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604
Subthreshold Stimulation of Purkinje Fibers
Interrupts Ventricular Tachycardia
in Intact Hearts
Experimental Study With Voltage-Sensitive Dyes
and Imaging Techniques
Guy Salama, Anthony Kanai,
Igor
R. Efimov
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Abstract The effects of subthreshold stimulation (STS)
delivered during right atrial pacing and ventricular tachycardia
(VT) were investigated in Langendorff-perfused guinea pig
hearts. The hearts were stained with a voltage-sensitive dye
(RH 421) to map the propagation of optical action potentials.
Sustained VT was reliably induced by 5-second trains (cycle
length [CL], 25 to 50 milliseconds; duration, 0.5 to 10 milliseconds; and voltage, 2x threshold voltage) of impulses (n=12
hearts) or a single premature beat (n=6). The location of
extrastimuli was not critical to the induction of VT, but the
diameter of the heart had to be .14.5 mm. During VT, heart
rate increased from 200 to 600 beats per minute; action
potential durations decreased from 112 to 175 milliseconds to
60 to 105 milliseconds, with no diastolic interval. Activation on
the epicardium spread anisotropically, but VT decreased the
"apparent" maximum conduction velocity (Om.) by 68% and
altered the orientation of the major axis from beat to beat.
Activation patterns and Omax measured during VT were similar
to patterns recorded during direct pacing of the ventricle and
indicated that Purkinje fibers no longer propelled ventricular
excitation. STS (CL, 25 to 50 milliseconds; duration, 0.5 to 25
milliseconds; and voltage, 0.5 x to 0.8 x threshold; trains of 2.0
to 2.5 seconds) interrupted VT when applied to Purkinje fibers
lining the endocardium (n=6) but failed to interrupt VT when
applied to the epicardium (n=8). In atrial pacing, STS delivered to the endocardium increased Omax from 2.44+0.32
(mean±SEM) to 3.63±0.21 m/s in a local region surrounding
the first activation sites (n=4). Alternatively, VT could be
terminated by reducing Omax (=55%) with procainamide (10
gmol/L) (n=6). STS terminates VT by synchronizing ventricular excitation most likely by increasing local conduction
and/or improving the coupling between Purkinje and ventricular cells. (Circ Res. 1994;74:604-619.)
Key Words * subthreshold stimulation * ventricular
tachycardia * action potentials * cycle length * apparent
maximum conduction velocity * anisotropic propagation .
reentry * voltage-sensitive dyes
n the clinical setting, human ventricular tachycardia
(VT) can be terminated by subthreshold stimula-
vation by mapping with multiple surface electrodes, STS
(10 to 100 Hz) terminated VTs most effectively when
applied at the "early" activation site of the tachycardia
on the endocardium.'0
Several mechanisms have been proposed to explain
the termination of VT and/or VF by STS. The proposed
mechanisms did not clearly distinguish between VT and
VF and were based on the interruption of reentry
pathways by reducing the size of the excitable gap. For
instance, STS may interrupt reentry by (1) prolonging
repolarization (ie, action potential [AP] duration) and
causing a local increase in the refractory period,'1 (2)
initiating an early partial depolarization, resulting in
supernormal conduction velocity at the stimulation
site,12 and/or (3) inducing a local release of catecholamine, which would enhance local conduction velocity.10 Difficulties in understanding the mechanism(s)
whereby STS interrupts VT are compounded by the
lack of a common definition of VT, the different physiological states of the hearts in various studies, and the
different methods used to initiate VTs. Thus, the interruption of VT by STS may well depend on the nature of
the VT. In turn, the most common cause of VT is
reentry, and at least three mechanisms have been
proposed to account for reentry. (1) An activation wave
front could propagate repeatedly around a cut (anatomic block) made in the center of the atrial muscle.'3 Two
tion (STS) applied with catheter electrodes inserted in the left ventricle through the aorta. These
findings are potentially important in the development of
effective low-energy defibrillation techniques. However,
the mechanism(s) whereby STS terminates VT and the
precise location of the electrodes applying STS have yet
to be elucidated.
Stimulation with low-level noncapturing impulses was
shown to block normal heart beats, first in amphibian'
and then in canine2 and human3 ventricular muscles.
STSs of different frequencies were subsequently tested
in attempts to interrupt arrhythmias. High-frequency
(333 Hz) STS blocked excitation waves elicited by
premature extrastimuli in canine ventricles.4 Low-frequency (13.3 to 20 Hz) STS was equally effective at
interrupting VT in the clinical setting,5 atrial tachycardia,6 and triggered activity7'8 and in blocking the induction of ventricular fibrillation (VF).9 In studies of actiFrom the University of Pittsburgh School of Medicine, Department of Physiology, Pittsburgh, Pa.
Previously published as a preliminary report in abstract form
(Circulation. 1990;82[suppl III]:III-98).
Correspondence to Dr Guy Salama, Department of Cell Biology
and Physiology, University of Pittsburgh, W1442 Biomedical Science Tower, Pittsburgh, PA 15261.
Salama et al Subthreshold Stimulation Interrupts VT
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factors were necessary for repeated reentry or circus
movement around the loop: block and slowed conduction. The anatomic barrier provided a track around
which a propagating wave could loop. Slowed conduction made it possible for the tissue to repolarize and
recover from refractoriness before the return of the next
activation wave front. The time interval between the
repolarization and the arrival of the next activation
wave front was called the excitable gap. Factors that
increase conduction velocity or the refractory period of
the muscle decrease the excitable gap and thus increase
the likelihood of extinguishing the wave front. The
anatomic block could be caused by myocardial infarction or ischemia, whereas slow conduction may result
from a rise in resting membrane potential. In circus
movement, activation wave fronts propagate repeatedly
around an anatomic barrier from beat to beat, resulting
in monomorphic electrogram signals. (2) Anisotropic
reentry is based on the heterogeneous propagation of
APs, with fast and slow conduction velocities along
directions parallel and transverse to the longitudinal
axis of ventricular cells. This form of reentry was
demonstrated in hearts with infarcted left ventricles
caused by a coronary occlusion. After a healing period,
the infarction resulted in a two-dimensional thin layer
of surviving tissue that sustained reentry loops through
pathways of fast and slow conduction that aligned with
the longitudinal and transverse axes of the underlying
fibers.14,15 As for circus movement, anisotropic reentry
requires a pathological injury and results in monomorphic electrogram signals. (3) In the absence of hypoxia
or ischemia (ie, in "healthy" heart muscle), leading
circle tachycardia was first demonstrated in rabbit atrial
muscle.'6 The difference between leading circle and
circus movement is the lack of an excitable gap; ie, the
instant a region of tissue is repolarized, the next wave
arrives to depolarize it. In leading-circle reentry, the
refractory period set the wavelength of the circuit, and
slight changes in refractoriness result in beat-to-beat
alterations of activation patterns. As a result, electrogram signals appear polymorphic.
The present report describes a nonhypoxic nonischemic model of VT in intact mammalian hearts and
confirms that STS terminates VT under controlled
experimental conditions. More important, activation
patterns were mapped at high spatial and temporal
resolution by use of voltage-sensitive dyes and imaging
techniques to examine the mechanism(s) whereby STS
terminates VT. On the basis of optical maps of normal
and arrhythmic activation patterns, it appears that
Purkinje fibers, which normally drive the excitation of
the myocardium, become uncoupled and fail to synchronously drive the activation of the myocardium during
VT. When STS is delivered to the endocardium, it
appears that the coupling between the Purkinje network
and the ventricular mass is reestablished, which interrupts such arrhythmias.
