Measurement of Action Potential Generation in Isolated Canine Left Ventricular Midmyocardial Myocytes

Daniel M. Johnson1, Leyla Hussein2, Roel L.H.M.G. Spätjens1, Jean‐Pierre Valentin2, Paul G.A. Volders1, Najah Abi‐Gerges2

1 Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands, 2 Safety Assessment UK, AstraZeneca R&D, Macclesfield, Cheshire, United Kingdom
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 10.14
DOI:  10.1002/0471141755.ph1014s55
Online Posting Date:  December, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Proarrhythmic side effects are a major limitation during the drug development process for cardiac and non‐cardiac compounds. Because changes in cardiac action potential (AP) are undesirable, the evaluation of the effects of test compounds on the AP is essential before advancing new compounds to clinical testing. However, an increase in repolarization duration alone is not always proarrhythmic, and newer surrogate markers have been suggested to better predict the occurrence of arrhythmia. Described in this unit is a protocol for assessing changes in AP duration in canine ventricular myocytes utilizing optical imaging techniques. This protocol can be used at an early stage of drug discovery due to its relatively fast throughput. Additionally, a protocol is presented for assessing the occurrence of after‐depolarizations, as well as a novel parameter for proarrhythmic risk, beat‐to‐beat variability of repolarization. This protocol can be used at a later stage of the drug discovery process to assess proarrhythmic potential. Curr. Protoc. Pharmacol. 55:10.14.1‐10.14.23. © 2011 by John Wiley & Sons, Inc.

Keywords: left ventricular midmyocardial myocyte; action potential; nonclinical safety; early after‐depolarization; QT interval prolongation; arrhythmogenic potential

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Test Compound‐Induced Changes in Action Potential Parameters in Canine Midmyocardial Myocytes
  • Alternate Protocol 1: Arrhythmogenic Effects of a Test Compound on Beat‐to‐Beat Variability of Repolarization and Occurrence of Early and Delayed After‐Depolarizations
  • Alternate Protocol 2: Arrhythmogenic Effects of a Test Compound on Beat‐to‐Beat Variability of Repolarization Under Conditions that Mimic Long QT 1 in the Presence of β‐Adrenergic Stimulation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Measurement of Test Compound‐Induced Changes in Action Potential Parameters in Canine Midmyocardial Myocytes

