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EFFECT OF COMBINATION POT.-i\SSIL'bI CHANNEL BLOCKERS ON CARDIAC REFEb$.CTORINESS IN ISOLXÎED RABBIT HEARTS AND NTACT GUI'IE.4 PIG HE.4RTS

A thssis submitted in conformit>.with the requiremrnts for the degres of hlastrr of Science Graduate Depanment of Medical Science Universit). of Toronto

O Copyright by Nikolaos Marnalias 200 1 National Libraiy Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliogrâphic Senrices services bibliographiques

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The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts from it Ni la thèse ni des extraits substantiels may be printed or otherwise de ceiieîi ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. EFFECT OF COMBINATION POTASSIUM CHANNEL BLOCKERS ON CARDIAC REFRACTORiNESS IN ISOLATED RABBIT HEARTS AND iNTACT GUiNEA PIG HEARTS

NIKOLAOS MAM.4LIAS MASTER OF SCIENCE 200 1 GRADUATE DEPARTMENT OF MEDICAL SCIENCE LJNIVERSITY OF TORONTO

ABSTRACT

Dofetilide and sotaiol prolong ventricular refractorinrss by blocking the rapidly açtivating delayrd rectitier potassium channel (Ikr). In vitro studies suggest that dofetilide blocks the Ikr channel in the open state. whereas sotalol may block the closed state. We tested whethrr this potential dittrrential statr-dependent blockade could br pharmacologically distinguishrd bu altering Ikl currrnt density. Sotalol (4 PM) and dofetilide (8 nM) were given alone and in combination with 3 pM barium (Ikl blocker)

to isolated perfused Langendorff rabbit hearts (n=8 per group). Ventricular effective

refractory penod (VERP). action potential duration. vèntricular tibrillation cycle length. and defibrillation threshold were measured pre and post drug'vehicle treatment. The

VERP was prolonged with dofetilide. sotalol. barium. combination dofetilide plus

barium. and sotaloi plus barium by 10?6%. 8e%. 7+3%. I-l+j0'o. and 824%.

resprctively (~~0.01 ). The cffect of dofetilide plus barium on VERP was additive.

whereas sotalol plus barium was no different than each drug alone: this interaction was

qualitatively the same for al1 other variables measured. This is the fint study to show

that different Ikr blockers have differîng effects when combined with another potassium

channel blocker. ACKNOWLEDGEMENTS

The completion of rhis thesis is my grearest scholastic accomplishmenr to dore. and I ivould like to ocknowledge the conrribirtions of severul indiricfurrls to rhis research.

It is wirh sincere gratitlrde rhat I rhank Dr. Paul Dorian for providing an environment conducive [O learning and scientifc inquiry. His scientific reachings und kind, genuine. strong moral support isill never be forgotfen. He rvas not only o mentor, but also a role mode1 thut exposed me [O the medical world oitfside the specificity of'my research. Throughout my gradirate rraining he helped me develop criricul thinking und problem solving skills. ivhich I ivillfind inwliruble horh in a crrreer in medicine. und rvhile encounrering chrrllrnges txprrirncrtl in lifè.

I would dso like tu rhonk Drs. David Yrwman anJ Perer Buck fur fheir gui~krnce as Program Advisory Cumrnitter membrrs for the diirariun ofhy reseurch. Their inqtrisitive nature und consrnrcrive !kedbock grearly broadened rhe scope ofrny knoivledge. It was rheir conrinuoirs encourugemenr thut hrlprd nie upprouch nerv chullenp wirh conjidence.

The help of Dr. .Yiungqinn Qi rvill rrhvcrjs h rememhered. us he sirpentsed tny rrork in rhe rinimui luborurory. He rvas u greur terrcher nor only of'rirrgicaf mrrhods, hirt rrlso of anest iesia. ctrrdiology, unJ concepts in rlr~*trophy.siolo p. Hi.s cissis~rin~~fi~rnied rht. groirndrivrk of'rstuhlishing the jirsr elecrrophysiologic infacfgttineu pig hrurr rnocirl. The ncrrurr o#:hispedtrgo~~ iius wirh good hrrrnoiir und enthir.sirrsm. ivhich helped me rnzhrace my rwrk dzrri>~prhe musi diffictrfr rimes.

I um very graiejirljor the bcisic science rmhings in cdiac e1ectrophysiulog-y uf' Ur. Rafid Rumirez (Ph.D. ctrndiciurr) mdfor rhe crssistunce in rhe oninial lcihoruroq. of !W.Cumeron Smirh (,Cf.Sc.cuntlidure~ over rhe l~isrycrr oj'mv resrcirch. I.fC.rl privilrpd ro itoiw collrburared ri+i!lithese two j~tniorrcicntisrs who gwdy wlire rhr tool 91' research us a meuns of-de fermining the ~rzrrh.

I thank Drs. Gil Gross. Perer Pennefirther, Robert Tsirshirnu. and Sirsan Belo /or rhe ir r ivocio us prrrt icipu ion in my M.Sc. Oral Ermninarion. Their us.sis~unceut rr rnornenr 's no rice. und rheir inrerest in my resecrrch lefi rin impression !rit il1 not soon forger.

The rrajj'of 'SI. Mchd S Hospital Reseurch CÏwrirrm >vusrhe sole pro vider of' onimul cure and laborutory safety. The experinrenis presented in this thesis woitld not have been possible ivirhout their splendid dedicution to proper luborutory practices.

Finally, I rioirld iike ro express my grorirzrde roivords the Grathrare Depurzmenr 01' rhe Insrirzrre of Medicol Science nt rhe Lniversih*of' Torontojbr proiiding me with the opporrunihfro ohtuin my .\laster of'Scirnce degrre. and to shme the knoii.ledge g~rinrd over rhe coirrse of my research ivirh scientisrs across the continent. As a new grahrire. I will enduringiy ucr as an advocate ojthe Deparmenr and rhe University. TABLE OF CONTENTS

.. Abstract ...... il Ac knowledgements ...... iii List of Tables ...... xi List of Figures ...... xiv... List of Abbreviations ...... xvrii Dedication ...... 'cxi

1.1 The Cardiac Action Potential ...... 1

1.1.1. The Resting Membrane Potential ...... 2 1.1.2. Phases of the Action Potential ...... 2 Il3. The Delayed Rectifier Potassium Current ...... 5 1 .1. 4. The Inward Rectifier Potassium Current ...... II 1 1 Determinanis of Impulse Propagation (Conduction) ...... 1 1 1 .l.6. Detenninants of Ventricular Refractoriness ...... 1-3 1 .l. 7. Measures or' Ventricular Refractoriness ...... 14

1.2. Mechanisms of Arrhythmias ...... 13

1 .2. 1 . Abnomal Automaticity ...... 16 1.2.1. Triggered Activity ...... 16 1.23. Rrrntry ...... 17

1.3. Modulating Cardiac Refractoriness ...... 19

1 3.1 . Antiarrhythmic Drugs ...... 19 1.3.2. The Effect of .4 ntiarrhythmic Dnigs on Cardiac Refractoriness ...... 21 1.3.3. The Effrct of Class 1 Antiarrhythmic Drugs on Cardiac Refractoriness ...... 23 1.4. The Effect of Class II Antiarrhythmic Drugs on Cardiac Rrfractorinrss ..... 24 1.3.5. The Effect of Class III Antiarrhythrnic Drugs on Cardiac Refractoriness .... 3 1 6.The Effect of Class IV Antiarrhythmic Dnigs on Cardiac Refractonness ... 38

1 .4. Ventricular Fibrillation - A Reeatrant Arrhythmia ...... 29

1.4.1. Initiation of Ventricular Fibrillation ...... 29 1.4.2. Maintenance of Ventricular Fibrillation ...... 31 - . . 1A.3. Ventricular Detibnllation ...... 32

1.4.3.1. The Critical Mass Hypothesis ...... 33 I .4.3.2. The Upper Limit of Vulnerability Hypothesis ...... 34 1.4.3.3. The Refiactory Penod Extension Hypothesis ...... 34 1.4.3.4. The Threshold of Synchronous Response Hypothesis ...... 35

1.4.4 . The Effect of Antiarrhythrnic Dmgs on Ventricular Fibrillation and De fibrillation ...... 35

1.5. Potassium Channels as Targets for Treatment of Arrhythmias ...... 36

1 .5 .1 . Antiarrhyhmic Potential of Selective Potassium Channel Blockers ...... 37 1.5. Proarrhythmic Potrntial of Selective Potassium Channel Blockers ...... 39 1.5.. The Effect of Blocking Multiple Cardiac Ion Channels ...... 40

1.6. Rationale ...... 42

1.7. Hypothesis ...... 45

2 . METHODS ...... 46

2.1. lsolated Langendorff Rabbit Heart Experiments ...... 46

2.1.1 . Langendorff Preparation ...... 16 2.1 .1. Hemodynamic Measurements ...... 47 2.1.3. Detemination of Electrophysiological Parameters ...... 48

2.1.3.1. Experimental Setup ...... 48 2.1 3.2. VF Induction and DFT Measurements ...... 50 2.1 3.3 Mrasurements of Ventricular Fibrillation Cycle Length ...... --57 2.1 .3.4. Measurements of Ventricular Effective Refractory Period ...... 52 2.1.3.5. Measurements of the Action Potential Duration ...... 55- - 2.1.3.6. Measurement of QRS Duration ...... 33

2.2. Intact Guiaea Pig Heart Experiments...... 57

2.2.1 . Guinea Pig Preparation ...... 57 2.1.2. Surgical Methods ...... 58

2.3. A Cuinea Pig Model Assessing Defibrillation Threshold and Cardiac Refractoriness ...... 59

2.3. Determination of Electrophy siological Parameters ...... 59

2.3.1 -1. VF Induction and DFT Measurements ...... 59 2.3.1.2. Measurements of Ventricular Effective Refractory Poriod ...... 60

2.4. A Cuinea Pig Model Assessing Cardiae Refractoriness: ERP and Dynamic Restitution ...... 61 2.4.1 . Measurements ofVentricular Effective Refractory Period ...... 6 1 2.4.2. Measurements. . . of .Activation-Recovery Intervals and Dynamic Restrtution Kinetics ...... 61 2.4.3. Measurement of QRS Duration ...... 61

2.5. Drug Administration ...... 64

2.5. Isolated Langendom Rabbit Hean ...... 64

2.5.1 -1. Dnig Treatment Groups ...... 63

2.5.1.1. l . Dofetilide . Sotalol. and Barium Croups ...... 65 2.5.1.1.2. Combination Dofetilide + Barium and Sotalol + Barium Groups ...... 66

Intact Guinra Pig Heart ...... 66

2.5.1. Control Group ...... 67 3.5 2 .2 . Drug Treatmrnt Group ...... 67

2.5.2.2.1. Dofetilide.. Group ...... 67 2.5.2.2.3. HMRlx6 Group ...... 68 2.5.2.7.3. Combination Dofetilide and HMRi556 Group ...... 68

2.6. Statistical Analysis ...... 69

1.6.1 . Isolated Langendofl Rabbit Heart ...... 69 2-62 Intact Guinea Pig Heart ...... 70

3. RESULTS ...... 73

3.1 tsolated Langendorff Rabbit Heart Model ...... 73

3.1 1 . Control Experiments ...... 73

3.1. 1 Electrophysiological Measurements ...... 73 3.1.1.2. Measurements of Cardiac Function and Other Measurements ...... 74

3.1 .2 . Dotetilide .4 dministration in Isoiated Rabbit Hearts ...... 76

3.1 2.1. Effect of Varying Dofetilide Concentration on Electrophysiologic iMeasurements ...... 76

3.1.2.1.1. Monophasic Action Potential Duration at 90% Rrpolarization ...... 76 3.1.2.1.2. Ventrïcular Effective Rçfractory Period ...... 78 3.1 1 3 Vrntricular Fibrillation Cycle Length ...... 80 3.1.2.1.4. Defibrillation Threshold ...... 82

3.1.2.2. Effect of 8 nM Dofetilide on Electrophysiologic Measurements ...... 84 3.1 2.3. Effect of Dcfktilide on Cardiac Function and Other Measurernents ...... 84

3.1.3. S~talolAdministration in Isolated Rabbit Hearts ...... 88

3.1.3.1 . Effect of Sotalol on Electrophysiologic Measurements ...... 88 3.1.3 .2. Effect of Sotalol on Cardiac Function and Other Measurernents ...... 88

3.1.4. Barium Administration in IsoIated Rabbit Hems ...... 91

3.1.4.1. Effect of Barium on Elrcirophysiologic Mrasurements ...... 91 3.1.4.2. Effect of Barium on Cardiac Function and Other Measurements ...... 91

