused under normoxic conditions. Within this
period, no visible shrinking or swelling occurred as
verified by microscopic control of the muscle diameter.
Production of Ischemia
Ischemia was produced by interruption of the arterial
perfusion with a tap, which resulted in a total drop of
perfusion pressure within 10-15 seconds. To avoid
diffusion of oxygen from the artificial atmosphere into
the muscle during ischemia, the gas content of the
chamber (94% O 2 -6% CO2 during normoxic perfusion)
was changed to a mixture of 94% N 2 -6% CO2 1 minute
prior to perfusion arrest. This interval was necessary to
 Kleber el al Electrical Uncoupling in Myocardial Ischemia
achieve complete mixing in the recording chamber.
During ischemia, the residual partial O2 pressure was
less than 15 mm Hg in the first 4 experiments and less
than 5 mm Hg in the rest of the experiments. This
difference had no detectable effect on the electrical
measurements. Only one ischemic period was pro-
duced per heart.
Measurement of lntracellular and Extracellular
Longitudinal Resistances and Conduction Velocity
Both detailed description and a critical evaluation of
the method used to determine passive cable properties
have been published elsewhere.16 In essence, passive
cable properties and conduction velocity were assessed
with two sequential measurements. First, the extracel-
lular voltage drop between two extracellular electrodes
(E, and E2) was recorded during application of a
subthreshold constant current pulse (20 msec duration)
that was applied extracellularly between the apical
electrode and the electrode at the base (electrical
ground, see Figure 1). The electrode E, was placed at
a distance of 1.2 mm or greater from the insertion of
the tendon. At this site, the subthreshold current that
is injected into the extracellular space at the apical end
is distributed regularly between the extracellular and
intracellular compartments, and membrane current has
become negligible. Therefore, the longitudinal tissue
resistance r,, which is composed of r, (intracellular
longitudinal resistance) and r0 (extracellular longitu-
dinal resistance) in parallel, can be obtained from Vo,
Downloaded from http://ahajournals.org by on September 30, 2022
Ax, and I:
'— = r = -Hl2_(n/cm) :D
Ax-1 r+r
where Vo is the subthreshold voltage drop between E,
and E2, Ax is the interelectrode distance, and I is the
strength of the subthreshold current (see Figure 5 of
Kle"ber and Riegger16).
For the second set of measurements, an excitatory
current pulse (0.5-1.0 msec in duration, double
threshold strength, 30 msec after the subthreshold
pulse) was applied to activate the apical end of the
muscle. The cycle length between stimuli ranged
between 500-800 msec among different experiments
and remained constant during an experiment. During
excitation, the extracellular bipolar electrogram was
measured between the electrodes E2 and E,, and the
transmembrane potential was measured between an
intracellular floating microelectrode E, and the extra-
cellular electrode E, (Figure 1). Conduction velocity 0
was obtained from the interval between the deflection
and the inflection of the bipolar electrogram and the
interelectrode distance (for linearity of propagation,
see K16berand Riegger16). The ratio of extracellular to
intracellular longitudinal resistance q was obtained
from the absolute values of the amplitudes of the
extracellular bipolar electrogram (AVo) and the trans-
membrane action potential (AVm):
273
q = = — (2)
AVm I - r,
where the values for r0 and r, (in ficm) were calculated
from equations 1 and 2. The values for the intracellular
longitudinal specific resistance R, (ficm), extracellular
longitudinal specific resistance Ro (ficm), and specific
tissue resistance R, (Q cm) were calculated during
normoxia from the measured cross-sectional area of the
muscle and from an assumed extracellular: intra-
cellular space ratio of l:3. 1 8 During ischemia, the
measurement of the specific resistances was not pos-
sible because of the lack of morphometric information
on volume changes in the extracellular and intracellular
compartment. Therefore, the results were expressed in
percent of the r0 and r, values during normoxic
perfusion. The paired t test was used for comparison
of results after a given duration of ischemia and of
results during normoxic perfusion. Summarized results
are expressed as mean ± SEM.
