Download Optical mapping of atrioventricular node reveals

Optical mapping of atrioventricular node reveals
a conduction barrier between atrial and nodal cells
BUM-RAK CHOI AND GUY SALAMA
Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh,
Pittsburgh, Pennsylvania 15261
rabbit atrioventricular node; compact node; atrioventricular
nodal delay; decremental conduction; atrioventricular node
conduction; photodiode array; voltage-sensitive dyes;
4-[b-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium
(AV) node was first described by
Tawara (25) in 1906 as the only electrical connection
between the atria and the ventricles in mammalian
hearts. The node is located between the interatrial
septum (IAS) and interventricular septum (IVS) in a
region called the triangle of Koch. It is composed of a
spindle-shaped compact network of small cells that was
found to be essentially the same in various species. The
main function of the AV node is to delay depolarization
(e.g., activation) between the atria and the ventricles
and thereby coordinate their contraction. Histological
analysis of the AV node indicated that the node consists
of five morphologically distinct cell types: 1) transitional cells commingled with 2) atrial cells; 3) midnodal
THE ATRIOVENTRICULAR
cells, 4) lower nodal cells, and 5) cells of the penetrating
AV bundle embedded within the central fibrous body
(17). Transitional cells are distinguished from atrial
cells by their smaller size, pale staining reaction, and
extensive connective tissue. Midnodal cells are closely
packed, have little intervening connective tissue, and
form the ‘‘compact’’ node. Lower nodal cells are elongated, smaller than atrial cells, and form a bundle
parallel to the AV ring.
The AV node of the rabbit heart (,1.5 mm in length)
has been extensively studied to characterize action
potentials (APs) and activation delays in various regions of the AV node. AV nodal cells were divided into
three zones on the basis of their electrophysiology (18):
atrionodal (AN), nodal (N), and nodal-His (NH) cells.
The N zone is an area of slow conduction and slow AP
upstrokes, the AN zone is a transitional region between
fast-conducting atrial muscle and the N zone, and the
NH zone is a transitional zone between the N zone and
the His bundle. This classification was not strict and
was further extended on the basis of the AP response
following a premature atrial stimulation (3). AN cells
were further subdivided into AN and ANCO cells
because, at fast pacing rates, APs of ANCO cells
exhibited two components, or a notch, on the AP
upstroke. Premature stimulation and pacing at faster
rates also served to distinguish N from NH cells. To
correlate a particular AP response to the morphology of
the cell, APs were recorded with microelectrodes filled
with potassium ferricyanide (24) or cobalt-containing
KCl (1) to selectively stain cells that fired a particular
type of AP. Such studies suggested that AN potentials
emanate from transitional cells and NH potentials
emanate from the lower nodal cells. It should be noted
that the diffusion of the stain to neighboring cells made
it difficult to demonstrate unequivocally that N-cell
APs originate from anatomically defined midnodal cells
(1).
Activation delays across the AV node were measured
with intracellular microelectrodes (3, 4); however, a
detailed spread of activation within the midnodal and
lower nodal zone could not be determined because
markedly different AP characteristics and activation
times could be measured at any location (but at unknown depths), with some cells activating early and
others late in the same region. As a result, it was not
possible to detect a wave of depolarization within the
node and thereby measure a conduction velocity in the
AV node.
Hoffman and Cranefield (9) introduced the concept of
decremental conduction to explain propagation delays
at the AV node. AV node cells and, in particular, N cells
have high intracellular resistance and reduced intercel-
0363-6135/98 $5.00 Copyright r 1998 the American Physiological Society
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Choi, Bum-Rak, and Guy Salama. Optical mapping of
atrioventricular node reveals a conduction barrier between
atrial and nodal cells. Am. J. Physiol. 274 (Heart Circ.
Physiol. 43): H829–H845, 1998.—The mechanisms responsible for atrioventricular (AV) delay remain unclear, in part
due to the inability to map electrical activity by conventional
microelectrode techniques. In this study, voltage-sensitive
dyes and imaging techniques were refined to detect action
potentials (APs) from the small cells comprising the AV node
and to map activation from the ‘‘compact’’ node. Optical APs
(124) were recorded from 5 3 5 mm (,0.5-mm depth) AV
zones of perfused rabbit hearts stained with a voltagesensitive dye. Signals from the node exhibited a set of three
spikes; the first and third (peaks I and III) were coincident
with atrial (A) and ventricular (V) electrograms, respectively.
The second spike (peak II) represented the firing of midnodal
(N) and/or lower nodal (NH) cell APs as indicated by their
small amplitude, propagation pattern, location determined
from superimposition of activation maps and histological
sections of the node region, dependence on depth of focus, and
insensitivity to tetrodotoxin (TTX). AV delays consisted of t1
(49.5 6 6.59 ms, 300-ms cycle length), the interval between
peaks I and II (perhaps AN to N cells), and t2 (57.57 6 5.15
ms), the interval between peaks II and III (N to V cells). The
conductance time across the node was 10.33 6 3.21 ms,
indicating an apparent conduction velocity (QN ) of 0.162 6
0.02 m/s (n 5 9) that was insensitive to TTX. In contrast, t1
correlated with changes in AV node delays (measured with
surface electrodes) caused by changes in heart rate or perfusion with acetylcholine. The data provide the first maps of
activation across the AV node and demonstrate that QN is
faster than previously presumed. These findings are inconsistent with theories of decremental conduction and prove the
existence of a conduction barrier between the atrium and the
AV node that is an important determinant of AV node delay.
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A CONDUCTION BARRIER REGULATES AV DELAY
MATERIALS AND METHODS
Experimental protocol. This study describes data from nine
experimental groups of rabbit AV node preparations for a
total of 44 hearts. In group 1 (n 5 3) hearts, the atrial-His
bundle (AH) interval and AV delays were measured before
and after the hearts were stained with the voltage-sensitive
dye to examine possible pharmacological effects of the dye. In
group 2 (n 5 4) hearts, APs were simultaneously recorded
with microelectrodes and optical mapping techniques to help
identify the origin of peak II (see Isochronal maps and movies
of activation). In group 3 (n 5 9) hearts, activation across the
IAS and the crista terminalis, conduction velocity across the
compact node, and AV delay were measured. In group 4 (n 5
4) hearts, AP signals were analyzed as a function of depth of
focus. In group 5 (n 5 4) hearts, optical recordings of APs were
carried out, and then the tissue was labeled with fiducial
marks to superimpose activation maps on histological sections of the tissue. In group 6 (n 5 3) hearts, AV node
preparations were used to test the effects of tetrodotoxin
(TTX). In group 7 (n 5 3) hearts, the effects of cutting the His
bundle were investigated, and in group 8 (n 5 5) hearts, the
effects of exposure to acetylcholine (ACh) were tested. In
groups 1–8, the hearts were allowed to beat at their intrinsic
rates, which were controlled by the sinoatrial (SA) node or
primary pacemaker; a crush of the SA node interrupted the
normal heart rate and, after a few minutes, the AV node
became the primary pacemaker. In group 9 (n 5 9) hearts, the
SA node was intentionally dissected and the heart was paced
on the right atrium (1–2 mm below the SA node) to measure
changes in AV delay and optical APs as a function of heart
rate. Alternatively, the SA node was dissected to measure
activation patterns with the AV node as the primary pacemaker. This investigation conformed with the Guide for the
Care and Use of Laboratory Animals published by the National Institutes of Health [DHHS Publication No. (NIH)
85–23, Revised 1985, Office of Science and Health Reports,
Bethesda, MD 20892].
AV node preparation. New Zealand White rabbits (2.0–3.0
kg) were anesthetized with pentobarbital sodium (Nembutal;
35 mg/kg) by intravenous injection in an ear vein. The chest
was opened, and heparin (200 U) was injected in the inferior
vena cava. After a few minutes, the heart was removed and
perfused through the aorta in a modified Langendorff perfusion. The perfusate contained (in mM) 130 NaCl, 12.5
NaHCO3, 1.2 MgSO4, 4.75 KCl, 1.0 CaCl2, and 20 dextrose.
Solutions were continuously gassed with 95% O2-5% CO2. The
pH was adjusted to 7.4 with NaHCO3. Input of perfusate at
the aorta was controlled with a peristaltic pump (Minipuls 2,
Gilson, Middleton, WI) and was connected to a graduated
manometer to obtain a physiological mean aortic pressure of
80 mmHg. Aortic pressure was monitored with a manometer
and/or a pressure transducer (P10, Statham, Waltham, MA).