3.1.5. Combination Dofetilide and Barium Experirnents In lsolated Rabbit Hearts ...... 94

3.1.5.1. Effect of the Combination of üoktilide and Barium on Elrctrophysiologic Measuremenrs...... 94 3.1.5.2. Effect of Cornbination Doîètilide and Bariurn on Cardiac Function and Other Measurements...... 94

3 1 6 Combinarion Sotalol and Barium Esperiments ...... 97

3.1.6.1 . Effect of Combination Sotalol and Barium on Elrctrophysiologic Measurements ...... 97 3.1.6.2. Effect of Combination Sotalol and Barium on Cardiac Function and Other Measurements...... 97

3 1.7. Sumrnary of Langendorff Rabbit Heart Esperiments ...... 100

3.1 .7.1. Drue effects on DFT and VFCL ...... 100 3.1.7.1. Dmg effects on VERP and MAPDqo ...... 100

3.2. Establishing an Intact Cuinea Pig Hearî Mode1...... 105

3.2.1. Control Experiments ...... 105

3.3. A Guinea Pig Model Assessing Defibrillation Threshold and Cardiac Refractoriness...... 105

3 .3. 1. Saline Administration ...... IO5

3 3.1 .1. Electrophysiological Measurements ...... IO5

vii 3 ...Hemodynamic and Other Measurements ...... 106

3.3.2. Combination PEG400 and DMSO .4dministration ...... 1 12

3 .XI.1. Combination 0.6 mL PEG400 and 0.2 mL DMSO ...... 1 i2

3.3.2.1 -1. Electrophysiological Measurements ...... 1 12 3.2.- 7 1 2 Hemodynarnic and Other Measurements ...... 1 12

3.3.2.2. Combination 0.3 mL PEG400 and 0.1 inL DMSO ...... 1 13

q q J .J 2.2.1 . Electrophysiological Measurements ...... Il3 9 9 J .J .2.2 .2. Hemodynarnic and Other Measurements ...... II4

3.3.2.3. Combination 0.15 mL PEG-100 and 0.05 mL DMSO ...... 114

-q77 J ..J .1 . Electrophysiological Measurements ...... 1 II 7 3 3.33 2 . Hemodynamic and Other Measurements ...... Il5

3.3.2.4. Summary of Combination PEGJOO and DMSO Experimrnts ...... Il5

J.J.J.31- Administration of 8 and 4 pgikg Doktilidr ...... 121

3.3.1 Effects of 8 pgkg Dofetilide on Elrctrophysiological bkasurernents ...... 131 3.3. Effrct of4 :.ig/k/kg Dofetilide on Elrctrophysiological Meastirements ...... 121 3.333 Effects of 8 pgkg Dofetilide on Hemodynamic and 0th 33 Measurements ...... 1- -. 3.34. Effects of 4 pgkg Dofetilide on Hemodynamic and Other Measurements ...... 123

3.4. A Guinea Pig Mode! Assessing Cardiac Refractoriness: ERP anci Dynamic Restitution ...... 126

3.4.1. Control Experiments ...... 126

3.4.1 . 1. Elrctrophysiologica1 Measurements ...... 116 3.4.1 .1. Dynamic Restitutian Kinetics of Control Experirnrnts ...... 128 3 4 1 3 Hemodynarnic and Other Mrasurrmcnts ...... 118

3.4. Dofetilide Experiments ...... 135

3 A.2. 1. Administration of 8 p-d/kg Dofetilide ...... 135 Effects of 8 pg/kg Do fetilide on Electrophysiological Measurements ...... I35 Dynamic Restitution Kinetics of the 8 pglkg Dofetilide Experiment ...... 136 Effects of 8 pgkg Dofetilide on Hemodynamic and Other Measurements ...... 137

3 .4 .2.2. Administration of 4 pgkg Do fetilide ...... I43

3.4.2.2.1. Effects of 4 pgikg Dofetilide on Electrophysiological Measurements ...... 143 3 A.2.2.2. Dynamic Restitution Kinetics of the 4 pgkg Dofetilide Experiment ...... 144 3 4 .2.2.. Effects of 4 ugkg Dofetilidr on Hernodynamic and Other Measurements ...... 145

3.4.3. Administration of 1.5 mg/kg HMR1556 ...... 151

Effects of 1.5 m_&g HMR 1556 on Electrophysiological Measurements ...... 131 Dynamic Restitution Kinetics of the 1.5 mg'kp HMRl556 Espcrimrnt ...... 152 Effects of 1.5 mg'kg HMR 1556 on Hemodynamic and Other Measurements ...... 152

3.4.4. Administration of Combination 4 pgkg Dofetilide and 1.5 mgkg .. HMRlx6 ...... 160

3 A.4.1. Effects of Combination 1 pgkg Dofetilide and I .5 rngikg HMR 1 556 on Electrophysiological Measurements ...... 160 3.4.1.2. Dynamic Restitution Kinetics of the Combination 4 &kg Dofetilide and 1.5 mgkg HMRl556 Experiment ...... 161 5.4.4.3. Effects of Combination 4 pgAg Dofetilide and 1 .5 mgkg HMR 1556 on Hrmodynamic and Other Measurements ...... 162

1. DISCUSSION ...... 168

4.1. Isolated Langendodf Rabbit Hearts ...... 168

Dofetilide Dose-Response Determination ...... 168 Selectivity of Ikr Block with Dofetilidr . SotaloI . and Barium ...... 169 Effect of Ikr or ikl Block on EIectrophysiologic Variables ...... 170 Effect of Ikr or Ik 1 Block on Hernodynamics and QRS duration ...... 171 Combination Ikr and Ikl Blockade ...... 173 Mechanisms of Combination Drug Effects ...... 173 PossibLe Mechanisms of Combination Ikr and Ik l Block ...... I74 4.1.7.1. Binding Site Cornpetition ...... 1 75 4.1 .7.2. Ailosteric Interactions ...... 176 4.1.7.3. Compensatory Current Densities ...... 176

41.8. Relevance of Ikr block in the Presence of Dimished Ikl current ...... 178

4.2. Intact Guinea Pig Hearts ...... 179

4.2.1. Measures of Cardiac Refractoriness in Control Expenments ...... 180 42.2. Selectivity of Agents for Potassium Channel block ...... 180 42.3. Effect of Ikr and/or Iks Block on DFT . ERP. and FFF ...... 180 4.2.4. Effect of [kr and/or Iks Block on Dynamic Restitutim Kinetics ...... 182

4.3. Limitations ...... 185

4.4. Conclusions ...... 188

4.5. Recommendstions for Future Research ...... 189

5 . REFERENCES ...... 190 LIST OF TABLES

Table 1 A, Electrophysiologic Measurements of Isolatrd Rabbit Hrms of the Control Group ...... 75

Table 1 B. Weights and Pararneters of Cardiac Function of Isolated Rabbit Hearts of the Control Group...... 75

Table II A. Electrophysiologic Measurements of Isolated Rabbit Hearts in the Control and Dofetilide-Treated Groups ...... 86

Table II B. Weights and Pararneters of Cardiac Function of Isolatrd Rabbit Hearts in the Control and Dofetilide-Treated Groups...... 86

Table III A. Electrophysiologic Measurements of Isolated Rabbit Hearts in the Control and Sotalol-Treated Groups ...... 90

Table III B. Weights and Pararneters of Cardiac Function of Isolated Rabbit Heans in the Control and Sotalol-Treated Groups...... 90

Table IV A. Electrophysiologic Measurements of Isolated Rabbit Hearts in the Control and Barium-Treated Groups ...... 93

Table IV B. Weights and Pararneters of Cardiac Function of Isolated Rabbit Hearts in the Control and Barium-Treated Groups...... 93

Table V A. Electrophysiologic Measurements of Isolatrd Rabbit Hearts in the Control and Combination Dofetilide and Barium-Trctatrd Groups ...... 96

Table V B. Weights and Pararneters of Cardiac Function of Isolated Rabbit Hearts in the ControI and Combination Dofetilide and Barium- Treated Groups...... 96

Table VI A. Electrophysiologic Measurernents of lsolated Rabbit Hrans in the Control and Combinat ion Sotalo1 and Barium--Treatsd Groups ...... 99

Table VI B. Weights and Parameters of Cardiac Function of Isolated Rabbit Hearts in the Control and Combination Sotalol and Barium-Treated Groups ...... 99

Table VII. Electrophysiologic Measurernents of Isolatrd Rabbit Hearts in All Groups ...... 1 02

Table VI11 A. Electrophysiologic Measurements of DFT. VFCL. and QRS Duntion of Intact Guinea Pig Hearts in the Control Group ...... 108 Table VI11 B. Electrophysiologic Measurements of VERP at Multiple Cycle Lengths of Intact Guiiiea Pig Hearts in the Control Group ...... 1 08

Table VITI C. Physical Measurements of Intact Guinea Pig Hearts in the Control Group ...... 108

Table VI11 D. Measurements of Hemodynarnics and Blood Gases of Intact Guinea Pig Hearts in the Control Group ...... 108

Table IX -4. Electrophysiologic Measurements of Intact Guinea Pig Hearts in the Control Groups (Saline and Organic Solvent Combinations)...... 1 17

Table IX B. Hernodynamic Measurements of Intact Guinea Pig Hearts in the Control Groups ...... 1 18

Table IX C. Physical Measurements of Intact Guinea Pig Heans in the Control Groups ...... 1 1 8

Table X A. Elcctrophysiologic Measurements of Intact Guinea Pig Hrans in the Control and Dofetilidt-Treated Groups ...... 124

Table X B. Blood Gas and Hemodynamic Measurements of Intact Giiinea Pig Hearts in the Control and Dofetilide-Treated Groups ...... II4

Table X C. Physicdl bltasurements of Intact Guinea Pig Hearts in the Control and Dofrtilidr-Treated Groups ...... 1 25

Table XI A. Ventricular Effective Refractory Periods of Intact Guinea Pig Hearts in the Control Group ...... 130

Table XI B. Activation-Recovery Intervals of Intact Guinea Pig Hearts in the Control Group ...... 1 30

Table XI C. Blood Gas and Hemodynamic Measurements of Intact Guinea Pig Hrarts in the Control Group ...... 13 1

Table XI D. Physical Measurements of Intact Guinea Pig Hearts in the Control Group ...... 13 i

Table XII A. Ventricular Effective Refractory Periods of Intact Guinea Pig Hearts in the Controi and 8 pg/kg Dofetilide Groups ...... 138

Table XII B. Activation-Recovery Intervals of Intact Guinea Pig Hearts in the Control and 8 pg/kg Dofetilide Groups ...... 138 Table XII C. Hemodynarnic Measurements of Intact Guinea Pig Hearts in the Control and 8 pg/kg Dofetilide Groups ...... 1 39

Table XII D. Physical Measurements of Intact Guinea Pig Hems in the Control and 8 &kg Dofetilide Groups ...... 139

Table XII1 A. Ventricular Effective Refractory Periods of Intact Guinea Pig Hearts in the Control and 4 pg/kg Dofetilide Groups ...... 146

Table XIII B. Activation-Recovery Intervals of Intact Guinea Pig Hearts in the Control and 4 pg/kg Dofetilide Groups ...... 146

Table XII1 C. Hemodynamic Measurements of Intact Guinea Pig Hearts in the Control and 4 pgkg Dofetilide Groups ...... 147

Table XIII D. Physical Measurements of Intact Guinea Pig Hearts in the Control and 4 pgkg Doktilidc Groups ...... 1-17

Table XIV A. Elecuophysiologic Measurements of Intact Guinea Pig Hcarts in the Control and 1 .j mg/kg HMR1556 Groups ...... 155

Table XIV B. Activation-Recovrry Intervals of Intact Guinea Pig Hearts in the Control and 1 .j mgikg HMR 1556 Groups ...... 155

Table XIV C. Hemodynamic Measurernents of Intact Guinea Pig Hsarts in the Control and 1.5 mgkg HMR1556 Groups ...... 156

Table XIV D. Physical Measurements of Intact Guinea Pig Hearts in the Control and 1.j m@kg HClR1556 Groups ...... 156

Table XV A. Ventricular Effective Refractory Prriods of Intact Guinea Pig Hearts in the Control and Combination 4 pgkg Dofetilide and 1.5 mgkg HMR1556 Groups ...... 163

Table XV B. Activation-Recovery Intervals of Intact Guinea Pig Hems in the Control and Cornbination 4 pdkg Dofetilide and 1.5 mgkg HMRlm6- - Groups ...... 163

Table XV C. Hemodynamic Measurements of Intact Guinea Pig Hearts in the Control and Combination 4 pgkg Dofetilide and 1.5 mL&g HMRlx6- - Groups ...... 164

