Download Atrio-Ventricular Conduction with and without AV Nodal Delay: Two

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in PresS. Am J Physiol Heart Circ Physiol (May 11, 2007). doi:10.1152/ajpheart.00115.2007
Atrio-Ventricular Conduction with and without AV Nodal Delay:
Two Pathways to the Bundle of His in the Rabbit Heart
Short Title: AV conduction without AV delay
By
William J. Hucker, BS1, Vinod Sharma, PhD2, Vladimir P. Nikolski, PhD1,2, Igor R.
Efimov, PhD1
Affiliations: 1 Biomedical Engineering Department, Washington University, St. Louis,
Missouri; 2Medtronic, Minneapolis, Minnesota
Correspondence author: Igor Efimov, Biomedical Engineering Department, Washington
University, 1 Brookings Drive, St. Louis, MO 63130. Email: [email protected]
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Copyright © 2007 by the American Physiological Society.
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Abstract
Background: The electrophysiological properties of atrioventricular nodal dual pathways
have traditionally been investigated with premature stimuli delivered with right atrial
pacing. However little is known about the functional characteristics of AV nodal inputs
outside of this context.
Methods: Superfused rabbit triangle of Koch preparations (n=8) and Langendorffperfused hearts (n=10) were paced throughout the triangle of Koch, and mapped
electrically and optically for: activation pattern, electrogram and optical action potential
morphologies, stimulation thresholds, and stimulus-His (S-H) intervals. Optical mapping
and changes in His electrogram morphology were used to confirm the activation
pathway.
Results: Pacing stimuli 2mm above the tricuspid valve (TV) caused fast pathway
activation of the AV node and His with a threshold of 2.4±1.6mA. An area directly below
the coronary sinus had high thresholds (8.6±1.4mA) that also resulted in fast pathway
excitation (P<0.001). S-H intervals (81±19ms) for fast pathway activation remained
constant throughout the triangle of Koch, reflecting the AV delay. Stimuli applied <2mm
from the TV resulted in slow pathway (SP) excitation or direct His excitation
(4.4±2.2mA threshold, P<0.001 compared to fast pathway). For SP/His pacing, S-H
intervals showed a strong dependence on the distance from the His electrode, and were
significantly lower than S-H intervals for fast pathway activation. SP/His pacing also
displayed characteristic changes in His electrogram morphology.
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Conclusions: Optical maps and S-H intervals for SP/His activation suggest that AV
conduction via SP bypasses the compact AV node via the lower nodal bundle, which may
be utilized to achieve long term ventricular synchronization.
Keywords: Resynchronization therapy, Atrioventricular node; dual pathway
electrophysiology; optical mapping; slow pathway.
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Introduction
In normal physiology, the atrioventricular node (AVN) serves as the gateway to
the His-Purkinje system and is responsible for the delay between atrial and ventricular
excitation. The AVN is located near the apex of the triangle of Koch (24), and it has at
least two inputs, each with unique electrophysiological properties (22). The fast pathway
(FP) input to the AVN, near the triangle apex, has a relatively short conduction delay and
long refractory period, while the slow pathway (SP) input, in the isthmus between the
tricuspid annulus and the coronary sinus ostium in both rabbits and humans (14; 16; 20),
possesses a relatively long conduction delay and relatively short refractory period (16).
The distinct functional characteristics of these pathways are critically important in
clinical AV nodal reentrant tachycardia (AVNRT).
Because of the clinical importance of dual pathway electrophysiology in AVNRT,
studies investigating AV nodal inputs, and the SP in particular, have done so in the
context of differential conduction between the FP and SPs, through the use of premature
stimuli (S2 beats) applied in the high right atrium or on the crista terminalis (CrT) (20;
29). SP activation in such a manner engages the normal atrial myocardium, transitional
cells, and the SP itself, with the end result of slower conduction to the His than through
the FP. Without a premature S2 stimulus, the SP has very little involvement in AV
conduction, normally acting as a dead end pathway (1). Therefore, there have been few
studies which have investigated SP physiology without premature stimuli, though it has
been established that the SP possesses unique pacemaking properties (8).
It has been commonly accepted that each AV nodal input funnels into a common
pathway to the His bundle (21). However the recent demonstration of “His electrogram
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alternans” by Zhang, et al (29) suggests that there may in fact be two separate pathways
to the His bundle. This study revealed characteristic changes in His electrograms which
were caused by whether the His bundle was excited by the SP or the FP, which argues
that the influence of the activating pathway spreads farther than the AVN to the His itself.