Extracellular electrodes (50 pirn tungsten wire) and
intracellular electrodes (floating glass microelectrodes,
see K16ber and Riegger16) were connected to high input
impedance buffer amplifiers (Analog Device 515), and
signals were amplified by differential instrumentation
amplifiers. Current strength was measured in the
feedback loop of an operational amplifier introduced
between the preparation and ground. The amplified
traces (current strength and extracellular and intracel-
lular voltage) were stored digitally in a signal memory
recorder (Max Meyer Electronics, Switzerland) at a
sampling rate of 100 yusec (subthreshold events) or 30
/usec (suprathreshold events). The data were analyzed
by a Hewlett-Packard Model 9617 computer and were
displayed on a graphic plotter. The interelectrode
distances were measured with a micrometer in the
eyepiece of the binocular microscope (magnification,
25 x ) .
Results
Extracellular and Intracellular Resistivities and
Potentials During Normoxic Perfusion
Figure 2 shows recordings during flow of sub-
threshold current (left panel) and after excitation of the
apical end of the muscle (right panel) when the muscle
was arterially perfused. The distance Ax between the
extracellular electrodes was 2.24 mm in this experi-
ment. The apical extracellular electrode was placed
1.32 mm from the end of the muscle, which is about
three times the estimated space constant (Kle"ber and
Riegger"). With this position of the electrodes (remote
from the zone of transmembrane current flow during
application of the subthreshold current), the longitu-
dinal tissue resistance r, is obtained directly from the
subthreshold voltage displacement, the current
strength, and Ax. The ratio ro: r, (1.38) was calculated
from the amplitudes of intracellular and extracellular
potentials (98 and 57 mV, respectively). In this case,
the resistance values, expressed in terms of specific
 274
2M*
10 mi
FIGURE 2. Left panel: Subthreshold current pulse (upper trace)
of20-msec duration flowing longitudinally through muscle and
producing subthreshold wltage drop (lower trace) between
extracellular recording electrodes E, and E2. Longitudinal
tissue resistance r, (consisting of r, and r, in parallel) is
calculated from current strength, subthreshold voltage, and
interelectrode distance. Right panel: Bipolar extracellular
electrogram (upper trace) and initial portion of transmembrane
action potential (lower trace). Ratio of extracellular to intra-
cellular longitudinal resistance r,: rt is calculated from ampli-
tudes of extracellular and intracellular signals. Valuesfor ro and
r, are obtained from r, and ratio r,' rr Conduction velocity is
calculated from conduction time (time between deflection and
inflection of extracellular electrogram) and interelectrode
distance.
longitudinal resistances, were R, = 97 Qcm, Ro = 70
Qcm, and R,= 115 f2cm. Expressed this way, the
values are independent of fiber diameter. Conduction
velocity was 65 cm/sec. Mean values of these param-
Downloaded from http://ahajournals.org by on September 30, 2022
eters from 8 experiments are shown in Table 1. They
agree well with earlier results obtained with this
preparation.l6 Note that, in contrast to findings obtained
in superfused preparations,2•"•" the extracellular lon-
gitudinal resistor has the same order of magnitude as
the intracellular longitudinal resistor, as is evident from
the ratio ro: r,.
Changes of Extracellular and Intracellular
Longitudinal Resistances During Ischemia
The change in the resistances of the tissue compart-
ments are expressed in relative changes in ro and r,. This
was necessary because calculation of the specific
resistances (Ro and R,) requires an assumption of the
actual ratio of extracellular: intracellular space. This
ratio changes during ischemia14 but has not been
assessed in our preparation.
Figure 3 shows the mean values from 8 experiments
of changes in the extracellular longitudinal resistance.
In this and subsequent figures, mean values were taken
from measurements within successive 2-minute time
Table 11. Electrical Constants From Arterially Perfused Rabbit Papillary Muscle
AVm AV 0
(mV) (mV)
n 8 8 8
Mean± SEM 102 ± 3 53+4 1.1+0.1
AVmand AV0. amplitudes of transmembrane action potential and extracellular electrogram, respectively; r o :r,. ratio of
extracellular: intracellular longitudinal resistance. Calculation of longitudinal specific resistances for whole tissue (R().
intracellular space (R,), and extracellular space (Ro) were based on assumed extracellular volume fraction of 0.25. l 8 0 .
conduction velocity.
