Microelectrode Demonstration
of Wedensky Facilitation in Canine
Cardiac Purkinje Fibers
By John C. Bailey, Joseph F. Spear, and E. Neil Moore
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
ABSTRACT
Microelectrode techniques were used to examine changes in excitability of canine
Purkinje fibers at sites distal to complete conduction block. Immediately distal to a
site where impulse propagation failed, it was possible to record subthreshold depolarizations. Intracellular stimulation of the Purkinje cells exhibiting these subthreshold
responses with constant-current pulses of subthreshold intensity enabled full-amplitude,
propagated action potentials to develop when the subthreshold stimuli were timed
to occur during the slow upstroke of the subthreshold depolarization. This period of
a decreased current threshold requirement for stimulation observed during the first
40—50 msec of the subthreshold depolarization was followed by a period during
which suprathreshold stimuli were necessary to evoke propagated responses. Propagated
action potentials which interrupted the subthreshold response during its repolarization
phase demonstrated reduced maximal rising velocities. We concluded that summation of appropriately timed subthreshold stimuli could induce full-amplitude, regenerative, all-or-none responses and that the changes in excitability of subthreshold responses were not solely voltage dependent. Sodium inactivation appeared to be
responsible for the depressed rate of action potentials that interrupted the subthreshold
response during its repolarization phase and might participate in the coincident
depressed excitability.
KEY WORDS
subthreshold depolarization
conduction block
regenerative depolarization
excitability
electrotonus
threshold
cardiac arrhythmias
sodium inactivation
• In 1886, Wedensky (1) demonstrated, in a
neuromuscular preparation, that a strong stimulus
produced a prolonged period of enhanced excitability of the nerve. Later, in a blocked neural
preparation, he reported that excitability was
temporarily increased distal to the site of conduction block (2). These two phenomena have been
referred to as the Wedensky effect and Wedensky
facilitation, respectively. The Wedensky effect has
been demonstrated in canine cardiac tissue by
Goldenberg and Rothberger (3) and in the human
heart by Castellanos and co-workers (4). It has
been suggested that Wedensky facilitation might
account for certain cardiac arrhythmias in man
(5-7), but its existence in the heart has never been
proven. The experiments presented in this paper
were designed to examine Wedensky facilitation in
canine cardiac false tendons, and microelectrode
techniques were used.
From the University of Pennsylvania, School of Veterinary
Medicine, Comparative Cardiovascular Studies Unit, Philadelphia, Pa. 19174, and the Krannert Institute of Cardiology,
Marion County General Hospital, and the Department of
Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202.
This work was supported in part by U. S. Public Health
Service Grants HL-04885, HL-06308, and HL-05749 and by
grants from the American Heart Association, the Herman C.
Krannert Fund, the Marion County Heart Association, and
the Indiana Heart Association.
Dr. Bailey's permanent address is Department of
Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202.
Dr. Spear is an Established Investigator of the American
Heart Association.
Received January 22, 1973. Accepted for publication
May 2, 1973.
Methods
Adult mongrel dogs of either sex were anesthetized
with sodium pentobarbital (30 mg/kg, iv), and their
hearts were immediately removed through a lateral
thoracotomy and placed in Tyrode's solution. Unbranched Purkinje false tendons 10-15 mm long and
1-2 mm in diameter were excised from either ventricle
and pinned to the floor of a 50-ml muscle chamber. The
preparation was superfused with modified Tyrode's
solution (2.7 mM potassium), bubbled with 95% O2-5%
CO2, and maintained at a temperature of 35°C. An
illustration of the preparation and the placement of
stimulating and recording electrodes is presented in
Figure 1A. Bipolar stimulating electrodes were located
on the proximal (St r ) and distal (Std) ends of the
false tendon perpendicular to the long axis. A third
48
Circulation Research, Vol. XXXIII, July 3973
49
TEMPORAL SUMMATION
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
bipolar electrode (not shown) was positioned on the
midportion of the false tendon so that the poles of the
electrode spanned the breadth of the false tendon. A
zone of complete bidirectional conduction block 2-3 mm
long (stippled area, Fig. 1A) was produced by
electrocauterization of the false tendon via this third
extracellular bipolar electrode; block was monitored by
recording intracellular microelectrodes located in cells
of the proximal (Rp) and distal (Rd) portions of the
false tendon. A third intracellular microelectrode for
both recording (Rm) and passing current (Stj) was
inserted into the false tendon immediately distal to the
area of conduction block. The method of using a single
microelectrode for simultaneous intracellular stimulation, recording, and current measurement has been
reported previously (8). The microelectrodes, drawn
from 1-mm soft glass tubing, had tip diameters of less
than 0.5ju, and, when filled with 3 M KC1, had tip
resistances of 15-30 megohms. Conventional methods of
signal amplification and capacitance neutralization
were utilized. Data were displayed on a Tektronix 565
memory oscilloscope and recorded on an Ampex FR1300 analog tape recorder and on 35-mm film using a
Tektronix 565 oscilloscope.
