From the Department of Cardiology, Cleveland Clinic Foundation,
Cleveland, Ohio.
Correspondence to Todor Mazgalev, PhD, Department of Cardiology, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195. E-mail mazgalt{at}cesmtp.ccf.org
Methods and Results—Structural and functional complexity,
combined with the absence of adequate experimental techniques, has
complicated attempts to directly evaluate the three-dimensional
electrical activity of the AVN. Thus, despite a century of research by
conventional electrophysiologic and histologic methods, even the
existence of conduction through AVN is still debated.
Conclusions—Using a novel combination of microelectrode
recordings and high resolution fluorescent imaging with
voltage-sensitive dyes, we have for the first time clearly demonstrated
three-dimensional conduction through the AVN.
The application of voltage-sensitive dyes and optical imaging
techniques to studies in cardiac electrophysiology is becoming
increasingly common. However, this new technology is being used mostly
to map electrical responses of thin layers of epicardial
cells3 or isolated cells and cellular
cultures4 that are considered morphologically and
functionally "flat" two-dimensional fields. Because the AVN in the
rabbit heart is located immediately below the endocardial
surface2 and, judging by microelectrode
experience and histologic sections,5 the deepest
cellular layers are less than 500 µm away, we suggested earlier
that multicomponent optical recordings from the AVN area
observed in our previous experiments may carry signatures of electrical
activity from several layers at the same time.6
We hypothesized that three-dimensional mapping of the AVN might be
feasible if high-resolution fluorescent imaging and
microelectrode recordings were combined.
The preparations were paced at a cycle length of 300 ms, at twice
diastolic threshold voltage, by a bipolar electrode located
in the sinus node area. Bipolar electrograms were continuously
recorded from low crista terminalis and the bundle of His. Optical
recordings of electrical activity were performed
simultaneously from 256 sites with a state-of-the-art
photodiode array imaging system.6 8
To test this hypothesis, we impaled a glass microelectrode to
record AVN cellular action potentials (AP). The narrow depth of
field and large numerical aperture of the optical lens and the
transparency of the glass microelectrode minimized optical
interference. Deeper impalement provided an AP typical for a distal
nodal cell2 (Figure 1
When the microelectrode was slightly withdrawn vertically, a more
superficial transitional cell was impaled (Figure 1
The above observations were reproduced in all five hearts and confirmed
that the optical signal contained information from at least two wave
fronts: one representing the activation of the superficial
envelope of transitional cells and a second one
representing the ensuing depolarization of the deeper
compact nodal layers (Figure 1
The primary advantage of optical imaging can be appreciated when
mapping of the spread of activation is attempted. Microelectrodes
cannot accomplish such a task. In fact, successful high-resolution
intranodal mapping of AVN activation has not been previously reported.
Figure 2
We constructed activation maps of conduction through the AVN by using
either only the first or only the second of the two optical signal
components. Pairs of such activation maps are shown in Figure 3
This creates an exciting possibility to gain a deeper understanding of
the many still controversial properties of AVN conduction. Among those
are the filtering role of the AVN during high-rate irregular rhythms
such as atrial fibrillation and the fundamental issue of whether or not
the AVN truly conducts or is an electrotonically modulated
oscillator.9 Furthermore, application of the
optical imaging technique may help to visualize dual pathway structures
(if they exist) and to correlate specific bipolar potentials that were
clinically described10 with the AVN cellular
responses. Finally, the combination of microelectrode
recordings with optical techniques offers a powerful new
approach for studying electrical conduction in other complex biologic
nonhomogeneous structures with a multilayer
architecture.
Received November 12, 1997;
revision received December 22, 1997;
accepted January 23, 1998.
2.
Meijler FL, Janse MJ. Morphology and electrophysiology
of the mammalian atrioventricular node. Physiol
Rev. 1988;68:608–647.
3.
Davidenko JM, Pertsov AV, Salomonsz R, Baxter W,
Jalife J. Stationary and drifting spiral waves of excitation in
isolated cardiac muscle. Nature. 1992;355:349–351.[Medline]
[Order article via Infotrieve]
4.