  Materials
  • Female beagle dog (≥9 kg, 9‐35 months)
  • Normal Tyrode's solution (see recipe), calcium‐free and containing 0.2 and 1.8 mM CaCl 2
  • Bovine albumin serum (BSA, Sigma‐Aldrich)
  • Pentobarbitone
  • 1 M CaCl 2
  • Collagenase A (Roche Diagnostics)
  • 70% and 100% (v/v) ethanol in water
  • Di‐4‐ANEPPS (Sigma‐Aldrich)
  • Test compound
  • Reference compound (e.g., dofetilide, a class III antiarrhythmic agent)
  • Dimethyl sulfoxide (DMSO; Sigma)
  • Equipment and supplies for myocyte isolation (Fig. , left panel) plus:
    • Micro‐osmometer (e.g., Advanced Instruments model 3300)
    • pH meter (e.g., SevenMulti, Mettler Toledo)
    • 1‐liter sterile Schott Duran bottles (LabPlanet, IL, USA)
    • Gassing system to deliver medical O 2 (99.5%, type F cylinder)
    • Oxygenating bubbler
    • Surgical scissors, straight, S/S, 105 mm (4‐1/8 in.)
    • Mayo dissection scissors, straight, B/B, 140 mm (5‐1/2 in.)
    • Medium dissecting forceps, straight, 160 mm (6.30 in.)
    • Aesculap Iris dissection forceps (Ted Pella)
    • 100‐, 200‐, and 500‐ml beakers
    • Sewing needle and thread
    • Waste tissue bag
    • 50‐ml BD Plastika syringe
    • Polyethylene tubing
    • Heating coil (Radnoti)
    • DC50‐B5 heating circulator (HAAKE, Thermo Fisher Scientific)
    • Perfusion system (R3603, Tygon, Saint‐Gobain Performance Plastics Corporation)
    • Cannula (vessel dilator 1.65 × 0.53 mm; Cordis, J&J Company)
    • 10‐ml syringes
    • 130‐mm dissection dishes without rubber silicone base
    • Cell dissociation sieve tissue grinder kit (Sigma‐Aldrich) with Screens for CD‐1 (100‐µm mesh)
    • Inverted microscope (e.g., Nikon Eclipse TS100)
  • Equipment and supplies for recording optical AP (Fig. ), including:
    • Antivibration table (Scientifica) and Faraday cage (Type II, Scientifica), covered with materials that prevent entry of light
    • Inverted microscope (e.g., Nikon Eclipse TE200) with 40× oil objective (1.30 NA, Nikon)
    • FHD microscope chamber system (IonOptix)
    • Cell MicroControls mTCII temperature controller and heater (IonOptix, Dublin, Ireland)
    • CF‐8vs valve assembly (IonOptix)
    • cFlow controller (IonOptix)
    • Immersion oil for fluorescence and general microscopy (Cargille Laboratories)
    • Long‐pass (>700 nm) and narrow band‐pass (540‐580 and 600‐640 nm) filters
    • Chromatic reflectors (<590 and <700 nm)
    • Dual‐emission photometry system with two photomultiplier tubes (PMTs), each with an amplifier and high‐voltage power supply (Cairn Research)
    • Xenon arc lamp power supply associated with arc lamp + housing (Cairn Research)
    • Cairn shutter supply associated with xenon arc lamp + housing (Cairn Research)
    • TH‐10Kmp‐thermistor probe (Cell MicroControls)
    • Bath‐mounted platinum wire electrodes
    • Infrared camera (Watec) and Vista 14‐inch monitor (Norbain SD)
    • HSE stimulator P (Hugo Sachs Elektronik) or MyoPacer Field Stimulator (IonOptix)
    • MultiClamp 700A amplifier (Molecular Devices)
    • Digidata 1322A digitizer (Molecular Devices)
    • Computer with pClamp 10 software (Molecular Devices) for acquisition and analysis of electrical signal
    • Corning cover glass (no. 1, 25‐mm sq; Corning Life Sciences)
    • Vacuum pump (Rena Air 200, Planet Rena)
    • Lens cleaning tissue (Thermo Fisher Scientific)

Alternate Protocol 1: Arrhythmogenic Effects of a Test Compound on Beat‐to‐Beat Variability of Repolarization and Occurrence of Early and Delayed After‐Depolarizations

  Materials
  • Isolated LVMMs (see Basic Protocol)
  • Normal Tyrode's solution (see recipe) containing 1.8 mM CaCl 2
  • Test compound
  • Reference compound (e.g., dofetilide, a class III antiarrhythmic agent)
  • Dimethyl sulfoxide (DMSO; Sigma)
  • 3 M KCl
  • Equipment and supplies for microelectrode recording of AP (Fig. ), consisting of:
    • Antivibration table and Faraday cage (e.g., TMC)
    • Inverted microscope (e.g., Nikon Eclipse TE200) with 10× eyepieces and 40× oil objective
    • Perfusion chamber for myocytes (e.g., BT‐1[‐SY], Cell MicroControls)
    • Perfusion system with water‐jacketed heat exchanger for inflow of superfusate to myocyte chamber (e.g., Radnoti)
    • Micromanipulator for coarse and fine positioning of microelectrodes (e.g., Narishige)
    • Headstage with microelectrode holder (e.g., Molecular Devices)
    • Glass microelectrodes (see unit 11.2 for fabrication of electrodes)
    • Fiber optic light source (e.g., Schott KL1500, Schott)
    • Programmable stimulator and computer with software for driving stimulator (e.g., Tabor pulse generator, Tabor Electronics)
    • MultiClamp 700A or Axoclamp 2B amplifier, Digidata 1322A digitizer (Molecular Devices)
    • Computer with pClamp 10 software (Molecular Devices) for acquisition and analysis of electrical signal
    • Video camera and monitor (e.g., Pulnix TM‐640, JAI, and CEM‐12A‐II, CBC Europe)
    • Video edge‐motion detector (e.g., Crescent Electronics VED‐105)
    • 3‐ml syringe and 22‐G spindle needle (World Precision Instruments, Germany)