Materials and Methods
Preparation
Guinea pigs (450 to 550 g, either sex) were anesthetized with
an intraperitoneal injection of Nembutal (30 mg/kg), and their
hearts were rapidly excised, cannulated at the aorta, and
retrogradely perfused in a modified Langendorif apparatus.
Input of perfusate at the aorta was controlled with a peristaltic
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pump (Miniplus 2, Gilson, Middleton, Wis) and was connected
to a graduated manometer to obtain a physiological mean
aortic pressure of 80 mm Hg. The flow rate of the pump
determined the flow of perfusate delivered to the coronary
vessels and was adjusted for each heart to take into account
differences in muscle mass. For each heart, the coronary flow
rate was set at the beginning of each experiment to obtain 80
mm Hg for aortic pressure and was thereafter kept constant. A
perfusion system with constant coronary flow rate ensured that
oxygen delivery to the myocardium remained constant at all
times, irrespective of the contractile state of the heart, coronary vascular tone, and arrhythmic conditions. Flow rates were
in the range of 12 to 18 mL/min for the various hearts used in
the present study. From the pressure-flow relation, changes in
aortic pressure were used to detect possible changes in coronary resistance that might occur as a consequence of hypoxia,
ischemia, or edema. Once the desired flow rate was determined, hearts selected for the present study had stable aortic
pressures; if the pressure rose above 90 mm Hg, the data were
discarded. The perfusate contained (mmol/L) NaCl 130,
NaHCO3 12.5, MgSO4 1.2, KCl 4.75, glucose 50, and CaCl2 1.0,
at 35-+±1°C. Solutions were continuously gassed with 95%
02/5% CO2 with the pH adjusted to 7.4 with NaHCO3.
The present study describes data from a total of 30 animals
(n=30). Activation patterns were measured during right atrial
pacing and then during VT induced by a train of extrastimuli
(n=12) or by a single precisely timed stimulus (n=6), for a
subtotal of n= 18. The termination of VT by STS delivered at
various locations on the heart was measured in the same 18
hearts. The interruption of VT by procainamide was repeated
in another 6 hearts, and the effects of STS during right atrial
pacing were assessed in another 6 hearts. In most hearts, the
induction and termination of VT were repeated several times,
typically five times. However, n refers to the number of hearts
tested, not the incidence of VT induction and termination.
The numbers of successful inductions and terminations of VT
were considerably larger since the process was typically repeated four or five times with each heart.
Guinea pigs weighing >450 g were used because their hearts
were sufficiently large (circumference, >45 mm) to induce
sustained VT. With smaller hearts, self-sustained VT could
not be induced by single premature beats or trains of extraimpulses. The critical requirement that hearts have a minimum
size to induce VT strongly suggested that reentry was the
predominant mechanism and that this criterion defined the
minimum length of the reentry pathways.
Staining Procedure
Hearts were stained with voltage-sensitive dye (RH 421,
Molecular Probes, Eugene, Ore) by injecting 50 ,L of a 1
mmol/L stock solution of dye (dissolved in ethanol) into a
5-mL bubble trap placed above the aortic cannula. The dye
was administered in a series of four bolus injections (12.5 ,L
each) made over a 5- to 10-minute period, which maintained
the dye concentration in the coronary vessels at :10 ,umol/L.
Ethanol from one injection was washed out before the next
injection such that the peak ethanol concentration in the
coronary perfusate was <0.25%. Control injections of 250 ,L
ethanol in the bubble trap produced a transient increase in
ethanol concentration of up to 5% in the coronary perfusate.
Such a bolus addition of ethanol had no adverse effects on left
ventricular pressure or surface bipolar electrogram signals.
Dye delivered through the coronary perfusate produced a
deep and homogeneous staining of the myocardium, which
made it possible to record optical action potentials for 2 to 4
hours without restaining the heart. For longer experiments,
the hearts were effectively restained by following the same
procedure. Stained hearts exhibited voltage-dependent fractional changes in fluorescence of -9.2±3% with signal-tonoise ratios of -250: 1.
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Circulation Research Vol 74, No 4 April 1994
Placement of Stimulating Electrodes
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Bipolar stimulating electrodes were constructed by use of
250-,um-diameter Teflon-coated silver wires with a 50-gm
interelectrode distance and exposed tips 0.5 mm in length.
Three sets of bipolar Ag+/AgCl stimulating electrodes were
positioned on the heart: (1) For atrial pacing, an electrode was
glued to the right atrium with cyanoacrylate adhesive. (2) For
endocardial pacing, a bipolar (250-,um-diameter Tefloncoated wires with a 50-gm interelectrode distance) electrode
with a 2-mm hook was inserted transmurally across the free
wall and then pulled back to remain implanted on the endocardium. The tips of the bipolar electrode were pushed against
the endocardium without penetrating the wall to avoid direct
current injection and excitation of deeper cells of the subendocardium. When the bipolar electrode was pulled ever so
slightly into the wall, cells of the subendocardium and the
midwall were directly excited; this excitation was readily
distinguished because activation patterns that emerged on the
epicardium were dramatically altered. Endocardial pacing
most likely excited both Purkinje and adjacent myocardial
cells. However, local excitation of nearby endocardial cells had
little effect on activation sequences because of the dominant
role of Purkinje fiber-driven excitation. (3) For epicardial
pacing, an electrode was positioned at the center of the
epicardium (ie, the center of the field of view of the photodiode array). The epicardial stimulating electrode could be
readily repositioned to other locations within or outside the
field of view of the array. After placing the stimulating
electrode, hearts were mounted in a fluid-filled Plexiglas
chamber designed to measure optical maps of AP propagation
and to abate the curvature of the left ventricle and gross
movements of vigorously contracting hearts. Details of the
chamber, spatial homogeneity of temperature on the preparation, and control conditions regarding effects of the chamber
on AP characteristics were previously reported.17
Pacing Protocols
Hearts were paced at a basic drive cycle length (CL) of 300
milliseconds (=200 beats per minute) (S1), with a square-wave
pulse of 5-millisecond duration, at twice the diastolic threshold (ie, 2x capture voltage). The rate was within the physiological range for guinea pigs (200 to 300 beats per minute at
41°C). Stable sinus (sinoatrial nodal) rhythm was mimicked by
pacing the right atrium at 200 beats per minute, which
overcame the intrinsic rate, at 35°C. During endocardial or
epicardial pacing, a second electrode was placed on the
endocardium or epicardium and was paced in synchrony with
right atrial stimuli to prevent out-of-phase sinoatrial nodal
beats from capturing the ventricles and interfering with activation patterns elicited by pacing the endocardium or epicardium. Refractory periods were measured using conventional
premature beat techniques. Heart rate was controlled by
synchronous pacing (S,) of the right atrium and the ventricle.
The premature stimulus (S2) was delivered to the ventricle at
the test site once for every 10 S, pulses. Starting with long S1-S2
intervals, S1-S2 was systematically decreased in 1-millisecond
steps. S2 elicited an extra beat that propagated across the
myocardium until S2 failed to elicit a propagated response. The
refractory period was defined as the shortest S1-S2 interval at
which a propagated response to S2 occurred. Both S, and S2
stimuli were 5-millisecond square pulses at 2 x diastolic
threshold voltage.