The flow rate of the pump determined the flow of perfusate
delivered to the coronary vessels and was adjusted to 7
ml · min21 · g wet wt21 at the beginning of each experiment
and kept constant thereafter. A perfusion system with ‘‘constant coronary flow rate’’ instead of the more typical ‘‘constant
aortic pressure’’ ensured that the flow of perfusate through
the myocardium remained constant and homogeneous during
changes of the contractile state and/or coronary resistance.
The perfusate was not recycled through the heart, and only
preparations with stable aortic pressures were selected for
the study; initial pressures were in the range of 80–100
cmH2O, and final pressures at the end of the experiments
changed by #5% of initial pressure. The free walls of the right
ventricle and atrium were dissected open to expose the IAS
and IVS and were pinned down on a Sylgard-coated horizontal chamber. The chamber was water-jacketed to control
temperature, which was continuously monitored with a thermistor placed near the optical field of view. A heating coil in the
chamber was used to continuously adjust the temperature of
the bath via a feedback system. Surface electrograms were
recorded with bipolar electrodes (Teflon-coated platinum wires,
250 µm and 1 mm apart) placed at key sites on the preparation: 1) near the SA node, 2) on the IAS near the AV ring, 3)
close to the His bundle, and/or 4) on the IVS.
Staining procedure. The heart was stained with the voltagesensitive dye 4-[b-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium (di-4-ANEPPS; Molecular Probes, Eugene, OR) by
gradual injection of 200 µl of a stock solution of dye [2 mM in
dimethyl sulfoxide (DMSO)] into the bubble trap over a
period of 5–10 min.
Other voltage-sensitive dyes, RH-421, di-8-ANEPPQ, and
di-12-ANEPPQ (Molecular Probes, Eugene, OR), were also
tested in attempts to obtain the highest possible signal-tonoise ratio for APs from the AV node. All four dyes were tested
by preparing stock solutions in DMSO or 1:1 mixtures of
DMSO and Pluronic acid. Each stock solution of dye was
tested by either injecting the dye in the coronary perfusate or
adding dye to the bathing solution (1–2 mM) for 30–45 min.
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lular coupling compared with atrial and ventricular
cells. The high coupling resistance could explain the
basic conduction delay of the AV node. Such decremental conduction would also predict a gradual delay
across the N zone so that delay is distributed across the
cell network. However, Billette (3) demonstrated that
the conduction delay is not decremental in space following a premature stimulus but seems more localized in
N cells, where conduction stagnates. Studies from
several investigators (1, 3, 4, 8) have led to the realization that slow conduction in the AV node cannot be
solely explained by active properties of N cells, such as
the maximum rate of rise of the AP upstroke (dV/dtmax ),
and that both passive and active properties are responsible for the inhomogeneous potential spread in the N
zone.
In the present report, we applied voltage-sensitive
dyes and optical imaging techniques to map electrical
activity across the AV node in attempts to elucidate the
mechanisms responsible for the AV node delay. Voltagesensitive dyes have been extensively used by various
investigators (7, 20, 22, 23) to measure optical APs in a
variety of cardiac muscle preparations. Simultaneous
recordings of transmembrane potential by optical and
microelectrode techniques have validated the high fidelity of optical APs compared with microelectrode recordings and demonstrated that optical APs detected the
classic features of atrial, pacemaker, and ventricular
APs (14, 23). Optical techniques also face important
limitations because the absolute value of membrane
resting potential cannot be obtained unless calibrated
with a microelectrode, the downstroke of the AP can be
distorted by movement artifacts, and the optical AP
represents the sum of APs from cells within a region of
tissue and not the AP of a single cell. Despite these
limitations, the technique offers important advantages
in mapping the inputs to the node, detecting the
activation sequence in the compact node, and identifying zone(s) of conduction delay.
A CONDUCTION BARRIER REGULATES AV DELAY
Fig. 1. Bipolar surface electrodes were used to measure atrial-His
(AH) intervals before (A), during, and after (B) perfusion with
4-[b-[2-di-n-butylamino)-6-naphthyl]vinyl]pyridinium (di-4-ANEPPS).
One bipolar electrode was placed on the interatrial septum (IAS)
halfway between the sinoatrial (SA) and atrioventricular (AV) nodes
to detect atrial depolarization [atrial bipolar electrogram (BE)]. A 2nd
electrode was placed at junction of tendon of Todaro (TT) and crista
terminalis (CT) to detect His bundle and ventricular depolarizations
(His BE).
from two 100-W tungsten-halogen lamps was collimated,
passed through a 520 6 20-nm interference filter, and focused
on the triangle of Koch. Fluorescence emission from the
stained tissue was collected with a camera lens (50 mm,
f1:1.4, Nikon), projected through a 630-nm cut-off filter
(RG-645, Schott Glass), and focused to form an image of the
preparation on the surface of a 12 3 12 photodiode array
(Centronic, Newbury Park, CA). Five diodes from each corner
of the array were ignored such that optical signals were
monitored from 124 of 144 diodes. The image of the AV node
was focused on the array at a magnification of 30.36 such
that each diode detected fluorescence APs from a 0.46 3 0.46
mm area of epicardium. The depth of field of the collecting
lens restricted the fluorescence measurements to a layer of
cells ,100 µm from the surface. The depth of field of AP
recordings was estimated by varying the staining protocol
and by empirical calculations based on the magnification of
the image, the numerical aperture of the lens, and the
wavelength of the emitted light (19). The image of the heart
focused on the array was reflected by a mirror onto a
custom-made graticule with the exact dimensions of the array
(Graticules, 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 was accomplished by focusing
and aligning the image of the heart on the graticule. The
photocurrents from 124 diodes were fed to a current-tovoltage converter, amplified, digitized (1.56 kHz per channel,
12-bit resolution per sample), and stored in a memory buffer
of an IBM PC 486DX/4 100-MHz computer. The sampling rate
(1.56 kHz per channel) was set to the maximum rate of the
data acquisition processor (DAP 1200e, Microstar Laboratories, Bellevue, WA). A data acquisition scan consisted of 128
simultaneously recorded traces: 124 optical plus 4 instrumentation channels. A scan consisted of a continuous recording of
these 128 channels for 1.2–3 s.
Simultaneous AP recordings with microelectrodes and voltage-sensitive dyes. In some experiments, conventional 3 M
KCl-filled microelectrodes with a resistance of 20–40 MV
were used to record APs from the AV node to identify the APs
that fired in synchrony with the second spike of the optical
recordings. Optical APs were recorded from the AV node zone,
and then the diodes on the array that detected three spikes
were used to identify the mid- and lower nodal zone, which
was then impaled with a microelectrode to simultaneously
record APs by the two techniques. These measurements were
challenging because the short working distance between the
lens and the preparation made it difficult to obtain stable
microelectrode impalements at a shallow angle of penetration.
Data analysis. Several criteria were used to assess the
‘‘health’’ of AV node preparations: 1) the rapid rise time of
atrial and ventricular AP upstrokes, 2) the short AH intervals
[,80 ms at 500 ms cycle length (28)], 3) the rapid propagation
of APs in the atrium (,0.1 m/s), and 4) the duration of
ventricular APs, because short AP durations are indicative of
ischemic injury. The activation time point at each diode was
taken as the time point of the maximum rate of rise of the
fluorescence (F) AP upstroke (dF/dtmax ), which represents the
time when most of the cells depolarized (21, 22). Optical
recordings from each channel were normalized and passed
through a Butterworth filter, and the first derivative of each
fluorescence signal (dF/dt) was calculated by a numerical
differentiation method using a three-point Lagrangian interpolation. The dF/dtmax time point was accepted as an activation time if dF/dtmax was greater than the standard deviation
(SD) of background noise. The analysis of optical APs was
automated using in-house software written with IDL 3.6.1b
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For all four dyes, staining the hearts by injections in the
coronary circulation was markedly more effective than bathing the preparation in dye solution such that greater amplitudes of optical AP upstrokes, signal-to-noise ratios, and more
homogeneous staining were obtained. Di-4-ANEPPS gave the
best signal-to-noise ratio and was used for the experiments
described in this study. In one experimental group (n 5 3), AH
intervals were measured with bipolar surface electrograms
before, during, and after perfusion of the hearts with dye
solution to examine the possible pharmacological effects of
DMSO with or without dye on AV conduction delays. Figure 1
shows the AH measurements from an AV node preparation.