Because of the clinical sequelae associated with right ventricular pacing (18; 26),
direct His bundle pacing has emerged as a cardiac resynchronization therapy (6; 7; 25;
27) and therefore the clinical relevance of His activation has increased. As its name
implies, direct His bundle pacing stimulates the His bundle with a pacing catheter placed
on the His bundle, at the apex of the triangle of Koch. However to our knowledge no
studies have investigated pacing stimuli applied within the triangle of Koch, directly
stimulating the SP and FP. We hypothesized that pacing stimuli applied in this region
may reveal whether or not two pathways to the His bundle exist. In this study we
mapped conduction, electrogram morphologies, and pacing thresholds for pacing stimuli
applied throughout the triangle of Koch. Our data suggest that the SP can be paced in a
relatively large area of the right atrium to produce His activation in the rabbit, while
avoiding the AV delay. However the direct clinical relevance of this study will have to
be proven in the human in the future.
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Methods
New Zealand white rabbits (n=18, 2.5 – 3 mos. old, 2-3 kg) were anaesthetized
with 100mg/kg pentobarbital and 1000 IU heparin intravenously, after which a midsternal
thoracotomy was performed and the heart removed. The heart was Langendorff perfused
with oxygenated (95% O2, 5% CO2) Tyrode’s at 37oC and received 50 µL of 5µ
Di-4-
ANEPPS (Molecular Probes, Eugene OR) over five minutes. We conducted a study in
superfused isolated AV junctions (n=8) and in Langendorff-perfused hearts (n=10) with
the AV junction exposed via right atriotomy (a more complete description of the
Langendorff perfused preparation is provided in the online data supplement). For
superfused experiments, the AV junction was dissected in cold Tyrode’s (0oC), and the
sinoatrial node removed. The preparation was superfused at 30mL/min with Tyrode’s
containing 15mM of the excitation-contraction uncoupler 2,3-butanedione monoxime
(BDM) (Sigma, St. Louis, MO) to prevent motion artifacts (4). A 16x16 photodiode
array was used with an optical mapping system that has been described previously (9).
Optical signals were sampled at 1.5 kHz, averaged and lowpass filtered at 120 Hz.
Optical activation maps display the optical signal derivative, which corresponds to
wavefronts of excitation.
Electrogram Recordings
Electrodes were placed on the interatrial septum (IAS) and crista terminalis (CrT),
and a quadrupole electrode on the His bundle recorded both the superior and inferior His
electrogram (SHE and IHE, respectively) to monitor fast-His and slow-His excitation.
Changes in His excitation and His electrogram morphology can occur due to changes in
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the AVN excitation pathway, previously referred to as His electrogram “alternans” (28;
29) due to the specific pacing protocol and alternating conduction pathways. If the AVN
and His are excited by the SP (i.e. slow-His excitation), the IHE has a larger amplitude
than when the AVN is activated by the FP. Conversely, when the FP excites the AVN
and His (i.e. fast-His excitation), the SHE has a larger amplitude than with slow-His
excitation. The location of each electrode is shown in Figure 1A.
A fourth roaming electrode was used to record electrograms throughout the
triangle of Koch. A schematic of the triangle of Koch is shown in Figure 1A. The
reference lead was located 3mm from the electrode tip. The Teflon-coated 0.13mm Pt/Ir
wire tip had ~0.07mm stripped to mimic a clinical hemispheric tip. This electrode was
mounted on a force transducer (FORT25, WPI Inc., Sarasota, FL) to control contact
force, ensuring that contact force was minimal and consistent between locations. The
electrode was moved in 1mm increments by a motorized micromanipulator throughout
the triangle of Koch (grid in Figure 1A). Electrograms were recorded at 1.5 kHz
(National Instruments, Austin TX) and each electrode location was digitally
photographed.
Stimulation Protocol
The preparation was paced from two electrodes: the IAS electrode and the
roaming electrode (Figure 1A). IAS pacing was constant at 2x threshold (~2mA), with
2ms pulses and 300ms cycle length (Figure 1B). IAS pacing simulated sinus pacing and
was used to mask the AV junctional rhythm which originates in the SP (8), as well as to
maintain tissue excitability in a consistent state as the roaming electrode was moved from
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location to location. Stimulation thresholds were determined using a ramp of unipolar
pulses (0.5ms pulses, 300ms cycle length, amplitude ranging from 0.33mA to 10mA
increasing by 0.33mA with each pulse, Figure 1B). Each ramp pulse was delivered 4560ms before an IAS pacing pulse (Figure 1B). The pacing ramp was delivered from the
roaming electrode for anywhere from 14-24 locations throughout the triangle of Koch
(grid in Figure 1A) allowing quick determination of threshold for each location. Pacing
threshold was defined as the amplitude of the ramp pulse that caused a shift in the
activation pattern from IAS pacing to stimulation beginning from the roaming electrode.
Statistics
Statistical correlation were determined with the Pearson’s correlation
coefficient R using a two-tailed student t-test to determine whether the null
hypothesis of R=0 was true, indicating that the two variables were not correlated
(13). Group comparisons were performed using a two-sample student t-test. Data
are represented as mean ± standard deviation.
