Expanded Materials and Methods
In vivo rabbit preparation
Female New Zealand white rabbits (2-3.5 kg) were anesthetized with 60 mg/kg sodium
pentobarbital, i.v. [1,2] during surgery and additional doses were administered as
necessary to maintain the depth of anesthesia (determined by eye lid reflex).
A
tracheotomy was performed and animals were mechanically ventilated with room air (5
mL/ kg) throughout the experiment. A femoral artery was cannulated to measure blood
pressure and two femoral veins and one jugular vein were cannulated for drug delivery.
Surface ECG electrodes were arranged in the standard lead II configuration. Animals
were left to stabilize for 5-10 min before control recording began.
Pre-treatment
Experiments:
Control
animals
were
continuously infused
with
methoxamine (20 µg/kg/min) after 10 minutes of control recording and clofilium infusion
(300 nmol/kg/min) was started after a further 10 mins (Fig. 5A). Both infusions were
continued for a further 15 minutes. In test animals, continuous infusion of RSD1235
(0.1, 0.3, or 1 µmol/kg/min) was started after 5 minutes of control recording followed by
methoxamine and clofilium as in controls. As a result, 0.1, 0.3, and 1 µmol/kg/min
RSD1235 was infused for a total of 30 minutes corresponding to cumulative doses of 1.2,
3.6, 11.6 mg/kg RSD1235, respectively.
Acute Intervention Experiments:
As first described [3], animals were infused with
methoxamine and clofilium in the same manner as for the control group in the pretreatment experiments (Fig. 5A).
In test animals, infusion of 1 or 3 µmol/kg/min
RSD1235 was started immediately after the first episode of TdP and continued for 5 min
1
(resulting in cumulative doses of 1.2 and 5.8 mg/kg RSD1235). In control animals, no
intervention was made.
ECG Recording. ECG was continuously recorded and analyzed using Labview data
acquisition and analysis software (National Instruments). An ECG waveform was
considered to show TdP when it consisted of >5 consecutive beats of polymorphic
ventricular tachycardia (see Fig. 5). The interval between the Q and the trough of the T
wave was recorded as the QT interval. QT interval was corrected to take into account
changes in heart rate (rate corrected interval is termed QTc) according to the method
described by [3] for rabbits using the following equation: QTc = QT - 0.479(RR - 250).
Rabbit Purkinje Fibers
Female New Zealand White rabbits were anaesthetized with sodium pentobarbital (60
mg/kg) and then euthanized by cervical dislocation. Hearts were rapidly excised and
placed in an ice cold modified Krebs’ solution (NaCl 118 mM, KCl 3 mM, Glucose 10
mM, NaHCO3 25 mM, KH2PO4 1 mM, MgSO4-7H2O 1.2 mM, CaCl2 1.8 mM) saturated
with carbogen (95% O2/5% CO2). A cut was made along the left-ventricular side of the
interventricular septum, extending to the apex of the heart. The heart was spread open
with pins and placed in a 37°C bath containing Krebs’ solution, bubbled with carbogen.
This preparation was used to test for interactions between dofetilide and RSD1235
affecting APD (Fig. 1).
We used the technique as we found that the AP duration was
very stable between preparations with this technique, as suggested by the very small error
bars on APD50 and APD90 in Figure 1 panels C and D. We used a single impalement
throughout experiments, and discarded preparations if the impalement was lost.
2
In control experiments with vehicle solution done at the same time as the experiments
with RSD1235 and dofetilide, we found that the mean action potential duration was 221
± 21 ms after the one-hour equilibration period, and was 232 ± 22 ms at the end of the
experiment, at 2.5 hours of impalement. APD was monitored every 15 min and the mean
maximum duration reached was 243 ± 38 ms and the minimum reached was 214 ± 21 ms.
These data are the mean data from eight preparations.
In experiments in which EADs were induced (Fig. 2), and in some APD experiments
where RSD1235 was used (see text to Figure 1) a PF was isolated from the left ventricle
by separating small pieces of ventricular tissue at the junctions of either side of the
Purkinje fibers and then transferred to the 37°C bath (Warner automatic temperature
controller TC-324B). The ventricular tissue was pinned into a perfusion bath (flow rate
of 10 mL/minute; bath size 5 mL) and the fiber was gently stretched to provide an
adequate surface for impalement with a floating microelectrode. Microelectrodes were
pulled using a horizontal electrode puller (Sutter Instruments, CA) from a filamented
glass blank with resistances between 20 and 30 Megohm and filled with 3 M KCl. Both
isolated PFs and whole-heart PFs were activated using local stimulation of the fiber ends
via a bipolar AgCl electrode (Dagan S-900 Stimulator, S-910 Stimulus Isolator). The
preparations were allowed to equilibrate for at least 1 hour while being stimulated via
suprathreshold stimuli at a frequency of 1 Hz. Drugs were not administered until APs
exhibited stable morphologies. APs were considered stable when they had a resting
membrane potential between -80 and -100 mV, a minimum action potential amplitude of
3
100 mV, although this was usually higher (Fig. 2) and an action potential duration at 90%
repolarization (APD90) of at least 200 ms. Changes in membrane voltage were measured
with an Axoclamp-2A amplifier and analog signals were converted to digital signals by a
Digidata 1200 interface. Data was recorded and analyzed using pClamp8 software (Axon
Instruments).
