REPOLARISATION ASSAYS

SINGLE VENTRICULAR MYOCYTES

ACTION POTENTIAL RECORDINGS IN SINGLE VENTRICULAR MYOCYTE

Figure 1 Representative record of an action potential recorded from a single ventricular myocyte.

Figure 1 shows typical recording made by scientists at OCP action potential taken from a single left ventricular myocyte under our standard experimental conditions used for repolarisation assays.

Standard conditions used for the repolarisation assay using single ventricular myocytes

Action potentials are recorded using conventional glass microelectrodes filled with 1 M KMeSO₄ and 10 mM KCl. Electrode resistances are in the range of 40 to 60 MW.  An Axoclamp 2B (Axon Instruments Inc) amplifier is used for electrical recording.  Action potentials are stimulated by application of brief (1-2 ms) current stimuli applied via the intracellular microelectrode in 'bridge' mode.  Following penetration of the cell with the microelectrode, a stabilisation period of at least 5 min is allowed before compounds are applied. The frequency of stimulation will normally be 1Hz, but for determination of possible frequency dependence effects of test compounds additional frequencies (e.g. 0.2 and 0.5 Hz) can be used.  Recorded signals are digitised at a sampling frequency of 100 kHz (i.e. 10 µs per point) and stored for subsequent off line analysis.  Action potential signals are continuously monitored online using an oscilloscope (Tektronix digital storage oscilloscope) to check for development of drug effects.  All recordings are made at 36˚C.

Action potential configuration is accessed by measuring the following parameters

• Resting potential
• Action potential amplitude
• Action potential duration
• Maximal rate of depolarisation

Resting potential is normally measured over a 5-10 ms period before the stimulus artefact.  Values for the resting potential normally fall between -75 to -85 mV.

Action potential amplitude is measured following the stimulus artefact and values normally fall between 120 to 130 mV.

Action potential duration is routinely measured at 50 % and 90 % repolarisation levels (APD₅₀ and APD₉₀).  Additionally, the time taken to reach any degree of repolarisation (e.g. 20%) can be measured.

Maximal rate of depolarisation is measured during the upstroke of the action potential following the stimulus artefact (See Figure 2) and values normally fall in the region of 400 to 600 V/s (measured at a sampling frequency of 10 µs per point).

Figure 2  Stimulus artefact and measurement of maximal rate of depolarisation

Action potentials are elicited by a 1-2 ms stimulus.  Under conditions where appropriate compensation is made for the electrode resistance and capacitance, the stimulus artefact resembles that shown in Figure 2 and in green in Figure 3.

The maximal rate of depolarisation can then be measured in the time period following the stimulus artefact shown in red. Resting potential is normally measured over a 5-10 ms period before the stimulus artefact shown in yellow.