A custom-designed, precision digital pulse generator1
permitted stimulation of one or both ends of the false
tendon with either identical or different stimulus
sequences. Concomitantly, a constant-current pulse,
with amplitude, duration, and time of occurrence
independently controlled, could be delivered to the
impaled cell immediately distal to the zone of block.
Figure IB illustrates the stimulus programs utilized in
this study. The bipolar stimulating electrodes on the
proximal (Stp) and distal (Std) ends of the false
tendon allowed simultaneous stimulation of both ends
of the false tendon five times (ST-S5) at constant basic
cycle lengths of 500-700 msec; then the train sequence
was automatically repeated. Excitability of the central
cell could be determined at selected times in the
interval between S4 and S t ; the S5 stimulus was omitted
at either the distal or both ends when intracellular test
stimuli were delivered. S5 was applied to the proximal
end of the false tendon via Stp to test orthograde
conduction block, whereas retrograde conduction block
was assessed by delivering S6 to the distal end only.
Once bidirectional failure of conduction had been
established, diastolic current threshold requirements
were determined for the cell immediately distal to the
block (Rm in Fig. 1). The Ss pulse was omitted from
both ends of the false tendon so that a steady, maximal
diastolic transmembrane potential could be recorded
from the central cell (R m ). Then, constant-current
pulses 4 msec in duration were delivered intracellularly
to that cell via Stj, and a stimulus intensity was determined that was slightly subthreshold for the entire
diastolic interval of the central cell. We have termed
this stimulus the "control subthreshold stimulus." To
test changes in excitability of the cell distal to the
conduction block, S5 was delivered to the proximal end
of the false tendon via Stp but was omitted from the
1
Constructed by Murray Bloom, 116 Elmwood Avenue,
Narberth, Pa. 19072.
Circulation Research, Vol. XXXIII, July 197}
distal end. This procedure resulted in the occurrence of
a subthreshold depolarization at Rm. The control
subthreshold stimulus was then delivered to the central
cell via St, at numerous intervals following S5, and
responses to the control subthreshold stimulus were
monitored by Rm and Rd. Then, current threshold
requirements at Rm were determined at numerous
intervals following delivery of S5 to the proximal end of
the false tendon via Stp. Finally, propagated action
potentials were initiated at Std and timed to arrive at
Rm at numerous intervals before and after delivery of S3
via Stp. The maximal rising velocity of phase 0 of these
action potentials was recorded by electronic differentiation of phase 0.
Results
Figure 2 illustrates the activation sequence
following establishment of bidirectional block. S4
and Si stimuli were delivered simultaneously at
both the proximal (St^) and distal (Sta) regions of
the false tendon. All-or-none action potentials
developed in all three Purkinje cells (first and third
responses) consequent to the S4 and Si stimuli. S5
was applied only to the proximal end of the false
tendon via Stp, and this stimulus resulted in an allor-none action potential only at Rp. Therefore,
conduction of this impulse failed within the zone of
conduction block, and the eel] immediately distal to
the area of block (R m ) responded to SB with only a
1st,
S«n
7\
/
st f
I
«m
R
d
B
FIGURE 1
A: Diagram illustrating the false tendon and the arrangement of stimulating and recording electrodes. The stippled
area represents the zone of conduction block. St p and Stfl
are proximal and distal bipolar stimulating electrodes, respectively. Rp and Rd are proximal and distal recording
microelectrodes, respectively. ^ is the recording microelectrode impaled immediately distal to the zone of block.
Stj indicates that an intracellular stimulus can be delivered
to the cell through the recording microelectrode (Rm) by the
use of rapid stimulate, record-switching circuits. B: Stimulus programs utilized in this study. Sj-S 5 could be delivered
simultaneously to both ends of the false tendon via the
proximal and distal stimulating electrodes (Stv and Stj.
Intracellular stimuli (Stj could be applied at varying times
between Si and St to the cell distal to the block (R^J. See
text for discussion.
50
BAILEY, SPEAR, MOORE
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
5 0 mV
125 msec
FIGURE 2
Documentation of orthograde conduction block. The S4 and
Sj stimuli were delivered simultaneously at both ends of the
false tendon via the proximal (Stp) and distal (Std) extracellular stimulating electrodes. The S 5 stimulus was applied
only via the proximal (Stp) stimulating electrode. The cell
proximal (Rp) to the site of conduction block responded to
this S5 stimulus with a full-amplitude depolarization. The
cell immediately distal (Rm) to the site of conduction block
responded with a slowly rising, low-amplitude, subthreshold
depolarization. The most distal cell, Hd, was not excited.