Circulation Research Vol 61, No 2. August 1987
+100%-
+50*
0 5 10 15 20
Time after onset of ischemia (mm)
FIGURE 3. Changes of extracellular longitudinal resistance
after arrest of coronary flow. Mean values (bars indicate
standard error) from 8 experiments (8 hearts). First stepwise
increase within first minute is followed by slower continuous
increase. Changes are significantly different from control
(p<0.0l) at all time intervals.
intervals. Following arrest of coronary flow, there is an
immediate increase in extracellular resistance of ap-
proximately 30%. In later stages of ischemia, a second
gradual increase occurs to levels of + 8 0 % after 20
minutes. The immediate increase in r0 coincides with
the drop in perfusion pressure and the resulting fall in
intravascular volume. Figure 4 shows that the loss of
the hydraulic stiffness ("garden hose effect"20) also
gives rise to a considerable fall in actively developed
tension. This fall in tension associated with the arrest
of perfusion, as measured in three experiments, was
31 ± 4 % .
The changes in intracellular longitudinal resistance
are shown in Figure 5. In Figure 5A, the changes are
plotted vs. time elapsed after the arrest of coronary
perfusion. In the first 10—12 minutes, intracellular
resistance remains constant; in later stages, there is a
steep increase. In individual experiments, this increase
occurred very rapidly at times varying between 12-18
minutes after interruption of coronary flow. To elim-
inate this variability and to visualize the average time
course of the onset of uncoupling, the same data were
replotted in Figure 5B by superimposing the individual
values between + 2 0 and + 3 0 % into one window on
a relative time scale. This shows that there is a mean
increase in intracellular longitudinal resistance of
+ 200% within 5 minutes.
The calculation of r, and ro requires linear cable
properties, which implies uniform continuous propa-
gation of the impulse. At a certain time and a certain
R. Ri Ro 0
(Qcm) (ftcm) (flcm) (cm/sec)
8 8 8 8
126±11 194 + 31 66 + 6 62+1
 Kliber et al Electrical Uncoupling in Myocardial Ischemia
Interruption of arterial perfusion
10 mN
20 mmHg [
30 s
FIGURE 4. Changes of tension (upper trace) and perfusion
pressure (lower trace) after interruption of coronary flow. Arrest
of perfusion is associated with marked decrease of twitch tension
from 12.8 to 8.8 mN and slight decrease in resting tension,
which bear similar time course as the decrease in pressure.
degree of uncoupling (as indicated by the dot in Figure
5A), propagation became discontinuous, which pre-
vented calculation of r, and r0. This discontinuity was
apparent from irregularities (notches) on the upstrokes
of intracellular potentials and/or fragmented extracel-
lular electrograms. Since measurements of the sub-
threshold events were independent of propagation, a
further and rapid uncoupling was indicated by the
continuous increase of total longitudinal tissue resis-
tance r, beyond this point (not shown).
Downloaded from http://ahajournals.org by on September 30, 2022
+ 200X
HOOX
.1 Z 1 2 i~=^
5 10 15
Time after onset of ischemia (min)
+ 200
° +100-
-15 -10 -5
Time (min)
275
The changes in extracellular and intracellular resis-
tances have consequences for the propagation of the
impulse and for the magnitude of extracellular potential
changes during the flow of injury current across an
ischemic boundary. Local current produced by a
propagating wave front flows through a resistor that is
composed of the intracellular and extracellular resis-
tances in series. During ischemia, this series resistance
increases immediately following arrest of coronary
flow. As shown in Figure 6 (top trace), during the first
10 minutes of ischemia, this increase is entirely
accounted for by the change in the extracellular
resistance, while the change in intracellular resistance
only contributes thereafter. In the lower part of Figure
6, the ratio ro: r, is plotted. This ratio increases from 1.1
to 2.1 in the first 10 minutes, reflecting the increase in
ro. Subsequently, the increase in r, causes the ratio to
decrease. These changes must be taken into account for
the interpretation of extracellular potential changes
(TQ- and ST-segment shifts) during regional ischemia
(see "Discussion").
Each muscle was reperfused after 25 minutes of
ischemia. This produced rapid visible swelling, inter-
stitial accumulation of erythrocytes, and contracture.
Assessment of cable properties was not possible
because of the irregular shape of the extracellular
electrograms, which may reflect either inhomogenei-
ties in the extracellular resistor or persistence of cellular
uncoupling. However, action potentials of normal
FIGURE 5. Top panel: Changes of intracellular longi-
tudinal resistance in acute myocardial ischemia (mean
\>alues±SEM from 8 hearts). Values at and after 15
minutes are significantly differentfrom control (p<0.05).
