The Action Potential of the Purkinje Fiber: An In Vitro Model for Evaluation of the Proarrhythmic Potential of Cardiac and Noncardiac Drugs

Sandra Picard1, Sonia Goineau1, Rene Rouet2

1 Porsolt and Partners Pharmacology, Boulogne‐Billancourt, 2 Universite de Caen, Caen
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 11.3
DOI:  10.1002/0471141755.ph1103s33
Online Posting Date:  July, 2006
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Abstract

The proarrhythmic potential of new chemical entities can be investigated using in vitro electrophysiological techniques measuring the cardiac action potential in isolated Purkinje fibers. Different types of arrhythmias may occur as early afterdepolarizations (EADs), which are favored by action potential duration lengthening and bradycardia, or as delayed afterdepolarizations (DADs), which are facilitated by tachycardia. The effects of a test compound on the occurrence of these arrhythmias, thought to be responsible for the development of torsades de pointes in the clinic can be studied using the experimental protocols described in this unit.

Keywords: cardiac risk assessment; proarrhythmic potential; Purkinje fiber; action potential; safety pharmacology

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of Changes in the Action Potential Parameters in the Purkinje Fiber During Superfusion with a Test Compound
  • Alternate Protocol 1: Evaluation of the Proarrhythmic Risk of a Test Compound on Epinephrine‐Facilitated Early Afterdepolarizations
  • Alternate Protocol 2: Evaluation of the Proarrhythmic Risk of a Test Compound on the Occurrence of Delayed Afterdepolarizations and/or Triggered Activities
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Changes in the Action Potential Parameters in the Purkinje Fiber During Superfusion with a Test Compound

  Materials
  • Methylene blue, optional
  • Cardioplegic Tyrode's solution (see recipe)
  • Male New Zealand albino rabbit weighing 2 to 2.5 kg
  • Normal Tyrode's solution (see recipe)
  • Test compound
  • 3.7% (v/v) HCl, prepared by diluting 37% (v/v) HCl with water
  • Experimental organ bath (see Fig. ), consisting of:
    • Temperature‐controlled water circulation system capable of maintaining a temperature of 36.5° ± 0.5°C in the superfusion chamber (e.g., Polystat; Fisher Scientific)
    • Plexiglas superfusion chamber (3‐ml capacity) with rubber silicone base (homemade; see step annotation) coupled to a Plexiglas tank (20 × 10 × 12 cm)
    • Peristaltic pump
    • Vacuum pump
    • Gassing system to deliver 95% (v/v) O 2/5% (v/v) CO 2
    • Polyethylene tubing for connecting bath components
  • Stereomicroscope
  • Stimulation equipment, consisting of:
    • Computer with software (e.g., Bio‐Stim, Bio‐Logic Research & Development) for driving the stimulation
    • Programmable stimulator (e.g., Bio‐Logic SMP‐311; Bio‐Logic Research & Development)
    • Bipolar stimulation electrode (homemade; see step annotation)
  • 100‐, 200‐, and 500‐ml flasks
  • Dissection materials, including:
    • Dissection dishes with and without rubber silicone base
    • Fine wire dissection tools
    • Fine pins
  • Recording equipment, consisting of:
    • Computer equipped with software for acquisition and analysis of the electrical signal (e.g., Iox, Emka Technologies, http://www.emka.fr)
    • Oscilloscope
    • High‐impedance capacitance‐neutralizing amplifier (e.g., Emka Technologies, http://www.emka.fr)
    • Reference Ag/AgCl electrode (E205, Phymep, http://www.phymep‐sarl.com)
    • Ag/AgCl wire filament electrode (E255, Phymep, http://www.phymep‐sarl.com)
    • Glass microelectrode (see unit 11.2) filled with 3 M KCl
  • Microelectrode holder attached to a micromanipulator (e.g., Harvard Apparatus)
  • Antivibratory plate (e.g., Harvard Apparatus)
  • Additional reagents and equipment for exsanguination (Dumotier et al., )

Alternate Protocol 1: Evaluation of the Proarrhythmic Risk of a Test Compound on Epinephrine‐Facilitated Early Afterdepolarizations

  • 0.1 µM epinephrine, prepared fresh in normal Tyrode's solution (see recipe) on the day of use
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Figures

Videos

Literature Cited

Literature Cited
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   Adamantidis, M.M., Lacroix, P., Caron, J.F., and Dupuis, B.A. 1995. Electrophysiological and arrhythmogenic effects of the histamine type 1‐receptor antagonist astemizole on rabbit Purkinje fibers: Clinical relevance. J. Cardiovasc. Pharmacol. 26:319‐327.
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