VT was induced by either a brief train of extrastimuli or a
single premature beat. The most reliable procedure to induce
an arrhythmia consisted of pacing the right atrium at a basal
rate of S, (CL, 300 milliseconds; duration, 5 milliseconds;
voltage, 2x threshold) and then turning on a burst of extrastimuli (S2) consisting of 5-second trains (CL, 25 to 50 milliseconds; duration, 0.5 to 10 milliseconds; and voltage, 2x
threshold). S2 applied as a train of impulses was equally
effective at eliciting an arrhythmia when delivered either on
the endocardium or epicardium. To induce an arrhythmia with
a single stimulus (S2), S, was synchronously applied on the
right atrium and the left ventricle (either on the endocardium
or epicardium), and the cycle length was set at 300 milliseconds. An extrastimulus (S2) was then applied after every
eighth S, at progressively shorter (S1-S2) coupling intervals in
10-millisecond decrements until the induction of VT or the
tissue became refractory to S2 stimuli. In most cases, VT was
elicited when SI-S2 was 5'65% of the CL (ie, '-195 or z162.5
milliseconds for S, cycle lengths of 300 and 250 milliseconds,
respectively). If the tissue was refractory to S2, the S1-S2
coupling interval was increased by 10 milliseconds longer
than the refractory period and then decreased in 1-millisecond
decrements to induce VT. When the extrastimulus was delivered on the endocardium, S2 rarely failed to initiate VT (<5%
failure rate). When delivered on the epicardium, S2 failed to
induce an arrhythmia in 15% to 20% of the hearts. During
sustained VT, hearts were paced on the right atrium at Sl, and
S2 was turned off. Electrode leads similar to those used to
induce VT were used to deliver STSs at various sites on the
endocardium or epicardium. To deliver STS, the settings on
the stimulator were changed to deliver a 2- to 5-second train
of impulses with a CL of 25 to 50 milliseconds, a duration of 0.5
to 25 milliseconds, and a voltage of 0.5 x to 0.8 x threshold.
For hearts in our modified Langendorff apparatus, coronary
flow rate remained constant before and after VT, and transient changes in vascular resistance were monitored through
changes in perfusion pressure. The onset of VT occasionally
caused a transient decrease in coronary resistance as monitored by a 10 to 20 mm Hg drop in perfusion pressure from a
control of 80 mm Hg. The decrease in coronary resistance
could be due to a metabolic vasodilation1819 or ventricular
relaxation at the start of VT.
Note that the term STS has become established in the
literature and is used here for the sake of consistency. Subthreshold stimulation can be more precisely defined as the
injection of current stimuli too low to elicit a regenerative
response or AP propagation in normal resting ventricular
muscle. An alternative nomenclature might be "subcapture"
stimulation, because local current responses are elicited but
fail to elicit the spread of APs.20 Two other terms are used in
the STS literature: "low"- and "high"-frequency STS are used
to refer to stimulation rates of .100 and >100 Hz, respectively, in line with the terminology used in previous reports.
Optics
Light from a 45-W tungsten-halogen lamp (PTI, Princeton,
NJ) was collimated with a parabolic reflector and cooled with
a heat filter. The excitation beam was controlled with an
electronic shutter (Melles Griot, Irvine, Calif) to illuminate
the heart only during optical recordings. The beam was passed
through an interference filter (520+20 nm, Omega Optical,
Brattleboro, Vt), reflected by a 45°C dichroic mirror (600-nm
cutoff, Corion, Holliston, Mass), and focused on the stained
heart with an epi-illumination lens (50 mm, 1:1.8 E series,
Nikon, Garden City, NJ). Epifluorescence was collected,
passed through a 645-nm cutoff filter (Schott Glass RG 645),
and focused onto a 12x12-element photodiode array (Centronic, Newbury Park, Calif). Five diodes in each corner of the
array were ignored such that optical signals were recorded
from 124 of 144 diodes. The magnification of the image
focused on the array was kept at x 1.5 such that each diode
received light from a 1 x 1-mm area of tissue. The depth of field
of the collecting lens restricted the fluorescence measurements
to a layer of cells 144 ,um from the surface.21 The image
focused on the array was reflected by a mirror onto a custommade graticule with the exact dimensions of the array (Graticules Ltd, Tonbridge, UK) located on a plane parafocal with
the plane of the array. Precise focusing and aligning of the
heart with respect to the array were accomplished by focusing
Salama et al Subthreshold Stimulation Interrupts VT
and aligning the image of the heart on the graticule. Details
regarding the optical setup were previously reported.'7'21
Data Acquisition
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Photocurrents from each of 124 photodiodes were passed to
current-to-voltage converters, AC-coupled with a 3-second
time constant, and amplified x100; transient signal changes
were digitized with eight-bit precision and stored via direct
memory access in a DEC PDP 11-73 computer (Digital
Equipment Corp, Woburn, Mass). Signals from each diode
were sampled every 0.768 millisecond such that electrical
activity from the heart was scanned at a rate of 1302 frames
per second. A frame for the array consisted of sequential
sampling of 128 analog signals, 124 optical signals, and 4
instrument channels. The instrument channels were used to
simultaneously monitor the basal pacing impulse, atrial and
ventricular bipolar electrograms, and extrastimuli. The instrument channels were sampled and digitized in synchrony with
the 124 optical recordings. The sampling rate of the analogto-digital (A/D) converters was three times faster than the
highest frequency component of APs (ie, more than twice the
Nyquist frequency). As a result, the digitized data reproduced
the analog signals without loss of information.22 The highfrequency cutoff of the analog circuits was 1 kHz, but the
effective frequency cutoff was limited to the sampling rate of
the A/D multiplexer, set at 1.3 kHz per channel for the present
experiments. Fast analysis and display programs made it
possible to view optical APs and activation patterns within 10
seconds after data acquisition. Selected data were transferred
from RAM memory to RL02 hard disk and then to magnetic
tape for archival storage. Data acquired under the RT-11
operating system (DEC) were transferred to a computer
operating under UNIX MVME 147 CPU (Motorola, Phoenix,
Ariz) to analyze the data in greater detail and generate
Postscript files for hard-copy laser-printer displays.
Signal Analysis
Scans were taken at various times during the course of an
experiment, and the maximum duration of a scan was 34
seconds at a rate of 1302 frames per second (limited by the
RAM memory available on the DEC computer). To analyze
an activation pattern for a particular cardiac beat, six optical
APs (from the apex, middle, and base of the heart) were
displayed on a monitor, and a cursor was used to select a
window of data representing one heart beat. An algorithm
calculated the first derivative of all 124 APs for the chosen
beat. Maximum dF/dt (Vm..) was taken as the time point when
most of the cells viewed by a given photodiode depolarized.
The earliest activation event among the 124 APs in the same
beat was assigned an activation time point of t=0.00. Activation at different diodes was measured relative to the first site
to fire an AP; ie, each diode was assigned a relative activation
time point with respect to the first site(s) to fire an AP. The
activation time point of each AP was labeled with a "tick"
mark that could be verified and corrected by the operator
using an interactive program. Occasionally, a brief noise spike
on an AP caused the algorithm to miscalculate and misplace
the tick mark identifying the relative activation time point. An
interactive program made it possible to correct the error by
canceling the mark and recalculating the depolarization time
point. The algorithm was highly reliable and rarely required
correction by the investigator (.1% of activation events). The
upstroke of optical APs was not distorted by movement
artifacts from vigorously contracting hearts, because they
preceded the onset of muscle shortening by 60 to 100 milliseconds. As a result, motion artifacts did not pose a problem for
the detection of activation times and activation patterns. The
repolarization time point of each AP was defined as (d2F/
dt2)m,x of the AP downstroke, and the duration was the time
interval between activation and repolarization time points.23
607
Patterns of AP propagation were displayed as isochronal
maps based on the relative activation times of the 124 APs.