AH intervals averaged over a period of 5 min before hearts
were stained were 42.7 6 2.4 ms (Fig. 1A); after hearts were
stained, AH intervals were 43.3 6 2.5 ms when averaged over
5 min of dye washout (Fig. 1B; n 5 3 hearts). Thus the present
staining conditions with the use of a DMSO stock solution of
di-4-ANEPPS caused no detectable changes in AV node
conduction.
To avoid motion artifact to optical recordings, 10 mM
diacetyl monoxime (DAM) solution was used to block contraction during data acquisition. Hearts were perfused in Tyrode
solution containing DAM for 10–12 min and then perfused
with standard Tyrode to avoid prolonged exposure to DAM,
which produced time-dependent changes in AP characteristics. Exposure to DAM (10 mM) did not appear to alter AV
conduction during brief periods of DAM perfusion as used in
these experiments, but prolonged perfusion (.45 min) produced a gradual increase in coronary resistance in total AV
delays followed by inexcitable atrial and ventricular tissue.
Optical apparatus. Details of the optical and recording
apparatus have been described elsewhere (22). The horizontal chamber was mounted on an X-Y-Z micromanipulator to
accurately select the zone of tissue viewed by the photodiode
array and to control the location of the focal plane of the
optical apparatus as a function of depth in the tissue. Light
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A CONDUCTION BARRIER REGULATES AV DELAY
by the operator using an interactive program. The detection
of activation time points using dF/dtmax was highly reliable
and rarely required correction by the investigator (,1% of
activation events). The upstrokes of optical APs from the AV
node were distorted by movement artifacts from vigorously
contracting hearts because atrial contractions interfered with
the small signals from the AV node zone such that the use of
DAM (as described in Staining procedure) was essential to
abate movement artifacts.
Histology. Optical electrophysiological recordings were
made to identify the zone of tissue that fired three sequential
spikes, and then the tissue was marked with fiducial points at
sites that corresponded to the edges of the array. The fiducial
marks were made by impaling the tissue with a microelectrode and then placing a silk surgical thread suture at those
sites to ensure that the fiducial mark was clearly identifiable
even after the histological processing. The tissue containing
the AV node region was excised and fixed in Bouin’s solution
overnight. The fixed tissue was embedded in paraffin, and
serial sections 5 µm thick were taken starting from the
endocardial surface, and every tenth section was mounted on
a glass slide. Mounted sections were progressively stained
with Mayer’s hematoxylin, counterstained with eosin, and
placed under a glass coverslip (15). Stained sections were
examined under a microscope and captured in TIFF images
(in 24-bit true color) using a charge-coupled device camera.
Shrinkage caused by fixation (,35%) was automatically
taken into account by aligning the fiducial marks on the
sections with their respective locations on the edges of the
array. The isochronal maps derived from optical APs were
superimposed on images of the tissue derived from histological sections using CorelDraw 7.0.
RESULTS
Optical recordings from AV node. Figure 2A shows an
anatomic sketch of the AV node region delineated by the
tendon of Todaro and the crista terminalis forming the
triangle of Koch and the central fibrous body. A symbolic map of the array is superimposed on the AV node
to identify the region of tissue mapped by the array. The
orientation of the array relative to the AV node was
arbitrary but was kept constant in the present study.
Figure 2B shows a set of 124 optical signals recorded
from the AV node region as well as simultaneously
recorded atrial and ventricular bipolar electrogram
(BE) recordings. Figure 3, A–D, shows the four types of
signals recorded from the AV node region and the
temporal relationship between these signals and the
surface electrograms. Each panel of Fig. 3 represents a
more detailed tracing of recordings from diodes a–d in
Fig. 2A. Each panel contains three traces, the atrial
and ventricular BEs plus an optical recording from one
of the diodes (a–d) viewing different sites on the
preparation. Diode a (see Fig. 2A) viewed the IAS and
detected atrial APs with negligible contribution from
other cell types such as nodal or ventricular cells (Fig.
3A). Diode b (see Fig. 2A) viewed the IVS and detected
ventricular APs (Fig. 3B). At border zones between the
IAS and IVS, diode c (see Fig. 2A) detected voltagedependent optical responses with two upstrokes per
cardiac beat (Fig. 3C). In these zones, the IAS overlapped ventricular tissue such that both cell types
contributed to the signal recorded by the same diode,
resulting in the sequential firing of atrial, and then
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(Interactive Data Language, Research Systems, Boulder, CO)
and Borland C11 4.0.
Isochronal maps and movies of activation. Optical signals
from atrial and ventricular muscles exhibited the expected
shapes and characteristics of atrial and ventricular APs. In
contrast, signals from the AV node region (the region delineated by the tendon of Todaro and the crista terminalis)
exhibited a set of three sequential spikes indicative of three
distinct depolarization events separated by marked time
delays (see Figs. 2–4). The first and third spikes (peaks I and
III) were coincident with atrial and ventricular depolarizations, respectively. The second spike (peak II) was only
detected in the AV node region and was the smallest in
amplitude. Thus three cell types were found to overlap in the
same zone of AV node tissue, and activation patterns were
independently generated for the three spikes. The combined
activation patterns represented atrial, nodal, and ventricular
activation that corresponded anatomically to the atrial, nodal,
and ventricular regions of the preparation. Isochronal maps
were generated for the three wave fronts using a linear
triangulation method (Tecplot-3DV, Amtec Engineering, Bellevue, WA). To map activation in the AV node zone, the region
that exhibited peak II was extracted first and was typically
delineated by 12–18 photodiodes, or areas of 4 3 3 or 6 3 3
diodes, on the array. Activation time points for all peak II
were triangulated, isochronal lines were calculated for every
1-ms interval using the values of the three edges of the
triangles by linear interpolation, and then points were connected by lines. With linear triangulation, 10–12 isochronal
lines were typically generated in a zone delineating 2–3 mm
of nodal tissue. Conduction velocity was determined from the
time delays between isochronal lines and the distance traveled by the wave front detected in the two-dimensional (2-D)
field of view of the array. These measurements represent
‘‘apparent’’ conduction velocities given that the precise pathways of the wave fronts are approximate, because the node is
a three-dimensional (3-D) structure (0.75–1.25 mm thick
according to histological analysis) and the pathway is determined from 2-D maps. As a result, conduction velocities may
be underestimated if the pathway propagation spreads in
depth across the thickness of the node as well as in the 2-D
field of view.
The spread of depolarization was animated to visualize the
spread of excitation waves. The animation program was
written with g11 2.7.2 and YORICK 1.2 by scaling the
amplitudes of the optical recordings into a range of 8-bit,
256-color levels. The signal-to-noise ratio was sufficiently
high so that filtering of the normalized signals was not
necessary. One frame of the byte-scaled data from the 12 3 12
array was converted to a 120 3 120 pixel image that consisted
of 12 3 12 squares (the level of depolarization was color coded
and filled an array of 10 3 10 pixels). The sequence of
excitation images was displayed on the monitor using X
Windows System (X Consortium) ‘‘pixmaps.’’ The typical
animation runs at 20 frames/s. Mapping and animation were
performed on a Pentium (100 MHz) running Linux (kernel
version 2.0) or an Indy workstation (Silicon Graphics, Mountain View, CA) running Irix 5.3. Movies of AP propagation
across the AV node can be seen at our web site (Salama, Guy.
Lab and current research: AV node conduction. [On-line]
Dept. of Cell Biology and Physiology, Univ. of Pittsburgh.
http://www.cbp.pitt.edu/la gs.htm); QuickTime movie player
is required.
The activation time point for each fluorescence AP was
taken at dF/dtmax, when most of the cells viewed by a diode are
depolarizing. Activation time points for all 124 APs were
labeled with ‘‘tick marks’’ that could be verified and corrected
A CONDUCTION BARRIER REGULATES AV DELAY
ventricular, APs. The interpretation of the cell types
responsible for these optical signals was validated by
the electrogram recordings in that the upstrokes of
atrial and ventricular optical APs were coincident with
the atrial and ventricular electrograms, respectively
(Fig. 3, A–C). In addition, atrial and ventricular APs
were recorded optically at sites consistent with the
anatomy of the preparation: they had the expected
shape and time course of atrial and ventricular APs,
and the delay between the firing of the two upstrokes
was consistent with the expected AV delay. More interesting was a unique feature of optical signals from the
AV node region shown in Fig. 3D. Signals recorded from
a narrow zone between the tendon of Todaro and the
crista terminalis (diode d; see Fig. 2A) exhibited a set of
three sequential depolarizations per cardiac beat, peaks
I, II, and III (Fig. 3D). By superimposing signals
detected by other diodes and surface electrodes, peak I
was again coincident with atrial depolarization and
peak III was coincident with ventricular depolarization. Peak II consistently fired at intermediate time
points and was only observed in a narrow (1 3 2 mm)
region of the preparation.