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Results
Identifying the Slow Pathway
The schematic in Figure 1A indicates the approximate location of the inputs
to the slow pathway (SP) and fast pathway (FP) with arrows. While the anatomic
substrate of the SP is often thought to be the INE (16; 20), the anatomic substrate of
the FP is less well defined and consists of transitional cells (TCs) that overlay the
compact AV node (CN).
Figure 2A-B compares electrograms and optical action potentials (OAPs)
recorded during IAS pacing. Each trace begins at the moment of IAS pacing
(pacing artifact shown in the electrograms), is 300ms long, and color coded to
distinguish different signal morphologies. Figure 2C directly compares
electrograms and OAPs recorded from the same site. During IAS pacing, the AVN
is activated by the FP, and the SP acts as a dead end pathway. Yellow electrograms
all have a sharp signal immediately following the pacing artifact, which signifies
fast conduction through the atrial myocardium. Yellow OAPs have atrial action
potential morphology. Blue electrograms contain both a fast signal directly after
the pacing artifact (similar in timing to that seen in yellow traces) and another sharp
spike ~ 80ms later, which reflects His excitation. Blue OAPs have two humps
which are the summation of atrial and His excitation. The first hump corresponds
to atrial activation, while the second hump has the distinct plateau phase of His
activation. Brown and purple electrograms contain several components. The first
component matches in time the signals seen in the yellow electrograms, and is
followed by complex low amplitude biphasic recordings that are consistent with
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slow conduction during the interval between atrial and His excitation. Brown
electrograms are located near the anatomic location of the AVN, and brown OAPs
have a nodal morphology. Purple electrograms and OAPs are located along the SP.
There is overlap in the characteristics of the brown and purple electrograms and
OAPs. Traces shown in black were difficult to classify because they share
characteristics of two types of activation. White traces were recorded from dead
ventricular tissue and therefore contain only noise.
In Figure 2C, the sharpest spikes in the electrograms correspond to the
dF/dtmax of the fluorescent optical signals. In the nodal and SP traces, the slow
conduction characteristics of the electrograms correlate well with the OAPs from
the same location. One exception is the spike seen in the nodal electrogram which
occurs during the plateau of the OAP. Based on the timing of this spike relative to
the bipolar inferior His electrogram (IHE), this spike most likely reflects excitation
of the NH region of the node, or the lower nodal bundle (LNB), where excitation
accelerates into the His bundle (2). Data supplement Figure 1 shows the
electrograms and OAPs from a second preparation to show that the signal
morphologies were consistent between preparations.
Pacing stimuli applied within the triangle of Koch produced different
activation patterns, depending on where the stimuli were delivered. Figure 3A
provides a schematic of the location of the conduction system in this preparation for
clarity, similar to the schematic shown in Figure 1A. In this schematic, the
approximate location of transitional cells (TC) has been marked with the
crosshatched area. Figure 3B illustrates the activation pattern caused by IAS
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pacing. Activation originated from the IAS electrode and spread rapidly across
atrial tissue and TCs that lie above the conduction system in the triangle of Koch,
which activated the FP of the AVN. Because the activation map displays dF/dt,
there is no conduction visible from approximately 50-70ms when there is
exclusively slow AV nodal conduction (which has a low amplitude dF/dt). After
this period His excitation occurred, which actually began in the LNB region (shown
in orange). The corresponding superior His electrograms (SHE) is shown within the
panel which shows that the interval between IAS pacing and His excitation was
approximately 70ms.
At many pacing locations, pacing from the roaming electrode caused an
atrial/fast pathway (FP) activation pattern similar to that seen with IAS pacing.
Figure 3C, illustrates FP activation of the AV node with a pacing stimulus applied
close to the SP on the lower crista terminalis (CrT). From this location, a fast wave
of excitation originated from the location of the roaming electrode (square pulse on
lower left of 3C), spread across the atrial tissue and transitional cells (TCs) that lie
above the conduction system in the triangle of Koch, and then produced His
excitation after a period where no conduction was visible. In the SHE trace below,
the His electrogram did not change morphology from IAS pacing, maintaining a
fast-His morphology.
When the roaming electrode was placed within ~2mm of the tricuspid valve
within the triangle of Koch, it excited the SP (Figure 3D). SP activation appeared as a
slow, narrow activation pattern which moved towards the apex of the triangle of Koch.
Upon reaching the AVN region, excitation continued toward the His bundle without
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pause (which can be seen in the timing of the SHE trace in panel D with His excitation
occurring 43ms after stimulation) and also spread retrogradely across the IAS. In the
SHE trace, the His electrogram morphology changed to a slow-His morphology with a
smaller amplitude during SP pacing (compare SHE morphology in panels B and D), and
returned to its original morphology after the ramp ended. Notice that the interval
between stimulation and His excitation (the S-H interval) is paradoxically longer for fastHis excitation than for slow-His excitation. This phenomenon is explored further in
Figure 7. Slow-His electrograms were observed in 6 of 8 superfused experiments.