The first to last isochrones depicted the earliest to the latest
regions of tissue to activate, and the time delay between
isochrones was in units of milliseconds. Each diode was
depicted as a box in a symbolic map of the array that was filled
with the appropriate AP tracing detected by that diode and its
relative activation time.
The rise times of optical AP upstrokes were taken as the
time difference between points at 10% and 90% of the AP
amplitude.
Detailed maps of fiber orientation were previously measured as a function of depth in guinea pig right and left
ventricles.21 Our measurements of fiber orientations at different depths of guinea pig free ventricular walls were in excellent
agreement with detailed histological studies in dog hearts.24
Histological analyses of fiber orientations were also superimposed on optical maps of activation and repolarization from
the same tissue. Each heart was paced at different sites to
compare activation patterns and time delays produced by the
different stimulation protocols. Conduction velocities were
measured from the activation time delays along the epicardium, and the mean±SEM conduction velocity was calculated
for perfused hearts paced under the same stimulation protocol. Mean±SEM conduction velocities were determined and
compared from the same hearts paced at different sites.
Conduction Velocities
A completely automated analysis of propagation velocity
was developed that did not require us to identify directions of
fast and slow propagation and to select diode pairs to calculate
propagation velocities. A computer algorithm calculated the
average activation (or repolarization) velocities from the vectorial average of 124 local velocity vectors oriented between 0
and Ir radians. Average velocities along different directions
were calculated from the projection of the average vector onto
a set of 36 orientations from 00 to 1800, using an averaging
algorithm with a bin width of 5°. The maximum and minimum
scalar amplitudes of vectors oriented from 00 to 1800 represented the fastest and slowest conduction velocities, respectively, in the tissue. Computer-automated calculations of maximum and minimum conduction velocities (0.. and Oi,
respectively) (based on the 124 local velocity vectors) were
compared with manual conventional measurements of conduction velocity (based on the selection of 20 diode pairs along
directions of fast and slow propagation). The values of Omax.
and Omin reported in these studies were obtained with the
automated algorithm, since it represented a more thorough
analysis of all the AP recordings. In the case of multiple
activation sites, the automated analysis underestimated conduction velocities because the averaging routines canceled
vectors of similar magnitudes but opposite directions. Multiple
breakthrough sites occurred rarely during atrial pacing and
more often during VT. Modifications of the algorithm avoided
subtraction of vectors oriented in opposite directions, as
occurs during colliding wave fronts, and made it possible to
calculate average propagation velocities even in the presence
of multiple activation (or repolarization) breakthrough sites.
The following algorithm was used to determine distribution
of the local conduction velocity vectors. First, a gradient vector
for activation (GA) was calculated for each diode. The horizontal component of the gradient vector (i) was calculated
from the activation time delays between a diode and its two
adjacent neighbors (for boundary diodes, only one neighbor
was taken into account) in the same row of diodes divided by
the distance 2d. The vertical component (j) was calculated
from the time delays between the diode and its adjacent
neighbors on the same column divided by 2d. The distance d
represented the distance (center to center) between tissue
patches viewed by the diodes (see Fig 1; interdiode distance d
equals 1.5 mm divided by the optical magnification). In the
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Circulation Research Vol 74, No 4 April 1994
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FIG 1. The relative activation and repolarization times were
determined for each diode as described in "Materials and
Methods." For a diode located at position i,j, a local gradient
vector of activation (GA) was determined from the averaged
activation time delay (t) between that diode and its nearest
neighbors divided by the distance (d) between the two recording
sites: GA=Gx+Gy, where Gx=1/2[(ti+1,i-ti,)/dd+(ti, -ti-,,i)/
and
d]i= (t+1,j-ti ,1j)I2di,
GY= 1/2[(t,j+1 -t,j1)/d+ (t,j-t,j 1)/d]j
=(ti,j+1-ti,j 1)2dj, where i and j are the horizontal and vertical
components of the gradient vector, respectively. The local
velocity vector U was defined as a vector with the same orientation in space and magnitude that is the reciprocal of IGAI:
IU-=1/IGAI=1/(GX2+Gy2)1/2. The direction of GA is represented by
a unitary vector n-=iGx/(Gx2+Gy2) 2+jG /(Gx2+G12)1/2 For each
diode, the local conduction velocity yector (UA) was calculated
as follows: UA= UJC1 =iGx/(GX2+Gy2) +jGy/(GX2+GY2)
present experiments, distance d=1 mm for optical magnification of 1.5. The local gradient vector was the resultant of the
vertical and horizontal components: GA=GX+GY. These 124
gradient vectors point in the direction of greatest change in
activation and have units of time per unit distance of myocardium; ie, they are normal to the local propagating wave front.
Local velocity vectors (units of meters per second) were defined
as vectors with orientations and magnitudes equal to the
reciprocal absolute values of the gradient vectors (see Fig 1).
Results
Patterns of Activation Under Various
Stimulation Protocols
Fig 2 illustrates a map of 124 simultaneously recorded
APs during a single cardiac beat. The optical response
from each diode is drawn in its appropriate location in
a symbolic map of the array. Each diode is identified by
a number on the upper left corner of the box that
defines its location. In the present experiments, the
array viewed 12 x 12-mm regions of left epicardium such
that the base, apex, and anterior and posterior regions
were respectively aligned with the top, bottom, left, and
right edges of the array. The optical tracings were
analyzed to determine the first site(s) to fire an AP(s)
and were assigned an activation time point (t=0.0). All
other sites fired at later time points, and for each diode,
the relative activation time delays were recorded above
the AP tracing (in milliseconds). From the relative
activation time delays, isochronal maps were plotted to
better visualize activation patterns.
During right atrial pacing, activation began on the
anterior and spread rapidly to the posterior region of
the epicardium (Fig 3A). In physiological sinus rhythm
and in atrial pacing, the specialized conduction system
of the heart elicited the synchronous excitation of the
ventricular free walls. As a result, the first sites to
activate represented "breakthrough" sites emerging
from the endocardium to epicardium. The pattern propagated rapidly across the surface and was highly reproducible whenever Purkinje fibers drove the excitation of
the tissue. For instance, when Purkinje fibers were
directly paced with an electrode paced on the endocardium, activation patterns were identical (in orientation
and activation time) to patterns observed during sinus
rhythm or right atrial pacing (Fig 3B). Whenever Purkinje fibers triggered ventricular excitation, activation
patterns that emerged on the epicardium represented
the breakthrough of excitation from the endocardium to
the epicardium. In atrial and subendocardial pacing
(Fig 3A and 3B), the "apparent" Omax was 2.64+0.24 m/s
(n= 18), which was attributed to the rapid conduction
velocity of Purkinje fibers.21 In contrast, direct stimulation of the epicardium produced elliptical patterns with
Omax (0.84±0.15 m/s) and 0m.i (0.44±0.06 m/s) (n=6)
oriented parallel to the longitudinal and transverse axes
of epicardial fibers (Fig 3C). Conduction velocities
during epicardial pacing represented velocities intrinsic
to the myocardial tissue, in the absence of Purkinje
fiber-driven activation. To verify this interpretation, the
endocardial pacing electrode that produced rapid Purkinje fiber-dependent activation (Fig 3B) was displaced
to the subendocardium to avoid direct stimulation of the
Purkinje fibers. In subendocardial pacing, activation on
the epicardium propagated with slow velocities compared with atrial pacing, and the major isochronal axis
of the ellipse was rotated by 900 (Fig 3C). Activation
driven by the specialized conduction system was dramatically different from activation obtained by direct
electrical stimulation of the myocardium in both orientation and velocity, and these patterns were highly
reproducible from heart to heart (n = 18). Unless stimulated directly on the epicardium, conduction velocities
measured from activation time delays on the epicardium
represented apparent conduction velocities, because
activation waves could propagate transmurally, ie, in a
direction perpendicular to the epicardial surface. Depolarization times for left ventricles under atrial pacing
or sinus rhythm were 8±4 milliseconds (mean±SD,
n= 18). The activation sequence always started from the
anterior and spread to the posterior edge of the ventricles. During pacing at the center of epicardia, activation
spread anisotropically from the stimulus site, resulting
in isochronal lines that formed ellipses with major axes
oriented at 135+±3. The orientation of the major axis
was parallel to the longitudinal axis of epicardial fibers.21 During epicardial pacing at the center of left
ventricles, activation times across the ventricles were
20±5 milliseconds (mean±SD, n=18), which was considerably slower compared with activation times (8±4
milliseconds) measured during sinus rhythm. These
differences in activation time delays were compiled from
18 guinea pig hearts. For each heart, there was a
consistent and significant increase (3.0±+ 0.4 times
[mean±+SD], n=18) in activation time delay during
epicardial pacing compared with Purkinje fiber-driven
excitation of the ventricles.
