Arrhythmogenic substrate in deep intra-trabecular structures of
RVOT endocardium in canine model of Brugada syndrome
Alexander Burashnikov PhD,1,2 Charles Antzelevitch
PhD., FACC1,2,3
1Lankenau Institute for Medical Research, Wynnewood,
PA, 2Sidney Kimmel College of Medicine,3Thomas Jefferson University, Philadelphia, PA,3Lankenau Heart Institute, Wynnewood, PA.
Short running title : Endo RVOT triggers VF in BrS
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
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).
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).1,2 These
observations have provided an understanding of why ablation of the RVOT
Epi exerts an ameliorative effect in patients with
BrS.3-8 However, endocardial (Endo) ablation of the
RVOT has also been reported to be effective in preventing BrS-related VF
in some patients.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 (4oC) Tyrode’s solution containing
8.5 mM [K+]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,
NaH2PO4 0.9, NaHCO3 20,
CaCl2 1.8, MgSO4 0.5, and D‑glucose 5.5,
bubbled with 95% O2 and 5% CO2.
(37.0±0.50C). The initial temperature of the coronary
perfusate was 300C and gradually warmed to
370C over a period of 5-6 min. The temperature was
maintained at 370C (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 MΩ 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.
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 µM; ≥ 1.5 hour),
was used to pharmacologically mimic the genetic defects associated with
BrS.13 APs were
recorded under baseline conditions and following 5-8 µM terfenadine. In
order to record Epi, the preparations were flipped over. Spontaneous
arrhythmias were continuously monitored using ECG recordings.
Drug: Terfenadine (Sigma-Aldrich, St Louis MO) was dissolved in
ethanol as a 5 mM stock solution.
Statistics: Statistical analysis was performed using unpaired
t-test. All tissue data are expressed as mean ± SD.