Similar optical activation patterns for SP and atrial/FP activation were observed in
the AV junctions of Langendorff perfused hearts. The whole heart preparation is shown
in Figure 4A. Activation maps of SP pacing are shown in Figure 4B-4C, with the
corresponding IHE trace below panel C. Similar to Figure 3D, SP pacing in the whole
heart produced a slow narrow activation pattern followed without pause with His
activation (Figure 4B). In the intact heart, His activation was followed by a robust
ventricular optical signal which could be seen through the overlying atrial tissue (Figure
4C). Atrial activation preceded ventricular activation by ~10-15ms (light blue activation
in 4C).
IAS pacing (Figure 4D-4F) produced a fast wave of excitation that spread
across the atrial tissue and transitional cells (Figure 4D). Following atrial
activation, excitation spread from the AV nodal region in two directions: the
wavefront of His excitation spread toward the His electrode and a wavefront of
decremental conduction spread down the SP and died out (Figure 4E). Ventricular
activation followed His excitation (Figure 4F). A small amplitude fast-His potential
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is seen in the IHE with IAS pacing (i.e. the IHE His deflection is larger with SP
pacing than with IAS pacing).
Pacing Within the Triangle of Koch
Figures 2-4 illustrate that the SP location can be identified with electrograms
and OAP morphology, and SP vs. FP activation can be differentiated with
activation patterns and His electrogram morphology in both the superfused and
whole heart preparations. Using both activation patterns and His morphology, we
identified which activation patterns occurred for multiple pacing locations
throughout the triangle of Koch. Figure 5 displays a summary of where the
different activation patterns occurred and the pacing thresholds for each in for the
superfused experiments. Online data supplement Figure 2 shows a representative
example of pacing stimuli and activation patterns recorded from many pacing
locations throughout the triangle of Koch. Generally, pacing stimuli applied within
~2mm of the tricuspid valve excited the SP (similar to Figures 3D and 4B), or the
His directly when pacing near the apex of the triangle of Koch. Direct His
stimulation was defined as fast conduction directly after stimulation which had a
stimulus-His interval <10ms and occurred in a small area near the His electrode. On
average, the pacing threshold for SP/His pacing was 4.4±2.2mA. Pacing stimuli
applied further away from the tricuspid valve generally excited atrial tissue,
producing FP excitation of the AV node with an average stimulation threshold of
2.4±1.6mA (P<0.001 compared to SP/His pacing thresholds). Additionally, there
was one area between the coronary sinus and the tricuspid valve which had very
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high pacing thresholds (8.6±1.4mA; P<0.001 compared to atrial/FP thresholds).
Pacing this region excited atrial tissue and the FP of the AV node. OAPs from this
region were quite noisy and very dissimilar from the large amplitude atrial OAPs
recorded from other areas of the triangle of Koch (see online data supplement
Figure 2). High pacing thresholds and very low OAP amplitudes suggest that few
excitable cells are located in this region.
There were several instances where different activation patterns occurred at
different thresholds, typically with atrial activation followed by SP or His excitation
at higher stimulus intensities. Very similar pacing thresholds for each activation
type were observed in the Langendorff perfused hearts (see online data supplement,
and data supplement Table 1), with the SP/His threshold statistically the same as the
RV pacing threshold.
It is important to note that once SP activation of the His bundle occurred, the
SP would excite the His bundle throughout the duration of the stimulation ramp in a
1:1 fashion. However, this was not the case for all pacing locations which produced
FP excitation. As shown in Figure 6, pacing near the superior border of the AVN
often disrupted AV conduction by causing 2:1 AV block or prolonged AV
conduction. In the example shown in Figure 6, IAS pacing conducted 1:1 to the His
before the stimulation ramp crossed threshold, seen in both the electrograms and the
OAP recorded above the His bundle. Once the stimulation ramp crossed threshold,
it excited atrial tissue (seen in the shift in both the OAP and the CrT trace) but
excitation did not propagate to the His. In the next beat, excitation propagates to
the His (seen in the OAP and the IHE), and conduction continues in this 2:1
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fashion. Therefore, AV conduction was disrupted by pacing in this location.
Pacing stimuli delivered to this area of the triangle of Koch caused 2:1 AV block or
prolonged AV conduction in 5 of 8 experiments. Once AV conduction was
disrupted, it did not fully recover in any of the 5 experiments, which suggests that
the effect of pacing in this area was not neurologically mediated.
The interval between the stimulation artifact and the His electrogram (S-H
interval) was measured for each pacing location. The S-H interval usually
stabilized 3-5 pulses after threshold was reached, and these stable values were
measured. S-H intervals at high stimulus intensities, where a greater amount of
tissue was presumably depolarized by far field stimulation, were discarded because
the S-H interval at the end of the ramp was generally different than the stable S-H
interval. Rarely, S-H intervals did not stabilize and linearly decreased throughout
the ramp, in which case the range of S-H intervals was noted. Also, we discarded
from the analysis any S-H interval that was recorded after AV conduction was
disrupted as shown in Figure 6. Data from one representative experiment is shown
in online data supplement Figure 3A.