Induction and Termination of Sustained VT by
Burst Stimulation
Fig 4 illustrates the induction and termination of VT
using a train of extrastimuli followed by a train of STS.
The top panel shows five tracings of continuous (34-
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FIG 3. Activation patterns recorded under different stimulation
protocols. A, The heart was paced (cycle length [CL], 300
milliseconds) with an electrode on the right atrium to control
heart rate and mimic activation patterns during physiological
sinus rhythm. B, The left endocardium was paced (CL, 300
milliseconds) in synchrony with the right atrium. Impulses from
the right atrium encountered refractory tissue because of atrioventricular nodal delay such that the activation pattern was
initiated by the stimulation of the endocardium. In panels A and
B, activation emerged at the center and anterior region of the
epicardium (solid dot) and propagated with similar orientation
and apparent conduction velocity. C, The epicardium was paced
(CL, 300 milliseconds) in synchrony with the right atrium, and the
activation pattern was initiated by the stimulus site (solid dot) on
the epicardium. Activation spreads according to the intrinsic
propagation velocity and orientation (1350) of the longitudinal
axis of epicardial fibers.21 D, The endocardial electrode in panel
B was displaced in the ventricular wall to avoid contact with
Purkinje fibers lining the endocardium. Activation was initiated
from a stimulus site on the endocardium, and the first site to
emerge on the epicardium (solid dot) was shifted slightly compared with panel B. A subendocardial site received the initial
excitation and propagated on the same layer of ventricle and
transmurally according to the fiber orientations as a function of
depth. The pattern that emerged on the epicardium (panel D)
was rotated by 700 to 900 compared with panel C because of the
transmural clockwise rotation of ventricular fibers from endocardium to epicardium. Activation began at solid dot and spread
according to isochronal line (shown 1 millisecond apart).
second) recordings of cardiac electrical activity. The top
three tracings (Nos. 15, 49, and 94) monitored optical
signals from three diodes on the array corresponding to
3 (of 124) separate sites on the heart. These diodes were
arbitrarily selected to represent APs from the base (No.
15), middle (No. 49), and apex (No. 94) of the left
epicardium. These numbers referred to our diodenumbering scheme that identified the location of the
diode on the array (as shown in Fig 2) and thus the area
of tissue monitored. The tracings labeled IC1 and 1C3
(indicating instrument channel 1 and instrument channel 3, respectively) monitored bipolar electrograms located on the left ventricle and atrium, respectively. The
heart was paced on the right atrium at a constant CL of
300 milliseconds, which mimicked stable physiological
sinus rhythm activation of the heart.
Fig 4 shows VT induced by extrastimuli (left "time
bar" between IC1 and 1C3) consisting of a 5-second
burst of stimuli (CL, 50 milliseconds; duration, 10
milliseconds; and voltage, 2x threshold) delivered on
the endocardium. To visualize APs before and during
the induction of VT, a 3-second interval of the five
tracings was displayed at a faster sweep speed (Fig 4,
lower left tracings). The first two APs in tracings 15, 49,
and 94 exhibited the expected upstroke plateau and
repolarization phases of guinea pig ventricular APs. The
last set of APs that fired under right atrial stimulation
(solid arrow, ASt) received an extrastimulus that produced a transitional premature beat (solid arrow,
TRANS) immediately after the repolarization phase of
the previous APs. The subsequent six cycles of depolarization (solid arrows, 1 to 6) depicted the onset of VT.
Each cycle of VT was analyzed to map the spread of
depolarization in greater detail in Fig 5. VT was monitored for 25 seconds and then was terminated by a
2.5-second train of STS (CL, 50 milliseconds; duration,
10 milliseconds; and voltage, 0.8 x threshold; right "time
bar") delivered on the endocardium. To visualize the
electrical events during sustained VT and its termination during STS, the tracings were displayed at a faster
sweep speed (Fig 4, lower right tracings). The solid
arrows (below channel 94, right tracings) labeled the
cycles of depolarization from 6 to 1, which occurred just
before the interruption of VT and recovery back to
atrial rhythm. Activation patterns in cycles 6 to 1 were
analyzed and displayed as isochronal maps in Fig 6.
During VT, AP upstroke velocities and amplitudes
decreased, and AP durations decreased from 112 to 175
milliseconds to 60 to 105 milliseconds (n=6). Electrical
activity recorded at all 124 sites appeared as waves of
depolarization, and the moment a region of tissue
repolarized, the next wave arrived to depolarize it with
no diastolic interval (n=6). Each activation sequence at
a particular site was associated with a set of depolarizing
waves recorded at the other 123 sites, such that the
spread of activation could be identified and mapped for
each cycle of VT. For instance, the 110th (Fig 4, solid
arrow labeled 110 under tracing 94) of 265 cycles of VT
was analyzed and displayed in Fig 6, to illustrate the
spread of activation during VT.
Fig 5 illustrates activation patterns during the induction of VT. Control activation patterns were measured
during right atrial pacing (Fig SA), the first transitional
beat at the onset of a train of extrastimulation (Fig SB),
and the first to the sixth beat of VT (Fig SC through
5H). These isochronal maps record the sequence of
consecutive cardiac cycles labeled in Fig 4 by solid
arrows (bottom left). The last normal atrial beat (Fig
SA) before VT exhibited the standard activation pattern measured when the specialized conduction system
(ie, Purkinje fibers) drives the excitation of the ventricles, as in Fig 3A and 3B. The transitional beat propagated more slowly and at a different orientation compared with the last atrial pacing beat (Fig SB). After the
induction of VT, heart rate increased from 200 to ;600
beats per minute, and the apparent Omax decreased by
two thirds compared with normal sinus rhythm. The
decrease in apparent conduction velocity presumably
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A. Atrial Stimulation
C. VT beat #1
E. VT beat #3
G. VT beat #5
B. Transitional beat
D. VT beat #2
F. VT beat #4
H. VT beat #6
FIG 5. Activation patterns during the induction of ventricular tachycardia (VT). lsochronal maps were derived from the consecutive
of depolarization that occurred during right atrial rhythm, just before VT, during a transitional cycle of depolarization, and for six
cycles of depolarization after the induction of VT (arrows labeled 1 through 6, bottom left in Fig 4). A, Activation pattern of the last normal
beat was typical for atrial pacing. Consistent with Fig 3A and 3B, activation emerged on the anterior segment of left epicardium (solid
dot) and propagated toward the posterior region. B, Activation pattern of the transitional beat is shown. Activation emerged on the
anterior region (solid dot), as in panel A, and propagated at slower apparent velocity and at an orientation similar to subendocardium
pacing shown in Fig 3D. C through H, During VT, apparent conduction velocities decreased by -60% (from -2.6 to -0.8 m/s), rate
increased from 200 to 600 beats per minute, and wave fronts aligned with the longitudinal axis of fibers of the subendocardium (B and
F), midwall (G), or epicardium (C, D, E, and H).21 Activation began at the solid dot and spread according to isochronal line (shown 1
millisecond apart).