Effective Refractory Period (ERP) measurements: ERP in whole-heart PFs was
determined following each treatment arm. 15 S1 pulses were delivered at a frequency of
1 Hz and this train was followed by an S2 pulse after a variable interval. The interval
was set to be greater than the refractory period and it was reduced in 10 ms increments
until an S2 response could no longer be elicited. The shortest duration which was able to
generate an S2 response was termed the ERP.
Whole-cell Patch Clamp Recordings
Nav1.5 channels were expressed in a stable line of HEK cells.
Coverslips
containing cells were placed in a superfusion chamber (volume 250 µl) containing the
control bath solution at 25°C (Warner automatic temperature controller TC-324B).
External solution contained (mM): NaCl 130; dextrose 10; HEPES 10; MgCl2 1; CsCl 5;
CaCl2 1 (pH adjusted to 7.4 with NaOH). Whole-cell current recordings and analysis
were made using an Axopatch 200B amplifier and pClamp8 software (Axon Instruments,
CA). Patch electrodes were pulled from thin-walled borosilicate glass (World Precision
Instruments, FL) on a horizontal micropipette puller (Sutter Instruments, CA). Electrodes
had resistances of 1.5-3.5 Megaohms when filled with control filling solution, in mM:
CsCl 130; Na2ATP 4; MgCl2 1; HEPES 5; EGTA 10 (pH adjusted to 7.2 with NaOH).
4
Analog capacity compensation and 75% - 85% series resistance compensation were used
in all whole cell measurements. Membrane potentials were not corrected for junction
potentials that arise between the pipette and bath solution. Data were sampled at 10-20
kHz and filtered at 5 to 10 kHz.
A step/ramp protocol (-100 mV step to 20 mV for 100 ms, then ramp back to -100
mV over 100 ms) was used to examine the three main components of Na+ current active
during an action potential [4]. After achieving the whole-cell configuration, the cell was
left to equilibrate for 5 to 10 minutes at a holding potential of -80 mV. This equilibration
period allowed complete dialysis of the internal pipette solution and was critical to
achieving stable electrophysiological signals. Since the sustained and late currents were
small (<60 pA), changes in leak current during the recording distorted the measurements
and the subsequent digital subtraction. Holding the cell at -80 mV for 5-10 minutes
before running the voltage protocol resulted in recordings with stable leak current and
undistorted currents of interest. 30-150 control traces (1 Hz) were obtained using the
step/ramp voltage clamp protocol and traces were averaged offline. Cells were then
perfused with either 30 µM RSD1235 or 30 µM lidocaine. 30 µM TTX was applied
when the amplitude of the peak early current had become stable for approximately 30150 traces. Following 30-150 traces of complete block, both drugs were washed out.
Analysis involved digital subtraction to identify the RSD1235-, lidocaine- and TTXsensitive currents.
30-150 consecutive traces were averaged before drug addition,
following perfusion with either RSD1235 or lidocaine, and following 30 µM TTX
perfusion.
Trace averaging significantly improved the signal-to-noise ratio.
5
The
RSD1235-, lidocaine-, and TTX-sensitive traces were obtained by digitally subtracting
the averaged trace in the presence of drug from the pre-drug averaged trace.
Drugs
Sodium pentobarbital (Somnotol) was obtained from MTC Pharmaceuticals. Clofilium
tosylate (MP Biomedicals) and methoxamine hydrochloride (Sigma) were dissolved in
saline for IV infusion. Dofetilide was obtained from Pfizer and dissolved in DMSO.
Lidocaine (Sigma) was dissolved in H2O.
6
Reference List
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clofilium in a modified in vivo model of torsade de pointes. Br J Pharmacol
2002;135:1003-1012.
[2] Bril A, Gout B, Bonhomme M, Landais L, Faivre JF, Linee P et al. Combined
potassium and calcium channel blocking activities as a basis for antiarrhythmic
efficacy with low proarrhythmic risk: experimental profile of BRL-32872. J
Pharmacol Exp Ther 1996;276:637-646.
[3] Carlsson L, Drews L, Duker G, Schiller-Linhardt G. Attenuation of proarrhythmias
related to delayed repolarization by low-dose lidocaine in the anesthetized rabbit. J
Pharmacol Exp Ther 1993;267:1076-1080.
[4] Clancy CE, Tateyama M, Liu H, Wehrens XH, Kass RS. Non-equilibrium gating in
cardiac Na+ channels: an original mechanism of arrhythmia. Circ 2003;107:22332237.
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