S-H intervals were divided into two groups, those associated with atrial/FP
activation and those associated with SP/His activation. When plotted against the
horizontal distance from the His electrode, atrial S-H intervals showed no distance
dependence (P=NS, Figure 7A) but instead remained at a constant level of
81±19ms. This constant interval between an atrial stimulus and His excitation is the
AV delay. Surprisingly, S-H intervals associated with SP and His excitation
exhibited a strong correlation with distance from the His electrode (P<0.001; Figure
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7B). If SP excitation experienced the AV delay, one would expect a nonlinear jump
from direct His excitation at small distances from the His electrode to values at or
above the AV delay of 81±19ms. Moreover, the S-H intervals during SP pacing
remained well below the AV delay measured from atrial/FP activation (81±19ms
delay for FP excitation and 53±25ms delay for SP excitation
4mm from the His
electrode; P<0.001). S-H intervals for SP/His excitation from each superfused
experiment are shown in online data supplement Figure 3B.
Figure 7C shows an example of S-H intervals measured during SP pacing
and IAS pacing with the last ramp stimulus, which excited the SP, and the first IAS
pacing pulse after the ramp. The S-H interval was 31ms for the last ramp stimulus,
and increased to 64ms when the His bundle was excited by IAS pacing.
Paradoxically, the S-H interval for the SP was shorter than the S-H interval for the
FP. The shift from slow-His to fast-His potentials is seen in both the IHE and SHE
traces. Similar S-H interval trends for both atrial and SP excitation were observed
in Langendorff perfused hearts, with SP S-H intervals shorter than FP S-H intervals
on average (see online data supplement Figure 4).
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Discussion
Our results support previous findings that several layers of conduction exist in the
triangle of Koch (10). Now we further demonstrate that these layers can be differentially
engaged with varying stimulus strengths and pacing locations. We found that stimulation
thresholds for atrial excitation are significantly lower than for SP/His activation, with the
exception of one area beneath the CS. Using the activation pattern documented by
optical mapping, as well as slow-His electrograms, we have verified SP excitation and
found that not only do S-H intervals decrease linearly as the stimulus location approaches
the His bundle for SP excitation, while S-H intervals associated with atrial/FP activation
remain nearly constant, but also that paradoxically S-H intervals for the SP are shorter
than those recorded for FP activation.
Due to the different modalities used to study the AV junction, there is a “lack of
common terminology” for its components (15). For instance, many previous studies,
including studies from our group, have referred to the AV nodal extension located in the
isthmus between the coronary sinus and the tricuspid valve as the “posterior nodal
extension” (8; 16). However, a naming task force has indicated that “inferior nodal
extension” (INE) more accurately describes its location when the heart is anatomically
oriented (5). Previous studies have described layers of atrial and transitional cells (TCs)
overlying the components of the conduction system in the triangle of Koch, such as the
INE, AVN, and His bundle (1). Functional studies have provided evidence that the INE
is the anatomic substrate of the SP (16; 20), however there is debate regarding whether
the INE itself, the transitional cells overlying the INE, or a combination are the true
substrate of the SP (19). Our results can not distinguish which cell layer or structure
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produced SP excitation; however our optical mapping results confirm that this pathway
can be reliably excited by pacing stimuli delivered within ~2mm of the tricuspid valve
within the triangle of Koch.
Plotting S-H intervals of atrial excitation against the distance from the His
electrode revealed no correlation (Figure 7A). The S-H interval of atrial activation is
composed of two intervals: conduction time in the atrial layer and FP (S-AVN interval),
and AV nodal conduction to the His bundle (the AV delay). Because conduction in the
atrial layer is fast (~35 cm/sec) (23), small changes in the distance between the
stimulating electrode and the His electrode change the S-AVN interval minimally.
Therefore the main determinant of the S-H interval is the AV delay, which remains
essentially constant.
On the other hand, plotting S-H intervals of SP excitation versus distance from the
His electrode showed a strong correlation (Figure 7B). A greater dependence on distance
would be expected for SP activation because, as the name implies, the SP conducts
slowly (~7cm/sec) (23). Therefore one would expect the S-AVN interval to decrease
slightly as the distance between the stimulus and the AVN decreased. However, once
conduction time in the SP is minimal, the S-H interval should be determined by the AV
delay, which is 81±19ms according to the atrial activation data (Figure 7A). As the His
bundle is approached further, the S-H interval should then jump to a very small value
when direct His activation occurs. Instead, the raw data (shown in data supplement
Figure 3) and the pooled data in Figure 7B show S-H intervals almost entirely less than
81ms and a linear decrease of the S-H interval as the His electrode is approached.