AP propagation within AV node. To elucidate the
origins of peak II, intracellular microelectrodes and
optical APs were simultaneously recorded from the
zone that exhibited the sequence of three depolarizations. As shown in Fig. 4A, the firing of peak II was
coincident with APs with the characteristic shape of N
and/or NH cells because of the slow diastolic depolarization and slow upstroke (n 5 4 hearts). In contrast,
microelectrode recordings of atrial, AN, His, and ventricular APs were not coincident with peak II depolarization (not shown). More detailed analysis of the spatiotemporal characteristics of peak II was carried out to
reinforce the interpretation that peak II originated
from the midnode and/or lower node region and to map
electrical conduction across the AV node. Figure 4B
shows the approach used to calculate the time delays t1
and t2 from the time points of dF/dtmax for peaks I–III.
Maps of electrical activation generated from activation time points (as in Fig. 4A) were highly reproducible
from beat to beat of the same heart and from heart to
heart. Figure 5 shows an experiment from a spontaneously beating heart with a cycle length of 417.92 6 0.96
ms (mean 6 SD). The mean AV delay (6SD) measured
with surface electrograms was 88.75 6 0.37 (n 5 5
beats). As shown in Fig. 5, the time points of dF/dtmax
for peaks I–III were detected separately and used to
map the spread of activation in the IAS (Fig. 5A), the
AV node (e.g., the zone of tissue detecting peak II) (Fig.
5B), and the IVS (Fig. 5C). Figure 5D depicts the
superimposition of all three activation patterns, showing that the three maps overlap in a small region
identified as the compact node. Activation across the
IAS occurred in 15 ms (Fig. 5A), and, after a substantial
delay, the AV node signal appeared at 43 ms (Fig. 5B)
and spread across the node in 8 ms. After another delay
(e.g., the time to propagate from the His bundle to the
apex and back to the base of the ventricle), the IVS fired
APs at 93 ms and spread in the field of view in 4 ms
(Fig. 5C). Such maps of conduction across the AV node
were highly reproducible in both their patterns and
temporal relationships. Table 1 lists the composite
analysis of nine rabbit preparations. The means 6 SD
for t1 and t2 were calculated for rabbit AV nodes (n 5 9)
under sinus rhythm, with an intrinsic cycle length of
301.64 6 8.41 ms. The time for peak II to propagate
across the compact node (i.e., midnode) was 10.33 6
3.21 ms, which predicts a conduction velocity of 0.162 6
0.024 m/s. The conduction velocity measured by optical
techniques is faster than that inferred from intracellular microelectrode recordings because the latter measurements were not based on multiple recordings within
the node and included a substantial component of t1 or
AN delays.
In Fig. 6, an activation pattern across the AV node is
shown as a sequence of activation maps captured at
different time points during a single cardiac beat. Each
map is a pseudocolor map of the array for which the
extent of depolarization at each site is color coded from
violet to red (from least to most depolarized potential).
The first map (at time t 5 0.0 ms) is violet or blue (i.e.,
the tissue is at resting potential). The next map, at 3.2
ms, shows the initial firing of the atrium that spreads
along the IAS in the subsequent maps (from 3.2 to 25.6
ms). After a partial repolarization of the IAS (from 25.6
to 41.6 ms), the firing of nodal APs begins in the lower
left zone of the array (44.8 ms) and spreads along a
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Fig. 2. A: sketch of AV node preparation superimposed on a symbolic
map of photodiode array to delineate AV node region being viewed.
Anatomic landmarks of AV node region are identified to correlate
optical signals obtained from each site with origins of signals.
Compact node, or midnodal region, is delineated by a speckled outline
and is bounded by TT and CT, surrounded by IAS above and interventricular septum (IVS) below, and adjacent to central fibrous body
(CFB). Orientation of array relative to AV node zone was arbitrary
but was kept the same for all experiments shown. B: simultaneously
recorded optical action potentials (APs) from 124 sites on AV node
zone. A symbolic map of array is shown as 124 square boxes, each
identifying the location of individual diodes. The region of tissue
viewed by each diode corresponds to map in A. The optical trace
recorded by each diode is shown in its respective location in a
compressed time base (400 ms). Inset: BE recordings, 1 located on IAS
2 mm above TT (A; outside field of view of array; atrial BE) and 1 on
IVS near apex of ventricle (ventricular BE), were simultaneously
recorded along with 124 optical signals. Optical APs recorded by
diodes at locations a–d (A) are shown in Fig. 3.
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A CONDUCTION BARRIER REGULATES AV DELAY
narrow zone (1–2 diodes wide), at first slowly and then
faster, into the His bundle (from 44.8 to 54.4). After a
delay (from 60.8 to 80.0 ms), the IVS depolarizes and
ventricular signals overlap the AV node zone (from 80.0
to 86.4 ms). Video animation of these maps is more
effective for visualization of the propagation pathway
and can be accessed from our web page (see Isochronal
maps and movies of activation).
Depth resolution and amplitude of AV node APs. The
AV node is a 3-D structure located near the surface of
the IAS, with the majority of cells comprising the
compact node found ,0.06–0.5 mm below the surface of
the IAS (6). Microelectrode studies have identified
zones of AN that overlap zones of N and NH cells on the
basis of their AP characteristics (6). The 3-D nature of
this structure implies that the focal plane of the
imaging system should be located below the surface of
the preparation to obtain maximum signal amplitudes
for AV nodal APs. As shown in Fig. 7, a given diode will
detect light from different volumetric zones of tissue as
the focal plane of the optical apparatus is shifted below
the surface. This is a natural consequence of optical
detection because light from cells located on the plane
of focus is transferred to the diode with the highest
efficiency, whereas light from cells above and below the
focal plane is transferred to the diode with decreasing
efficiency. The efficiency of light transfer as a function
of depth (at any given wavelength of light) depends on
the ‘‘energy transfer function’’ of the collecting lens,
measured at a particular optical magnification (21).
The AV node was optically sectioned by recording APs
from the surface and then from deeper optical sections
by shifting the plane of focus into the tissue in 250-µm
steps. Changes in the relative amplitude from peak I to
peak II were used to estimate the depths of the cells
responsible for the generation of peak II.
As shown in Table 2, the relative amplitude from
peak II to peak I increased with increasing depth of the
focal plane for 0, 0.25, and 0.5 mm and then decreased
for 0.75 and 1.0 mm. The most likely explanation for
these depth-dependent changes is that peak I emanated from the surface and peak II emanated from cells
,0.5 mm below the surface. In all experiments, the
focal plane was first adjusted to maximize peak II
relative to peak I at the beginning of each experiment.
This approach made it possible to maximize the signalto-noise ratio for peak II, delineate the 2-D distribution
of the cells that fire peak II, and map the spread of
activation of peak II.
Activation sequence of pacing AV node. A key feature
of the AV node is that, in the absence of SA node
activity, N or NH cells can become primary pacemak-
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Fig. 3. Classification of optical signals from AV node
region. Optical APs from AV node region shown in Fig.
2B exhibited 1 of 4 possible characteristics depending
on location of recording. Each class of optical AP was
temporally correlated with atrial and ventricular BEs
to show firing sequence of APs. A–D each show a set of 3
traces, the atrial (top) and ventricular (bottom) BEs
plus an optical recording (middle), from diodes labeled
a–d, respectively, in Fig. 2A. A: optical AP recorded from
atrial cells located on IAS by diode a. B: ventricular AP
recorded from IVS by diode b. C: optical APs from AV
boundary were recorded by diode c. At such sites, atrial
and ventricular cells overlapped, resulting in optical
signals with 2 spikes, synchronous with atrial and
ventricular BEs, respectively. D: optical recordings from
AV node at diode d invariably consisted of 3 sequential
depolarizations (peaks I–III). Peaks I and III were
coincident with atrial and ventricular BEs, and peak II
fired at an intermediate time point, indicating that AV
node was origin of signals that exhibited 3 sequential
spikes. DF/F, fractional fluorescence change.