20 25
Dot indicates time and level of uncoupling at which
propagation becomes discontinuous (limit for linear
cable analysis). Bottom panel: Time course of uncoupling
after interruption of coronary flow. Values shown in
Figure 6, top panel, have been replotted. For each
experiment, the value between + 20 and + 30% was
assigned to relative time 0 to visualize average time
course of uncoupling. Note threefold increase within 6
minutes.
+5
 276
0 5 10 15 20
Time after onset of ischemia (min)
FIGURE 6. Top panel: Changes of series resistor (r, + r,) after
induction of ischemia. Lower area indicates contribution ofro;
upper area indicates contribution of rr Changes at all time
intervals were significantly different from control (p<0.05).
Bottom panel: Changes of ratio ro: r, in ischemia. Changes after
1-14 minutes were significantly different from control (p<0.01
between 1-12 minutes. p<0.05 between 12-14 minutes).
amplitudes could consistently be recorded after reper-
fusion.
Downloaded from http://ahajournals.org by on September 30, 2022
Changes in Transmembrane Potentials
Figure 7 shows extracellular bipolar electrograms
(upper panel) and transmembrane potentials (lower
panel) from an experiment in which it was possible to
maintain the intracellular impalement for 25 minutes
and to place the extracellular reference electrode as
close as 40 /xm to the intracellular microelectrode.
The delayed activation of the impaled cell during
ischemia is clearly visible. The increased conduction
time is apparent from the increased interval between
downstroke and upstroke of the bipolar electrogram.
After 25 minutes of ischemia, action potential ampli-
tude had decreased by 18 mV. At this time, the upstroke
50 mV
bipolar
electrogrom
control 19 mm 25 mm
50 mV
transmembrane
potential
5 ms
Circulation Research Vol 61. No 2. August 1987
was composed of 2 components, probably indicating
discontinuous conduction.21
The mean values for changes in action potential
amplitude and resting membrane potential are shown
in Figure 8. Resting membrane potential values were
obtained in 6 experiments in which the transmembrane
potential was also recorded on an inkwriter at a low
paper speed. The mean values were obtained from
measurements in successive 4-minute time intervals.
Action potential amplitude values were obtained from
8 experiments and plotted in 2-minute intervals. Both
the degree of depolarization and the reduction in action
potential amplitude following arrest of coronary per-
fusion were consistently smaller in this preparation than
in intact hearts with regional or global ischemia.922
Changes in Conduction Velocity
In Figure 9, the mean percent change in conduction
velocity in the 8 experiments is plotted. Within the first
minutes, there was no significant change. After 20
minutes of ischemia, however, conduction velocity had
decreased by 51 % from a control value of 62 ± 1 to
32 ± 0 . 3 cm/sec.
For a linear cable, conduction velocity should be
inversely related to the square root of the series resistor
ro + r, (see Jack et al15). The contribution of the
resistance change was calculated from the results
shown in Figure 6 and is represented by the interrupted
line of Figure 9. In the first 4-5 minutes, conduction
velocity was higher than predicted by the increase in
the series resistor; after 15-20 minutes, it was lower.
This indicates, as expected, that conduction velocity is
further determined by other factors such as changes in
action potential upstroke and changes in excitability.
Discussion
The methods used in the present study were designed
for the assessment of electrical activity and of cable
properties in densely packed, arterially perfused ven-
tricular muscle. The preparation used offers two
advantages: 1) It allows measurements of the effects of
ischemia, and 2) it excludes the effects of an extracel-
lular shunt resistance made up by a small layer of
superfusion fluid in in vitro preparations or by cavitary
FIGURE 7. Extracellular bipolar electrograms (top
traces) and transmembrane action potentials (bottom
^m traces). Positions of extracellular and intracellular elec-
trodes are shown on inset. Recordings during control and
after 19 and 25 minutes of ischemia are superimposed.
2 mm Initial portions of transmembrane action potentials were
obtained from single impalement; intracellular electrode
was impaled 40 yjn apart from extracellular floating
microelectrode. Discontinuity of conduction is indicated
by notch in upstroke of action potential at 25 minutes.
This excludes determination of ratio r,' r, at this stage of
ischemia.