Abbreviations are the same as in Figure 1. See text for discussion.
slowly rising, low-amplitude subthreshold depolarization (second, small response in the middle tracing Rm). The distal cell (R^) was not excited, thus
demonstrating that complete orthograde conduction
block was present between Rp and R^. When S5
was delivered via Std alone (not shown), the
distal cell (R d ) was activated, followed by fullamplitude depolarization of the middle cell (R m ).
In this circumstance the proximal cell (R p ) was not
activated, because retrograde conduction failed in
the zone of block.
We attempted to characterize the voltage and the
time course of the subthreshold depolarizations
resulting from failure of propagation of impulses
originating proximal to the zone of conduction
block. Only those experiments in which the central
cell membrane demonstrated a maximal diastolic
potential between —80 mv and —90 mv were
considered. Because the width of the blocked zone
was not known precisely and because impalement
of the first fully excitable cells distal to the block
was not assured, we can make only certain general
statements about the subthreshold depolarization.
The extent of subthreshold depolarization of the
central cell ranged from 5 mv to 10 mv, and
restoration of the maximal diastolic potential was
attained after 200-230 msec. Maximal depolarization
was reached after approximately 50 msec; repolarization required 150-200 msec. The maximal subthreshold depolarization diminished with distance,
although subthreshold responses could still be
recorded as far away as 5mm. In no circumstance
did more distal cells demonstrate subthreshold
responses of greater magnitude than those of the
cells nearer the site of conduction block. When
more severe cauterization of the false tendon was
applied, subthreshold responses were never recorded in the distal segment.
In Figure 3, S5 was applied only to the proximal
end of the false tendon in a preparation in which
bidirectional block had been established; subthreshold depolarization of the central cell ensued
(middle, low-amplitude response). During the
subsequent three sweeps of the oscilloscope, the
control subthreshold stimulus was delivered intracellularly (Sti) to the central 'cell prior to (A),
during (B), and following (C) the subthreshold
depolarization. When the stimulus was applied
Three superimposed recordings from the middle intraceUular
electrode (RnJ used both to stimulate and record. The first
action potential followed delivery of S, via the distal extracellular stimulating electrode (St^). The middle, subthreshold
depolarization was consequent to delivery of Ss via the
proximal extracellular stimulating electrode (Stp). The third
record of superimposed action potentials was due to delivery
of S, via Std. If the control subthreshold stimulus was applied intracellularly at times A or C, excitation resulting
in an all-or-none response did not occur. However, if that
stimulus was delivered at time B, an all-or-none response
occurred. There was subsequent cycle length-dependent
foreshortening of the action potential consequent to St. See
text for discussion.
Circulation Research. Vol. XXXlll,
July
I97i
TEMPORAL SUMMATION
51
The minimum current intensity necessary to excite
the resting membrane was 0.58 x 10~6. However,
13.5 msec after the onset of subthreshold depolarization, at a time when transmembrane potential
had declined 3.2 mv, threshold requirements were
reduced to 0.38 X 10~e. Once maximum depolarization was attained, there followed a period of
relative refractoriness, when currents suprathreshold for the resting membrane were required to
elicit all-or-none responses. Thus, a current stimulus Sufficient to excite the cell at a given
transmembrane potential during depolarization was
insufficient to excite the membrane at the same
transmembrane potential during repolarization of
the subthreshold response.
Figure 5 demonstrates the changes observed in
the maximal rate of rise for all-or-none responses
interrupting a subthreshold depolarization of 7.8
mv. Propagated action potentials, initiated at Std,
were timed so that they arrived at the central cell at
various intervals during the time course of its
subthreshold depolarization. The maximal rate of
9
a
320300-
6
280260-
3
2
LI
240-
O.M.O.P.
1.0
220M.RV. (
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
prior to the occurrence of the subthreshold
depolarization (A), the cell was not excited.
Similarly, when the intracellular stimulus followed
repolarization of the subthreshold response (C),
excitation did not ensue. However, when the
control subthreshold stimulus was delivered near
the peak of the subthreshold response (B), a fullamplitude depolarization of the central cell occurred, followed by conduction to the distal cell
(not shown). If the subthreshold depolarization
was omitted by deleting Ss at the proximal
stimulation site (St p ) and the intracellular stimulus
was applied at the same time as in B, no response at
either the middle or the distal recording site (Rm or
Rd) was observed. The zone of summation and
facilitation began approximately 5 msec after
initiation of the subthreshold response and persisted
until maximal subthreshold depolarization was
attained. Thereafter, as transmembrane potential
returned to resting values, the control subthreshold
stimulus was ineffective in producing summation.