Alternate Protocol 2: Arrhythmogenic Effects of a Test Compound on Beat‐to‐Beat Variability of Repolarization Under Conditions that Mimic Long QT 1 in the Presence of β‐Adrenergic Stimulation

  • 10 mM isoproterenol (Sigma) in 30 µM ascorbic acid (prepare immediately before use and store in the dark at 4°)
NOTE: Stock and working solutions of isoproterenol should be wrapped in tin foil and kept closed whenever possible to prevent degradation.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure 10.14.1 Isolation of canine LVMMs. Left: Apparatus for isolating LVMM cells from a canine heart, including solution reservoir (A), heated coil (B), cannula (C), and heart (D). Top center and top right: Cannulation (C) of the left anterior descending (LAD) coronary artery for perfusion of the left ventricle (LV). The catheter is placed through the aorta into the LAD (E). Bottom right: A section through the heart shows the midmyocardial region from which myocytes are isolated (striped area). Bottom center: A single isolated LVMM (F).
  •   FigureFigure 10.14.2 Optical experimental setup and tissue bath. Epifluorescence recordings are made from LVMM cells as previously described (Hardy et al., ). The setup includes a Faraday cage (A), HSE stimulator P (B), Cairn shutter (C), cFlow controller (D), Vista 14‐inch monitor (E), dual‐emission photometry system (F), MultiClamp 700A (G), Digidata 1322A (H), antivibration table (I), vacuum waste bottle (J), inverted microscope (K), long‐pass filter (L, >700 nm), CF‐8vs valve assembly (M), superfusion tubing (N), connection to iris (xenon arc lamp, O), narrow band‐pass filters (P, 540‐580 and 600‐640 nm), infrared camera (Q), chromatic reflectors (R, <590 and <700 nm), temperature controller (S), outflow (T), thermistor (U), chamber (V), oil objective (W), inflow (X), electrodes (Y), and heater (Z).
  •   FigureFigure 10.14.3 Measurement of changes in the AP during superfusion with reference compounds. (A) Representative optical AP of a canine LVMM cell and parameters measured during the experiment. APD50, APD70, and APD90: action potential duration optically measured at 50%, 70%, and 90% repolarization. (BD) Representative optical APs recorded using di‐4‐ANEPPS‐based method with vehicle solution (0.1% DMSO) and in the presence of dofetilide (class III antiarrhythmic; 1 µM), cisapride (multiple ion channel blocker; 0.1 and 10 µM), and terfenadine (multiple ion channel blocker; 0.1 and 10 µM). B‐D reprinted from Hardy et al., ( J. Pharmacol. Toxicol. Methods 60(1):94‐106) with permission from Elsevier
  •   FigureFigure 10.14.4 Effects of nine reference compounds on optically measured APD. Graphs show mean % change in APD50 and APD90 induced by (A) dofetilide (n = 6 [1 dog] for each set), (B) E4031 (class III antiarrhythmic; n = 6 [1 dog]), (C) D‐sotalol (class III antiarrhythmic; n = 8 [2 dogs] for first set, n = 6 [1 dog] for second set, n = 3 [1 dog] for E4031 during second set), (D) ATXII (agonist of late INa; n = 5 [1 dog]), (E) cisapride ( n = 5 [1 dog]), (F) terfenadine ( n = 