A CONDUCTION BARRIER REGULATES AV DELAY
ers. To investigate the spread of activation in hearts, we
removed the SA node so that the AV node became the
primary pacemaker (n 5 4). Figure 8A shows the
optical signals detected from the AV node zone when
pacemaker activity was initiated at the AV node. In this
case, the initiation of the heart beat begins in the node
[AV node (midnodal)] at a time delineated by the time
line labeled a (Fig. 8A). As a primary pacemaker, the AV
node trace (or peak II) fired first, and the atrial and
ventricular BE are delayed (,28 ms) and are coincident
with the time line labeled c (Fig. 8A). Note that, as
expected, the optical recording of ventricular AP was
coincident with the ventricular BE. On the other hand,
the recovery of atrial AP was coincident with the time
line labeled b (Fig. 8A) and precedes the atrial BE
because the optical signal recorded APs near the AV
node at an early time of activation, whereas the BE was
located on the IAS, near the SA node. When the AV node
is the dominant pacemaker, the amplitude and slope of
the first depolarization was markedly decreased as
expected because peak II is now the leading wave of
depolarization originating from the AV node. In Fig. 8B,
activation maps indicate that impulses were initiated
in a narrow zone within the triangle of Koch, and the
first site to activate fired in the node as indicated by the
asterisk. The signal spread in 10 ms within the node,
and after a substantial delay (27.96 ms; see arrow in
Fig. 8B), activation spread across the IAS. The zone of
activation delineated by peak II during sinus rhythm
(not shown) was the same as that delineated by the
leading wave of AP upstrokes when the AV node became
the pacemaker (Fig. 8B). The marked conduction delay
from the AV node zone to the IAS (Fig. 8B) indicates
that propagation is not smooth but is discontinuous in
both the anterograde and retrograde directions. This
barrier to conduction (arrow) between the compact
node and the IAS is dramatically evident in all the
activation maps (Fig. 8B). The activation pattern within
the ventricular tissue was intentionally omitted from
the activation maps of Fig. 8B so that AV nodal
activation would not be obscured.
Superimposition of activation maps and histological
sections of AV node. In four hearts, optical maps of APs
were recorded from the AV node region and the tissue
was labeled with fiducial marks, fixed, embedded in
paraffin, serially sectioned, and mounted on microscope
slides for histological analysis. Figure 9A shows the
anatomic landmarks of the AV node seen from longitudinal cross sections taken from the endothelial surface
at increasing depths in the tissue. The conventional
anatomic landmarks of the AV node are all resolved at a
depth of 100 µm (Fig. 9A, left), namely, the IAS, the pale
cells of the compact node, the IVS, and the His bundle.
As shown in Fig. 9, A and C, zones of fat cells are found
adjacent to the AV node region. These zones of adipose
cells are typical of mammalian AV nodes and extend
continuously at all depths of the preparation. In this
heart, the dimensions of the AV node zone were largest
at a depth of 150 µm (Fig. 9A, middle) and decreased at
a depth of 200 µm (1-mm scale at right applies to A–C).
Activation maps recorded from the same preparation
are shown in Fig. 9B as a set of maps taken every 3.2
ms. In this case, the SA node was removed and the AV
node became the primary pacemaker. The superimposition of isochronal maps derived from optical recordings
and the histological section taken at a 150-µm depth
showed that the compact node is the tissue underlying
the optical map-identified activation patterns across
the AV node. Moreover, the earliest sites to fire APs in
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Fig. 4. Detection of activation time points in AV node. A: simultaneous optical and microelectrode recordings from AV node. Optical
signals detected a sequence of 3 depolarizations, and peak II was
coincident with an AP recorded from a microelectrode impaled in a
midnodal or distal node cell, either an N or NH cell. The shape and
time course of the microelectrode AP show that peak II fired in
synchrony with an N and/or an NH cell. B: 1st derivatives of all 4
classes of signals (dF/dt) were taken to analyze spread of activation
across AV node and to determine time point of activation at each site.
For signals from AV node, dF/dt identified 3 separate activation time
points. Top: DF/F recorded from a diode viewing AV node exhibited a
set of 3 upstrokes (arrows) per cardiac beat (peaks I–III). Bottom: delays
between peaks I and II (t1 ) and between peaks II and III (t2 ) were
determined from time points of maximum dF/dt (dF/dtmax ) of voltagesensitive fluorescent signals. dF/dt of peak II allowed identification of
activation time points for cells comprising AV node that were used to
map activation patterns across node.
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A CONDUCTION BARRIER REGULATES AV DELAY
these maps were located in the proximal region of the
compact node (i.e., posterior), and subsequent APs fired
in the distal zone of the AV node (i.e., anterior), followed
by the His region, with increasingly faster propagation
velocity. The superimposition of optical isochrones and
histological images (Fig. 9C) provides compelling evidence that these optical signals originate from the
firing of APs in the AV node and delineate the shape and
boundaries of the AV node. Figure 10 shows another
example of activation isochrones across the AV node
toward the His bundle and depicts the marked increase
in conduction velocity from the proximal to the distal
portion of the node. The traces (Fig. 10A) show the
Table 1. Analysis of voltage-dependent fluorescent
signals from atrioventricular node
Parameter
t1 , ms
t2 , ms
Conduction time of peak II, ms
QN , m/s
49.50 6 6.59
57.57 6 5.15
10.33 6 3.21
0.162 6 0.024
Values are means 6 SD for n 5 5 optical signals recorded from 9
rabbit atrioventricular (AV) node preparations. Delays between
peaks I and II (t1 ) and between peaks II and III (t2 ), conduction time
of peak II, and AV node conduction velocity (QN ) were calculated and
grouped for rabbit hearts under normal sinoatrial node activation,
with a cycle length of 301.64 6 8.41 ms. QN was determined from time
delay and distance measured from isochronal maps of midnodal
region of AV node as shown in Fig. 5 and did not include lower nodal
or His bundle region shown in other experiments (see Figs. 9 and 10).
QN , conduction velocity, and total time to propagate within node were
highly reproducible from heart to heart.
kinetics of optical signals recorded from these two
regions. The top trace (Fig. 10A), recorded from the
proximal region of the AV node trace, exhibited the
typical sequence of three depolarizations. Here, peak II
originated from the midnodal region, which initiated
waves of depolarization (Fig. 10A, top arrow). The
bottom trace, recorded from a more distal region,
exhibited a sequence of two depolarizations, a His
followed by a ventricular depolarization.
Effect of TTX on AV node signals and conduction. The
major depolarizing current of N and NH cells is a slow
inward current (most likely Ca21 current) that is
insensitive to the voltage-gated Na1-channel blocker
TTX (12). In contrast, atrial, AN, His, and ventricular
cells are somewhat sensitive to TTX, which, at 5–10
µM, decreases dV/dtmax. We investigated the effect of
TTX (10 µM) on the propagation times across the AV
node, that is, of peak II and His bundle depolarization,
in the same manner as in Fig. 10A (n 5 3). Figure 10B
shows an example of activation maps measured from
the node before (left) and after (right) 20 min of TTX
perfusion. TTX reduced the rise time of atrial and
ventricular APs (not shown) but did not alter the
spatiotemporal characteristics of peak II (compare the
proximal zone of the AV node in Fig. 10, A and B). On
the other hand, TTX produced a marked decrease in
apparent conduction velocity in the distal zone of the
activation map (Fig. 10B, right, TTX-sensitive zone).
The sensitivity of the distal zone to TTX supports its
identification as a His bundle region from the histological analysis (Fig. 9) and the spatiotemporal distribu-
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Fig. 5. Isochronal maps of activation across AV node.
From activation time points obtained as described in Fig.
4, activation sequences of atrial (peak I), nodal (peak II),
and ventricular (peak III) regions were triangulated individually and isochronal lines of activation were drawn 1
ms apart. Gray scale from bright to dark represents
‘‘early’’ to ‘‘late’’ activation times. Axes are length in
millimeters. A: depolarization sequence of IAS generated
from peak I. B: activation sequence of the AV node
generated from peak II. C: activation sequence of IVS
generated from peak III. D: composite isochronal map of
AV node region generated from superimposition of activation maps in A–C reveals details about supraventricular
activation and spread of activation across AV node and
IVS. Note that from last depolarization of peak I to first
depolarization detected through peak II, there is a 30- to
33-ms interval during which firing of APs is not detected
anywhere in the preparation.
