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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


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

PDF or HTML at Wiley Online Library

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
PDF or HTML at Wiley Online Library


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

  • 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,
    • Oscilloscope
    • High‐impedance capacitance‐neutralizing amplifier (e.g., Emka Technologies,
    • Reference Ag/AgCl electrode (E205, Phymep, http://www.phymep‐
    • Ag/AgCl wire filament electrode (E255, Phymep, http://www.phymep‐
    • 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
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Abi‐Gerges, N., Small, B.G., Lawrence, C.L., Hammond, T.G., Valentin, J‐P., and Pollard, C.E. 2004. Evidence for gender differences in electrophysiological properties of canine Purkinje fibres. Br. J. Pharmacol. 142:1255‐1264.
   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.
   Antzelevitch, C. and Sicouri, S. 1994. Clinical relevance of cardiac arrhythmias generated by afterdepolarizations. Role of M cells in the generation of U waves, triggered activity and torsade de pointes. J. Am. Coll. Cardiol. 23:259‐277.
   Brosch, S.F., Studenik, C., and Heistracher, P. 1998. Abolition of drug‐induced early afterdepolarizations by potassium channel activators in guinea‐pig Purkinje fibres. Clin. Exp. Pharmacol. Physiol. 25:225‐230.
   Burashnikov, A. and Antzelevitch, C. 1998. Acceleration‐induced action potential prolongation and early afterdepolarizations. J. Cardiovasc. Electrophysiol. 9:934‐948.
   Burashnikov, A. and Antzelevitch, C. 2006. Late‐Phase 3 EAD. A unique mechanism contributing to initiation of atrial fibrillation. Pacing Clin. Electrophysiol. 29:290‐295.
   Davidenko, J.M., Cohen, L., Goodrow, R., and Antzelevitch, C. 1989. Quinidine‐induced action potential prolongation, early afterdepolarizations, and triggered activity in canine Purkinje fibers. Effects of stimulation rate, potassium and magnesium. Circulation 79:674‐686.
   Dumotier, B.M., Adamantidis, M.M., Puisieux, F.L., Bastide, M.M., and Dupuis, B.A. 1999. Repercussions of pharmacologic reduction in ionic currents on action potential configuration in rabbit Purkinje fibers: Are they indicative of proarrhythmic potential? Drug Dev. Res. 47:63‐79.
   Farkas, A., Lepran, I., and Papp, J.G. 2002. Proarrhythmic effects of intravenous quinidine, amiodarone, D‐sotalol, and almokalant in the anesthetized rabbit model of torsades de pointes. J. Cardiovasc. Pharmacol. 39:287‐297.
   Fazekas, T., Krassoi, I., Lengyel, C., Varro, A., and Papp, J.G. 1998. Suppression of erythromycin‐induced early afterdepolarizations and torsades de pointes ventricular tachycardia by mexiletine. Pacing Clin. Electrophysiol. 21:147‐150.
   Goineau, S., Picard, S., and Lacroix, P. 2004. Species‐ and gender‐related effects of moxifloxacin on cardiac repolarization in isolated Purkinje fibers. J. Pharmacol. Toxicol. Meth. 49:217‐239.
   Jones, D.L., Petrie, J.P., and Li, H.G. 2001. Spontaneous, electrically, and cesium chloride induced arrhythmia and afterdepolarizations in the rapidly paced dog heart. Pacing Clin. Electrophysiol. 24:474‐485.
   Joshi, A., Dimino, T., Vohra, Y., Cui, C., and Yan, G.X. 2004. Preclinical strategies to assess QT liability and torsadogenic potential of new drugs: The role of experimental models. J. Electrocardiol. 37 suppl:7‐14.
   Kondo, M., Tsutsumi, T., and Mashima, S. 1999. Potassium channel openers antagonize the effects of Class III antiarrhythmic agents in canine Purkinje fiber action potentials. Jpn. Heart J. 40:609‐619.
   Lu, H.R., Vlaminckx, E., Teisman, A., and Gallacher, D.J. 2005. Choice of cardiac tissue plays an important role in the evaluation of drug‐induced prolongation of the QT interval in vitro in rabbit. J. Pharmacol. Toxicol. Methods. 52:90‐105.
   Ooie, T., Takahashi, N., Saikawa, T., Iwao, T., Hara, M., and Sakata, T. 2000. Suppression of cesium‐induced ventricular tachyarrhythmias by atrial natriuretic peptide in rabbits. J. Card. Fail. 6:250‐256.
   Orth, P.M., Hesketh, J.C., Mak, C.K., Yang, Y., Lin, S., Beatch, G.N., Ezrin, A.M., and Fedida, D. 2006. RSD1235 blocks late I(Na) and suppresses early afterdepolarizations and torsades de pointes induced by class III agents. Cardiovasc. Res. 14: in press.
   Patterson, E., Scherlag, B.J., and Lazzara, R. 1997a. Early afterdepolarizations produced by d,l‐sotalol and clofilium. J. Cardiovasc. Electrophysiol. 8:667‐678.
   Patterson, E., Scherlag, B.J., Szabo, B., and Lazzara, R. 1997b. Facilitation of epinephrine‐induced afterdepolarizations by class III antiarrhythmic drugs. J. Electrocardiol. 30:217‐224.
   Rubart, M., Pressler, M.L., Pride, H.P., and Zipes, D.P. 1993. Electrophysiological mechanisms in a canine model of erythromycin‐associated long QT syndrome. Circulation 88:1832‐1844.
   Sato, T., Hirao, K., and Hiejima, K. 1993. The relationship between early afterdepolarization and the occurrence of torsades de pointes—An in vivo canine model study. Jpn. Circ. J. 57:543‐552.
   Sheridan, R.D., Turner, S.R., Cooper, G.J., and Tattersall, J.E. 2005. Effects of seven drugs of abuse on action potential repolarisation in sheep cardiac Purkinje fibres. Eur. J. Pharmacol. 511:99‐107.
   Sicouri, S. and Antzelevitch, C. 1991. Afterdepolarizations and triggered activity develop in a select population of cells (M cells) in canine ventricular myocardium: The effects of acetylstrophanthidin and Bay K 8644. Pacing Clin. Electrophysiol. 14:1714‐1720.
   Sicouri, S., Moro, S., and Elizari, M.V. 1997. d‐Sotalol induces marked action potential prolongation and early afterdepolarizations in M but not empirical or endocardial cells of the canine ventricle. J. Cardiovasc. Pharmacol. Ther. 2:27‐38.
   Verduyn, S.C., Vos, M.A., Van der Zande, J., Kulcsar, A., and Wellens, H.J.J. 1997. Further observations to elucidate the role of interventricular dispersion of repolarization and early afterdepolarizations in the genesis of acquired torsade de pointes arrhythmias. A comparison between almokalant and d‐sotalol using the dog as its own control J. Am. Coll. Cardiol. 30:1575‐1584.
   Viskin, S. 1999. Long QT syndromes and torsade de pointes. Lancet 354:1625‐1633.
   Yan, G‐X., Wu, Y., Liu, T., Wang, J., Marinchak, R.A., and Kowey, P.R. 2001. Phase 2 early afterdepolarization as a trigger of polymorphic ventricular tachycardia in acquired long‐QT syndrome. Circulation 103:2851‐2856.
PDF or HTML at Wiley Online Library