5 [1 dog]), (G) alfuzosin (multiple ion channel blocker; n = 6 [1 dog] and n = 4 [1 dog] for ATXII for experiments in which D‐sotalol did not have an effect on optically measured APD), (H) diltiazem (inhibitor of ICa,L; n = 7 [2 dogs]), and (I) pinacidil (opener of IKATP; n = 4 [2 dogs]) at a pacing frequency of 1 Hz. Data expressed as mean ± SEM. * P < 0.05, $ P < 0.01, and # P < 0.001 compared to values from 0.1% DMSO. Statistical analysis is to be ‘within cell’, with each concentration of each reference compound within each cell compared to vehicle (0.1% DMSO). Change from vehicle analyzed and the geometric mean change (%) from vehicle is to be reported, together with 95% confidence limits for the mean change. Effects are to be reported as statistically significant if P < 0.05. Increases or decreases are to be considered equally likely, leading to a two‐sided testing approach. The data are log transformed prior to analysis and each parameter is analyzed separately. All comparisons are with the vehicle group. Reprinted from Hardy et al., ( J. Pharmacol. Toxicol. Methods 60(1):94‐106) with permission from Elsevier
  •   FigureFigure 10.14.5 Sharp‐electrode experimental setup. The center panel is a schematic drawing of the assembly of components in the outer photographs, including inverted microscope (A), microscope table with perfusion chamber (B), perfusion system with water‐jacketed heat exchanger for inflow of superfusate (C), fiber‐optic light source (D), micromanipulators for coarse and fine positioning of microelectrodes (E), video camera (F), headstage (preamplifier) with holder and microelectrode (G), vibration‐isolated work station (H), stimulator (I), microelectrode amplifiers (J), video edge‐motion detector (K), analog‐to‐digital converter (L), and computer (M) with pCLAMP 10 software for automated waveform control and data acquisition.
  •   FigureFigure 10.14.6 Effects of test compounds on APs recorded from a canine LVMM using the SE technique. Effects of (A) HMR1556 (inhibitor of IKs; 0.5 µM), (B) dofetilide (class III antiarrhythmic; 1 µM), and (C) ATX II (augmenter of late INa; 0.02 µM) on BVR and APD90 at 1 Hz pacing frequency. Representative AP recordings showing the minimum, maximum, and median beats are shown for each condition. Poincaré plots for each condition are also shown. Reprinted from Johnson et al., ( J. Mol. Cell. Cardiol. 48(1):122‐130) with permission from Elsevier
  •   FigureFigure 10.14.7 Effect of β‐adrenergic stimulation on a canine LVMM after blockade of the IKs current with 0.5 µM HMR1556. (A) Consecutively paced APs at 1 Hz pacing frequency, together with Poincaré plots during initial perfusion of HMR1556. (B) Addition of β‐adrenergic stimulation increases APD and BVR before DADs are seen. (C) The occurrence of DADs increases APD and BVR further, and DAD‐triggered APs and EADs are subsequently observed (D). Arrows indicate timings of paced beats and asterisks indicate EADs. Reprinted from Johnson et al., ( J. Mol. Cell. Cardiol. 48(1):122‐130) with permission from Elsevier