Current threshold requirements for evoking an
all-or-none action potential were determined at
numerous times during the time course of subthreshold depolarization. Figure 4 illustrates a representative strength-interval curve for a subthreshold
depolarization of 8.5 mv. Qualitatively similar
results were obtained in 12 separate experiments
0.9-
200160-
08160b 0.7
x
140"
a. 0.6-
120-
0.5-
100-
0.4-
600
0.5
0.2-
700
80O
900
1000
TIME (msec.)
1100
1200
FIGURE 5
500
600
700
TIME ( msec. )
800
900
FIGURE 4
Strength-interval curve for a subthreshold depolarization of
8.S mv. During the upstroke of the response, current threshold requirements were reduced. During repolarization, suprathreshold stimuli u>ere required to elicit all-or-none depolarization. M.D.P. = maximal diastolic potential.
Circulation Research, Vol. XXXIII, July 1973
Maximal rising velocity (M.R.V.) of propagated action potentials plotted against membrane potential of a subthreshold
depolarization of 7.8 mv. The action potentials were evoked
at the distal stimulating site at various times so that they interrupted the subthreshold depolarization at different times.
The maximal rising velocity declined during repolarization
of the subthreshold response. M.D.P. = maximal diastolic
potential.
52
BAILEY, SPEAR, MOORE
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
rise of phase 0 of each of these interrupting all-ornone action potentials was recorded. When the
propagated action potential arrived prior to the
onset of or following the termination of the
subthreshold response, maximal rising velocity
averaged 281 ± 6 v/sec. During the upstroke of the
subthreshold depolarization, rising velocity remained essentially the same as it was for the resting
membrane (271 v/sec). However, the maximal
rising velocity of the action potential arriving 50
msec after the onset of subthreshold depolarization,
when the membrane had depolarized 7.8 mv, was
reduced to 129 v/sec. The maximal depression of
rising velocity (114 v/sec) was attained 75 msec
after the onset of the subthreshold response.
Thereafter there was a gradual return of maximal
rising velocity to control values. Similar changes in
maximal rising velocity were recorded in ten
separate experiments.
Discussion
In 1903, Wedensky (2) described a temporary
increase in excitability of a nerve distal to a site
where impulse propagation had been arrested.
Although the impulse fails to engage actively the
distal segment, the enhanced excitability nevertheless is a consequence of the blocked impulse. This
phenomenon is commonly known as Wedensky
facilitation. Blair and Erlanger (9) have subsequently demonstrated, in a blocked peripheral
nerve, that two appropriately timed subthreshold
stimuli delivered proximal to the block can activate
the distal nerve by temporal summation during this
period of increased excitability. Hodgkin (10) has
confirmed these observations and demonstrated that
electrotonic currents ahead of the blocked impulse
produce displacement of the transmembrane potential toward threshold in the distal segment. These
extrinsic potentials, so named because they resemble applied electrotonic potentials, are not propagated; rather, they decline exponentially along the
nerve,
Similarly, in our experiments utilizing segmentally blocked canine Purkinje false tendons, we also
recorded increases in excitability of cells immediately distal to the site of conduction block. The period
of enhanced excitability coincided with the slow
upstroke of a subthreshold depolarization of the cell
distal to the block. Furthermore, if a subthreshold
stimulus was delivered during this time of enhanced
excitability, a propagated action potential ensued.
Thus, these data demonstrate that Wedensky
facilitation can occur in cardiac tissue.
The subthreshold depolarizations that we recorded distal to a blocked impulse in some ways
resembled the extrinsic potentials of Hodgkin (10).
In our studies these responses were never sufficient
to elicit regenerative responses, although such an
event is possible. Moreover, the magnitude of the
potential and the net changes in excitability
declined in cells more distal to the blocked impulse
(11). The onset of subthreshold depolarization
occurred immediately following block of the
propagated impulse, and the initial rise of the
subthreshold response was roughly exponential.
Finally, the increase in excitability during the
upstroke of these responses appeared to be voltage
dependent, i.e., less current was required to fully
excite the membrane because the transmembrane
potential was nearer the threshold potential.
The subthreshold responses that we recorded
were further characterized by a period of partial
refractoriness; during this time a stronger suprathreshold stimulus was required to elicit full
excitation. Thus, at a given transmembrane potential during the depolarization phase of the subthreshold responses, a stimulus subthreshold for
the resting membrane was sufficient to excite,
whereas, at the same transmembrane potential
during repolarization of the response, a stimulus
suprathreshold for the resting membrane was
necessary to produce propagated action potentials.