waves
decreased the synchrony of activation since during VT
the same region of ventricle depolarized at 30 milliseconds (Figs 5G and 6C) compared with 4 to 5 milliseconds during atrial pacing (Figs 3A and 5A). Activation
patterns (Fig 5C through 5H) had similar apparent
propagation velocities and orientations compared with
those measured during direct electrical pacing of the
ventricle, with Omax of 0.84±0.06 m/s. The major isochronal axis of the transitional (Fig 5B) and the fourth
beat (Fig 5F) were oriented at a 225°/45° angle, which
suggested that activation was initiated deep in the
subendocardium, as in Fig 3D. The major isochronal
axis of beats 1 (Fig 5C), 2 (Fig SD), 3 (Fig 5E), and 6
(Fig SH) were oriented at 135°/3150, which indicated
that the epicardial fiber orientation2' guided the propagation of these wave fronts, as in Fig 3C. The bipolar
electrograms indicated that extrastimuli induced a polymorphic VT (Fig 4, tracing ICI) while retaining a stable
atrial rhythm (Fig 4, tracing 1C3).
The termination of VT was reliably achieved by STS
delivered to the Purkinje fibers on the surface of the
endocardium (Fig 4, right tracings). Fig 6 described the
isochronal maps for beat 110 in the middle of VT, six
beats preceding the termination of VT, and the recovery to atrial pacing. The 110th beat depicted a pattern
with two activation wave fronts that propagated and
collided at the center of the ventricle (Fig 6A). The
third beat before atrial rhythm revealed another example of two activation breakthrough sites. The spread of
activation during the interruption of VT was consistently slower than during atrial rhythm (Fig 6B through
6C), and the first normal beat exhibited the rapid
activation and orientation of activation pattern observed during Purkinje fiber-driven excitation of the
ventricles (Fig 6H).
Sustained VT could be induced by 5-second trains of
burst stimulation (CL, 25 to 50 milliseconds; duration,
0.5 to 10 milliseconds; and voltage, 2x threshold)
applied to the endocardium or epicardium or by a single
properly timed premature beat (duration, 5 milliseconds; voltage 2x threshold) as illustrated in Fig 10.
However, sustained VT could only be induced in hearts
isolated from guinea pigs weighing 2450 g, most likely
because the larger physical dimensions of their hearts
were required to maintain reentrant pathways (n=36).
The hearts of 450-g guinea pigs weighed, on average,
2.5 +0.15 g wet weight (n= 12), with a circumference of
44.5+2.8 mm during diastole. VT could not be induced
by extrastimulus protocols in hearts with smaller dimensions (n= 12).
VT could be terminated by STS (CL, 25 to 50
milliseconds; duration, 0.5 to 25 milliseconds; and voltage, 0.5 x to 0.8 x threshold) applied at various sites on
the endocardium, but not on the epicardium. To illustrate the latter result, VT was induced by a train of
extraimpulses applied on the endocardium as in Fig 4
(not shown) and was monitored for 45 seconds; then
STS was delivered to the epicardium. As shown in Fig 7,
Salama et al Subthreshold Stimulation Interrupts VT
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FIG 6. Activation patterns during termination of ventricular tachycardia (VT). lsochronal activation maps were derived from transmembrane depolarizations for the 1 1 0th cycle of VT (A), consecutive cycles of VT during the delivery of subthreshold stimulation (B through
F), and the recovery to atrial rhythm (H) (arrows, bottom right, Fig 4). The 1 1 0th beat of VT (A) and the third beat before termination (E)
each show two early activation sites that resulted in wave-front collisions. The major axis of the wave fronts of most beats (A through C
and E through G) aligned with the longitudinal fiber axis of the epicardium. Termination of VT resulted in the immediate recovery of the
atrial pacing activation pattern (H). Activation began at the solid dot and spread according to isochronal line (shown 1 millisecond apart).
four optical recordings of electrical activity (Nos. 19, 21,
43, and 47) and a ventricular bipolar electrogram were
not significantly altered by the firing of STS delivered to
the epicardium (bottom tracing). In this case, the electrode delivering STS was located at diode 45 between
diode tracings 43 and 47, shown in Fig 7. Fig 7a depicts
an activation pattern recorded during VT, and Fig 7b
depicts a pattern recorded during VT plus STS applied
at diode 45 (square pulse). The location of the STS
electrode was changed to various sites on the epicardium; however, STSs at all epicardial sites were equally
ineffective at interrupting VT. More precisely, STS
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FIG 7. Subthreshold stimulation
(STS) delivered to the epicardium
failed to interrupt ventricular tachycardia (VT). VT was induced in a
guinea pig heart as described for Fig
4, and attempts were made to terminate VT by STS delivered to the epicardium. Recordings from arbitrarily
chosen diodes (Nos. 19, 21, 43, and
47) and a recording of a ventricular
bipolar electrogram (BE) detected
multiple cycles of VT. The logic
pulses denoting the delivery of STS
on the epicardium (bottom tracing)
indicated the onset of STS (50 milliseconds between pulses). a, The activation pattern measured during a
cycle of VT (arrow a on tracing 43)
propagated according to the epicardial fiber axis. b, The activation pattern measured during a cycle of VT
plus STS shows a slight disturbance
of the pattern near the STS electrode
site (square pulse) but did not interrupt VT, even when the stimulation
voltage was increased to 6x threshold. Activation began at diode No.
102 (*) and spread according to
isochronal line (shown 1 millisecond
apart).
614
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FIG 9. Effects of subthreshold stimulation (STS) on the epicardium during atrial pacing. Bipolar pacing leads were attached to
the right atrium and epicardium. A, Activation pattern measured
during atrial pacing had the expected apparent conduction
velocity and orientation. B, Simultaneous application of atrial
pacing and endocardial STS (at square-pulse symbol at diode
102) caused a local increase in apparent velocity. This resulted
in an increase in the number of photodiodes detecting earlier
activation times not seen with atrial stimulation alone. On cessation of STS, apparent velocity and the activation pattern
reverted immediately back to the control value as in panel A.
Numbers indicate activation time delays in milliseconds.
failed to interrupt VT when the STS electrode was
located at various positions on the apex, base, and
anterior and posterior regions, as well as the center of
the left and right epicardia. Six hearts were thus tested
with STS electrodes located at a minimum of 24 sites on
the epicardium.
STS Increases Local Apparent Conduction Velocity
To elucidate the mechanism whereby STS interrupts
VTs, STS was applied on the endocardium under right
atrial pacing and during VT to examine the spatial
heterogeneities of electrical responses induced by STS.
