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\begin{document}

\title{Arrhythmogenic substrate in deep intra-trabecular structures of RVOT
endocardium in canine model of Brugada syndrome}



\author[1]{Alexander Burashnikov}%
\author[1]{Charles Antzelevitch}%
\affil[1]{Sidney Kimmel Cancer Center}%


\vspace{-1em}



  
  \date{May 22, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\emph{Introduction:} A prominent action potential (AP) notch in the
epicardium (Epi) of the RVOT is known to predispose to the development
of closely-coupled phase 2 reentrant extrasystoles, capable of
precipitating ventricular tachycardia and fibrillation (VT/VF) in the
setting of BrS. Ablation of this Epi substrate exerts an ameliorative
effect. In some BrS patients, Endo ablation of the RVOT is effective as
well. The prime objective of this study was to examine the
electrophysiological basis for premature beats originating from the
endocardium (Endo) of the right ventricular outflow tract (RVOT) in
experimental models of Brugada syndrome (BrS). \emph{Methods:} Canine
coronary-perfused cardiac preparations incorporating the RVOT (n=15)
were studied using standard microelectrode techniques. Terfenadine, a
sodium and calcium channel blocker, was used to pharmacologically mimic
the effects of the genetic defects associated with BrS. \emph{Results:}
Under baseline conditions, a prominent AP notch was recorded in Epi and
in the deep intra-trabecular structures of RVOT Endo, but not in the
smooth Endo surface of the RVOT. Terfenadine markedly accentuated the AP
notch in the deep intra-trabecular structures of RVOT Endo leading to
the development of closely-coupled phase 2 reentrant extrasystoles
capable of triggering polymorphic VT/V. Still, Epi RVOT region was more
likely to develop extrasystoles than Endo RVOT. VT/VF was recorded in
12/15 preparations. \emph{Conclusions:} Our findings suggest that the
deep intra-trabecular structures of RVOT Endo harbor the substrate for
the development of phase 2 reentrant extrasystoles capable of triggering
VT/VF. Our data may help to explain the effectiveness of Endo RVOT
ablation in some BrS patients.%
\end{abstract}%



\sloppy


\textbf{Arrhythmogenic substrate in deep intra-trabecular structures of
RVOT endocardium in canine model of Brugada syndrome}

Alexander Burashnikov PhD,\textsuperscript{1,2} Charles Antzelevitch
PhD., FACC\textsuperscript{1,2,3}

\textsuperscript{1}Lankenau Institute for Medical Research, Wynnewood,
PA, \textsuperscript{2}Sidney Kimmel College of
Medicine,\textsuperscript{3}Thomas Jefferson University, Philadelphia,
PA,\textsuperscript{3}Lankenau Heart Institute, Wynnewood, PA.

\emph{Short running title} : Endo RVOT triggers VF in BrS

\emph{Address for correspondence:}

Alexander Burashnikov, PhD

Lankenau Institute for Medical Research

100 E. Lancaster Ave Wynnewood, PA 19096 Phone: (315) 404-3798 E-mail:
BurashnikovA@mlhs.org

Word count: 3660

\textbf{Acknowledgment:} We gratefully acknowledge grant support to CA
from NHLBI (HL152201), the W.W. Smith Charitable Trust (AB and CA) and
the Martha and Wistar Morris Fund (CA).

\textbf{Disclosures:} None

ABSTRACT

Introduction: A prominent action potential (AP) notch in the epicardium
(Epi) of the RVOT is known to predispose to the development of
closely-coupled phase 2 reentrant extrasystoles, capable of
precipitating ventricular tachycardia and fibrillation (VT/VF) in the
setting of BrS. Ablation of this Epi substrate exerts an ameliorative
effect. In some BrS patients, Endo ablation of the RVOT is effective as
well. The prime objective of this study was to examine the
electrophysiological basis for premature beats originating from the
endocardium (Endo) of the right ventricular outflow tract (RVOT) in
experimental models of Brugada syndrome (BrS).

Methods: Canine coronary-perfused cardiac preparations incorporating the
RVOT (n=15) were studied using standard microelectrode techniques.
Terfenadine, a sodium and calcium channel blocker, was used to
pharmacologically mimic the effects of the genetic defects associated
with BrS.

