The Mechanism of Pause-Induced Torsade de Pointes in Long QT Syndrome

Jinqiu Liu, M.D.; Kenneth R. Laurita, Ph.D.

Disclosures

J Cardiovasc Electrophysiol. 2005;16(9):981-987. 

In This Article

Methods

Experiments were performed in accordance with Public Health Service guidelines for the care and use of laboratory animals. The methods used for isolation and perfusion of the arterially perfused left ventricular wedge preparation are detailed in previous studies.[17] Wedge preparations (n = 23) with dimensions of 2.0 × 1.0 × 0.5 cm were dissected from left ventricles of mongrel dogs weighing 18 to 22 kg. Each wedge was perfused with Tyrode's solution of the following composition (mmol/L): NaCl 135, KCl 4.03, NaH2PO4 0.9, NaHCO3 20, CaCl2 1.8, MgSO4 0.492, and Dextrose 5.5, buffered with HEPES 10 and 100% oxygen (36 ± 1°C). To record transmural action potentials, the wedge was perfused with the voltage sensitive dye di-4-ANEPPS (15 μM) for 10–15 minutes and placed into a Tyrode filled Lexan imaging chamber. The temperature was maintained at 36 ± 1°C by a heat exchanger and a temperature controller. A transmural ECG was recorded using extracellular silver chloride electrodes placed near the epicardial and endocardial surfaces of the preparation. Perfusion pressure was maintained between 40–50 mmHg with a digitally controlled flow pump.

Optical mapping techniques described previously[17] were used to measure transmural action potentials from hundreds of sites with high temporal and spatial resolution across the transmural wall. Briefly, excitation light obtained from a 180 W quartz tungsten halogen lamp light source (Oriel Corp., Stratford, CT, USA) was directed to the heart using a light guide. Fluoresced light from the heart was collected by a tandem lens assembly[18,19] and directed to a 16 × 16 element photodiode array. Signals recorded from each photodiode and ECG signals were multiplexed and digitized with 12-bit precision at a sampling rate of 1,000 Hz per channel (Microstar Laboratories Inc., Bellevue, WA, USA). For the present study an optical magnification of 1.24 was used which resulted in a total mapping field of 1.4 cm × 1.4 cm with 0.89-mm spatial resolution. To view, digitize, and store the position of the mapping array relative to anatomical features, a mirror was temporarily inserted between the lenses of the tandem lens assembly to direct reflected light to a CCD video camera.

The IKr blocker d-sotalol (100 μmol/L) was used as a model of LQT2 and ATX-II (5 nmol/L) was used as a model of LQT3. The validity of these pharmacological agents as surrogates for congenital syndromes has been demonstrated in a variety of experimental studies.[7,20] In all experiments, Cytochalasin D (cyto D 2 μmol/L) was used to inhibit contraction so that action potentials and EADs could be detected in the absence of motion artifact.

Unipolar stimulation was delivered from the endocardium at a pulse width of 2 msec and at a strength of twice diastolic threshold. Short-long cycle length pacing consisted of 10 beats of steady state baseline pacing (1,000 or 600 msec) followed by a premature stimulus (S2) and then a delayed stimulus (S3) delivered over a wide range of pauses (1,200, 1,400, 1,700, 2,000, and 3,000 msec). This protocol was repeated with the addition of a second premature stimulus (S4) following the delayed stimulus (S3). The coupling interval of all premature beats was equal to the effective refractory period (ERP) plus 10 msec. All pacing protocols were delivered with and without drug administration. In addition, during and after drug administration optical mapping recordings were made while the preparation was allowed to beat spontaneously and achieve steady state. The ECG, perfusion pressure, flow, and temperature were monitored continuously throughout each experiment to verify steady state conditions. At the end of each experiment, tissue viability was confirmed using 10 mL of TTC (14 mg/mL) staining.

Action potential depolarization and repolarization times were determined from the maximum first derivative during the upstroke and maximum second derivative during the repolarization phase, respectively. M-cells were defined functionally as where APD is longest for an average cycle length >2,000 msec. The midmyocardium was defined anatomically as approximately 2 mm from the endocardial surface and 2 mm from the epicardial surface. DOR, as measured by the range of APD across the transmural wall, was determined for baseline pacing and for the beat following the pause (S3). The method of Bayly et al.[21] was modified for optically recorded action potential maps to accurately quantify the direction and magnitude of the local repolarization gradient at each recording site. TdP was defined as three or more beats of polymorphic ventricular tachycardia that terminated spontaneously. EADs were defined as a sudden increase in transmembrane potential during the end of phase 2 or phase 3 of the action potential. EAD-induced triggered activity is a term used to describe an extrasystole caused by an EAD. During spontaneous rhythm, pause-dependent EAD-induced triggered activity or TdP was only considered when the pause was 100 msec longer than the basic cycle length.

Data are expressed as mean ± SD. Statistical analysis of the data was performed with Student's t-test for paired data and unpaired t-test and ANOVA for unpaired data, as appropriate. Percentages were compared using the chi-square test. A Fisher's exact test was used to determine significant differences in EAD and EAD-induced triggered activity between groups. Significance was defined as a value of P < 0.05.

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