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.