 Kliber et al Electrical Uncoupling in Myocardial Ischemia
100
-60
f important roles. No information is available on even-
-80
0 5 10 15
Time ofter onj«t of ischemia (mln)
FIGURE 8. Changes of action potential amplitude (AVJ and
resting membrane potential (RMP) after onset of ischemia. For
both measurements, changes were significantly different from
control at all time intervals (p<0.05).
blood at an endocardial boundary in vivo.23 For these
reasons, normal values for cable parameters'6 show a
much higher value than previously reported for the
extracellular longitudinal resistance, which is slightly
higher than the value for intracellular longitudinal
resistance. This finding implies that changes in elec-
trotonic interaction between ventricular cells are de-
pendent to an equal degree on the resistances of
intracellular and extracellular spaces.
Arrest of coronary flow resulted in an almost
immediate increase in extracellular resistance. This
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increase appears to not be related to metabolic changes
since it has also been observed in fibers exposed to an
atmosphere containing 94% O2.16 It is most likely due
to the fall in perfusion pressure and to the associated
decrease in intravascular volume. The mechanical
consequence of arrest of perfusion, which in our
preparation was a reduction of twitch tension by 30%,
has been termed loss of hydraulic stiffness or garden
hose effect in hearts in vivo.20 It is responsible for the
early systolic bulging of ischemic myocardium in intact
hearts following coronary artery occlusion. The mech-
anism of the slower secondary rise in extracellular
resistance is not directly evident from the present
investigation. It could very well be due to a decrease
in extracellular volume, which is expected to occur as
a consequence of cell swelling secondary to an increase
of intracellular osmolality of ischemic cells.14
Our experiments show that ischemic myocardial
cells uncouple rapidly after an initial stable phase of
10-15 minutes, during which intracellular longitudinal
resistance remains unchanged. This finding adds fur-
ther support to the views that the ionic homeostasis of
acutely ischemic cells is not markedly disturbed and,
particularly, that there is no marked increase of
intracellular free Ca2+ during an initial phase of about
10-15 minutes after coronary artery occlusion. In line
with this result are findings of maintained low intra-
cellular Na+ activity in guinea pig hearts22 and of low
resting tension in early ischemia in rabbit interventric-
ular septa.17 Functional uncoupling after 10-15 min-
utes correlates well with the first morphologic changes
277
observed at the intercalated disk. They consist in a
migration of vesicular bodies toward the intercalated
disk and in a dissociation of the gap junctional
membranes.12 The reasons for cellular uncoupling,
which was not reversible after 25 minutes, can be
manifold. Both a decrease in intracellular pH7 and an
increase in intracellular calcium6 are likely to play
tual additional effects of metabolites produced during
ischemia on nexal resistance. An association with an
increase of free intracellular Ca 2+ , and probably Na 2+ ,
20 is most likely. Thus, the delayed increase in coupling
resistance in our experiments corresponds in time to the
delayed increase in resting tension in ischemic rabbit
interventricular septa,17 which may be caused by an
increase in free Ca 2+ . The relation with the breakdown
of ionic homeostasis is further suggested from mea-
surements of cellular K+ loss. Accumulation of extra-
cellular potassium occurs in two distinct phases.1324
The second increase (indicating onset of irreversibility)
occurs at a time similar to the increase in intracellular
resistance.
The change in the ratio ro: r, may have important
implications for the interpretation of the magnitude of
extracellular potentials recorded across an ischemic
boundary in hearts with regional ischemia. The in-
stantaneous difference in transmembrane potentials
between normal and ischemic cells provides the driving
force for the injury current flowing through the intra-
cellular and extracellular resistances across an ischemic
border. This driving force is divided between the
intracellular and extracellular resistances. The voltage
drop over the extracellular resistance builds up the
extracellular field.2 At a given driving force, the initial
increase in the ratio ro: r, observed in our experiments
will result in an increase in the extracellular component
(TQ-segment depression and ST-segment elevation).
While the subsequent decrease of this ratio will
decrease the changes in the extracellular field, i.e., the
amplitudes of the extracellular electrograms.
The reduction in action potential amplitude and
-100X
5 10 15 20
Time ofter oniet of ischemia (min)
FIGURE 9. Changes of conduction velocity in percent after
arrest of coronary perfusion. Within the first 4 minutes,
conduction velocity remains unchanged. Afterwards, there is
significant decrease to 51% after 20 minutes (p<O.OJ at all time
intervals). The interrupted line indicates effect of rise in (r, + r,)
on conduction velocity as predicted from linear cable theory.