A CONDUCTION BARRIER REGULATES AV DELAY
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Fig. 6. Pseudocolor maps of activation across AV node region. A sequence of array images recorded at different time
points during a single cardiac beat was generated as an alternative approach to display activation maps. Levels of
local depolarization were linearly converted to rainbow color map (color scale 0–255), where least to most
depolarized levels produced a color shift from blue to red, respectively. Successive images taken every 3.2 ms were
generated from optical recordings as described in MATERIALS AND METHODS. Inset: orientation of images.
tion of the signals (Fig. 10A). Thus the posterior zone
consists of TTX-insensitive cells (most likely N and/or
NH cells), and the anterior zone contains TTX-sensitive
cells (most likely His and perhaps NH cells), in line
with intracellular microelectrode recordings.
Effect of His bundle cut. Figure 11A shows the atrial
and ventricular BEs and optical recordings from the
IAS, the AV node, and the IVS following a cut of the His
bundle. The His bundle forms the only electrical connection between the AV node and the ventricle such that
cutting fibers below the AV node effectively disconnected electrical coupling between the ventricular myocardium and inputs from the AV node. As shown in Fig.
11A, severing the His bundle eliminated the synchronous firing of ventricular BE and the detection of
ventricular APs. This finding reinforced our interpreta-
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A CONDUCTION BARRIER REGULATES AV DELAY
tion that APs from the lower left zone of the preparation
originate from ventricular cells. Under our experimental conditions, the His bundle cut produced a 2:1 block
between atrial and nodal activation (Fig. 11A, node). As
a result, some atrial activation waves (Fig. 11A, I)
failed to entrain an AV node activation (Fig. 11B; note
the lack of AV node activation), whereas the next beat
(Fig. 11A, II) was coupled to an AV node activation (Fig.
11C). Not all activity ceased in the ventricular tissue
because ectopic beats were occasionally observed, but
these were out of phase with atrial and nodal APs. An
advantage of His bundle cuts is the absence of ventricular APs, which interfered with our ability to detect the
repolarization phase of N-cell APs.
A conduction barrier between atrial and nodal cells
regulates AV delay. AV delay is physiologically regulated by heart rate, parasympathetic, and sympathetic
activity. To investigate the mechanisms responsible for
the physiological regulation of the AV node delay,
activation maps across the node were analyzed as a
Table 2. Ratio of peak II to peak I as a function
of depth of focus
Depth, µm
Peak II/Peak I
0
250
500
750
1,000
0.301 6 0.011
0.331 6 0.036
0.490 6 0.108
0.424 6 0.154
0.387 6 0.136
Values are means 6 SD of ratios of peak II to peak I from
normalized signals recorded from 4 rabbit AV node preparations,
with 3 cardiac beats averaged per preparation. Optical APs were
recorded with focal plane at various depths below surface of AV nodal
zone. Amplitudes of peaks I and II were measured from data
printouts. Ratio of peak II to peak I first increased with increasing
depth of focus (from 0 to 0.5 mm) then decreased (from 0.5 to 1.0 mm).
Initial increase in ratio was due to an increase of peak II with
increasing depth of focus rather than a decrease of peak I and was
statistically significant (P , 0.05) by analysis of variance.
DISCUSSION
The study presents the first measurements of electrical propagation across the AV node using voltagesensitive dyes and imaging techniques. A number of
technical difficulties were overcome, including signal-tonoise ratio, depth of focus, detection and analysis of
three sequential optical upstrokes, and display of activation maps. Several lines of evidence were presented
to support the interpretation that peak II represented
AV nodal AP and could be used to map activation within
the node. 1) The occurrence of peak II was anatomically
correlated with the location of the midnodal and lower
nodal regions (Fig. 9). 2) The temporal relationship
between peak II and electrograms recorded from the
IAS and IVS indicated that peak II originated from cells
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Fig. 7. Ratio of peak II to peak I as a function of depth. Schematic of a
transverse histological section of rabbit AV node indicates different
layers of cell types found as a function of depth in tissue [adapted
from Janse et al. (10a)]. Volumetric elements (shaded cubes) superimposed on section represent volume of cells that are primary sources of
fluorescence signals (F) detected by a diode when focal plane is
shifted below surface in a stepwise manner. At different focal planes,
different layers of cells become primary sources of signal. hn, Excitation light.
function of cycle length. The heart rate was controlled
by removing the SA node and pacing the right atrium
with bipolar electrodes placed near the SA node. In this
experimental group (n 5 9), cycle length was varied
from 270 to 350 ms; shorter cycle lengths made it
difficult to temporally separate the overlapping peaks
I–III for analysis, and longer cycle lengths were possible except that extra beats occasionally interfered
with the measurements. In this range of cycle lengths,
activation maps across the IAS, AV node, and IVS
maintained the same pattern. AV delay measured with
atrial and ventricular electrograms changed with cycle
length, as shown in Fig. 12, and was highly correlated
with changes in t1, with a correlation coefficient of 0.98,
but correlated poorly with changes in t2, with a correlation coefficient of 0.42 (each data point corresponds to
the mean 6 SD).
Heart rate and AV delay are regulated physiologically by sympathetic and parasympathetic innervation
to the heart. Although much is known regarding the
mode of action of neurotransmitter at the SA node, the
mechanisms responsible for AV node regulation are less
well understood. In a separate experimental group (n 5
5), we altered AV delay by perfusing the heart with ACh
(0.1 µM). In all five experiments, the major change in
the optical map was an experimentally significant
increase in t1 by 19 6 2.3 ms and a slight increase in the
time to activate the node [e.g., a decrease in apparent
conduction velocity in the node region (QN )]. Figure 13,
A and B, depicts the maps of activation of the IAS
before and after perfusion with ACh, respectively. Note
the similarities in activation patterns across the IAS
and the node; the only marked change caused by ACh is
an increase in the delay between atrial and nodal
activation, with t1 increasing by 39.4% from 44.2 to 61.6
ms (Fig. 13, A and B). The prolongation of t1 with a
slight decrease in QN suggests that ACh hyperpolarizes
N cells such that the atrial current injected across a
high-resistance barrier is less effective at reaching
suprathreshold potential in the AV node. Thus a longer
interval of current injection is required to reach threshold and elicit AV node activation. The increased AV
delay caused by an ACh-induced hyperpolarization can
be attributed to an injection of current across a highresistance barrier with passive electrical properties.
A CONDUCTION BARRIER REGULATES AV DELAY
H839
firing in the correct time frame for AV nodal APs. 3) The
spread of activation analyzed from peak II reproducibly
(in .28 hearts) produced a zone of slow conduction
distinguishable from either atrial or ventricular propagation. 4) Under conditions in which the AV node was
the primary pacemaker, peak II became the first upstroke to fire during a cardiac beat and produced the
same spatiotemporal zone of activation as that observed when the AV node is driven by atrial inputs. 5)
After the His bundle was cut, ventricular activation
was blocked, but the spatiotemporal characteristics of
peak II did not significantly change. 6) The signal
amplitude of peak II as a function of depth of focus was
consistent with the interpretation that peak II originated from the firing of APs by cells ,0.2–0.5 mm
below the surface of the preparation. 7) Peak II signals
(Fig. 3D) recorded from the posterior zone of the
activation map were insensitive to TTX. Late signals
(observed in ,50% of the preparations) consisting of a
small depolarization preceding a ventricular AP represented the firing of His bundle APs and were TTX
sensitive (Fig. 10).
Peak II is not movement artifact. A major concern was
the possibility that peak II was primarily caused by
movement artifact (MA) rather than voltage-dependent
fluorescence signals. Extensive measurements were
carried out to negate that possibility.
First, all measurements were obtained with the
judicious use of DAM, an uncoupler of excitationcontraction coupling. It was important to find suitable
conditions for the use of DAM (see Staining procedure)
because perfusion with DAM for .30 min prolonged AV
delays and reduced excitability of the preparation, and
continued DAM perfusion eventually blocked all electrical activity (measured optically or with electrodes). On
the other hand, DAM effectively blocked contractions
after 8–10 min and could be washed out to reverse its
effects. In this way, DAM perfusion for 10 min gave us
15–20 min to acquire data before washing out DAM.
Second, the dF/dt of peak II produced a sharp, unique
time point, whereas analysis of MAs by the same
signal-processing technique produced neither a sharp
maximum dF/dt (see Fig. 4) nor a single maximum time
point so that an activation map could not be generated
from MAs.
Third, analysis of activation patterns from peak II
were highly reproducible (.27 hearts) in the anatomic
location, the direction of propagation, and the propagation velocity. MAs are typically unpredictable, and such
a coincidence is statistically negligible.