Fast VG, Kleber AG. Microscopic conduction in cultured
strands of neonatal rat heart cells measured with voltage-sensitive
dyes. Circ Res. 1993;73:914–925.
5.
Imaizumi S, Mazgalev T, Dreifus LS, Michelson EL,
Miyagawa A, Bharati S, Lev M. Morphological and
electrophysiological correlates of
atrioventricular nodal response to increased vagal
activity. Circulation. 1990;82:951–964.
6.
Efimov IR, Fahy GJ, Cheng YN, Van Wagoner DR, Tchou
PJ, Mazgalev TN. High resolution fluorescent imaging of rabbit
heart does not reveal a distinct atrioventricular nodal
anterior input channel (fast pathway) during sinus rhythm. J
Cardiovasc Electrophysiol. 1997;8:295–306.[Medline]
[Order article via Infotrieve]
7.
Cheng Y, Mowrey KA, Efimov IR, Van Wagoner DR, Tchou
PJ, Mazgalev TN. Effects of 2,3-butanedione monoxime on the
atrial-atrioventricular nodal conduction in isolated
rabbit heart. J Cardiovasc Electrophysiol. 1997;8:790–802.[Medline]
[Order article via Infotrieve]
8.
Efimov IR, Cheng YN, Biermann M, Van Wagoner DR,
Mazgalev T, Tchou PJ. Transmembrane voltage changes produced by real
and virtual electrodes during monophasic defibrillation shock delivered
by an implantable electrode. J Cardiovasc
Electrophysiol. 1997;8:1031–1045.[Medline]
[Order article via Infotrieve]
9.
Meijler FL, Fisch C. Does the
atrioventricular node conduct? Br Heart
J. 1989;61:309–315.
10.
Jackman WM, Beckman KJ, McClelland JH, Wang X, Friday
KJ, Roman CA, Moulton KP, Twidale N, Nazlitt HA, Proior MI. Treatment
of supraventricular tachycardia due to
atrioventricular nodal reentry, by radiofrequency
catheter ablation of slow-pathway conduction. N Engl J
Med. 1992;327:313–318.[Abstract]
11.
Knisley SB. Transmembrane voltage changes during
unipolar stimulation of rabbit ventricle. Circ Res. 1995;77:1229–1239.
12.
Girouard SD, Laurita KR, Rosenbaum DS. Unique
properties of cardiac action potentials recorded with
voltage-sensitive dyes. J Cardiovasc Electrophysiol. 1996;7:1024–1038.[Medline]
[Order article via Infotrieve]
13.
Efimov IR, Ermentrout B, Huang DT, Salama G.
Activation and repolarization patterns are governed by different
structural characteristics of ventricular
myocardium: experimental study with voltage-sensitive dyes
and numerical simulations. J Cardiovasc Electrophysiol. 1996;7:512–530.[Medline]
[Order article via Infotrieve]
© 1998 American Heart Association, Inc.
Basic Science Reports
High-Resolution, Three-dimensional Fluorescent Imaging Reveals Multilayer Conduction Pattern in the Atrioventricular Node
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—The
atrioventricular node (AVN) is the only normal
electrical link between the upper and lower chambers of the heart. The
AVN modulates transmission of impulses, thus coordinating the
contraction of the atria and ventricles.
Key Words: atrioventricular node • conduction • mapping • imaging
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Since the first
morphologic description by Tawara,1 the
atrioventricular node (AVN) has been one of the most
studied, yet least understood, structures of the heart. The
three-dimensional pattern of activation of the AVN has been mostly
speculative because of the exceptional architectural and functional
complexity of this rather small region2 and the
lack of appropriate experimental techniques. The glass microelectrode
has been the only reliable tool to visualize electrical responses of
AVN cells from different layers. However, microelectrodes cannot
provide a map of activation. This is because at a single position,
cells located closer to the surface may be activated
substantially earlier than deeper cells. Moreover, the depth of
microelectrode impalement is either unknown or very difficult to
verify. Finally, simultaneous use of more than two
microelectrodes has proved to be prohibitively
difficult.2
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
The isolated rabbit AVN preparation as well as the experimental
techniques have been previously described in
detail,6 thus will be outlined only briefly.