 278
resting membrane potential were considerably less than
that observed in intact hearts with global or regional
ischemia.'•22-23~27 An eventual contamination of the
gaseous atmosphere within the recording chamber by
diffusion of oxygen can be ruled out; oxygen pressure
was carefully monitored and found to be lower than 15
mm Hg in the first 4 experiments and less than 5 mm
Hg in the remaining 4 experiments. A major difference
between our preparation and thick-walled intact ven-
tricles during ischemia is the partial pressure of CO2 in
the tissue. It is known that pco2 rises rapidly and
continuously to values between 200 and 400 mm Hg
within 15 minutes following coronary artery
occlusion.M-M In our preparation, Pco2 was kept low by
the constant CO2 content of the atmosphere (6%)
around the muscle that was continuously exchanged. It
has been shown that both extracellular K+ accumula-
tion and depolarization of resting membrane potential
can be modulated by varying Pco2 in the subepicardial
layers of the globally ischemic guinea pig hearts.30 This
interaction appears to occur between CO2 (or H + ) and
passive K+ efflux because active K+ influx remains
unaffected during the early phase.3' The exact way a
change of pco2 or pH interferes with cellular K+ loss
in ischemia remains to be clarified, however.
The contribution of the changes in extracellular and
intracellular resistances on the changes in conduction
velocity was calculated according to linear cable
theory. The predicted overall effect was a steady
decrease in conduction velocity by 12% after 10
Downloaded from http://ahajournals.org by on September 30, 2022
minutes and by 23% after 20 minutes. A comparison
with the actually measured changes in conduction
velocity in our preparation indicates that the changes
in conduction are additionally related to other factors
in a complex way. In the first 4 minutes, there was no
significant change in conduction velocity, while a small
decrease was predicted by the increase in r0. In later
stages, measured conduction velocity was consistently
lower than could be expected from the increase in r0 + r,
alone. The present data do not allow the contribution
of the changes in action potential and excitability to be
assessed in a quantitative way. An initial decrease in
current requirement for excitation due to the shift of the
resting membrane potential towards the threshold
potential32 has been considered as a factor preventing
an early decrease in conduction velocity.I333J4 In later
stages of ischemia, changes in action potential upstroke
characteristics as well as a decrease in excitability are
likely to contribute to conduction slowing. In our
experiments, action potential amplitude decreased by
10-20%. A correlation of conduction velocity with
changes in maximal upstroke velocity could not be
carried out in our experiments. A precise measurement
of maximum upstroke velocity was possible in only a
few experiments (Figure 7) because of interference
with the large extracellular field.35 Distortion of the
action potential upstroke was noted whenever the
extracellular reference electrode was placed further
than about 80 fim from the impalement site.
The changes in conduction observed in intact ven-
tricles with acute ischemia are different from those in
Circulation Research Vol 61, No 2, August 1987
the present experiments. Within 4 - 5 minutes, longi-
tudinal conduction velocity decreases to 50% of the
control value, and conduction blocks occur within the
same period.34 Conduction block is cycle-length de-
pendent in the sense that zones which block the impulse
at short cycle lengths can conduct the impulse upon a
sudden increase in cycle length. This indicates that the
delayed, time-dependent recovery of excitability is the
most important determinant of conduction block. The
marked decrease in action potential amplitude and
upstroke velocity and the time-dependent recovery of
excitability occur only in ischemic cells that are
depolarized to a critical degree.27-3* The possible
reasons for the absence of this marked shift of resting
potential in our preparation have been discussed above.
In conclusion, the increase in extracellular resistance
in the first 10 minutes of ischemia contributes to a small
but significant extent to the decrease in conduction
velocity during the acute reversible phase of ischemia.
Reentrant excitation in the ventricular wall9 during this
phase will be predominantly caused by the effect of
ischemia on the electrical activity of the membrane
(changes in action potential upstroke and in excitabil-
ity). After 15-20 minutes, the rapid uncoupling of the
ischemic cells is likely to contribute to the disturbances
of impulse conduction, which may be important factors
for the occurrence of arrhythmias during this and
subsequent periods.
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