Fourth, in a few experiments, the Ca21 concentration
(0.2–5 mM) was varied to produce vastly different
levels of MAs, yet the spatiotemporal characteristics of
peak II did not vary significantly.
Fifth, when the AV node was the primary pacemaker,
peak II fired first and no interference from MA from
atrial and ventricular contractions was possible. In the
latter case, the signals produced maps of electrical
activity similar to those obtained when the AV node
fired after the atrial activation.
Finally, microelectrode impalements in the midnodal
zone recorded N- or NH-cell APs that were coincident
with the rise of peak II signals, which demonstrates
that peak II depolarization originated from the firing of
N and/or NH cells.
Taken together, these findings give a high degree of
confidence that peak II represented the electrical activity of a group of cells that fired APs in the correct
anatomic location and at the right time.
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Fig. 8. Optical recordings from a pacing AV node. SA node was removed so that AV node became primary
pacemaker. A: atrial and ventricular BEs were used to monitor firing of IAS and IVS and were temporally correlated
with optical APs recorded from various regions of AV zone preparation: IAS (atrial AP), posterior boundary of AV
node (transitional cells), AV node (midnodal cells), and IVS (ventricular AP). Earliest activation occurred at AV node
(time line a) and propagated to atrium (time line b), His bundle, and ventricle (time line c). B: activation maps of AV
node and IAS generated from atrial and nodal APs (axes are length in millimeters). Activation sequence in IVS was
intentionally omitted to allow visualization of conduction through node and delay between nodal and IAS
activation. Impulses initiated in node (*) and propagated across node in 10 ms and to IAS after a 17.96-ms delay.
Step delay in excitation wave occurred across a conduction barrier delineated by closely packed isochronal lines
(arrow). Isochronal lines were 2 ms apart. For web site access to QuickTime movie of propagation sequence, see
MATERIALS AND METHODS.
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A CONDUCTION BARRIER REGULATES AV DELAY
Identification of various AV node cell types. Previous
electrophysiological and morphological studies have
identified at least five different cell types in the AV node
zone. A major limitation of the optical technique is the
difficulty of resolving single cell APs by optical mapping. However, drug interventions such as TTX proved
to be useful in identifying the cell types comprising the
activation zone delineated by peak II and His depolarization (Figs. 9 and 10). For example, TTX had no effect
on propagation in the proximal region (Fig. 10A) but
slowed propagation in the distal region. This suggests
that peak II consists primarily of N and/or NH cells
with few TTX-sensitive AN or His cells, whereas the
distal region contained TTX-sensitive His cells. Another limitation of the optical techniques was the
inability to measure AP durations because signals from
the compact node consisted of superimposed atrial,
nodal, and ventricular APs. We attempted to resolve
the complete time course of N-cell APs (including the
repolarization phase) by increasing the optical magnification and varying the depth of focus. With the present
optical configuration, we failed to selectively measure
N-cell APs, even at the expense of decreased signal-tonoise ratio. An alternative approach used here was to
cut the His bundle to block ventricular APs and measure N-cell AP durations. However, cutting the His
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Fig. 9. Superimposition of activation maps and histological landmarks of AV node. A: charge-coupled device images
of 3 longitudinal sections of same AV node taken at different depths. Anatomic landmarks are IAS, IVS, CT, fat
deposits (F), midnodal region (N), and His bundle. B: activation maps recorded from same AV node showing
sequence of depolarization at 3.2-ms intervals, as in Fig. 6. Within a single cardiac beat, progression from earliest
(top left) to latest (bottom right) activation map shows sequence of depolarization. C: superimposition of activation
map derived from peak II depolarization on a longitudinal cross section of same AV node preparation. Optical and
histological maps were aligned as described in MATERIALS AND METHODS and indicate that a barrier to electrical
propagation forms a collar at posterior edge of AV node (may include zones of adipose tissue) and that a bridge with a
high electrical resistance appears to separate IAS from midnodal region.
A CONDUCTION BARRIER REGULATES AV DELAY
H841
bundle could change the electrotonic coupling between
NH and His fibers and thereby alter N-cell AP durations. Despite these limitations, optical mapping techniques offer a new approach for studying the organization and coupling of A, AN, N, NH, and His cells in ways
that cannot be resolved by conventional electrode techniques.
Activation patterns across AV node region. The depolarizations of peaks I–III delineate the spread of AP
propagation across the atrial, nodal, and ventricular
tissue, respectively. The earliest depolarizations (peak
I) were synchronous with the atrial electrogram and
mapped the sequence of depolarization across the IAS,
overlapping the AV node region. The time course,
distribution, and propagation velocity of these APs
identified them as atrial in nature, with AN characteristics as they overlap the AV node zone. After the last
AN cell depolarization, there was a delay of 30–40 ms
(during which no other cell depolarizations could be
detected) followed by depolarizations within the node,
detected by peak II. This delay between peak I and II
indicated that conduction was discontinuous between
AN and midnodal cells. The zone delineated by peak II
represented the firing of midnodal cells in the posterior
region of the node and lower nodal cells in the more
anterior region of the AV node (Fig. 9). In ,50% of the
preparations, His bundle APs were detected as small
depolarizations that preceded ventricular APs and de-
lineated a narrow track of electrically active tissue
contiguous with the lower nodal region. In line with
this interpretation, there was a progressive increase in
conduction velocity from the posterior region (midnode)
to the anterior track (His bundle) of the AV node. Thus,
for the first time, optical techniques provided detailed
maps of the sequence of activation across the AV node
and described the organization of the various cell types
comprising the node. The data also indicated that there
was a discontinuity in conduction, with a well-defined
anatomic location between atrial and midnodal cell
depolarization.
Mechanism(s) responsible for AH intervals. A number
of important conclusions can be extracted from the
present findings. One major finding is that the conduction through the AV node is not decremental but
discontinuous, and we conclude that a conduction barrier exists between the atrial and midnodal cells.
Features of this barrier are consistent with the presence of an inexcitable gap across which activation
proceeds electrotonically through a high-resistance
pathway. Although a barrier consisting of resistance
and capacitance components is consistent with the
data, we have not excluded the possibility of a barrier
with more complex electrical properties, with possible
modulation by neurotransmitters. The superimposition
of activation maps on longitudinal cross sections of the
AV node suggests that the electrical barrier surrounds
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Fig. 10. Effect of TTX (10 µM) on activation across AV node and His bundle. Excitation maps across AV node were generated from activation time points of peak II,
as in Fig. 3, and isochronal lines were
drawn 1 ms apart. A: in some preparations, activation maps generated from intermediate (peak II) depolarization revealed a pattern of depolarization across
midnodal, lower nodal, and His bundle
regions (left). Optical signals (right) measured from midnodal region (AV node)
exhibited typical response characteristics
of peak II. Signals from lower node region
(not shown) were similar to midnodal signals. Signals from His bundle were typically recorded from zones exhibiting 2
sequential depolarizations: upstrokes generated by His firing followed by firing of
ventricular cells. Time lines labeled A, N,
and H represent the depolarization time of
the IAS, node, and His bundle, respectively. B: to confirm interpretation of these
signals, TTX was used to identify nature of
cells producing optical signals. Activation
maps were drawn in absence of TTX (left)
and then recorded after 20 min of perfusion with 10 µM TTX. Note that conduction across posterior segment of node was
TTX insensitive, whereas conduction
through anterior segment slowed markedly, supporting interpretation that these
responses originated from His bundle.
H842
A CONDUCTION BARRIER REGULATES AV DELAY
the node and lies at the boundary of the midnode and
the IAS. Zones of fat deposits (adipose cells) adjacent to
the posterior and anterior zones of the AV node surround the AV node in 3-D, according to serial longitudinal sections, and are typically found in rabbit (6) and
Fig. 12. Correlation of t1 and AV delay at various cycle lengths. A
heart was paced from 270 to 350 ms with bipolar electrodes placed
near SA node; t1 and t2 were measured as described in Fig. 4. Total AV
delay (measured with surface electrograms), t1, and t2 were plotted
as a function of cycle length. Each set of data points was fitted with a
line drawn using polynomial curve fit. Step delay in conduction
between A (and/or AN) and N cells (t1 ) changed with rate, whereas t2
was within experimental error and rate independent. Correlation
coefficient between AV and t1 delay as a function of cycle length was
0.98, and that between AV and t2 was 0.39.
human AV nodes (personal communication, Dr. L. C.