Results: Under baseline conditions, a prominent AP notch was recorded in
Epi and in the deep intra-trabecular structures of RVOT Endo, but not in
the smooth Endo surface of the RVOT. Terfenadine markedly accentuated
the AP notch in the deep intra-trabecular structures of RVOT Endo
leading to the development of closely-coupled phase 2 reentrant
extrasystoles capable of triggering polymorphic VT/V. Still, Epi RVOT
region was more likely to develop extrasystoles than Endo RVOT. VT/VF
was recorded in 12/15 preparations.

Conclusions: Our findings suggest that the deep intra-trabecular
structures of RVOT Endo harbor the substrate for the development of
phase 2 reentrant extrasystoles capable of triggering VT/VF. Our data
may help to explain the effectiveness of Endo RVOT ablation in some BrS
patients.

Key words: Cardiac arrhythmias, Ventricular fibrillation, Extrasystole,
Electrophysiology, J wave syndrome.

CONDENSED ABSTRACT

The epicardium of the right ventricular outflow tract (RVOT) has long
been considered to be the source of the arrhythmogenic substrate in
Brugada syndrome (BrS), accounting for the ameliorative effect of
epicardial ablation. However, endocardial ablation has also been
reported to be effective in some patients. Using canine RVOT
preparations, we demonstrate for the first time an arrhythmogenic
substrate (prominent action potential notches) in the deep
intra-trabecular structures of RVOT endocardium, capable of generating
closely-coupled phase -2-reentrant extrasystoles capable of triggering
polymorphic VT/VF in the setting of BrS. Our findings may explain the
effectiveness of Endo RVOT ablation in some BrS patients.

INTRODUCTION

A prominent action potential (AP) notch in the epicardium (Epi) of the
right ventricular outflow tract (RVOT) predisposes to the development of
closely-coupled phase 2 reentrant extrasystoles, capable of
precipitating ventricular tachycardia and fibrillation (VT/VF) in the
setting of Brugada syndrome (BrS).\textsuperscript{1,2} These
observations have provided an understanding of why ablation of the RVOT
Epi exerts an ameliorative effect in patients with
BrS.\textsuperscript{3-8} However, endocardial (Endo) ablation of the
RVOT has also been reported to be effective in preventing BrS-related VF
in some patients.\textsuperscript{9-12} These observations suggest that
parts of Endo may also harbor a substrate contributing to
arrhythmogenesis in the setting of BrS. Transmembrane action potential
(AP) characteristics of the complex Endo structures of the RVOT have not
been fully explored. The present investigation was designed to examine
the AP characteristics of RVOT Endo in canine coronary perfused
preparations under baseline conditions and in the setting of BrS.

METHODS

The investigation was performed according to the Guide for Care and Use
of Laboratory Animals published by the US National Institute of Health.
The study was approved by the Institutional Animal Use and Care
Committee of the Lankenau Institute for Medical Research. Dogs weighing
12-20 kg were anticoagulated with heparin (200 IU/kg) and anesthetized
with pentobarbital sodium (35 mg/kg, i.v.). The chest was opened via a
left thoracotomy, the heart excised, placed in a cardioplegic solution
consisting of cold (4\textsuperscript{o}C) Tyrode's solution containing
8.5 mM {[}K\textsuperscript{+}{]}\textsubscript{o} and transported to a
dissection tray. Preparations containing the RVOT were excised,
cannulated and perfused from either right or left coronary arteries
using polyethylene tubing. All severed coronary branches were ligated
using silk thread. Non-perfused areas of the RVOT were dissected using a
razor blade. Preparations perfused from the right coronary artery
contained the entire right atrium and the rim of the right ventricle
including the RVOT. Preparations perfused from the left coronary artery
contained the RVOT and part of the septum.

The RVOT in the canine heart is supplied with blood from both the left
and right coronary arteries. Out of 22 preparations perfused from the
right coronary artery, the RVOT was relatively well-perfused
(approximately 40-60\% of RVOT) in 8 preparations and these were
selected for use in the study. All seven preparations perfused via the
left coronary artery had a relatively large proportion of the RVOT
perfused (about 50-75\%). Thus, 15 preparations perfused from right and
left coronary arteries were used and data from these preparations were
combined.