In Fig 8, VT was induced by a single properly timed
stimulus (not shown), and STS delivered on the Purkinje fibers lining the endocardium interrupted VT. Fig
8 compares the local effects of STS on depolarizing
waves on the epicardium at sites close and farther from
the stimulating electrodes. The STS electrode was
placed on the endocardium at a site opposite the
epicardium viewed by diode 69. Consequently, the optical tracings recorded by diodes 57, 69, and 81 corresponded to electrical activity near the STS site; those
recorded by diodes 15, 25, and 49 were distant to the site
of STS delivery. From the position of each diode on the
array seen in Fig 2, the magnification of the image
viewed by the array (ie, each diode viewed a lx 1-mm
area of tissue), and the thickness of the left ventricle
(1.8 mm), the activities recorded by diodes 69, 57, 81, 49,
25, and 15 were respectively 1.8, 2.04, 2.04, 4.82, 5.9, and
6.7 mm away from the STS electrode. As shown in Fig 8,
STS interrupted VT; however, this did not occur instantaneously but gradually as the electrical activity near the
site of STS exhibited a gradual increase in rate of rise
and amplitude of depolarizations. This implied that STS
increased the synchrony of depolarization and the apparent conduction velocity at local sites near the STS
electrode. Sites more distant from the STS electrode
were not altered by STS pulses, until the interruption of
VT. Approximately 15±3 diode recordings nearest the
location of the STS site exhibited increases in rate of
615
rise and amplitude (n=6). IC1 on Fig 8 recorded a
polymorphic ventricular electrogram during VT, which
abruptly changed to a standard right atrial rhythm
electrogram waveform with a concordant T wave (lower
right of bottom tracing).
The effects of STS delivered to the endocardium
during normal right atrial pacing rhythm are illustrated
in Fig 9. Two bipolar pacing leads were attached to the
heart, one on the right atrium (to mimic sinus rhythm)
and the other on the endocardium (square-wave symbol). In panel A, the right atrium was paced at a
300-millisecond CL, resulting in the expected epicardial
activation patterns (control) recorded during right atrial
pacing with a rapid apparent conduction velocity of
2.64+0.14 m/s (n=6). In panel B, the electrode placed
on the endocardium was turned on to deliver STS
(voltage, 0.5 x threshold; 20 Hz; and duration, 5 milliseconds) during right atrial pacing (as in panel A). This
resulted in a larger region of epicardium that fired
synchronous APs; ie, a greater number of photodiodes
detected APs that fired within the region of epicardium
delineated by the 0- and 1-millisecond isochrones compared with the initial sites of activation measured during
sinoatrial nodal stimulation alone (panel A). This demonstrated that STS induced a regional decrease in activation delays in a 4x4-mm patch of tissue, causing a local
increase in apparent conduction velocity (3.63±0.21 m/s,
n=6). On cessation of simultaneous STS, conduction
velocity and activation patterns immediately reverted back
to those seen with atrial stimulation alone (panel A). The
instantaneous change of activation patterns from those
shown in panels A and B on STS and the reversal from
panel A to B on cessation of STS suggested that these
changes were not caused by local release of catecholamine
at the site where STS was applied.
Termination of VT by Procainamide
In contrast to the local increase in apparent conduction velocity by STS, VT could also be interrupted by
slowing down AP upstrokes and propagation using
procainamide. The induction of VT with a single premature beat and its termination with a bolus administration of procainamide are illustrated in Fig 10. The
experimental protocol is similar to that shown in Fig 4,
except that VT was induced by a single properly timed
premature beat delivered during the relative refractory
period of APs on the epicardium (Fig 10, bottom
tracings, left). The firing of the extrastimulus is indicated by the solid arrow below 1C3 (Fig 10, bottom, left
tracing), which monitored the stimulator output. As in
Fig 4, the arrhythmia induced by a premature beat
produced a polymorphic ventricular electrogram (IC1),
whereas the atrial electrogram remained unaltered
(IC2). This indicated that the electrical perturbation
produced a VT and that the arrhythmia was not of
supraventricular origin. Procainamide (2 ,ug/mL of perfusate) was injected at the cannula inlet in a bolus of
perfusate (5 mL) at the time indicated by the arrow (Fig
10, bottom, right). VT was interrupted within 3 seconds
after the delivery of procainamide.
The termination of VT by procainamide within 3.0
seconds (Fig 10, bottom tracings, right) was remarkably
fast, since the time required for the drug to travel from
the injection site to the coronary circulation was --2.0 to
2.5 seconds. As a sodium channel antagonist, procain-
616
Circulation Research Vol 74, No 4 April 1994
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FIG 1 1. Procainamide decreases apparent conduction velocity
during atrial stimulation. The heart was paced synchronously on
the right atrium and the base of the left epicardium to measure
action potential propagation and activation patterns of epicardial
fibers. A, Epicardial excitation was initiated at the location of the
electrode pacing the tissue (solid dot) and spread from base to
apex as depicted by the isochronal lines of propagation. The
directions of the longitudinal and transverse conduction velocities were tangential and perpendicular to the isochronal lines,
respectively. B, After a 3-minute perfusion of the heart with
perfusate containing 10 ,umol/L procainamide, activation spread
at considerably slower velocities, :'2.3 times slower than control
conduction velocities. Numbers indicate activation time delays in
milliseconds.
amide produced the expected decreases in the rate of
rise of AP upstrokes and also decreased the amplitude
of optical APs. The decrease in AP amplitude occurred
because each photodiode recorded the sum of APs from
cells in a patch (1 x 1 x0.144 mm) of tissue, and procainamide decreased propagation and the synchrony of AP
firing (n=6).
When procainamide (10 ,umol/L) was administered to
hearts during epicardial pacing (S,, 300 milliseconds),
the refractory period on the epicardium did not change
significantly, increasing slightly from 88±1.0 to 92±2.0
milliseconds (n=6). On the other hand, the transverse
conduction velocity decreased by more than 50% from
0.57±0.15 to 0.25±0.08 m/s (Fig 11). These changes in
refractoriness and conduction velocity were reversed by
washing out the drug (not shown).
In the present experiments, VT induced by extraimpulses were self-sustained, continued for up to an hour,
and typically progressed to VF, unless interrupted by
STS or procainamide (n=6). These preparations could
remain normoxic and survived long periods of VT and
VF, because in Langendorff-perfused hearts, coronary
perfusion is artificially maintained by the peristaltic
pump and the perfusion pressure, which deliver perfusate to the coronary vessels. In the more physiological in
situ studies of VT, recoil of blood from the left ventricular ejection fraction provides the driving force for
coronary perfusion. Thus, in studies of arrhythmias in
situ, VT was shown to decrease, and VF stopped
cardiac output and coronary perfusion.25
Discussion
The main results are that arrhythmias (most likely
VT) can be reliably induced in guinea pig hearts by
premature extraimpulse(s) and terminated by STS applied on the endocardium but not on the epicardium.
The mapping of APs with voltage-sensitive dyes and
imaging techniques provided measurements of activation patterns from the epicardial surface with high
617
spatial and temporal resolution. Activation patterns
measured from two-dimensional surfaces on the epicardium were strongly dependent on the stimulation protocol. Control activation maps measured under atrial
stimulation were anisotropic with the orientation and
apparent propagation velocity typical of activation observed after the direct stimulation of Purkinje fibers
lining the surface of the endocardium. In right atrial
pacing, epicardial activation patterns were anisotropic
(ie, elliptical), and the major and minor axes (ie,
longitudinal and transverse conduction velocities) were
aligned according to the endocardial fibers, which receive synchronous excitation from Purkinje fibers.21 In
contrast, direct stimulation of the epicardium produced
anisotropic activation patterns with considerably slower
values for Omax and Omin, which were oriented parallel to
the longitudinal and transverse axes epicardial fibers,
respectively. During VT, activation patterns had orientations and propagation velocities similar to those measured during stimulation of the epicardium or intermediate layers within the ventricular free wall. A most
critical observation was that, during VT, activation
patterns indicated that Purkinje fibers failed to drive the
excitation of the ventricular free wall. Furthermore,
activation patterns during VT were most likely caused
by a reentry mechanism, since a minimum dimension of
the heart was critical to establish sustained arrhythmias.