Nichols, School of Medicine, University of Pittsburgh,
PA). These fat deposits likely form an effective electrical barrier that insulates the AV node from the IAS. As
a result, electrical coupling to the AV node can only
occur via narrow bridges: one bridge connects to the
IAS and the other to the cristae terminalis that are
unobstructed by fat deposits.
A mathematical simulation of optical signals was
developed to predict the signal characteristics under
different conditions such as synchronous, decremental,
or discontinuous conduction. The model consisted of 15
cells of equal dimensions firing optical APs of equal
amplitude and time course, and all 15 are detected with
one diode. When all 15 cells fired APs synchronously,
the resulting optical AP had a sharp upstroke with a
rise time similar to that of the single cell upstroke (Fig.
14A). When the cells fired with a smooth conduction
delay (50 ms across 15 cells), as in decremental conduction, the resulting optical AP had a slower rise time
indicative of the time-averaged upstrokes of asynchronous depolarizations (Fig. 14B). Only when there was
an abrupt delay (e.g., 45 ms) between APs from the first
10 cells and the last 5 cells did the resulting optical APs
exhibit two distinguishable upstrokes (Fig. 14C, arrow). Thus decremental conduction can be excluded as
an AV node delay mechanism, because the resulting
optical APs from the atrium and the node would blend
into a single upstroke (not 3 temporally distinct spikes)
representing the time-averaged sum of APs (Fig. 14B).
Decremental conduction is also incompatible with our
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Fig. 11. Effect of cutting His bundle on electrical
activity was investigated in 4 rabbit AV node preparations. A: simultaneous recordings of atrial and ventricular BEs and optical APs from IAS (atrium), AV
node, and IVS (ventricle) are shown after His bundle
was cut. As expected, His bundle cut blocked excitation to IVS, abolishing ventricular BEs and APs. In
absence of coupled ventricular depolarization, AV
node signals fired 2 sequential spikes, an atrial and a
nodal spike; the latter was not followed by a ventricular AP, revealing plateau and repolarization phases of
AV node AP. Atrial APs were synchronous with atrial
BE, but nodal APs were markedly reduced at every
other beat, suggesting a 2:1 block of AV conduction.
Activation maps of 2 successive beats (beats I and II
on nodal AP) are shown, respectively, in B and C.
Activation map produced by beat I produced an IAS
excitation pattern (B), whereas beat II mapped IAS
and nodal activation. Note that IAS activation patterns were similar for beats I and II. Isochronal lines
were 1 ms apart; axes are length in millimeters.
A CONDUCTION BARRIER REGULATES AV DELAY
H843
Fig. 13. Effect of neurotransmitter ACh on AV
node delay was investigated by perfusing heart
with 0.1 µM ACh. AV delay increased within
seconds after ACh perfusion, and data were
acquired 2–5 min after ACh injection (n 5 5).
Isochronal lines were 2 ms apart; axes are
length in millimeters. A: control activation
map recorded immediately before ACh injection. B: activation map recorded after 3 min of
perfusion with ACh. First nodal activation
occurred at 44.2 ms in control (A) and at 61.6
ms in presence of ACh (B).
node conduction (11, 13, 16). Discontinuous conduction
was also tested by applying a local perturbation [i.e.,
current (27), high K1 (5), freezing (26), or a sucrose gap
(2)] in otherwise uniform cardiac fibers (e.g., Purkinje
or ventricular). In such experimental models, the perturbation produced a step change in delay and ‘‘stagnation’’ similar to that observed in the node. Step delays
were caused by an interruption of active transmission
of an impulse as it arrived at the inexcitable gap. The
electrotonic transmission through the inexcitable gap
slowly charged distal cells until the resting potential of
the distal cells reached threshold and ignited active
transmission in the distal cells. It is critically important that the step delay is determined by the time
needed to inject the electrotonic current necessary to
induce active transmission in the distal cells. James et
al. (10) also argued against decremental conduction
because AV delays were not proportional to the size of
the AV node in various mammalian hearts. They proposed that pacemaker cells are involved in the AV delay
as coupled relaxation oscillators modulated by electrotonic atrial inputs.
The location and nature of the inexcitable gap and of
discontinuous conduction are still controversial. Early
studies (18) indicated that the major component of AV
delay occurred between the firing of late AN and early
N cells. Subsequent findings by Billette et al. (3, 4)
Fig. 14. An experimentally recorded atrial AP was used to simulate optical AP recordings during synchronous,
decremental, and discontinuous activation of 15 cells with equal dimensions and signal amplitudes. A: synchronous
activation of 15 cells. Optical recording is a single spike representing sum of 15 APs. B: decremental conduction of 15
cells. Cells were coupled through a high-resistance barrier, resulting in graded delays (50 ms) evenly distributed
from first to last cell. Optical AP is a single spike with a slow rise time caused by asynchronous depolarization of the
15 cells. C: discontinuous activation between first 10 cells (‘‘early activating cells’’) and last 5 cells (‘‘late activating
cells’’). This model was based on a single step delay due to a high-resistance, inexcitable barrier, resulting in
discontinuous conduction (10 early activating and 5 late activating cells). Time to propagate across early and late
activating cells was 5 ms, and there was a 45-ms delay between early and late cells. In discontinuous conduction,
optical AP will exhibit 2 distinct upstrokes that are temporally separated according to step delay across inexcitable
gap.
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measurements of QN (Table 1). For instance, if conduction through the node is decremental (with no ‘‘step’’
changes in conduction) and accounts for AV delays,
then QN should be at least 10 times slower than in the
atrium. From the analysis of optical signals, the total
AV delay was the sum of 1) the interval between A- and
N-cell activation (t1 ), 2) the time to propagate across
the node, and 3) the time between N- and V-cell
activation (t2; see Table 2). The conduction velocity in
the node was 0.162 6 0.024 m/s, approximately onethird that of atrial tissue (0.76 6 0.062 m/s). However,
the delay due to conduction through the node was #14
ms, an insufficient interval to account for the AV delay.
On the other hand, delay due to discontinuous conduction between peaks I and II was 44.24 ms or ,50% of AV
delays. On the basis of these results, it can be postulated that the major AV nodal delay is not due to the
slow conduction inside the node but to discontinuous
conduction between peaks I and II. Moreover, when the
AV node became the primary pacemaker, the conduction barrier at the posterior margin of the AV node was
still responsible for the major component of AV delay
during retrograde as well as anterograde propagation
(n 5 3).
The concept of an inexcitable gap in the node, resulting in discontinuous conduction, was inferred from
intracellular electrode studies (3) and simulations of AV
H844
A CONDUCTION BARRIER REGULATES AV DELAY
The authors are grateful for the inspiration of the late Richard A.
Lombardi. The authors thank Dr. Gregory Kloehn for technical
assistance and William Hughes, departmental machinist, for construction of the heart chamber and manipulators to adjust the focal plane
of the optical apparatus and to position stimulating and surface
recording electrodes. Thanks are also due Drs. Ronald L. Hamilton
and Lawrence C. Nichols (Dept. of Pathology, University of Pittsburgh, PA) for technical support and guidance regarding analysis and
interpretation of the AV node histology.
Address for reprint requests: G. Salama, Dept. of Physiology,
School of Medicine, Univ. of Pittsburgh, 3500 Terrace St., Room 2, 314
Biomedical Science Tower, Pittsburgh, PA 15261.
Received 7 February 1997; accepted in final form 22 August 1997.
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showed that, at short cycle lengths, the interval between an atrial electrogram and the latest AN cell had
not changed; the delay occurred between the latest AN
cell and the earliest NH cell. Moreover, at a short cycle
length (130 ms), N-cell AP dissociated into two components: the first component coincided with the AP upstroke of late AN cells, and the second component
coincided with the AP upstroke of the earliest NH cells.
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physiological control of AV delay. Prolongation of AV
delay by ACh is consistent with the notion proposed by
James et al. (10) that the AV node is a pacemaker
modulated by electrotonic inputs. The latter interpretation is based on a hyperpolarization of AV nodal cells by
ACh such that a greater input of current is required
across the inexcitable gap to obtain a suprathreshold
depolarization that can activate the AV node. Thus the
present data are incompatible with a decremental
conduction mechanism and provide direct evidence for
the existence of a conduction barrier, its specific anatomic location, and a measurement of the step delay (t1 )
involved in the physiological regulation of AV delay.
This study raises new questions regarding the mechanisms underlying t1 and AV delays and demonstrates
the potential of optical techniques to elucidate basic
problems in AV node physiology and salient clinical
problems.
A CONDUCTION BARRIER REGULATES AV DELAY
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