The coronary-perfused preparations were placed in a
temperature-controlled bath with the endocardial surface up (8 x 6 x 4
cm) and perfused with Tyrode's solution at a rate of 8-10 mL/min. The
Tyrode's solution contained (in mM): NaCl 129, KCl 4,
NaH\textsubscript{2}PO\textsubscript{4} 0.9, NaHCO\textsubscript{3} 20,
CaCl\textsubscript{2} 1.8, MgSO\textsubscript{4} 0.5, and D-glucose 5.5,
bubbled with 95\% O\textsubscript{2} and 5\% CO\textsubscript{2}.
(37.0\selectlanguage{ngerman}±0.5\textsuperscript{0}C). The initial temperature of the coronary
perfusate was 30\textsuperscript{0}C and gradually warmed to
37\textsuperscript{0}C over a period of 5-6 min. The temperature was
maintained at 37\textsuperscript{0}C (Cole Parmer Instrument. Co., IL).
The perfusate was warmed by circulation through a metal tube (Small
Parts Inc. Miami, Fl) immersed in a bath chamber before flowing to the
preparation so that the temperature of the perfusate matched that of the
bath. The perfusate was delivered to the artery by a roller pump, with
perfusion pressure of 40-50 mmHg throughout the experiment. An air trap
was used to avoid bubbles in the perfusion line. The preparations were
equilibrated in the tissue bath until electrically stable, usually 30
min, while pacing at cycle lengths (CL) of 500 to 800 ms. Basic
stimulation was applied using a pair of thin silver electrodes insulated
except at their tips.

Transmembrane APs (sampling rate 41 kHz) were recorded using floating
glass microelectrodes (filled with 2.7 M KCl, 10-25 M\selectlanguage{greek}Ω \selectlanguage{english}DC resistance)
connected to a high input impedance amplification system (World
Precision Instruments). The signals were displayed on oscilloscopes,
amplified, digitized and analyzed (Cambridge Electronic Design,
Cambridge, England) and stored on computer hard drive or CD. A
pseudo-electrocardiogram (ECG) was recorded using two electrodes
consisting of Ag/AgCl half cells placed in the Tyrode's solution 1.0 to
1.2 cm from the opposite ends of the preparation, thus measuring the
electrical field of the preparation as a whole. AP parameters were
measured from signals with the largest phase 0 amplitude in any given
set of conditions.

\textbf{Study Protocols:}The preparations were paced at a CL of 1000 ms,
using a pair of thin silver electrodes insulated except at their tips
(bipolar rectangular pulses of 2~ms duration and twice diastolic
threshold (DTE) intensity). Terfenadine, a sodium and calcium channel
blocker (5-8 \selectlanguage{greek}µ\selectlanguage{english}M; [?] 1.5 hour), was used to pharmacologically mimic the
genetic defects associated with BrS.\textsuperscript{13} APs were
recorded under baseline conditions and following 5-8 \selectlanguage{greek}µ\selectlanguage{english}M terfenadine. In
order to record Epi, the preparations were flipped over. Spontaneous
arrhythmias were continuously monitored using ECG recordings.

\textbf{Drug:} Terfenadine (Sigma-Aldrich, St Louis MO) was dissolved in
ethanol as a 5 mM stock solution.

\textbf{Statistics:} Statistical analysis was performed using unpaired
t-test. All tissue data are expressed as mean \selectlanguage{ngerman}± SD.

\textbf{RESULTS}

Under baseline conditions, APs from Endo were recorded from all
preparations (n=15) and APs from Epi from 8 preparations. A significant
spike and dome morphology of the AP was consistently recorded on the Epi
surface of the RVOT in all 8 preparations in which Epi was measured
(\textbf{Fig. 1} and \textbf{Table 1} ). In contrast to the relatively
homogeneous distribution of an AP notch (spike and dome morphology) on
Epi, a marked spatial heterogeneity of spike and dome morphology was
observed in the Endo of the RVOT, ranging from none to very prominent
(\textbf{Fig. 1} ). An AP morphology lacking a notch was commonly
recorded on the smooth surface of Endo, whereas a prominent notch was
recorded deep within the inter-trabecular structures (\textbf{Fig. 1} ).
The RVOT Epi displayed a prominent AP notch. However, phase 0 amplitude,
phase 1 magnitude, and phase 1 amplitude were larger in deep trabecular
Endo vs. Epi RVOT (\textbf{Fig. 2} , Table 1). No arrhythmias were
recorded under baseline conditions.