Table XV D. Physicai Measurements of Intact Guinea Pig Hearts in the Control and Combination 4 @kg Dofetilide and 1.5 rng/kg HMRlx6- - Groups ...... 164 LIST OF FIGURES

Figure 1. Ventncular action potential and time-dependent contribution of inward currents (ma. Ica-L). NdCa-exchange current. and outward currents (M. Ikr. Iks. Ito) ...... 3

Figure 2. Action potential and underlying membrane currents (Ikr. Iks.) ...... 6

Figure 3. Current-voltage relationships showing the r ffects of changing membrane potential on the ionic currents generated by inward and outward rectifying potassium channels ...... 7

Figure 4. States and voltage-dependent kinetics of the rapidly activating delayed rectifier potassium current, Ikr ...... 9

Figure 5. Escitability during the cardiac action potential ...... 13

Figure 6. A rrentrant circuit ...... 18

Figure 7. Chemical- - structures of do fetilide. d.1-sotalol. and the chromanol HMRlx6 ...... - ...... 27

Figure 8. Schematic representation of the srtup of the isolated Langrndorff rabbit hran ...... 19

Figure 9. Det'ibdlation threshold (DFT) determination ...... 5 1

Figure 10. Ventricular fibrillation cycle length (VFCL) determination ...... 33

Figure I 1. Determination of ventncular effective refractory period (VERP)...... 54

Figure 12. Measurements of monophasic action potential duration at 90% repolarization (MAPDqo)...... 56

Figure 1 3. Measurements of activation-recovery interval (AM) in intact guinea pig hearts...... 63

Figure 14. Dose-response curve with a linear dose scale for the effect of dofetilide on the percent change from baseline ( pre-do fetilide) in monophasic action potential duration at 90% repolarization (MAPDgO)at 400 mec pacing cycle length ...... 77

xiv Figure 15. Dose-response cune with a linear dose scale for the efkct of dofetilide on the percent change from baseline (pre-dofetilide) in ventncular effective refractory period (VERP) at 400 msec pacing cycle length ...... 79

Figure 1 6. Dose-response curve with a linear dose scale for the e ffect of do fetilide on the percent change from baseline (pre-dofetilide) in ventricular fibrillation cycle length (VFCL) ...... 8 1

Figure 17. Dose-response curve with a linear dose scale for the effect of dofetilide on the percent change from basrline (pre-dofetilide) in defibrillation threshold (DFT) ...... 83

Figure 18. Bipolar electrograms recorded from the le!? ventricular epicardium during fibrillation after treatmrnt with saline or dmg(s)...... 87

Figure 19. Individual measurements of MAPDqO.VERP. VFCL. and DFT in in the isolated Langendorff rabbit hem...... 1O3

Figure 20. Mean percent changes k SEM in MAPDqo, VERP. VFCL. and DFT from baseline to administration of 8 nM dofetilide. 4 pM sotalol. 3 pM barium. combination 8 nM dofetilide and 3 pM barium. and combination 4 pM sotalol and 3 FM barium. for each mrasurrment. respectivel y in isolated rabbi t hrans...... 1 04

Figure 2 1. Individual measurements of defibrillation threshold (DFT). ventncular tïbrillation cycle length (VFCL). and QRS duration in intact guinea

piew hearts ...... 109

Figure 22. Mean values and individual measurements of vrntricular effective refractory period (VERP) measured from the right ventncular endocardium of intact guinea pig hearts at each pacing cycle length at badine and afier saline...... 1 10

Figure 23. Individual measurements of systolic and diastolic blood pressure. heart rate. pC02. p02. and pH obtained at baseline and afier saline administration in the intact guinea pig hart...... 1 1 1

Figure 21. The effect of high (combination 0.6 mL PEGJOO and 0.2 mL DMSO). medium (combination 0.3 mL PEG400 and 0.1 mL DMSO).and low (combination O. 15 mL PEGJOO and 0.05 mL DMSO) doses of organic solvents on DFT in the intact guinea pig heart. measured at baseline and post solvent treatment ...... 1 19 Figure 25. The effect of varying combinations of PEGJOO and DMSO on the ventricular effective refractory period (VERP) and the percent change in VERP of the intact guinea pig heart from baseline to post solvent treatment ...... 120

Figure 26. The mean ventricular effective refractory period (VERP) rneasured at each pacing cycle length at baseline and after saline administration. Individual mrasurements of VERP obtainrd at each pacing cycle length at baseline and aher saline in the intact guinea pig heart ...... 132

Figure 17. The mean activation recovery intervals measured from maximum -dV/dt of the ORS deflection to the peak of the T wave (AMvAT) for each pacing cycle length at baseline and atier saline administration in the intact guinea pig hem Individual measuremcnts of ARI,,,ii. obtained at each pacing cycle length ...... 133

Figure 18. Dynamic restitution kinetics at baseline and afier saline administration of a control rxperiment in the intact guinea piç hem ...... 134

Figure 19. The ventricular effective te fractory period (VERP) and mran activation- recovery intervals (ARIpkr) at each respective paced cycle length measured at baseline and after administration of 8 pgkg doktilide in the intact guinea pig hcan...... 1 40

Figure 30. The effect of 8 pgkg dofetilide on the percent change in right VERP measured from badine in the intact guinea pig heart ...... 14 I

Figure 3 1. Dynamic restitution kinetics at baseline and after administration of 8 pgikg dofetilide in the intact çuinea pig heart ...... 142

Figure 32. The ventricular effective refractoy period (VERP) and mean activation- recovery intervals (ARIPcAT)at each respective paced cycle length measured at baseline and after administration of 4 pgkg dofetilide in the intact guinea pig hean ...... 148

Figure 33. The effect of 4 pgkg dofetilide on the percent change in right VERP measured from baselins in the intact guinea pie hem ...... 149

Figure 34. Dynamic restitution kinetics ai baseline and afrer administration of 4 p@kg dofetilide in the intact guinea pig han...... 150

Figure 3 5. The ventricular effective refractory period (VERP) and mean activation-recovery intervals (ARIpdT) at each respective paced cycle length measured at baseline. and after administration of 1.S mgkg HMR 1556 in the intact guinea pig heart ...... 1 57

xvi Figure 36. The effect of 1.5 mgkg HMRljj6 on the percent change in right VERP measured fiom baseline in the intact guiriea pig heart ...... 158

Figure 37. Dynamic restitution kinetics at baseline and after administration of 1.j mgkg HMR1556 in the intact guinea pig heart ...... 159

Figure 38. The ventricular effective refractory period (VERP) and mean activation- recovery intervals (ARIpcakT)at each respective paced cycle length rneasured at baseline. and atier administration of ccmbination 4 pgkg dofetilide and 1.5 mgkg HMR 1156 in the intact guinea pig heart ...... 165

Figure 39. The effect of combinarion 4 pg/kg dofetilide and 1.5 mgkg HMR1556 on the percent change in right VERP measured from baseline in the intact guinea pig heart ......

Figure 40. Dynarnic restitution kinetics ût badine and aftrr administration of combination 4 pgkg dofetilidr and 1.5 mgkg HMR1556 in the intact guinea pig heart ...... 167 LIST OF ABBREVIATIONS

AP action potential APD action potential duration AR1 activation-recovery interval AMpeidi~ activation-recovery interval measured from the maximum -dV/dt of the QRS deflection to the peak of the T wave bpm beats per minute CaC12 calcium chloride CASCADE Cardiac Arrest in Seattle: Conventional versus Arniodarone Dnig Evaluation Study closed ion channel siate degrees Celsius cm centimetre cm-7 centimetre squared CO2 carbon dioxide d- dextrorotatory isomer DC direct current DF defibrillation DFT defibrillation threshold DI diastolic interval DIAMOND Danish Trial in Acutr Myocrudial Infarciion on Dofetilide DMSO dimethy lsul foxide DR dynamic restitution EC 50 effective drug concentration producing 50% of maximal response ECG electrocardiogram Em, maximal drug response ERP effective re fractory period ESVEM Electrophysiologic Study Versus Electrocardiogaphic Monitoring F French FFF fastest follow frequenc y FFF,, pacing-induced fastrst follow frequency I gram Hz hertz 1 inactivated ion channel state [Ca calcium current I k delayed rectifier potassium current Ili 1 inward rectifier potassium current Ikr rapidly activating dclayed rectifier potassium current Iks slowly activating delayed rectifier potassium current INa sodium current INaiCa sodium/calcium exchange current Ito transient outward potassium current intrarnuscular intraperitoneal

xviii i.u. international unit i.v. intravenous K+ potassium KCI potassium chloride kg kilogram 1- lovorotatory isomer L litre L/min litre per minute MAP monophasic action potential M.4PDqa monopbasic action potential duration at 90% repolanzation M~?' magnesium ion MgCl? magnesium chloride mg/k milligram pet- kilogram mgikgîhr milligram per kilogram per hour min minute mL millilitre m L/hr millilitre per hour millilitre per minute miliimetre miliimetre of mercury millimolar millimetre per second msec millisecond mV millivolt PM micromolar micrometre sodium chloride sodium bicarbonate sodium di hydrogen p hosphatc amine group nanomolar non-signi ficant open ion channel state oxygen partial pressure of carbon dioside partial pressure of oxygen polyethylene~lycol400 molrcular wight first stimulus second stimulus standard deviation sec second SEM standard rrror of the mean subcutaneous Survival With Oral D-sotalol standard deviation of the vertical distance of data points From the fitted curve TdP torsades de pointes V volt VERP ventricular effective refractory period VF ventricular fibrillation VFCL ventricular fibrillation cycle length to mj~fbthrrDimitrios. and nymorher Panagiota. ivhose love. giiidrrnce. support. und patience have giren me the srrength ;O contintre ... 1. INTRODUCTION

Sudden death from lethal arrhythmias is a common cause of cardiac death. In an effort to reduce the incidence of such lethal arrhythmias. it is important to determine the electrophysiologic and pharmacologie factors responsible for their genêsis and maintenance. Arrhythmias resulting in abnormally fast heart rates (tachyarrhyhias) may in part result from reentry (of electrical impulses). of which ventricular fibrillation

(VF) is an important cxample. The behaviour of VF is determincd by the action potrntial duration (APD) of individual cells of the myocardium. which largely determines cardiac refractoriness. Cardiac refractoriness cm modulate reentry circuits in the hean. and in the last decade efforts in arrhythmia management have shified from altering myocardial conduction to chançing cardiac refractoriness. The duration of the action potential (AP) and cardiac refractoriness can be modulated by changes in ion channel activity. particularly repolarizing potassium (K-) channels. with the use of K' channel blockers.

However. selectivr K* channel blockrrs have not only been shown to be proarrhythmic

(resulting from early after-depolarizations). but also less efficacious than other agents blocking multiple targets. which prompts the investigation of combining selective antiarrhythmic agents. However. combinat ion dmg thrrapy is not well understood. although it has the potential to better treat arrhythmias than moriotherapy.

1.1. The Cardiac Action Potential

The cardiac AP describes the electrical changes of a ceIl as a function of the changes during the cardiac cycle. and these occur in conducting myocardial tissues. 1.1.1. The Resting Membrane Potential

The uneven distribution of ions and selective permeability across the cardiac ce11 plasma membrane produces a potential difference inside versus outside the cell. The membrane potential represents the voltage differences brtween the interior of the cardiac cell and the extracellular space. At rest. the membrane potential of each myocardial ceIl is largely determined by the electrochemical gradient for potassium. The electrochemical

gradient for potassium is maintained by the Na'-Ky-ATPase. responsible for pumping sodium out of the ceIl in exchange for potassium into the dl. Selective permeability to potassium allows these positively charged ions to move down their concentration gradient. and le& out of the cell. canying a positive charge across the plasma membrane.

The resultinp outward current creates an elrctrical potential difference across the plasma

membrane. where the interior of the ce11 is negatively chuged. Therefore. a balance is

establishrd betwern the electronegativity of the interior of the cell and the potassium

concentration gradient. which each favor potassium influx and ct'flus. respsctively.

1.1.2. Phases of the Action Potential

Cardiac cells. similar to other excitable cells. cm be stimulated to generate tiny

currents across the plasma membrane. Thsx currents are duc to the oprning and closing

of channels allowing the movement of ions across the membrane. resulting in net

increases or decreases in transmembrane potential. The combined currents through al1

the channels in one cardiac ce11 results in the cardiac AP (ser Figure 1 ). INa p-

Figure l. Ventricular action potential and time-dependent contribution of inward currents (l'Na. Ica-L). NafCa-exchange current. and outward currents ( Ik 1. Ikr. Iks. 110). Tracings are schcmatic representations of the time dependence of the contri bution of each current to the depolarization and repolarization process. Doanward detlrctions are representative of inward current. while upwrd detlections indicate outward current. The relative sire of the currents is not drawn to scale. (Reprinted from The Atlas of Heart Diseases. Arrhythmias: Electrophysiologic Principles. Volume 10. Eugene Braunwald ed., page 1.8. 1996. with permission from Current Medicine and from Dr. Eugene Braunwald) The ventricular cardiac AP is divided into five phases. The AP upstroke (phase O) is due to a large increase in Na' conductance of 1-2 ms in duration (Gettes and Reuter.