Activation patterns for SP pacing in Figure 3D and 4B show SP excitation proceeding
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linearly to the His bundle without pause. This data suggests that there is no dependence
of the SP/His S-H interval on the AV delay, implying that SP excitation avoids the AV
delay and excites the His bundle directly.
How can the slow pathway conduct to the His more quickly than the fast pathway?
Many studies have investigated the rate dependent properties of the SP vs. the FP
in the context of premature stimuli, most commonly with pacing stimuli applied in the
high right atrium or to the CrT (20; 29). Based on these studies, the SP was given its
name because excitation took longer to reach the His bundle than through the FP.
However we found that SP excitation reached the His bundle faster than FP excitation.
Despite this apparent paradox, our results are fully consistent with the large body of
evidence concerning the SP for two reasons. First, SP excitation in previous studies
traveled the full length of the SP, which our data indicate would have an S-H interval
similar to FP excitation (right side of Figure 7B). In the context of a premature beat, this
activation would take even longer. In the absence of pacing stimuli applied directly to
the SP, SP conduction to the His only occurs when a premature stimulus encounters a
refractory FP (Figure 8A). Because conduction traveling the full length of the SP takes
the same amount of time as the AV delay (or longer with premature stimuli), it would be
impossible to recognize that SP conduction avoided the AV delay. Only through pacing
the SP incrementally along its length, as we did in this study, would it become apparent
that SP excitation does not experience the same AV delay as FP excitation. Secondly, in
this study we engaged the SP directly, potentially avoiding any conduction delay that
may occur at the interface of the atrial tissue with the SP (Figure 8A). This interface may
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be responsible for an additional delay in conduction, which would slow SP conduction
even further (23).
In this study, we paced at 300ms within the triangle of Koch, close to the AVN
itself. Therefore one interpretation of our data is that because of the His bundle
proximity, pacing stimuli excited the His bundle directly when stimuli were delivered
within 2mm of the tricuspid annulus, and the SP was not involved. Direct stimulation of
the His bundle would certainly result in short S-H intervals. However this possibility is
unlikely for two reasons. First, SP activation was confirmed by being visualized with
optical mapping. Second, pacing stimuli applied much closer to the His bundle itself
(such as on the right side of Figure 5, and the right side of the grid shown in data
supplement Figures 2 and 3) produced atrial activation with an AV delay of ~80ms or
longer throughout the entire pacing ramp. Therefore it seems that the tissue area initially
captured by the pacing stimulus was rather small. Also the S-H intervals that were
analyzed were measured 3-5 beats after the pacing ramp reached threshold (i.e. 2X the
pacing threshold), which also limits the extent of tissue that was excited at that point in
the pacing ramp.
There is a growing consensus in the literature that the His, lower nodal bundle
(LNB), and the INE form a continuous structure distinct from the compact AVN tissue on
a morphological, molecular, and functional basis (1; 17; 20). We interpret our data to
suggest that instead of a final common pathway as classically suggested by Mendez and
Moe ,(21), there are instead two pathways to the His bundle: one through the compact
node and the other through the LNB. Medkour et al (20) and Khalife et al (16) put forth
similar arguments suggesting that the INE connects to the His bundle through the LNB,
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and the FP passes through the compact node. The concept of two pathways to the His is
supported by changes in His electrogram morphologies (i.e. slow-His and fast-His
electrograms). The distinct His electrogram signatures of FP or SP activation indicate
that activating pathway influences how the His bundle, or which part of the His bundle, is
depolarized (29).
Based on this dual pathway concept, we suggest that SP excitation begins in either
the inferior TCs, or the INE itself, and travels via the INE to the His bundle through the
LNB with a gradient of conduction velocities, as shown in the schematic in Figure 8B.
As excitation approaches the His, the level of Cx43 increases (17) and conduction
velocity increases until the His bundle is reached. FP activation from the atrial tissue
surrounding the AVN funnels into the compact node via the TCs that overlie the AVN.
The AV nodal delay is due to the green compact nodal tissue and its connecting TCs (3),
after which excitation passes to the His.
Interestingly, we identified a small region below the CS where stimulation
thresholds were significantly higher than the surrounding myocardium. This area may
serve as a localized area of block, contributing to anisotropy in the atrial myocardium and
may play a role in atrial flutter and fibrillation, similar to the block zone in the intercaval
region that can maintain typical atrial flutter (11).
Clinical Implications and limitations
The existence of a SP “bypass tract” in the right atrium expands the area that
potentially could be paced to achieve His bundle excitation with direct His bundle pacing
procedures, alleviating the difficulty of pacing the small His region located close to aorta
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(7). Locating the SP pacing location could be guided by the slow conduction
characteristics in SP electrograms, shown in Figure 2 and shown clinically (12), which
are used to guide SP ablations during AVNRT procedures.