Rabbits (n=5) of either sex weighing 2 to 3 kg were
anesthetized, and after a midsternal incision, the heart was
removed and placed onto a Langendorff apparatus, where it
was retrogradely perfused with oxygenated (95%
O2, 5% CO2)
room-temperature, modified Tyrode's solution. The heart was stained by
a bolus injection with 350 µL of 2 mmol/L solution of
di-4-ANEPPS in DMSO. After staining, the heart was removed from the
Langendorff apparatus and the right atrial AVN preparation
was dissected.6 The final preparation contained
right atrial tissues with the triangle of Koch and the AVN. This
preparation was pinned down on a thin silicon disk and superfused at
37.0±0.1 Co, with a flow rate of 30 mL/min;
12.5 mmol/L BDM was added to suppress contraction-induced
distortion of optical signals. As recently shown, this concentration of
BDM has no adverse effect on rabbit AVN
conduction.7
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Similar to previously described records6
from the AVN area, we found that optical signals recorded from the
distal AVN region, close to the bundle of His, consistently
exhibited multiple components. Thus in the time interval between an
atrial input activation and the subsequent activation of the bundle of
His, two distinct optical action potentials could be identified. We
hypothesized that the first of these components represented
depolarization of cells located in the superficial envelope of
transitional cells, whereas the second component
represented the delayed depolarization of cells in the
compact nodal region. 
, bottom left panel). The inscription of
this AP preceded or coincided with the bundle of His electrogram
activation and was absent when the latter was missing because of
conduction block (in this example, a 3:2 Wenckebach cycle was observed
and the second beat was blocked in the AVN). It is evident that the AP
of this deeper cell always coincided with the second component of the
optical signal. AVN block was always associated with the absence of
both the cellular action potential and the second component in the
optical signal. Finally, dissociation between the two optical
components clearly reflected the overall AVN delay. That is, a marked
dissociation in the first beat (blue strip) was due to the long delay
between the atrial and His activations. In contrast, the shorter
conduction delay in the third beat (yellow strip), which followed the
blocked one, resulted in less separation between the components in the
optical signal.
View larger version (53K):
[in a new window]
Figure 1. A multilayer structure is schematically drawn (top
panel) to represent a cross section of the
atrioventricular node (AVN) perpendicular to the
endocardial atrial surface. The compact node (red) resembles a slightly
flattened football5 covered with a superficial layer of
transitional cells (brown) that form an envelope, providing a
connection between the atrial tissue and the compact node. The compact
node, in turn, connects to the bundle of His (green). Connective tissue
protrusions are shown in black. Arrows show hypothetical wave fronts in
the transitional and compact nodal fibers. Optical recordings
obtained by a 16x16 photodiode array were accompanied by bipolar
electrograms from the atrial input to the AVN (*) and from the bundle
of His (*). Depth of optical recordings was probably limited to
300 to 500 µm.11 12 In addition, a roving glass
microelectrode was utilized to impale cells in different AVN layers
(black and white circles). Each of the lower panels illustrates three
consecutive heartbeats with simultaneously acquired atrial
input and His electrograms, along with optical and microelectrode
signals. See details in the text. 