1974). This depolarization increases the probability of ~a'channel activation (opening).

There exists a threshold potential. defined as the membrane voltage at which Na- channel opening produces sufficient Na- current to overcome the repolarizing outward K' currents. When the threshold potential is reached the Na' ions depolarize ventricular myocyles, increasing Ihe membrane potential to +4O mV. A period of early repolarization (phase 1) follows this peak of membrane potential. and is due to activation of the transient outward current (Ito). Ito has been shown to consist of two cornponents: a voltage-dependent. rapidly activating K* current with outward rectification. and a Ca' dependent chloride current. conducting the inward flow of chloride ions (Coraboeuf er trl.. 1982. Tseng and Hoffrnan. 198%. Activation of Ito couplrd with voltagr-deprndrnt inactivation of the drpolarizing Na* current. retums the membrane potrntial to approximately +10 mV. The plateau phase (phase 2)of the cardiac AP is dur to a balance between inward and outward currents. Inward ciments inciude Ca- currents. the

Na*-Ca' exchange current. and a slowly inactivating Na- current: outward currents include the delayed rectifier K- current (Ik). comprised of rapidly and slowly activating cornponents. Ikr and Iks resptxtively (Sanguinetti and Jurkirwicz. 1990). The late npid phase of repolarization (phase 3 ) is due to increasing outward K* conductance couplrd with progressive decay of Ca* currents. Early phase 3 repolarization is mainly due to Ikr. but there is a combined influence of the inwardly rectitjing potassium current (Ikl ) and the Na--K+ pump current that contributes to this phase of the AP. Ikl channels are progressively activated as repolarization procerds. and repolarize the ventricular cells

back to their restinp membrane potential of -80 to -85 mV.

1 -1.3. The Delaved Rectifier Potassium Current

The delayed rectifier potassium current. Ik. is largely responsible for the early to

late phase of repolarization during the cardiac AP. and is both voltage- and time-

dependent. It is drscribed as possessing two components. Ikr and Iks: the former is a

rapidly activating delayed rectifier. and the latter is a slowly activating delayed rectifier

(Sanguinetti and Jurkiewicz. 1990). The time constants for these channcls are species

dependent. Ikr is known to open more npidly (generally in the order of a few hundred

milliseconds (Courtney et al.. 1992)) and deactivate more slowly than Iks (Figure 1).

These are voltage dependent as each channel conducts ouhvard current at membrane

potentials positive to -86 mV (Figure 3).

The currènt densitirs of Ikr and Iks are not only specirs dependent. but also var).

within different regions of the samc hem (which is one of the underlying factors

involved in heterogenei ty of APD and cardiac re fractonness) (Cheng et a!.. 1 999).

Furthemore. the contribution of each of their current densities varies with hean rate.

perhaps resulting in reverse- and positive-rate dependence of APD or ERP prolongation

observed with Kcchannel blockers. Both currents are found in venuicular myocytes

from guinea pig (Sanguinetti and Jurkiewicz. 1990). mouse (Nuss and Marban. 1994).

sheep (Noble and Tsien. 1969). rabbit (Salata el al.. 1996). dog (Gintant. 1996. Han.

200 1). pig (Nabauer et ai.. 1997) and hurnans (Wang et al.. 1993. Li et rd.. 1996. Pereon

et al.. 2000). Figure 2. Action potential (AP) and underiring membrane currents (Ikr. Iks). Dashed lines show the time course of the AP to compare with corresponding currents. Solid arrows on the lefi y-ais indicate the zero refercnce (O pNpF for currents. -90 mV for the AP). The right y-mis indicates the membrane potential for the AP. Recordings were taken during the 10th pulse from rest during activity at 1 Hz in a mathematical mode1 of dog atrium. (Reprinted from American Journal of Physiology: Heart and Circulatory Physiology. Volume 279. Rafael J. Ramirez et trl.. Mathematical Analysis of Canine Atrial Action Potrntials: Rate. Rrgional Factors. and Electrical Remodeling. pages H 1767-H 1785.2000. with permission from The American Physiological Society and from Mr. Rafari Ramirez) / Icutward rectification /

1outward

----inward-- --- rectification --- - _ --- +120mV 1 I I I 1 Voltage

Current

Figure 3. Current voltage rrlationships showing the effects of changing membrane potential on the ionic currents generated by inward and outward rectifyiny potassium channels. Inward currents are domnward. outward currents are upwrd. Al1 potassium currents are zero at -86 mV (the Nernst potential for potassium). Outward rectification occurs when depolarization opens potassium channels. increasing outward current. Inward rectification occurs when depolarization causes potassium channels to close. decreasing outward current. (Reprinted from Pacing and Clinical Electrophysiology: PACE. Volume 18. David W. Whalley et al.. Basic Concepts in Cellular Cardiac Electrophysiology: Part 1: Ion Channels. Membrane Currents. and the Action Potential. pages 1 5%- 1 574. 1995. with permission from PACE and Dr. Augustus Grant) When Kt currents were found to decay during maintained depolarization. it was proposed that K' channels cmoccupy three states. expressed as closed. open. and inactivated (Miller. 1991. Kiehn ei cil.. 1999. Wang rr ul.. 1997) (Figure 4). Transitions between each of these states is bi-directional (Le. from closed. to open or vice-versa. and

fiom open to inactive or vice-versa), and is dependent on the membrane potential. The

Ikr channel is closed at the resting membrane potential. and a conformational change of the channel to the open statr occurs upon membrane depolarization. The time course for the conformational change fiom closed to open is quite slow compared to the rapid

inactivation of the channel. which occurs as soon as the channel has conformed to the open state. The three states of the delayed rectifier K* channe1 map physically correspond to different channsl conformations. The open statr allows iC ions to tlon

through the channel down their elrctrochemical gradient. The closed and inactive staies.

although non-conducting and do not permit ion pemeation. are distinctly different in that

the latter does not allow channel opening upon membrane depolarization ( Whalley et cd..

1995). The membrane potential cm influence transitions arnong the threr states (Zagotta

et al.. 1 994a. 1994b). The Ik chamel activates with a delay upon membrane

depolarization (Fedida et a!.. 1993). which indicates voltage drpendent transitions among

several non-conducting closed states before channel opening (Hoshi et 01.. 1994. Zagotta

et al.. 199Jb). The the tcikrn for transitions among closrd states contributes to the

overail de!ay in actual channel openinp.

The inactivated and open states of the delayed rectifier charnels are found in

equilibriurn. Furthemore. inactivation is rapid relative to activation. and therefore. during

the initial phases of the AP. Ik cha~elsare largely inactivated. OnIy during slow fast activation inactivation

slow deactivation

Figure 4. States and voltage-dependent kinetics of the rapidly activating drlayed rectifier potassium current. Ikr. The time course of activation and deactivation of the IE;r channcl are slow compared to the rapid inactivation kinetics. C = closed state. O = open state. I = inactivated state. the late phase of repolarization does recovery from inactivation permit conduction through this channel. Thus. inactivation limits Ik channel conductance until latr repolarization. Although the activation process (resulting in channel opening) is strongly voltage dependent. the inactivation process shows little voltage dependence (DeCoursey.

1990. Zagotta and Aldrich. 1990). The delayed rectifier channels display two types of inactivation: fast or N-type. and slow or C-type. both of which can exist in the same chmel. N-type inactivation is termed as such since it is causèd by amino acid residues in the NH2 terminal of the channel. These arnino acids in the NH2 terminal fonn a linked inactivation particle that cm interact with the open channel to cause inactivation (Hoshi et al.. 1990. Zasotta rr ui.. 1996). Channel inactivation has been rxplained with a 'ball- and-chain* rnodrl. in that once the channel has oprned. the inactivation particle. or 'bali'. with the comected polypeptide. or 'chain'. can bind to the intemal mouth of the pore and block the passage of ions (Benzanilla and Armstrong. 1997. Jan and Jan. 1992).

However. after deletion of the N-terminal. a slow inactivation still exists. trrmrd C-type inactivation (of which little is known). since the inactivation involves amino acid residues found close to the carboxyl teminal of Ik channels (Hoshi et di.. IWO). Unlike N-type inactivation which requires one inactivating subunit. C-epe inactivation has becn interpreted in tcrms of a Yoot in the door' mrchanism (Ogielska et tri.. 1995). This

'slow' inactivation is associated with an immobilization of the gating charge of the channel afier channel opening (Fedida et ul.. 1996). and entails conformational changes involving cooperation between the four subunits fonning the functional K- channe1

(Panyi rr ul.. 1995). 1. f .4. The Inward Rectifier Potassium Current

The current which is largely responsible for repolarization during late phase 3 of the AP is inward in rectification and is manifested by the inward rectifier potassium channel. Ik 1. This charnels is voltage dependent. maintaining the resting membrane potential near the equilibrium potential for potassium (o-86 mV) under physiological conditions (Counney el al.. 1992). Curtent through Ikl is strongly inwardly rectifying at membrane potentials greater than this equilibrium potential (Figure 3 ). Similady. the magnitude of the current is dependent on the K' gradient and intemal block by M~'*ions and polyamines (Vandenberg. 1995).

1.1.5. Deteminants of Impulse Propagation (Conduction)

The electrotonic currents that are involved with impulse propagation dong excitable cardiac tissue are quitc differcnt than the ionic currents across the plasma membrane that generate the cardiac AP. Although voltage-gated Na' channrls are responsible for drpolxization. the electrotonic currents propagated by this wavc of depolarization determine conduction velocity. Conduction velocity dong cardiac tissue is dependent on the rate of depolarization (otheniise referied to as C',,). and the amplitude of the AP. which are both determined by the inward depolarizing Na* current responsible for the upstroke of the AP. Elrctrical resistances of the tissues that transmit the electrotonic currents generated by the depolarizing ion currents are also drtenninants of conduction velocity. which are dependent on the state of the intercalated disks (gap junctions) connecting the plasma membranes of adjacent cardiac cells. Modification of the inward iNa curreni or gap junctions will thrreforc have important effects on conduction velocity. However. these are not manipulated in the experiments of this thesis.

1.1.6. Determinants of Ventricular Refractoriness

One distinct characteristic of cardiac cells (with the exception of sino-atrial nodal cells) is the plateau caused by the opening of calcium channels. This plateau in membrane potential leads to a prolonged APD and thiis prevents summation of consecutive contractions. or tetanization of cardiac muscle.

The inability to reexcite cardiac tissue for a penod of time following an AP is defined as refractoriness. and is classitied in three different States: absolute refractory.

relative refractory . and effective rr fractory periods ( Figure 5). The state of ventricular

refractoriness depends on the number of Na' channels that have recovered from

inactivation and are available to conduct a depolarizing current. and the ability of the

membrane to depolarizr to the threshold potential for activation of thesr available Ka-

channels. Recovery of Na* channels from an inactive state is dependent on both voltage-

and time-dependent propenies. which drtermine the duration of refractoriness. During

the plateau phase and early phase 3 repolarization of the ventricular AP. almost al1 Na-

channels are inactivated. The absolute rehtory period is definrd in this period of the

4P (specifically hmthe AP upstroke to a repolarizing membrane potential of

approximately -50 mV). during which time a delivered stimulus. regardless of its

magnitude. will not initiate membrane depolarization. A change in membrane potential

during repolarization from -50 mV to more negative potentials is followed by an increase

in the number of Na- channels that have recovered from inactivation (dur to their time

and voltage dependence (Ureidmann. 1955)). This penod defines the relative refractory L I I 1 a I a 50 100 150 200 250 300 350 400 action potential duration (msec)

Figure 5. Excitability during the cardiac action potential. The absolute refractory period (ARP). during which no stimulus regardless of its strength is able to iriitiate a propagated action potential. is followed by the relative refractory penod (RRP).and the effective re fractory period ( ERP). During the RRP stimuli that exceed normal pacing threshold (Le. greatrr than twice diastolic threshold) can cause a propagated action potential. The ERP is drfined as the shortrst duration at which an impulse of normal amplitude and conduction velocity can be induced with a stimulus similar in magnitude to the threshold stimulus required to induce an action potential in late diastole. (Reprinted frorn The Atlas of Heart Diseases. Arrh.ythmias: Electrophysiologic Principlrs. Volume 10. Eugene Braunwald ed.. page 1.6. 1996. with permission from Current Medicine and from Dr. Eugene Braunwald) period. during which APs elicited by a stimulus much larger than the normal threshold will be initiated. However. this AP will have a reduced amplitude and slower conduction velocity due to a large proportion of the Na' channels that remain inactivated. The effective refractory period (ERP) follows the relative refractory state. in that APs of normal amplitude and conduction velocity cmbe induccd with a stimulus similar in magnitude to the threshold stimulus required to induce an AP in latr diastole. Action potential duration is thus a determinant of refractoriness. and either Na- channel block (or decreased Na' current). andior decreased K- current cm increase refractory periods.