SP pacing may very well have lower pacing thresholds than that required for His
bundle pacing (27) due to the fibrous tissue surrounding the His bundle. Our experiments
in the rabbit revealed that SP pacing and direct His pacing had pacing thresholds that
were not statistically different. However in the rabbit, the endocardial side of the His
bundle is only covered by a small amount of connective tissue, whereas in the human, the
His bundle is encased in the fibrous tissue of the central fibrous body. Therefore it is
possible that SP pacing in the human will have a lower threshold than His bundle pacing.
Additionally, pacing thresholds for RV epicardial pacing and SP/His pacing were not
statistically different in the whole heart experiments (data supplement Table 1),
suggesting that clinical SP pacing thresholds may be close to pacing thresholds for RV
pacing. However, this study was conducted in normal rabbit AV junction preparations
where no attempt was made to disrupt AV conduction. Whether or not His capture from
the SP will be possible in a diseased AV junction remains to be shown.
Conduction curves are the gold standard used to investigate AV nodal dual
pathway electrophysiology. Due to the number of pacing locations investigated in this
study, it was not possible to construct conduction curves for each. Although conduction
curves are very useful clinical tools, optical mapping in our preparations confirmed the
distinct activation patterns of FP vs. SP activation (Figures 3 and 4), and our optical
mapping of SP excitation corresponds very well to SP optical mapping performed during
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standard S1-S2 protocols (23). Additionally, slow-His potentials provided another line of
evidence for FP vs. SP participation in conduction.
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Acknowledgements
This project was funded by Medtronic, Inc. and a Whitaker Foundation Graduate
Fellowship.
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Figure Legends:
Figure 1: A: Schematic of the triangle of Koch, with transitional cells omitted from the
schematic for clarity. IAS pacing was delivered from the location marked with a red box.
The roaming electrode was moved throughout the triangle of Koch, to the locations
marked with blue boxes. Locations of bipolar electrodes marked with gray circles, and
approximate location of entry to the fast pathway (FP) and slow pathway (SP)
are shown. B: 0.5ms unipolar pulses increasing in amplitude from 0.33mA to 10mA,
were applied from the roaming electrode. 2ms bipolar IAS pacing was constant at 2X
threshold (~2mA). Each ramp pulse was applied 45-60ms before an IAS pacing pulse.
CN: compact AV node; CrT: crista terminalis electrode; CS: coronary sinus; IAS:
interatrial septum; IHE: inferior His electrode; INE: inferior nodal extension; LNB: lower
nodal bundle; SHE: superior His electrode; TT: tendon of Todaro; VS: ventricular
septum.
Figure 2: Electrograms (A) and OAPs (B) in the triangle of Koch. Traces begin with
IAS pacing artifact and are color-coded with activation of atrial tissue-yellow, His-blue,
nodal-brown, SP-purple, ventricular-white, and intermediate traces-black. Each OAP
begins at time of pacing artifact. C: Comparison of selected electrograms and OAPs
recorded from the same site, with the dotted line marking the time of the bipolar IHE
electrogram. All traces are 300ms long. See Figure 1 for abbreviations.
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Figure 3: Schematic of the conduction system (A), similar to Figure 1A, is shown with
the approximate location of transitional cells (TC) shown as crosshatched area.
Activation patterns during IAS pacing (B), atrial activation from the roaming electrode
(C), and SP excitation (D), with the corresponding SHE traces shown below. Pacing
locations for A-C are indicated with square pulses. See Figure 1 for abbreviations.
Figure 4: Activation patterns in the whole heart preparation. A: photo of the whole
heart AV nodal preparation, with a schematic of the AV conduction system and
transitional cells as crosshatched area. B-C: Slow pathway (SP) pacing from the
roaming electrode located on the SP (SP electrode) produced SP and His excitation
(B) followed by atrial and ventricular activation (C). IHE trace is shown below,
starting with pacing artifact. D-F: IAS pacing of the high IAS first excited atrial
tissue (D), followed by AV nodal activation which spread both towards the His
bundle and into the SP where excitation died out (E). His excitation was followed
by ventricular activation (F). IHE electrogram is shown, starting with the IAS
pacing artifact. A, H, V: Atrial, His, and ventricular components of IHE,
respectively; RAA: right atrial appendage; RVFW: right ventricular free wall; see
Figure 1 for other abbreviations.
Figure 5: Summary plot of activation patterns and stimulation thresholds throughout the
triangle of Koch from 8 superfused experiments. Box marks optical mapping field of
view. See Figure 1 for abbreviations.
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Figure 6: A: Photograph of preparation with location of roaming electrode marked with
*. B: OAP recorded from the area of the blue box in A and electrograms from the IHE,
CrT, and IAS pacing electrodes. Ramp artifacts marked with *, which differ due to
aliasing (0.5ms pulses sampled at 1500 samples/sec). Before the ramp is above
threshold, the preparation is paced from the IAS, however when the ramp is above
threshold, 2:1 AV block occurs. See Figure 1 for abbreviations.