Videos

Literature Cited

Literature Cited
   Abi‐Gerges, N., Valentin, J.P., and Pollard, C.E. 2010. Dog left ventricular midmyocardial myocytes for assessment of drug‐induced delayed repolarization: Short‐term variability and proarrhythmic potential. Br. J. Pharmacol. 159:77‐92.
   Antzelevitch, C. 2007. Heterogeneity and cardiac arrhythmias: An overview. Heart Rhythm 4:964‐972.
   Bass, A., Valentin, J.P., Fossa, A.A., and Volders, P.G. 2007. Points to consider emerging from a mini‐workshop on cardiac safety: Assessing torsades de pointes liability. J. Pharmacol. Toxicol. Methods 56:91‐94.
   Bridgland‐Taylor, M.H., Hargreaves, A.C., Easter, A., Orme, A., Henthorn, D.C., Ding, M., Davis, A.M., Small, B.G., Heapy, C.G., Abi‐Gerges, N., Persson, F., Jacobson, I., Sullivan, M., Albertson, N., Hammond, T.G., Sullivan, E., Valentin, J.P., and Pollard, C.E. 2006. Optimisation and validation of a medium‐throughput electrophysiology‐based hERG assay using IonWorks HT. J. Pharmacol. Toxicol. Methods 54:189‐199.
   Fenichel, R.R., Malik, M., Antzelevitch, C., Sanguinetti, M., Roden, D.M., Priori, S.G., Ruskin, J.N., Lipicky, R.J., and Cantilena, L.R. 2004. Drug‐induced torsades de pointes and implications for drug development. J. Cardiovasc. Electrophysiol. 15:475‐495.
   Gallacher, D.J., Van de Water, A., van der Linde, H., Hermans, A.N., Lu, H.R., Towart, R., and Volders, P.G. 2007. In vivo mechanisms precipitating torsades de pointes in a canine model of drug‐induced long‐QT1 syndrome. Cardiovasc. Res. 76:247‐56.
   Gralinski, M.R. 2003. The dog's role in the preclinical assessment of QT interval prolongation. Toxicol. Pathol. 31:S11‐S16.
   Hardy, M.E.L., Pollard, C.E., Small, B.G., Bridgland‐Taylor, M., Woods, A.J., Valentin, J‐P., and Abi‐Gerges, N., 2009. Validation of a voltage‐sensitive dye (di‐4‐ANEPPS)‐based method for assessing drug‐induced delayed repolarisation in beagle dog left ventricular midmyocardial myocytes. J. Pharmacol. Toxicol. Methods 60:94‐106.
   Harmer, A.R., Abi‐Gerges, N., Easter, A., Woods, A., Lawrence, C.L., Small, B.G., Valentin, J.P., and Pollard, C.E. 2008. Optimisation and validation of a medium‐throughput electrophysiology‐based hNav1.5 assay using IonWorks. J. Pharmacol. Toxicol. Methods 57:30‐41.
   Hondeghem, L.M. 2008. QT prolongation is an unreliable predictor of ventricular arrhythmia. Heart Rhythm 5:1210‐1212.
   ICH (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use). 2005a. The nonclinical evaluation of the potential for delayed ventricular repolarization (QT interval prolongation) by human pharmaceuticals. ICH S7B CHMP/ICH/423/02, May 25, 2005.
   ICH (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use). 2005b. The clinical evaluation of QT/QTc interval prolongation and proarrhythmic potential for non‐antiarrhythmic drugs. ICH E14 CHMP/ICH/2/04, May 25, 2005.
   Jacobson, I., Carlsson, L., and Duker, G. 2010. Beat‐by‐beat QT interval variability, but not QT prolongation per se, predicts drug‐induced torsades de pointes in the anaesthetised methoxamine‐sensitized rabbit. J. Pharmacol. Toxicol. Methods 63:40‐46.
   Johnson, D.M., Heijman, J., Pollard, C.E., Valentin, J.P., Crijns, H.J., Abi‐Gerges, N., and Volders, P.G. 2010. I(Ks) restricts excessive beat‐to‐beat variability of repolarization during beta‐adrenergic receptor stimulation. J. Mol. Cell. Cardiol. 48:122‐130.
   Lindgren, S., Bass, A.S., Briscoe, R., Bruse, K., Friedrichs, G.S., Kallman, M.J., Markgraf, C., Patmore, L., and Pugsley, M.K. 2008. Benchmarking safety pharmacology regulatory packages and best practice. J. Pharmacol. Toxicol. Methods 58:99‐109.
   Schroeder, K., Neagle, B., Trezise, D.J., and Worley, J. 2003. Ionworks HT: A new high‐throughput electrophysiology measurement platform. J. Biomol. Screen. 8:50‐64.
   Szabo, G., Szentandrassy, N., Biro, T., Toth, B.I., Czifra, G., Magyar, J., Bányász, T., Varró, A., Kovács, L., and Nánási, P.P. 2005. Asymmetrical distribution of ion channels in canine and human left‐ventricular wall: Epicardium versus midmyocardium. Pflugers Arch. 450:307‐316.
   Thomsen, M.B., Verduyn, S.C., Stengl, M., Beekman, J.D., De Pater, G., Van Opstal, J., Volders, P.G., and Vos, M.A. 2004. Increased short‐term variability of repolarization predicts D‐sotalol‐induced torsades de pointes in dogs. Circulation 110:2453‐2459.
   Thomsen, M.B., Matz, J., Volders, P.G., and Vos, M.A. 2006. Assessing the proarrhythmic potential of drugs: Current status of models and surrogate parameters of torsades de pointes arrhythmias. Pharmacol. Ther. 112:150‐170.
   Volders, P.G., Stengl, M., van Opstal, J.M., Gerlach, U., Spätjens, R.L., Beekman, J.D., Sipido, K.R., and Vos, M.A. 2003. Probing the contribution of IKs to canine ventricular repolarization: Key role for beta‐adrenergic receptor stimulation. Circulation 107:2753‐2760.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library