1 .1.7. Measures of Ventricular Re fiactoriness

Although APD is a determinant of cardiac refractoriness. the duration of the cardiac AP at 90% repolarization (APDw) is conventionally used to closely estimate

.4PD. The duration of the .4P is in part dependent on the ability of cardiac repolarizing currents to drcrease the membrane potential to the resting. diastolic value. The ERP. like

APDgi,. also lürgely drpends on the kinrtics of repolarizing K+ currents. but does so in order for Na' channels to recover from inactivation and propagate anothcr AP. and as such. it is also a rneasure of ventricular excitability.

The 'fastest-follow-frrquenc" (FFF)is the fastest pacrd rate that allows for each pacing stimulus to induce an AP. Unlike rneasurements of ERP. detemination of the

FFF is a rneasure of ventricular functional refractoriness. It is determincd using prolonged pacing drive trains. and thus provides information on the dynamic properties of electrical restitution.

Restitution describes the behaviour of APD shortening as a function of increased heart rate. and cmbe measured after single or multiple beats. the latter resuiting in the measure of dynamic restitution (DR). In particular. DR kinetics are a measure of the degree of APD shortening following a shonening of the preceding diastolic interval (DI) as heart rate is increased. Assrssment of the steepest slope of DR kinetics in vitro gives important insights into the propensity to ventricular fibrillation (VF. a lrthal reentrant cardiac tachyarrhythmia) upon rapid myocardial stimulation (Koller rr ul.. 1998. Riccio et ai., 1999). If the DI is shortened (with increases in paced rhythm). an AP of short duration will follow. This will in tum lengthen the next DI. followed by a long APD. eliciting a short APD. resulting in a self perpetuating short-long series of APD oscillations. termed 'altemans'. The steeper the restitution relation of APD versus DI. the more marked the oscillation is cxprcted to become (Koller et al.. 1998). When this dope exceeds unity. these oscillations becorne chaotic and lrad to VF. Furthemore. interventions which decreasc the slope of the APD-DI relation are expected to protcct from VF (Weiss et al.. 1999).

The ventricular fibrillation cycle length (VFCL). the interval betwern succrssivc deflections of an electrocardiogram (ECG) or endomyocardiogram during fibrillation. has not only been determined to be a reliable index of refractoriness during ventricuiar fibrillation (Lammers rr rd.. 1986). both in hurnans (Misirr et rd.. 1995). and dogs

(Opthof er al.. 199 1 ). but has also been shown to correlate well with the APDso

(Murakawa et cd.. 1997). Therefore. VFCL is a measure of ventricular refiactoriness at the fastest possible sustainable ventricular rhythm.

1.2. Mechanisms of Arrhvthmias

An arrhythmia is the disruption of the orderly sequences of cardiac depolarization and repolarization. These may occur in the form of a rapid (tachyarrhythmic). slow (bradyarrhythmic), or asynchronous series of contractions. Some mhythrnias may precipitate more senous or even lethal rhythmic disturbances, such as torsades de pointes

(TdP), an arrhythmia characterized by excessive QT lengthening that can lead to VF.

Although many factors can increase the incidence of tachycardias (suc h as m y ocardial ischemia, acidotic or alkalotic alterations in pH. blood electrolyte disturbances. or increases in sympathetic tone). the mechanisms underlying the initiation of cardiac arrhythrnias are abnorrnal automaticitp. triggered activity. and reentry.

1.2.1. Abnonnal Automaticitv

Abnormal impulse formation is the least cornmon cause yet the most easily understood of the mechanisms of tachyarrhythmias. It involves the accelerated discharge of a ce11 anatomically closer to the ventricles than the dominant sino-atrial nodal pacemaker cells. This results in abnormal iiccrleration of phase 4 activity of the .4P that givrs rise to a premature systole if it is a single early discharge. or a tachycardia if it is a repetitive accelerated discharge (.4llessir er al.. 1973).

1.2.2. Trigeered Activitv

Triggered activity is a result of early or delayed afierdepolarizations which can cause premature systoles or tachyarrhythmias. These spontanrous vrntncular depolarkations are known to be generated by an overloûd of calcium influx. oscillating calcium release from the sarcoplasmic reticulurn. and chloridr ion emu. through ci'- activated chloride charnels (Charpentier el al.. 1 993). Tachyarrhythmias caused by triggered activity involve the movernent of positive ions into the cardiac crll (or negative ions out of the cardiac cell) causing an afterdepolarization during late phase 3 or early phase 4. resulting in a second AP. Tnggered afierdepolxizations may also be a result of a prolonged AP as seen in the long QT syndrome, or with drugs that prolong the APD. resulting in reactivation of calcium channels. thus causing calcium overload (January et al.. 1991).

1.2.3. Reentry

Reentry depends on the existence of two anatomically or physiologically distinct pathways. The substrates for reentry are an area of slowed conduction. and unidirectional block (see Figure 6). An arrhythmia resulting from reentry begins when an impulse enters a bundle of conducting tissue (myocardial or Purkinje fibres). and encounters the proximal end ofa bifurcation where antegrade conduction is blocked (Le. unidirectional block). Propagation of the impulse through the other normally conducting pathway can conduct the impulse around the area of block. The impulse then reaches a point distal to the depressed area. "rr-enters" the depressed area. and is propagatcd only in a retrograde fashion since unidirectional block of conduction is only in the antegrade direction. Since conduction is slowed in the depressed tissue area possessing the property of unidirectional block. retum of the retrograde impulse to the initial point of bifurcation is deinyed. Provided that the transient time through the circuit rxceeds the refractory period in the circuit. reexcitation of the myocardiurn may occur via this "short-circuiting" of the conducting tissue. causing a premature ventncular contraction (Euler and Moore. 1980).

The result is a premature systole generated at the site of the anatomical circuit. If this reentry mechanism becomes tepetitive. it results in a sustained ventricular arrhythrnia such as ventricular tachycardia (VT) or VF. Figure 6. A reentrant circuit. Prerequisites for a reentq circuit are an area of slowed conduction and unidirectional block (A). The effect of a class III antiarrhythmic agent is to increase the duration of the action potrntial and the refractorp prriod preventing an electrical impulse from foming a reentrant circuit (B). (Reprintrd from The Atlas of Heart Diseases. .r\rrhythmias: Electrophysiologic Principles. Volume 10. Eugene Braunwald ed.. page 2.14. 1996. with permission from Current Medicine and from Dr. Eugenr Braunwald) Antiarrhythrnic agents have been show to be effective in treating arrhythmias causrd by reentry circuits. Their rnechanisms in abolishing reentry are most likely bu: a) slowing conduction so that propagation around the reentrant circuit cannot occur. b) depressing membrane responsiveness. or c) increasing refractonness so that the reentrant wavefront impinges on refractory tissue. terminating its progress. The last of these threr mechanisms and the antiarrhythmic agents possessing properties of prolonging APD will be discussed in more detaii. as they most closely relate to the objectives of this thesis.

1.3. Modulatinp Cardiac Refractoriness

The behaviour of reentrant cardiac arrhythmias (of which VF is an important example) is partially detrrmined by the behaviour of APD in myocardial cells. As numerous antiarrhythmic dnigs act by altering cardiac refractoriness. it is important to fint introduce these agents. their classification. and thrir mechanisms of action.

1 3.1. Antiarrhvthmic Drues

The most widely accepteci classification of antiarrhythmic drugs was proposrd by

Vaughan-Williams ( Vaughan- Williams. 1970). In this classitïcation. antiarrhythmic agents are separated according to their effects on APD primarily determined from in virro rxperiments on Purkinje fibres.

Class I antiarrhyzhmic drugs block Na- channels. and thus depress conduction velocity in the myocardium. These agents have been separated into three subclasses in an attempt to differentiate between their subtle effects on the cardiac AP. Class Ia agents block Na' channels. and thus depress conduction in the atria. the ventricles. and the His-

Purkinje system. They also prolong cardiac refractoriness by prolonging the cardiac AP. Class lb dmgs also block Na' channels, more so when the cardiac cells are depolarized as opposed to a resting state. They differ tiom class Ia dmgs in that that they shorten APD.

Class Ic ilgents also inhibit Na- channels. but only have a slight effect on cardiac refractoriness. Class II antiarrhythmic dmgs are blockers of beta-adrenoreceptorç (P- blockers), which also results in a relative prominence of vagal effects on the heart. Class

III agents prolong APD by blocking K+channels. but have no significanr çffect on conduction velocity. Class IV dmgs directl) block calcium currents by inhibiting Ca* channel opening. In doing so, they decrease the inward current carried by Ca* across the ceIl membrane. decrease the rate of rise of phase 4 depolarization. and slow conduction in tissues such as the SA and AV nodrs (which functionally are highly dependent on calcium cunents).

However. manp of the antiarrhythmic agents available have actions relating to more thm one class. and not ail dmgs in the same class or subclass have identical effects according to the Vaughan-Williams classification. In an effort to precisrly detinr an antiarrhythmic drug's cfficacy. a new approach to classifying thrse agents has been proposed. termed the "Sicilian Gambit" (Task Force of the Working Group on

Arrh-ythmias of the European Society of Cardiology. 1 99 1 ). It attempts to idrnti fy a dmg that will best modifj a particular target known to affect a vulnerable panmeter ofa cardiac arrhythrnia. AIthough ail dmgs can potentially be charactrrized by their effect(s)

(or lack thrreof) on al1 channels. receptors. and pumps. limitations exist with this classifi cation. maidy since the exact mechanisms of mhythmias are poorly undentood. 1-32. The Effect of .htiarrhvthic Drugs on Cardiac Refractoriness

Antiarrhythmic dmgs which prolong cardiac refractoriness have variable effectiveness as a hnction of heart rate. Rate-dependence is a phenomrnon whereby a drug exerts greater effects at more rapid rates of stimulation. It is usually manifrst as greater drug-induced slowing of conduction at rapid than at slower rates. Rate-dependent phenomena can be explained by the modulated receptor hypothesis (Courtney. 1980).

For example. agents that target Na- channels during the open or inactivated state will be more effective blockcrs at high rates of stimulation. Drug dissociation occurs in a tirne- and voltage-dependent fashion. and less time for drug unbinding to occur results in an enhanced drug effect at fastgr rates of stimulation. Reverse ratedependence. on the contrary rxplains the progressive loss of druy effect at rapid hcan rates. This occurs with drugs that prolong APD by blocking K' channels. particularly Ikr.

It has been proposed that the progressive shortening of the cardiac AP at faster rates of stimulatioii occurs becausr Ikr currcnts do not dractivate complrtr.ly during diastole, but accumulate tkom beat to beat (Hauswirth et al.. 1972). Under this hypothesis. it is expected that selective blockers of Ikr (such as dofetilide and E-403 1 ) would be even more effective at faststrr pacing rates. because the contribution of Ikr to the total repolarizing cumnt should be increased under these conditions. However. the opposite is found. suggrsting that other sources of repolarizing currents. such as Iks. may become more prominent at shon cycle lengths. Thus. the phenomenon of reverse rate- dependence may be due to a relatively greater contribution of Iks. perhaps

'-compensating" for the decreased outward current resulting from Ikr block at rapid rates compared to slower rates. The kinetics of Iks channels are such that at fast rates they may not completely deactivate. resulting in an increased net repolarizing current

(Sanguinetti and Jurkiewicz. 1990). Drugs which exhibit little reverse rate-dependence. and thus maintain their effects on refractoriness at rapid rates. have been shown to be

more effective in arrhythmia management than those which lose their effect as rates

increase.

The difference between rate-dependence and "use-dependence" is quite markrd.

and should be noted in this rhesis. Use-dependence indicates that the more frequently the

channel is activated. the greater is the drgree of block. Because K' channel blockers

contribute to the repolarizing phase of the AP. use-de pendence is sometimes in ferred

from the effects of K' channel blockers on repolarization. Although in many cases use-

dependence at the chmnel level map translate into ratr-drpendrnce at the whole organ-

level (and vice-versa). the two do not always coincide (Jurkiewicz and Sanguinetti.