Under experimental conditions pharmacologically mimicking BrS (i.e.,
terfenadine-mediated block of I\textsubscript{Na} and
I\textsubscript{Ca}; 5-8 \selectlanguage{greek}µ\selectlanguage{english}M; [?] 1.5 hour), our principal focus in these
studies was to determine spatial differences in the AP characteristics
of Endo given the fact that we had a limited period of time to collect
these data prior to development of VT/VF. It is noteworthy that the
characteristics of right ventricular Epi AP and phase 2-reentry in the
setting of BrS are well documented.\textsuperscript{13-15} Out of the 15
preparations studied, high quality APs were recorded from the Endo
regions in 12 (due to an ``early'' onset of persistent VF in 3 of the
preparations). Terfenadine markedly accentuated the spike and dome
morphology of APs recorded from the intra-trabecular region of RVOT
Endo, but caused little change in the early phases of the AP recorded
from the smooth Endo surface (\textbf{Fig. 3; Table 2} ). Marked
heterogeneities were commonly observed in the AP notch recorded from the
intra-trabecular structures of the RVOT Endo, ranging from a significant
augmentation of the AP dome and delay in the onset of phase 2 to loss of
the AP dome (\textbf{Fig. 4} ). Under similar conditions, little
heterogeneity was observed in the characteristics of APs recorded from
the smooth surface of the RVOT Endo (\textbf{Fig. 4} ). Accentuation of
the AP notch leading to delay in the onset of phase 2 and in some cases
to loss of AP dome in the intra-trabecular Endo was associated with the
development of phase 2 reentrant extrasystoles (\textbf{Fig. 4} ). When
considering preparations in which the origin of the premature beats
could be reasonably determined (based on the polarity of the QRS in the
ECG traces; n=9 preparations; \textbf{Fig. 5} ), an Epi and Endo origin
of the extrasystole was observed in 8 and 5 preparations, respectively,
with 5 of these preparations displaying both Epi and Endo origin, and 4
and 1 preparations displaying only Epi or only Endo origin,
respectively.

In the setting of experimental BrS, closely-coupled extrasystoles were
recorded in 14/15 preparations, giving rise to polymorphic VT/VF in
12/15 preparations. The first episode of VT/VF appeared after 93-120
minutes of perfusion with terfenadine. We observed [?] 3 self-terminating
episodes of VT/VF in each of the 12 preparations and in 7 of them VT/VF
was persistent (\textgreater{} 1 hour). \textbf{Figure 6} depicts
examples in which VF was initiated by extrasystoles originating from
either Epi or Endo of the RVOT.

\textbf{DISCUSSION}

To the best of our knowledge, the present study is the first to
demonstrate that the deep intra-trabecular structures of the canine Endo
RVOT display APs with a prominent spike and dome morphology, that when
accentuated in the setting of BrS, could lead to the development of
phase-2-reentry and VT/VF.

Action potential spike and dome and phase-2 reentry

It is well established that in large mammals, including humans and dogs,
transient outward current (I\textsubscript{to}) density is much greater
in ventricular Epi vs. Endo cells and that this is responsible for a
more pronounced spike and dome morphology of the AP in ventricular Epi
than in Endo.\textsuperscript{16} Moreover, Epi of the RVOT is known to
display a more pronounced spike and dome AP morphology than Epi of the
rest of RV or LV.\textsuperscript{14,17}The prominent AP notch in the
RVOT Endo intra-trabecular structures is most likely due to the presence
of a prominent I\textsubscript{to}.

In previous studies, we and others have shown that a net outward shift
in the balance of current during the early phases of AP can lead to
accentuation of the AP notch, can result in loss of AP dome at some
sites but not others in RV and LV Epi.\textsuperscript{13-15} This is
observed following augmentation of I\textsubscript{to} or
I\textsubscript{K-ATP} (using NS5806 or pinacidil) or following
inhibition of I\textsubscript{Na} and/or I\textsubscript{Ca} (using
terfenadine, ajmaline or verapamil).\textsuperscript{13-15} When loss of
the dome is spatially heterogeneous, it can lead to propagation of AP
dome from regions in which it is maintained to regions where it is lost,
resulting in a closely-coupled extrasystole due to phase 2
reentry.\textsuperscript{13,15}

\textbf{Endocardium of RVOT as a source of triggers in BrS}

The epicardial surface of the RV, particularly the RVOT, has been shown
to be the site of origin of phase-2-reentry-induced extrasystoles
capable of trigging VT/VF in the in ventricular wedge and whole heart
models of BrS.\textsuperscript{13-15,17-19} Consistent with these
experimental observations, numerous clinical
studies\textsuperscript{3-8,12,20} as well as experimental
studies\textsuperscript{21} have demonstrated that ablation of the RVOT
Epi substrate can significantly reduce the occurrence of VT/VF in the
setting of BrS. Ablation of the RVOT Endo has also been shown to reduce
BrS-associated VT/VF in some clinical cases.\textsuperscript{9-12} These
data suggest that RVOT Endo can be involved in the generation of VT/VF
in patients with BrS.