Figure 7: S-H intervals associated with atrial activation (A) and associated with SP/His
activation (B) for all superfused experiments are plotted vs. the distance from the His
electrode. C: Electrograms recorded at the end of a stimulation ramp which excited the
SP, and the following beat which was paced from the IAS. The unipolar ramp generated
a much larger stimulus artifact in the electrograms than bipolar IAS pacing.
Figure 8: A: Schematic of the propagation of a premature S2 stimulus to the His bundle
through the slow pathway (SP). B: Schematic of the two pathways that lead to His
bundle activation. SP excitation passes through the INE and LNB directly to the His
bundle, while FP activation passes through transitional cells (TC) and the compact node
(CN), where the AV nodal delay occurs. See Figure 1 for abbreviations.
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Figure 1: A: Schematic of the triangle of Koch, with transitional cells omitted from the
schematic for clarity. IAS pacing was delivered from the location marked with a red box.
The roaming electrode was moved throughout the triangle of Koch, to the locations
marked with blue boxes. Locations of bipolar electrodes marked with gray circles, and
approximate location of entry to the fast pathway (FP) and slow pathway (SP) are shown.
B: 0.5ms unipolar pulses increasing in amplitude from 0.33mA to 10mA, were applied
from the roaming electrode. 2ms bipolar IAS pacing was constant at 2X threshold
(~2mA). Each ramp pulse was applied 45-60ms before an IAS pacing pulse. CN: compact
AV node; CrT: crista terminalis electrode; CS: coronary sinus; IAS: interatrial septum;
IHE: inferior His electrode; INE: inferior nodal extension; LNB: lower nodal bundle; SHE:
superior His electrode; TT: tendon of Todaro; VS: ventricular septum.
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Figure 2: Electrograms (A) and OAPs (B) in the triangle of Koch. Traces begin with IAS
pacing artifact and are color-coded with activation of atrial tissue-yellow, His-blue, nodalbrown, SP-purple, ventricular-white, and intermediate traces-black. Each OAP begins at
time of pacing artifact. C: Comparison of selected electrograms and OAPs recorded from
the same site, with the dotted line marking the time of the bipolar IHE electrogram. All
traces are 300ms long. See Figure 1 for abbreviations.
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Figure 3: Schematic of the conduction system (A), similar to Figure 1A, is shown with the
approximate location of transitional cells (TC) shown as crosshatched area. Activation
patterns during IAS pacing (B), atrial activation from the roaming electrode (C), and SP
excitation (D), with the corresponding SHE traces shown below. Pacing locations for A-C
are indicated with square pulses. See Figure 1 for abbreviations.
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Figure 4: Activation patterns in the whole heart preparation. A: photo of the whole heart
AV nodal preparation, with a schematic of the AV conduction system and transitional cells
as crosshatched area. B-C: Slow pathway (SP) pacing from the roaming electrode located
on the SP (SP electrode) produced SP and His excitation (B) followed by atrial and
ventricular activation (C). IHE trace is shown below, starting with pacing artifact. D-F:
IAS pacing of the high IAS first excited atrial tissue (D), followed by AV nodal activation
which spread both towards the His bundle and into the SP where excitation died out (E).
His excitation was followed by ventricular activation (F). IHE electrogram is shown,
starting with the IAS pacing artifact. A, H, V: Atrial, His, and ventricular components of
IHE, respectively; RAA: right atrial appendage; RVFW: right ventricular free wall; see
Figure 1 for other abbreviations.
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Figure 5: Summary plot of activation patterns and stimulation thresholds throughout the
triangle of Koch from 8 superfused experiments. Box marks optical mapping field of view.
See Figure 1 for abbreviations.
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Figure 6: A: Photograph of preparation with location of roaming electrode marked with *.
B: OAP recorded from the area of the blue box in A and electrograms from the IHE, CrT,
and IAS pacing electrodes. Ramp artifacts marked with *, which differ due to aliasing
(0.5ms pulses sampled at 1500 samples/sec). Before the ramp is above threshold, the
preparation is paced from the IAS, however when the ramp is above threshold, 2:1 AV
block occurs. See Figure 1 for abbreviations.
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Figure 7: S-H intervals associated with atrial activation (A) and associated with SP/His
activation (B) for all superfused experiments are plotted vs. the distance from the His
electrode. C: Electrograms recorded at the end of a stimulation ramp which excited the
SP, and the following beat which was paced from the IAS. The unipolar ramp generated a
much larger stimulus artifact in the electrograms than bipolar IAS pacing.
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Figure 8: A: Schematic of the propagation of a premature S2 stimulus to the His bundle
through the slow pathway (SP). B: Schematic of the two pathways that lead to His bundle
activation. SP excitation passes through the INE and LNB directly to the His bundle, while
FP activation passes through transitional cells (TC) and the compact node (CN), where
the AV nodal delay occurs. See Figure 1 for abbreviations.
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