1993). One reason for this lack of correspondence lies in the complex nature of

repolaization. where several EC channels contribute to repolarization. and the fact that

these outward currents are closrly balanced bg inward currents (such as Ica. Na. and

iNa/Ca. As a result. no one cumnt dominates repolarization (Luo and Rudy. 199 1 ).

In addition. some antiarrhythrnic drugs may display temporal or spatial

heterogeneous repolarization in a given mass of cardiac tissue. This "dispersion" of

repolarization includes differences in APD (from different cell types. and different

transmural locations within the heart) as well as conduction pattern and velocity of

depolarizing wavefronts. although its precise mechanism is not ~~11-undrrstood.The

propensity to reentrant arrhythmias is relatsd to instantaneous tissue excitability. which is

in tum dependent on APD, Na' channel recovery kinetics. as well as myocardial resistance. However. the concept of temporal and spatial dispersion of repolarization is beyond the scope of this thesis. and was not measured in the experirnents conducted.

1.3.3. The Effect of Class 1 Antiarrhythmic Drues on Cardiac Refractoriness

While the primary action of class 1 agents (of al1 three subclasses) is to slow conduction velocity by blocking sodium currents. they have bren found to have effects on cardiac refractoriness as measured by ERP and XPD at least in part because of K- channel block in addition to Na- channel block. Quinidine (class la agent) increased

ERP, leaving dispersion of refractoriness unaltered in dogs (Cha cr ai.. 1996) and in hurnans (Shechter et cri.. 1983). Quinidine was also found to increase APD and ERP in guinea pig papillary muscle (Berman and Dorian. 1993. Uematsu et rd.. 1989. Li and

Ferrier. 199 1 ). in doys. in vivo (Costard-Jacklc and Franz. 1989a. Costard-Jackle er al..

1 %Wb. Farges et rd.. 1978). md in viirc in perfused Purkinjr fibres (Montera et al..

1992). Furthemore. quinidine was found to prolong .\PD.but not ERP. in a reverse rate- dependent manner in dogs (Cotsard-Jackle et al.. 1989b) and humans (Haberman rr (11..

1993). Procainamide (another class Ia drug). much likr quinidine. was also found to

dose-dependenily increase ERP in Langendorff perfused guinea pig hearts (Inoue et ut..

1991). dog Purkinje fibres (Coyle er cal.. 1992). and rabbit ventricular papillary muscle

(Toyama and Futura. 1983). whiie also increasing APD in isolated rabbit hearts (Koller

and Franz. 1994). In vivo studies have show procainamide to increase ERP in dog

(Schoels et ai.. 1991j. rabbits (Qiu er al.. 1997). and humans (Jalil er al.. 1998). in a

reverse rate-dependent manner (Haberman et cri.. 1993). Overall. class Ia drues increase

ERP and the ERPIAPD ratio (post repolaization refractoriness). Their cffects on APD is

reverse rate-dependent. and on ERP is not reverse rate-dependent. Conflicting data exists arnong various species with class Ib-induced alterations in cardiac refractoriness. Lidocaine (class Ib agent) was shown to increase ERP and APD in canine (Zhu et al.. 1990. Kinnaird and Man. 1984) and porcine Purkinje Fibres (Winslow et al.. 1989), In addition. in vivo studies in dogs showed ERP prolongation (Lynch et al..

1990. Inoue et ai.. 1985. Costard-Jackle et ai.. 1989b). while increasing the ERP/APD ratio (Costard-Jackle and Franz. 1989a). Homever. lidocaine did not change ERP in

Langendorff perfused guinea pigs hrarts (Inoue et al.. 1994). possibly due to the prolonged pacing protocol of the study. rendering tissue viability questionable.

Furthemore. homogeneity of repolarization (measured by dispersion of refractoriness) was preserved with increases in ERP in the intact porcine heart (Simrns rt ui.. 1997). and in hurnans (Engel et cil.. 1976).

Studies of flecainide (class Ic drugs) are al1 in agreement of slightly increasing

ERP in a rate-dependent fashion in whole animal prrparations in pig (Wirth and

Knobloch. 200 1 ). dog (Kidwell et dl.. 1993. Usui et al.. 1993 Lang et ril.. 1989.

Dobmeyer et ui.. 1985). and humans (Watanabe er al.. 200 1 ). Ogawa et oi ( 1993) showed that flrcainide increased dispersion of ventricular retiactoriness in dogs. In

addition. in virro assays confirmed the development of postrepolarization refractoriness

in canine myocardium (Martin er ai.. 1 996) and guinea pig papillary muscle ( Winslow

and Campbell. 199 1 ). by displaying greater increases in ERP relative to .4PD in isolated

preparations.

1.3.4. The Effect of Class 11 Antiarrhthic Drugs on Cardiac Refiactoriness

Little data exists on the effects of bsta-adrenergic blockade on cardiac

repolarization. Acebutalol (P-blocker) was found to increase ERP slightly. yet significantly. in dogs receiving chronic oral drug therapy (Vincent et cil.. 1993).

Selective blockade of PI and adrenoreceptors by atenolol and ICI 1 1835 1 respectively. were found to prolong ERP in closed chest dog studirs (Takei et al.. 1992).

However, propranalol (a non-selective P-blocker) was found not to significantly alter

ERP in the same model. yet increased ERP under the condition of increased sympathetic activity (Lang et al.. 1989). Administration of 8-2395 (also a non-selective P-blocker) was found to significantly. yet slightly increase monophasic APD and ERP. without the development of postrepolarization refractoriness in anesthetized dogs (Guirnond et ul..

1983).

1.3 3. The Effect of Class III Antiarrhythmic Drus on Cardiac Refractoriness

The duration of the cardiac AP is mainly the sum of phases 2 and 3. as phases O and 1 are quite short. Theretore. the APD can be prolonged bp essentially prolonging phase 2 andior 3. The currents involvrd in phases 2 and 3 are yuitr differrnt. yet slightly overlapping: during the end of phase 2 and begi~hgof phase 3 gradually increasing Ikr and Iks are the main outward currents (as they are inactivated at positive potentials during the plateau phase): dunng laie phase 3. Ikl cumnt is involved in repolarization. as it is opened by rernoval of rectification (Viswanathm el ul.. 1 999). Therefore. çlass III agents can prolong phases 2 and 3 possibly by reducing Iks. Ikr. or Ikl currents.

E-403 1 (a selective blocker of the Ikr channel) has been show to increase APD.

ERP. and the ERPlAPD ratio in dogs t Takatsuki rr al.. 1998. Ogawa et d.. 1993). In vitro studies of canine and guinea pig isolated perfused hearts. and guinea pig isolated ventricuiar myocytes have also show E-103 1 to increase ERP in a reverse rate- dependent manner (Yamada et al., 1996. houe et al.. 1 994. Lynch er ul.. 1993). Experiments in isolatrd ferret papillary muscle have shown this drug to dose-dependently increase ERP in a reverse rate-dependent manner (Baskin and Lynch. 1998. 1994. Baskin et al., 199 1). The ERP and APD measured at 50 and 90% repolarization were also found to be prolonged dose-dependrntly with this agent in sheep Purkinje fibres (McIntosh et al.. 1 994). Furthemore. clinical studies have shown that E-JO3 1 increases ventricular

ERP in hurnans (Fujiki el al.. 1994).

Dofetilide (Figure 7A). an open-channel Ikr blocker. increases ERP in guinea pig ventncular myocytes (Lynch et al.. 1995. Yang er ni.. 1 Wa). rabbit and ferret papillary muscle (Kovacs rr ul.. 200 1. Baskin and Lynch. 19%. Baskin er trl.. 199 1 ). isolated canine myocytes (Martin et d..1996). and isolated human ventricl<:(Montero and

Schmitt. 1996) in a reverse rate-dependent manner. ln vivo pig studies have shown dofetilide to increase ERP (Wirth and Knobloch. 2001 ). and in dogs to do so in a reverse rate-dependent fashion (Bauer ri cri.. 1999). with a decreasr in dispersion of refractoriness and no change in post repolarization refractorhess (D'Alonzo et al.. 1995.

Cha et ai.. 1996). As in in virro and in vivo animal studies. dofetilide has also been show to reverse rate-dependently increase ERP in humans (Derakhchan et (11.. 100 1.

Yang rr rd.. 1 9% ).

Ail experimental observations of the effect of d.1-sotalol (referred to sotalol. unless otherwise stated) (Figure 78) on proloiigation of APD and ERP are consistent in the literature. with respect to isolated human venuicle (Montero and Schmitt. 1996). in vivo dog hearts (Kidwell er rd.. 1993). open-chrst rabbits (Novosel et ol.. 1993). guinea pig papillary muscle (Yang et al.. l992b). and i~ vivo in humans (Nademanee et al.. 1985

Hoffmann et al.. 1 993. Roden, 1993). However, sotalol has been show to increase Figure 7. Chemical structures of dofetilide (A). d.1-sotalûl (B). and the chromano1 HMRljS6 (C). monophasic APD and ERP in an unexpected rate-dependent manner in isolated human myocardium (Watanabe et al.. 2001). guinea pig papillary muscle (Berman and Donan.

1993) and isolated myocardium (Ishii et al.. 1999, contrary to the findings of other investigators who found reverse rate-dependent propenies of this dnig in riw in closed- chest canines (Freigang et 01.. 1997. Fei and Frarne. 1996). Sotalol was also found to develop postrepolarization refractoriness in isolated rabbit ventricular myocardiurn

(Cobbe et a/.. 1985).

The chromano12398 was found to block Iks channels in guinra pig cardiomyocytes (Busch et a/.. 1996) as well as Iks channels expressed in Xrnopus oocytes (Busch et al.. 19%. Suessbt-ich et cil.. 1996. 1997). It was found to prolong the ventricular AP in a rate-dependent manner in both guinea pig and human ventricular myocytes (Bosch rr til. 1998) at rui ECro of 1 FM. but \vas also found to block Ito currents in human ventricular myoc'es (ICso= 24 FM). and canine lefi ventricular cells

(Sun et al.. 200 1 ). Mathematical modeling of the chromano1293 B has postulated that it

blocks Iks in the open state ( Fujisawa el al.. 2000). More recently howevrr. the

chromanol HMRl556 (Figure 7 C) was developed in an etfort to increase the potency and

improve selectivity of blocking the Iks channel. Gogelein and colleagues found

HMR1556 to inhibit Iks channels expressed in Xenopus oocytes with an ICcoof 170 nhl.

Prolongation of APD was found in guinea pig papillary muscle and Langendorff perfused

guinea pig hearts at a range of pacing frequencirs (Gogelein et 01.. 7000).

1.3 -6. The EfTect of Class IV Antiarrhvthmic Druas on Cardiac Refractoriness

Class N dmgs mediate their effects through calcium channel blockade. and thus

affect the plateau (phase 2) of the M. Literature of the effects of calcium channd blockade alone on cardiac refractoriness is limited. Verapamil (class IV drug) has been shown to decrease ERP. while increasing heterogeneity of repo1,uization in vivo in canine

(Benardeau er al.. 2001). but also to increase ERP while decreasing APD in isolated canine Purkije fibres (Kinnaird and Man. 1984). Diltiazem (also a class IV antiarrhythmic agent). on the contrary. has been shown to have no significant effect on

ERP in dogs (Benardeau et al.. 2001 ). but was found to prolonp .4PD at 20 and 50% repolarization (Miyazai

1.4. Ventricular Fibrillation - A Reentrant Arrhvthmia

Ventricular fibrillation (VF) is a lethal cardiac tachyarrhythrnia responsiblr for approximately 400,000 cases of sudden cardiac death per year in Nonh America

(Myerburg er al,. 1 992). It is charactrrized by hemody narnic collapse coupled wiih complete irregularity and disorganization of QRS defltxtions of crirdiac electricai tracings. whether they br endocardial. epicardial. or surface ECGs obtained from iimb

leads. The irregular. continually undulating eiectrocardiographic deflections present during VF are representative of the electrical activity of the ventriclrs. which is random.

chaotic. and asynchronous in nature.

Mechanistic explanations of VF involve theories of circus movements to establish

a circuit favourable for the initiation and continuation of reentry. An external rlectrical

stimulus. or a spontaneous premature beat may initiate VF. However. the maintenance of

fibrillation is independent of the ini tiating cause. and is sel f-sustained.