The present study points to the presence of an arrhythmogenic substrate
in the deep intra-trabecular structures of the Endo of the RVOT in the
setting of BrS, similar to that previously described in the Epi of the
RVOT.\textsuperscript{22} Consequently, ablation of either surface of
the RVOT has the potential to reduce BrS-related arrhythmogenicity. When
applied to the ventricular endocardium, an ablation catheter is likely
to ablate both the ``smooth'' muscle and deep intra-trabecular
structures, thus exerting an ameliorative effect. An alternative
explanation for the effectiveness of Endo ablation in some patients is
that ablation of RVOT Endo may extend to the RVOT Epi, thus affecting
Epi BrS substrate (considering that the RVOT is relatively
thin).\textsuperscript{12,23}

To the best of our knowledge, this is the first study to systematically
examine the AP characteristics of RVOT Endo in the setting of BrS using
intracellular microelectrode techniques. Our data suggest that the deep
structures of RVOT Endo, in addition to the RVOT
Epi,\textsuperscript{14,17} can be the source of the substrate for
development of phase 2 reentry, capable of triggering VT/VF in BrS.
Interestingly, there are substantial electroanatomical abnormalities on
the Endo RVOT surface in patients with Brugada syndrome detected with
unipolar mapping, and these abnormalities correlate with VF
occurrence.\textsuperscript{24}

\textbf{Limitations}

Our study was performed using isolated coronary-perfused preparations
and the genetic defects responsible for BrS were pharmacologically
mimicked. Despite these limitations, coronary-perfused canine
ventricular models have been previously shown by us and others to be
capable of recapitulating the principal electrocardiographic arrhythmic
clinical manifestations of BrS, as well as the response to autonomic and
pharmacologic manipulation.\textsuperscript{2,25,26}

\textbf{CONCLUSION}

Our finding demonstrate for the first time that the deep
intra-trabecular endocardial structures in the region of the RVOT, in
addition to those in the epicardium of the RVOT, harbor the substrate
for BrS in the form of prominent AP notches that can serve as the source
of phase 2 reentrant extrasystoles capable of triggering VT/VF.

\textbf{REFERENCES}

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\textbf{Table 1. Characteristics of action potentials recorded from the
intra-trabecular and smooth surface of the Endo vs. those recorded from
the Epi surface of the RVOT under baseline conditions}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\textbf{Endocardium} (n=15)\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\textbf{Endocardium} (n=15)\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\textbf{Epicardium} (n=8)\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Intra-trabecular\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
``Smooth Surface''\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Phase 0 amplitude (mV)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
98\selectlanguage{ngerman}±6**\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
99\selectlanguage{ngerman}±5\#\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
84\selectlanguage{ngerman}±6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Phase 1 amplitude (mV)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
71\selectlanguage{ngerman}±4*\selectlanguage{english}+\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
91\selectlanguage{ngerman}±5\#\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
64\selectlanguage{ngerman}±3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Phase 2 amplitude (mV)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
89\selectlanguage{ngerman}±5*\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
92\selectlanguage{ngerman}±4\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
95\selectlanguage{ngerman}±4\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Phase 1 magnitude (mV)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
27\selectlanguage{ngerman}±7*\selectlanguage{english}+\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
8\selectlanguage{ngerman}±3\#\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
21\selectlanguage{ngerman}±4\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Phase 2 magnitude (mV)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
18\selectlanguage{ngerman}±4**\selectlanguage{english}+\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1\selectlanguage{ngerman}±1\#\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
31\selectlanguage{ngerman}±4\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

The AP with the most prominent spike and dome morphology recorded in
each preparation was taken for statistical analysis. CL = 1000 ms. *
p\textless{}0.05 vs. Epi. ** p\textless{}0.001 vs. Epi. \selectlanguage{english}+ p\textless{}
0.001 vs Endo ``smooth''. \# p\textless{}0.001 vs. Epi.