The present study demonstrates that extrastimuli reliably induce VT in a nonhypoxic nonischemic mammalian heart and that STS delivered on the endocardium
interrupts VT, whereas STS applied elsewhere failed to
terminate VT.
Leading Circle VT in the Nonischemic
Nonhypoxic Heart
The present data raise an important question regarding the nature of the arrhythmias initiated by premature
extrastimuli in nonhypoxic nonischemic guinea pig
hearts: did premature impulses induce a VT, a VF, or a
VT that progressed to VF? This question is not easily
resolved, because there are no unique methods to
distinguish VT from VF by standard electrogram recordings.26'27 An essential feature of VT is the induction
of one primary depolarizing wave front that propagates
repeatedly along a reentrant pathway. In one form of
VT (ie, circus movement), slow conduction around a
ring of tissue and an anatomic barrier are required to
maintain a single reentrant loop.13 An important feature
of circus-movement VT is that activation wave fronts
propagate repeatedly along the same path, resulting in
monomorphic electrogram signals. The concept of circus movement had prevailed as the dominant form of
VT until the demonstration of leading-circle or leadingedge reentry.16 Leading-circle reentry (single-wave
front VT) was characterized by the absence of an
excitable gap, because the moment a region of tissue
would repolarize, it would depolarize. Another important feature of leading-circle reentry is the lack of
anatomic blocks such that VT could be induced in
healthy (nonischemic) myocardium.'6 In contrast, VF
has been defined as multiple random reentrant wave
fronts with so many propagating wave fronts that the
mutual extinction of these wavelets becomes improbable.28 However, there were no estimates of the number
of simultaneous wavelets necessary to achieve VF.
618
Circulation Research Vol 74, No 4 April 1994
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In the present studies, the polymorphic ventricular
electrogram responses would be interpreted as VF.
However, the optical activation maps indicated the
occurrence of VT rather than VF, because each cycle of
depolarization produced a finite number (one to three)
of activation wave fronts that propagated across the left
epicardium. The activation map for each cycle of arrhythmia exhibited a different pattern of depolarization
but did not reveal a large number of simultaneous
propagating waves as postulated by Moe et al.28 Furthermore, these arrhythmias qualify as VT rather than
VF because precise activation patterns and propagation
velocities were measured for any given cycle of the
arrhythmia. Activation exhibited systematic time delays
from the first to last region to depolarize, which indicated that the first region to depolarize initiated a wave
of depolarization that propagated to the surrounding
tissue. Activation wave fronts propagated anisotropically with apparent Omax and Omin values similar to those
observed by direct stimulation of the subendocardium
(Fig 5B and 5F, Fig 6D), midwall (Fig 5G), or epicardium (Fig SC, SD, 5E, and 5H; Fig 6A, 6B, 6C, 6F, and
6G).21 Activation maps during VT indicated that the
Purkinje fibers failed to synchronize the ventricular
depolarization because of the longer activation time
delays across the epicardium and the shift in the orientation of isochronal lines. The orientation of activation
patterns varied from cycle to cycle, but the predominant
orientation indicated that activation was guided by the
epicardial fiber structure.
Optical APs indicated that the moment ventricular
cells repolarized, they depolarized with no detectable
excitable gap between waves of depolarization. Thus,
cells in the process of repolarization are reexcited near
their relative refractory period by the next wave of
excitation and depolarize almost immediately before
establishing a resting potential. This class of VT resulted in polymorphic electrogram recordings, because
activation pathways changed from beat to beat and on
occasion multiple wave fronts propagated simultaneously and collided. In contrast, VT produced by reentry
around an anatomic block (ie, circus movement) would
produce monomorphic recordings, because the singleactivation wave front travels along a fixed path, is
reproducible from beat to beat, and exhibits an excitable gap between cycles of depolarization. Put together,
the best interpretation is that these VTs fell into the
category of leading-circle VT.
The major limitation of the present study and of the
interpretation of the type of arrhythmia induced by a
premature beat is that the apparatus did not record
activity from other regions of the heart. Nevertheless,
activation maps from the left epicardium included a
significant part (40%) of the total epicardial surface and
were most likely representative of activation patterns
occurring elsewhere on the heart. Thus, it is unlikely
that more complex (ie, multiple, random) activation
wave fronts occurring on the right epicardium would not
influence measurements on the left epicardium over the
span of several hundred cycles of arrhythmia. Sustained
VTs were only obtained in hearts with minimum dimensions, which also indicated that a reentrant mechanism
was responsible for these rhythm disturbances and that
a minimum reentry path length was required to sustain
VT. Thus, premature stimuli delivered to normal guinea
pig hearts induced a form of reentrant arrhythmia, most
likely polymorphic leading-circle VT.
Termination of VT
STS delivered to the endocardium of guinea pig
hearts interrupted VT within 3 seconds but failed to
significantly alter VT when delivered to the epicardium.
By changing the location of the electrode delivering STS
to the endocardium, it appeared that STS delivered to
the Purkinje fibers lining the endocardium was required
to interrupt VT. STS frequencies previously reported to
be effective at interrupting VT were selected in the
present study (CL, 25 to 50 milliseconds). Different STS
frequencies were tested on the same hearts to interrupt
VTs. In this range of frequencies, there were no frequency-dependent effects in the likelihood of interrupting VT with STS. The actions of STS were also consistent with the generation of a local electrotonic
depolarization, which failed to capture and propagate
but enhanced the local conduction velocity. As shown in
Fig 8, there was an abrupt increase in the rate of rise
and amplitude of the upstrokes of APs recorded near
the STS site (Nos. 57, 69, and 81). In Fig 9, the
simultaneous application of STS during atrial pacing
(panel B) caused an increase in the number of photodiodes detecting earlier activation times that were not
detected with atrial stimulation alone (panel A). Alternatively, STS may elicit the release of catecholamine
from local neurons, which in turn increased the local
conduction velocity.29 Activation maps recorded during
atrial pacing (Fig 9A) were immediately modified by
turning on the endocardial STS (Fig 9B) and reversed
back to atrial activation patterns (as in Fig 9A) within
one beat (CL, 300 milliseconds), a time course too fast
to be attributed to catecholamine release. The latency
of onset of a catecholamine response after a burst of
sympathetic stimulation is '-1.0 to 1.5 seconds, whereas
the response lasts :20 seconds.3031 Even if catecholamine release is involved in the termination of VT by
STS, it is not a sufficient criterion because STS delivered to the epicardium should also elicit catecholamine
release yet fail to interrupt VT. Shenasa et al0 suggested that STS is only effective on the endocardium
because the endocardium is the site responsible for the
initiation of reentrant VTs. Although this hypothesis
cannot be entirely excluded, the patterns of activation
during VT indicate that the initiation site varies from
beat to beat and in many cases appears to originate at
the left epicardium.
Acknowledgments
This study
supported by American Heart Association
(AHA) grant 87-1065, the Western Pennsylvania Affiliate, Inc,
of the AHA, and the Whitaker Research Foundation (Dr
Salama). The authors would like to thank Dr Mohammed
Shenasa for suggesting these experiments, Richard A. Lombardi and William Bolish for the development of software to
analyze activation patterns, and William Hughes for machining
the experimental chamber and optical components.
was
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G Salama, A Kanai and I R Efimov
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Circ Res. 1994;74:604-619
doi: 10.1161/01.RES.74.4.604
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