1.41. Initiation of VentricuIar Fibrillation

Reentrant excitation leads to very rapid. self-sustaining. irregular electrical

activity through the conducting fibers of the heart. which is the hallmark of VF. Although reentry is the mechanism by which VF self-perpetuates. its initiation involves heterogeneity of repolarization as described by Moe and Abildskov (1959). They first postulated the Multiple Wavelet Hypothesis to rxplain the initiation of VF. by stating thrit an electrical impulse will propagate unifomly and rapidly in al1 directions tiom the site of origin; however, recovery from activation due to this impulse is not as uniform as impulse propagation, since the duration of refractoriness varies from one muscle fiber to the next. This. in tum. allows for some myocardial fibers to be excitable at a time when adjacent fibers are not. and a second impulse initiated at the primary site of origin will be irregularly propagated. This second impulse will be conducted by tissue in an advanced state ofrecovery. but will not be propagated through tissue in early stages of re frractoriness.

Temporal dispersion results from such a process of excitation/recovery. and increases in magnitude accordingly with each impulse initiated thereafier. and the sprcad of myocardial excitation will cease to exist in an orderly fashion. Thus. the resulting increasingly irregular wavefronts split. and divide around re fractory tissue. which is incapable of conducting an impulse. The resulting rnultiplicity of new wavefronts

(termed 'daughter wavelets') will propagate in the same fashion as the parent-impulse.

independently and non-uniformly. Daughter wavelets will then decelente or accelerate when encountrring tissue in an rûrlp state of refractoriness or advanced state of recovrr!.

respectivrly. Upon an encounter with refractory tissue. a daughter wavelet may becorne extinguished; however, it may divide again, or combine with neighbouring wavelets. and

in such a manner will change direction and fluctuate in size. Therefore. VF is defined as

a state in which many simultaneous wandering wavelets coexist. 1.4.2. Maintenance of Ventricular Fibrillation

Maintenance of VF requires the presence of multiple waveiets, which in tum requires a large mass of tissue. Two other properties necessary to sustain VF are an irregularly short refractory period. and persistent areas of slow conduction and local block, which are dynamic properties of rapidly and irregularly stimulated cells.

According to the wavelet theory of Moe and Abildskov. the larger the nurnber of wavelets. the less likely ir is that a segment of conducting rnyocardium will simultaneously become refractory or escitable. allowing the wavelets to collide and extinguish the arrhythmia. On the contrq, if the number of wavelets is small. there is an increased probabilitp that they may fuse. ailowing the rhythm to cxtinguish.

According to the physical properties of conductive tissue. a smallrr wavelrt

"wavelength" (7,) would result from decreased refractory period (rp)ancilor conduction velocity (cv)(I = rp x ci.). A larger number of waveltts of shorter wavelength cm therefore coexist in the presence of a short refractory period: a prolonged refractory period would render the entire ventricular rnyocardiurn rrfractory. and al1 wavelets would merge. thus terminating VF. The conduction velocity of wavefronts is also a determining factor of VF maintenance. since rapidly conducting impulses would increase the wavelet wavelength: this would niake it less likely for the genesis of daughter wavelets. and it would decrease the probability that the conducting wavrfront would be disorganizrd.

A larger myocardial mass will. by its anatomical nature. be able to support many more wavelets than a smaller mass of tissue. This supponed the observations of McWilliam

( 1 887) and Porter ( 1 894) that VF was more easily sustained in larger hexts. Garrey

(1 914) showed that pieces of ventricular muscle with a surface area of less that 4 cm' ceased to fibrillate when removed from a fibrillating heart. and that at least one quarter of the original myocardial mass was required for maintenance of VF. Other investigators have determined this minimum myocardial mass to be 6 (Szabuniewicz et al.. 1968). 8

(Garrey. 19 14). 12 (Ruiz and Valentinuni. 1994). 20 (Janse er cil.. 1995). 25 (Damiano er d..1 WO), and 2.5 g (Malkin et uZ., 1998). This may suggest that the mass of cardiac muscle required to maintain fibrillation is depcndent on the electrophysiologic propenies of the heart: a larger mass is required if conduction is not slow enough and refractoriness is not short enough to maintain reexcitation.

1.4.3. Ventricular De fibrillation

Applying an electrical current to the heart (either directly to the myocardiurn. or

transthoracically). termed 'defibrillation'. is the only known treatmctnt to terminaie VF.

Successful venvicular defibrillation is determined by the strength of the shock. the shock

waveform of the applied current. the electrode shape and size. and the mass of

myocardium encompassed by the defibrillating shock ( Shibata et ai.. 1988).

De fibrillation energy or voltage (V) requirements can be described by a dose-

response curve. which can predict energy values associated with rates of successhl

defibrillation. The nature of defibrillating shocks is therefore probabilistic. This may be

due to alteration in rlectrophysiologic characteristics of myocardial tissue in\.olved in the

initiation and continuation of VF. or the duration of VF before the first defibrillating

shock (Echt et al.. 1988. Fujimura er al., 1989). Nevertheless. the term ~detibrillation

threshold' (DFT) will be used to describe the minimum energy requirements necessary to

terminate VF (Davy et al.. 1987). althouph an dl-or-none threshold does not eaist. Numerous theories of defibrillation have been postulated including the critical mass hypothesis, the ULV hypothesis. the refractory period extension hypothesis. and the threshold of synchronous response hypothesis. All investigations attempting to elucidate the mechanisni of defibrillation agree that total extinction of fibrillatory activity is observed with high defibrillating energy levels. and that srnaIl shock energies do not alter

VF activity. The challenges in interpretation of defibrillation lies in the probabilistic nature of shock levrls that faIl in between these two extremes. Although many theories have been used in an attempt to explain the complex and controversial phcnomenon of defibrillation. the precise mechanism remains unknown.

1 -4.3. I . The Critical Mas Hvpothesis

According to the critical mass theory of deti brillation. activation wavefronts can be stopped only within a certain critical mass of myocardium. In terms of the multiple wavelet hypothesis. the applied defibrillating shock would teminate most of the wandering wavelers of VF. with the remaining ones encountering tissue in a drpolarizrd state (i.e. refractory). and terminate dur to an environment that is nor electrophysialogically conductive. Therefore. according to this hypothesis. defibrillation could succeed even if electrical activity penisted after the shock. Zipes and colleûgues

(1975) were the first to show this esperimentally. postulating that wavefronts of VF nerd only be terminated in 73% of a dog's ventricular mass. Studies that followed showed that the myocardial fraction of terminated wavefronts could be smaller (Zhou er dl.. 199 1.

Witkowski et ai.. 1990). 1.4.3.2. The Upper Limit of Vulnerability Hvoothesis

During sinus or paced rhythms. a critically timed stimulus during the cardiac cycle can induce VF. Specifically. this 'temporal window'. determined to be located on

the T-wave of the ECG. is termed the vulnerable period (Swerdlow et al.. 1997. Hoffman

et al.. 1955). It is also determined that there is an upper and lower strength stimulus

during the vulnerable period that is capable of inducing VF; shock strengths outside these

upper and lower limits are unsuccessful in generating the arrhythrnia (Winfree. 1990).

The upper limit of vulnerability hypothesis for defibrillation States that unsuccessful

shocks slightly weaker than necessq to de fibrillate will terminate al1 activation

wavefronts during VF: however. since this same shock stimulates regions of ventricles

during their vulnerable period. it gives riss to new activation wavefronts that reinitiatr VF

(Chen er al.. 1986). Thus. to be successful. the defibrillating shock strength must br

greater than or equal to the upper limit of vulnerability.

1 A.3.3. The Refracton: Period Extension Hypothesis

Defibrillation shocks drlivered to refractory tissue were first shown by Sweensy

and colleagues ( 1990) to depolarize tissue that is not refractory at the time of the shock.

and prolong the tissue's refiactory period. Successfùl defibrillation results from the

prevention of fibrillatory wavefronts from propagating aftrr a shock that has induccd an

extension of the refractory period (Dillon et cri.. 1992. Kwaku and Dillon. 1996).

Sweeney and colleagues also propsed that when refiactory penod extension is

homogeneous. it reduces the probability of reentrant excitation of postshock wavefronts

and prevents the delayed generation of refibrillating wavefronts (Sweenry rr oi.. 1996).

Other investigators have shown that the prolongation of repolarization and refractoriness. like the de fibrillation succers rate, increases with increasing shock strength (Tovar and

Jones. 1997. Sweeney et al.. 199 1. 1990). On the other hand. a low energy shock. or a shock given early in the refractory period of a myocardial ce11 wili be insufficient in prolonging the refiaciory penod, lcading to unsuccessfbl defibrillation. Furthemore. postshock VF activation was also studied (Shibata et al.. 1988). and electrical shocks were found to prolong myocardiûi refractorinrss which influrnced patterns of electrical propagation of VF afler the shock.

1.1.3.4. The Threshold of Svnchronous Response Hv~othrsis

The threshold of synchronous response hypothesis was developed by Fabatio

(1 967). postulating that the process of fibrillation induction and defibrillation are related through common electrophysiologic mechanisms. Induction of VF occurs when a shock dcsynchronizes electrical activation by stimulating some. but not al1 of the excitable cardiac tissue. A shock that is strong rnough to depolariztf al1 of the Ièast èxcitablti tissue would not induce VF. Relating this concept to de fibrillation. this hypothrsis States that this same minimum shock strength would also defibrillate the hran because in a regular rhythm. it would have been strong enough to depolarize al1 of the myocardial fibres.

1.4.4. The Effect of Antiarrhvthmic Dmgs on Ventricular Fibrillation and Defibrillation

The four Vaughan-William classes of antiarrhythmic drues have bern reported to have variable effects on defi brillation tl~reshold.

Class I antiarrhythmic dmgs (Na- channel blockers) either increase or have no effect on DFT. Quinidine (class la agent). propafenone (class Ib drue). and encainide and flecainide (class Ic agents) have al1 been teund to increase (Woolfolk el uf.. 1966. Babbs et al., 1979, Peters et al., 199 1, Fain et al.. 1986. Hernandez et al.. 1989) or have no effect on DFT (Dorian et al., 1986, Echt et al.. l989a. Stevens er al., 1996. Szabo er al..

1988) in dogs and hurnans. Class 11 antiarrhythmics (blocken of beta adrenoreceptors). such as propranalol and atenolol have been show to have no effect on DFT in dogs

(Deeb et al., 1983), possibly due to stabilizing membranes of cardiac cells (Ruffy er al..

1987). Class III agents (K' channel blockers). such as sotalol. have been shown to ciecrease energy requirements for de fibrillation (Echt et ul.. 198%. Wang and Dorian.

1989. Dorian and Newman. 1993. Murakawa et al..1997). Class IV antiarrhythrnics

(calcium channel blockers). such as nifedepine. diltiazem. and veraparnil have also been show to increase DFT both in dogs (Hite et al.. 1989) and hurnans (Jones et cal.. 199 1 ).

1.5. Potassium Channels as Targets for Treatmcnt of Arrhvthmias

Reentry as a mechanism of arrhythrnia cm be altered by altering the APD of a myocardial cell. Action potential duration can in turn be modulated by altering cardiac ion channel activity. Since repolarizinç K- channels are important to refractoriness. the ability of K- channrl blockers to modify refractoriness will help design pharmacological approaches to decreasing the incidence of reentry. in particular disorganizèd reentry such as VF.

Dispersion of repolarization (both temporal and spatial) have bsen corrdated with a decreased VF threshold (Han rr ul.. 1966). The only pharmacological means by which fibrillation could be potentially terminated is by refractory period prolongation or refractory period homogeneity. Thus. studies have been concentrated on exploiting the class III effects of antiarrhythmic drugs (narnely homogeneiry of spatial and temporal dispersion of repolarization and prolongation of the APD) in an effort to treat ventricular tachyarrhythias. particularly VT and VF. The mechanism underlying the prevention of such arrhythrnias is an increase in homogeneity and prolongation of refractoriness. thus preventing reentrant wavefronts from propagating in retrograde directions within myocardial conductive tissue. In addition. antiarrhythrnic therapy with drugs possessing ciass III effects have also been show to decrease DFT. Such antiarrhythmic drugs are adrninistered as adjunctive therapy in patients with implantable cardiovertedde fi bri 1lators

(ICDs). thus minimizing the voltage ntcessary for successful shock treatment of VF or

VT. This in turn minimizes shock induced myocardial damage to the hem. and prolongs device battery life, minimizing the need for recurrent surgical interventions for re- implantation. Ultimately. this may equate to increased rates of patient survival.

Iongevity. and quality of life.