\textbf{Table 2. Terfenadine-induced accentuation of the AP spike and
dome morphology in the Endo intra-trabecular regions of the RVOT}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
& \textbf{Intra-trabecular} & \textbf{Intra-trabecular} &
\textbf{``Smooth Surface''} & \textbf{``Smooth Surface''}\tabularnewline
\midrule
\endhead
& Control & Terfenadine & Control & Terfenadine\tabularnewline
Phase 0 amplitude (mV) & 99\selectlanguage{ngerman}±5 & 92\selectlanguage{ngerman}±7** & 100\selectlanguage{ngerman}±6 & 95\selectlanguage{ngerman}±5*\tabularnewline
Phase 1 amplitude (mV) & 71\selectlanguage{ngerman}±4 & 67\selectlanguage{ngerman}±5* & 92\selectlanguage{ngerman}±3 & 90\selectlanguage{ngerman}±4\tabularnewline
Phase 2 amplitude (mV) & 90\selectlanguage{ngerman}±4 & 95\selectlanguage{ngerman}±4* & 92\selectlanguage{ngerman}±4 & 93\selectlanguage{ngerman}±4\tabularnewline
Phase 1 magnitude (mV) & 28\selectlanguage{ngerman}±7 & 25\selectlanguage{ngerman}±7 & 7\selectlanguage{ngerman}±3 & 5\selectlanguage{ngerman}±5\tabularnewline
Phase 2 magnitude (mV) & 19\selectlanguage{ngerman}±5 & 27\selectlanguage{ngerman}±6** & 1\selectlanguage{ngerman}±1 & 3\selectlanguage{ngerman}±2\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

The AP with the most prominent spike and dome morphology recorded in
each preparation was taken for statistical analysis. CL = 1000 ms. N= 12
for each. * p\textgreater{}0.05 vs. control **. p\textgreater{}0.01 vs.
control.

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/658544/articles/662851-arrhythmogenic-substrate-in-deep-intra-trabecular-structures-of-rvot-endocardium-in-canine-model-of-brugada-syndrome}

\textbf{Figure 1.} Typical action potential (AP) morphologies recorded
from the Epi and Endo surfaces of the canine RVOT. A significant AP
spike and dome morphology giving rise to a prominent notch was present
throughout the Epi surface. On the Endo surface, manifestation of an AP
notch ranged from very prominent to none. A prominent AP notch was
recorded from the deep intra-trabecular Endo structures.

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/658544/articles/662851-arrhythmogenic-substrate-in-deep-intra-trabecular-structures-of-rvot-endocardium-in-canine-model-of-brugada-syndrome}

\textbf{Figure 2.} Typical action potential morphologies recorded in the
intra-trabecular Endo and Epi surfaces in canine RVOT. Arrows depict the
difference between the Endo and Epi.

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/658544/articles/662851-arrhythmogenic-substrate-in-deep-intra-trabecular-structures-of-rvot-endocardium-in-canine-model-of-brugada-syndrome}

\textbf{Figure 3.} Terfenadine-induced accentuation of spike and dome
morphology in the intra-trabecular region of Endo. CL= 1000 ms.

\textbf{Hosted file}

\verb`image4.emf`  available at \url{https://authorea.com/users/658544/articles/662851-arrhythmogenic-substrate-in-deep-intra-trabecular-structures-of-rvot-endocardium-in-canine-model-of-brugada-syndrome}

\textbf{Figure 4.} Dynamic appearance and disappearance of AP dome in
the intra-trabecular Endo and phase-2 reentry-induced propagated
extrasystole originating from the Endo of the RVOT in the presence of
terfenadine. Each panel shows simultaneously recorded ECG and action
potentials (AP). All recordings were obtained from the same preparation.
The stimulation electrode was located on the ``smooth'' Endo.

\textbf{Hosted file}

\verb`image5.emf`  available at \url{https://authorea.com/users/658544/articles/662851-arrhythmogenic-substrate-in-deep-intra-trabecular-structures-of-rvot-endocardium-in-canine-model-of-brugada-syndrome}

\textbf{Figure 5.} Endocardial and epicardial origin of extrasystoles in
the setting of experimental BrS. The Epi/Endo origin of the
extrasystoles were determined based on the polarity of R wave. The
tracings were recorded in the same preparation.

\textbf{Hosted file}

\verb`image6.emf`  available at \url{https://authorea.com/users/658544/articles/662851-arrhythmogenic-substrate-in-deep-intra-trabecular-structures-of-rvot-endocardium-in-canine-model-of-brugada-syndrome}

\textbf{Figure 6.} Extrasystoles originating from either Epi or Endo of
the RVOT can trigger VF in the presence of terfenadine (8 \selectlanguage{greek}µ\selectlanguage{english}M). Origin of
the extrasystole can be judged on the basis of the polarity of the R
wave recorded from the pseudo-ECG. The stimulation electrode was located
on the smooth surface of the Endo RVOT. The two traces were recorded
from the same preparation.

\selectlanguage{english}
\FloatBarrier
\end{document}