Moreover, this period of partial refractoriness
corresponded to a time when the maximal rate of
rise of phase 0 of entering action potentials was
severely depressed. Inasmuch as the maximal rate
of depolarization indicates the ability of the cell
membrane to undergo an increase in sodium
permeability (12, 13), the observation of a reduced
rate of depolarization strongly suggests sodium
inactivation during this period of relative refractoriness. We did not observe slowed or blocked
conduction of the action potentials which interrupted the subthreshold response at a time when
current threshold requirements were increased and
when the maximal rising velocity of the entering
action potentials was reduced. However, such
alterations of conduction might occur if the safety
factor for propagation of the entering action potential is diminished.
We have demonstrated that temporal summation
of two subthreshold events distal to a site of
conduction block in false tendons can elicit
propagated action potentials. It is possible that the
occurrence in cardiac tissue of subthreshold depolarizations demonstrating periods of enhanced, then
Circulation Research, Vol. XXXlll,
July 1973
TEMPORAL SUMMATION
depressed, excitability may prove to be important in
the genesis of certain arrhythmias, The period of
relative refractoriness may cause slowing or block
of impulses with low margins of safety for
conduction. The interaction of subthreshold stimuli
in a small area of structurally or functionally altered
cardiac tissue may give rise to nonreentrant,
nonautomatic, premature extrasystoles (5). Moreover, summation of this type may participate in
reentry at the level of syncytial connections (14)..
Acknowledgment
The authors are grateful for the expert technical assistance
of Mr. Ralph Iannuzzi.
References
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
1. WEDENSKY, N.E.: t)ber die Beziehung zwischen Reizung
und Erregung im Tetanus. Akademie der Wissentschaften, Berichte 54:96, 1886.
2. WEDENSKY, N.E.: Die Erregung, Hemmung und
Narkose. Pfluegers Arch 100:1-144, 1903.
3.
GOLDENBEBG, M., AND ROTHBERGER, C.J.:
TJntersuch-
ungen an der spezifischen Muskulatur des Hundeherzens. Z Gesamte Exp Med 90:508-528, 1933.
4.
CASTELLANOS,
A., LEMBERC,
L., JOHNSON,
D., AND
BERKOVITS, B.V.: Wedensky effect in the human
heart. Br Heart J 28:276-283, 1966.
5. SCHERF, D., AND SCHOTT, A.: Extrasystoles and Allied
Arrhythmias. New York, Grune & Stratton, 1953.
Circulation Research, Vol. XXXIU, July 1973
6. FiSCH, C , AND KNOEBEL, S.B.: "Wedensky facilitation"
in the human heart. Am Heart J 76:90-92, 1968.
7. SCHAMROTH,
L.,
AND FHIEDBERG,
H.D.:
Wedensky
facilitation and the Wedensky effect during high
grade A-V block in the human heart. Am J Cardiol
23:893-899, 1969.
8. MOORE, E.N., AND BLOOM, M.: Method for intracellular
stimulation and recording using a single microelectrode. J Appl Physiol 27:734-735, 1969.
9. BLAIR, E.A., AND ERI-ANGER, J.: Temporal summation
in peripheral nerve fibers. Am J Physiol 117:355-365,
1936.
10. HODGKTN, A.L.: Evidence for electrical transmission in
nerve; Parts I and II. J Physiol (Lond) 90:183-232,
1937.
11. WEIDMANN, S.: Electrical constants of Purkinje fibers. J
Physiol (Lond) 118:348-360, 1952.
12. HODGKTN, A.L., AND HUXLEY, A.F.: Dual effect of
membrane potential on sodium conductance in the
giant axon of Loligo. J Physiol (Lond) 116:497506, 1952.
13. WEIDMANN, S.: Effect of the cardiac membrane
potential on the rapid availability of the sodiumcarrying system. J Physiol (Lond) 127:213-224,
1955.
14.
CRANEFIELD, P.F., KLEIN, H.O., AND HOFFMAN, B.F.:
Conduction of the cardiac impulse: I. Delay, block,
and one-way block in depressed Purkinje fibers. Circ
Res 28:199-219, 1971.
Microelectrode Demonstration of Wedensky Facilitation in Canine Cardiac Purkinje Fibers
JOHN C. BAILEY, JOSEPH F. SPEAR and E. NEIL MOORE
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Circ Res. 1973;33:48-53
doi: 10.1161/01.RES.33.1.48
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1973 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/33/1/48
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/