, bottom right
panel). Although it cannot be proven that this cell had exactly the
same x-y coordinates as the previously impaled cell, it is
safe to estimate the horizontal dislocation to <100 µm. This
cell had a faster upstroke and depolarized earlier after the atrial
activation in each beat. In addition, it was activated even
when AVN block was present. Thus this was a typical AVN
transitional cell.2 Note that its AP coincided
with the first of the two components in the optical signal. Moreover,
both the cellular AP and the first of the optical components in each
beat were related to the atrial activation and significantly preceded
the bundle of His activation. ). The optical recordings were
also able to discriminate the occurrence and location of conduction
block. Note, for example, that in the left bottom panel of Figure 1, the optical signal in the second (blocked) atrial beat did not exhibit
an appreciable second hump, suggesting that the block of the wave front
occurred at a substantial distance before reaching the source of the
optical signal. In contrast, in the right bottom panel of Figure 1, a
small but distinct second hump in the optical signal in the second
(blocked) atrial beat is evident, suggesting that a fading wave front
stopped closer to the source of the optical signal. illustrates the individual
optical signals obtained in one preparation during propagation of a
single atrial beat. Note that the characteristic dissociation of the
optical signals into two components becomes progressively more
pronounced when moving from the input (left) to the output (right) of
the AVN. The ratio of the amplitudes of the two components is not
constant. This can be explained by the variable thickness of the
layer of transitional cells on the endocardium, which causes different
relative contributions to the total optical signal of the transitional
and the deeper nodal cells. Thus a relatively larger amplitude second
component should be expected at more distal (right) coordinates where
the late-depolarized deeper cells are located. On the other hand, a
larger-amplitude first component should be expected at the proximal
(left) coordinates, where the earliest-depolarized transitional cells
are abundant. At certain intermediate coordinates, the two components
are of comparable amplitude.
View larger version (83K):
[in a new window]
Figure 2. Two hundred fifty-six simultaneous
optical traces were obtained from a 6x6 mm area that includes the
atrioventricular node (AVN). Each trace was
recorded from a 320x320 µm spot. Different colors are used
to distinguish signals from the posterior approaches to AVN (blue), the
anterior approaches (green), and the compact nodal area (red). Signals
in black are from the area of the opening of the coronary sinus
and were ignored during the analysis. This scan was performed
during a premature atrial beat with a coupling interval of 140
ms. . First components in the distal node
were significantly (see Figure 2, rows 12 to 14, columns 12 to 16)
smaller relative to second components. However, it was of comparable
amplitude with first components recorded from the proximal node. It
is evident from Figure 3 that two distinct wave fronts were present
during the propagation of both the basic (left panels) and the
premature (right panels) beats. The first wave front (upper maps) ran
over the envelope of transitional cells and brought the excitation from
the pacing site to the AVN region. This wave front encircled the
opening of the coronary sinus (shown in black) and
activated the viewing area within 30 ms during the basic beat
and within 55 ms during the premature beat. The second wave
front (the bottom maps) was only traceable in a portion of the viewing
field that corresponds to the location of the compact node and the more
distal nodal region. Conduction in this deeper region of nodal fibers
was initiated after and probably by the superficial wave front. This
conduction was substantially slower. This slowing of conduction was
especially pronounced during premature beats. In Figure 3, it took 100
ms to transverse the deeper cellular layers; the entire surface was
activated within 55 ms. In addition, the area of slow
conduction, defined by the presence of secondary components in the
optical recordings, was consistently larger during
propagation of premature atrial beats. The phenomena described above
were consistently observed in all five preparations.
View larger version (68K):
[in a new window]
Figure 3. Activation maps during basic (left) and premature
(right) beats. The activation times at each individual location were
determined by -(dF/dt)max
(F=fluorescence).13 The isochronal lines were
drawn with a 5-ms resolution. Spread of activation is shown as
white-to-red gradient. The left pair of maps illustrates the conduction
of a basic drive beat at a cycle length of 300 ms; the right pair of
maps was obtained during the propagation of a premature beat with a
coupling interval of 180 ms. See text for details.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
The novel combination of microelectrodes and high-resolution
optical imaging with voltage-sensitive dyes can now be successfully
added to the arsenal of tools used to study the electrophysiology of
the AVN. This novel integration of techniques has helped to directly
demonstrate the previously deduced pattern of multilayer AVN conduction
and to provide the first detailed three-dimensional mapping of
transmission of basic and premature beats through the AVN. This has
been achieved by reconstructing the three-dimensional pattern from
two-dimensional optical mapping assisted by the functional information
obtained with microelectrodes and macroelectrodes.
Acknowledgments
This study was supported in part by a Northeast Ohio AHA
Affiliate Grant-in-Aid (210-BG) to Igor Efimov.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1.
Tawara S. Das Reizleitungssystem des
Säugetierherzens. Jena, Germany: Fisher, 1906.
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