1 .j.1. Antiarrhvthmic Potential of Selective Potassium Channel Blockers

By blocking Ikr anaor Iks charnels. antiarrhythmic agents can prolong ventricular refractorincss. and possibly reduce the incidence of tachyanhythrnia via mechanisms relatrd to terminating wave fronts during reentrg. Do fetilide. a class III antiarrhythrnic agent selective for blocking the Ikr channel, has displayed clinical effectiveness in decreasing DFT (Davis er cil.. 1999, Mehdirad et al.. 1999). Preliminary results of the Danish Investigators of Arrh'hmia and Monality on Dofetilide

(DIAMOND) trial showed that isolatèd block of Ikr by dofetilide in patients with congestive heart failure and left ventncular dyshinction produced neutral effects on total mortality. relatively low incidence of TdP. and increased facility in maintaining sinus rhythm in patients with atrial fibrillation and in thosr developing arrhythmia during follow-up (DIAMOND Investigators. 1997). Amiodarone (an antiarrhythmic agent possessing class 1. II. III. and IV effects) has been shown to control a wide spectrum of tachyarrhythmias. The drug exerts a powerful suppressant effect on VT and provides control in approximately 70% of recurrent susrained VT or VF (Singh. 1995). Amiodarone was associated with a significant reduction in the risk of cardiac monality. cardiac arrest. or syncope in patients resuscitated from VF in the CASCADE trial when compared with other anti-arrhythmic agents (CASCADE Investigators, 1993). This drug has also been extensively investigated in several large clinical trials such as CHF-Stat (Singh rr al.. 1995). EMIAT

(Julian et al.. 1997). and CAMIAT (Cairns et al.. 1997). examining its effects on lifr- threatening arrhythmias and/or rnortality. suggesting anti-arrhythmic efficacy with uncertain survival bene fit.

Sotalol (possessing class II and III effects) appears to be comparable to amiodarone in its effectiveness in treating patients with ventricular tachyarrhythmias. In the Electrophysiologic Study Versus Electrocardiographic Monitoring (ESVEM) trial involving patients with sustained VT or VF. sotalol was superior to six class t agents with respect to total rnortality. sudden death. and cardiac death (Manson. 1993b).

Although the class III rnethanesulfonanilidr E-403 1 has not been used sstensively clinically. has heen reported to be effective in treating supraventricular tachycardias in

humans (Fujiki el al.. 1994).

Aldiouyh a clinically effective Ikl biocker does not exist. rxperimental studies are

suggesting that Ikl block may benefit arrhythmia suppression. New agents are currently

being developed that in part block Ikl. with eKects on other potassium channels (such as

terikalant. known to block Ikr in addition to Ikl channels (Jurkiewicz et al.. 1996)). Block of Ik1 is thought to be effective in suppressing a ventricular injury potential present between ischernic and non ischemic regions in unhealthy hearts. which may be responsible for inducing VF (Ridley et al.. 1992). The mechanism involved rnay be slight depolarization of non-ischemic tissue with Ikl block. in order to reduce the flow of

injury potential during diastole (Rees and Curtis. 1993).

1.5.2. Proarrhythmic Potential of Selective Potassium Channel Biockers

By blocking Ik currents and prolonging the APD. early after depolarizations

(EAD) rnay be generated by spending too much time in the window voltage for calcium

channel reactivation (Janua~and Riddle. 1989. Januaq rr ut.. 199 1 1).or sodium channel

reactivation (Grant et ai.. 1976). the former resulting in an increase in intracellular

calcium ions resulting in triggered automaticity from DADs (Volders er rd.. 1997): the

latter facilitates the genesis of reentry arrhythrnias bp slowing the recovery of Na-

channels from inactivation. and providing more tiine for incompletrly recovrred or

slowed conduction (Starmer rr d.. 199 1 ). Furthemore. prolongation of the ..\PDas a

result of K~ channel block may magnify inhomogeneities in refractoriness and

repolarization that are common in diseased and scarred hearts. promoting reentry

(Restivo ef ai.. 1997).

The most proarrhythrnic action of antiarrhythmic agents with class III effects is

the initiation of TdP. which cm lead to VF and sudden death of patients (Janse and Wit.

1989. Surawicz. 1989. Taglialatela et ai.. 1998). This may arise from an incrcased risk of

EADs from triggered activity due primarily to excessive APD prolongation at slow hean

rates. 1.5 -3. The Effect of Blocking Multiple Cardiac ion Channels

Combination antiarrhythmic drug therapy has been shown to be beneficial. both in experîmental animals and ciinical arenas. and has been well studied with combination

~a and K' blocken. The combination of quinidine or procainamide (class la drugs) and sotalol (class III agent) has proven to be more effective than either drug alone in preventing recurrent sustainrd VT. Although sotalol has been shown to ex hibit reverse rate-dependence. this action was abolished when both drugs were administered in combination to humans (Lee et al.. 1997). Qunidine in combination with sotalol was also found to produce additive effects on prolonging the ERP in guinea pig papillary muscle

(Berman and Dorian. 1993).

Other drug combinations. such as mexiletine in combination with quinidine. were found to have additive effects on increasing ERP. and eliminaiing the reverse rate- dependence found with quinidine alone ( Costard-Jackle and Frrinz. 1 989a. Costard-Jackle et al.. 1989b).

Sotalol displays both class II actions (due to the P-blocking properties of the 1- isomer) and class 1 II actions (a result of the d-isomer) (Gomoll er d..1986). Clinically. d-sotalol has been assessed in antiarrhythmic rfficacy in the SWORD trial ( Waldo cr ul..

1996). which was terminated prematurely due to increased patient mortality. However. the combination class II and III effects of sotalol on decreasing DFT has made it one of the few drugs regularly administrred to patients fitted with ICDs (Wang and Dorian.

1989. Dorian and Newman. 1993. Donan et rd.. 1994). Furthemore. the cornbined P- and K- channel blocking effects of sotalol has demonstrated to be clinically more effective in the treatrnent of VT and VF than six class 1 dnigs studied, with respect to total mortality. sudden cardiac death and arrhythmia recurrence (Manson. 1993a. 1993b).

Drugs which have less reverse rate-dependent class III effects are expected to result in a lower risk of proarrhythmia. namel y TdP. Honiogeneous repolarization of refractoriness is also expected to reduce the propensity to arrhythmia. Both these mechanisms of action may explain the s fkctiveness of arniodarone in decreasing the propensity to proarrhythmia compared to other dnigs used clinically. Amiodarone as been shown to possess class 1.11.111 and IV effects by blocking beta-adrenoreceptors and multiple cardiac ion channels. including Ito. Ikr INa and Ica. It has been shown to be more effective than class 1 agents in reducing the risk of cardiac arrest (CASCADE

Investigators, 1993). It has also been show to decrease DFT, and is the most common agent administered as adjunctive therapy in patients fittrd with ICDs.

investigational drues such as azimilide (Ikr and Iks bloçker) (Salata and Brooks.

1997) and tedisamil (Iks and Ito blocker) (Dukes et al.. 1990. Donan and Newman 1997).

have been shown to decrease DFT with corresponding increases in VFCL. as weil as

increases in ERP (Qi et al.. 1999. Dorian and Newman. 1997) in open chest dog models

of VF. Azimilide has aiso been shown to prolong cardiac refractoriness with lrss reverse

rate-dependence than Ikr blockade alone with dofetilide. pointing toward the potential of

Iks block in eliminating the reverse-rate dependence of APD prolongation resulting frorn

Ikr block (Natte1 et al.. 1998). 1.6. Rationale

Cardiac repolarization is influenced by a nimber of potassium currents.

Therefore. the investigation of antiarrhythrnic agents which block such currents. both selectively and in combination. is benencial in detennining their effectiveness in prolonging repolarization and drcreasing propensity to arrhythrnia.

Dofetilide and sotalol are class III antiarrhythrnic agents that possess reverse rate- dependent properties on prolonging APD. As such, their propensity to inducing TdP is high. On the other hand. barium (also possrssing class III antiarrhythmic rffects) pnmm-ily blocks Ikl currents (althouph it has recently been shown to have moderate effects on Ikr cumnts (Weerapura es (11.. 2000)). Torsades de pointes with Ikl block has not been reported due to: a) a lack of clinically available Ikl blockers. and b) a lack in reverse rate-dependent APD prolongation of currently availabie experimental Ik 1 blockers. Funhermore. Ikl current density has ben nponed to be decreased in patients with terminal heart Mure (Beuckelrnann es al.. 1993). and Ik 1 block may therefore be a surrogate for studying a diseased state in norrnal hearts (Pogwizd er rd.. 200 1 ).

Antiarrhythmic drug therapy using a combination of channel blockers has been more promising in reducing the incidence of arrhythmias thsn single c hannel blockrrs

(Lee et al.. 1997, Berman and Dorian, 1993. CASCADE Investigators, 1993. Natte1 et rd..

1998). Different potassium currents are responsible for repolarizing the myocardial cells at different phases of the AP (Sanguineni and Jurkiewicz. 1990. Courtney et al.. 1992).

This has prompted an investigation of combining drugs that each block different K- charnels. Dofetilide and sotalol are selective blockers of the Ikr channel (responsible for early phase 3 repolarization). and bariurn is a relatively selective blocker of Ikl channels

(responsible for late phase 3 repolarization and maintaining resting membrane potential).

Dofetilide and sotalol are similar in the following respect: a) they are selective in blocking the sarne channel. b) they display similar pharmacodynamics (Kimura er (il..

1996, Le Coz et al.. 1995. Srdgwick et cal.. 1991, Singh et al.. 1987.). and c) they alter electrophysiologic propenies. both by prolonging ventricular APD and refractory periods in a reverse rate-dcpendent mmer (Jurkirwicz and Sanguinetti. i 993. Marschang er (il..

2000. Williams et al.. 1999). Howevrr. evidence suggests that both these agents may exert their effects by blocking different states of the Ikr channel (Carmeliet. 1993).

The 'downstrearn' effects of reduced ikl current in myocardial disrase may alter

Ikr current density. which would in tum alter the pharmacologic effectiwness of Ikr channcl blockers. As rabbit hearts are known to posses both Ikr and Ik 1 currents in

similar densities to that of humans. the rabbit hart was the species of choice for

experimental studies of Ikr and Ikl block in combinatinn. Funhrrmore. Iks currents in

rabbit hearts play no significant role in cardiac repolarization (Lengyel et al.. 7001 ). and

are not expected to provide a 'compensatory' effect on decreased Ikl and/or Ikr current

density with K* channel blockade. In addition. the denrrvated isolated prepantion of the

Langendorff mode1 allows for measuring dmg èffects under minimal autonornic

in tluences.

Furthemore. the reverse rate-dependent and proarrhythmic nature of selective Ikr

blockers is thought to be due to an accumulation of Iks current. This prompted the

investigation of combining Ikr with iks block to detennine the effects of Iks block on the reverse rate-dependent nature of prolonging APD with clinically available KAchannel blockers. Howeverl since Iks currents in rabbit hearts play no role in repolarization

(Lengyel et al., 2001) and the isolated rabbit heart preparation is prone to electromechanical dissociation during extensive pacing protocols (especially at high rates). an intact guinea pig heart rnodel (established specifically for this study) kvas a preparation most suitable for the study of Ikr and Iks block in combination. Furthemore. the interaction between Iks and Ikr block and the presence or lack of reverse rate- dependence of AP prolongation with combination block can feasibly be tested in this model since: a) Ikr currents are also present in guinea pig. and b) Iks channels have been

reported to play a significant role in cardiac repolarization in this species. In addition. the

intact heart model represrnts a more cornplex system than an isolated heart. In contrast

to the isolated Langendorff hrart model. the electrophysiology of the intact heart and

pharmacological effects of antiarrhythmic dmgs may be influenced by the autonornic

nervous system. which is an important source of variation. Drug therapy in this model

may have a plethora of electrophysiological effects influenced by dose. route of

administration. state of the myocardial tissue. and autonornic tone. effects that are

overlooked in an iso Iated heart model. 1.7. Hvpothesis

The central hypothesis of this thesis was rhat under conditions in which Ikl currents are diminished by block with bariurn. different selective Ikr blocken (dofetilide and sotalol) would both produce additive effects on cardiac refractoriness (measured by

APD. ventricular ERP and VFCL). and DFT in the isolated rabbit heart.

The secondary hypothesis of this thesis was that Iks block (with HMRI 556) in intact guinea pig hearts would display rate-dependent prolongation of cardiac refractoriness (measured by activation recovery-interval (ARI. a surrogatr to APD) and

ERP). Block of Ibr (with dofetilide) in combination with Iks block would produce additive. if not synergistic effects on cardiac refractorinrss. eliminating the reverse-rate dependent effects of Ikr block alone. 2. METHODS

2.1. Isolated Lan~endorffRabbit Heart Ex~eriments

Fony eight male New Zealand White rabbits weighing between 3.0 and 5.0 kg

(mean 3.8 k 0.5 kg) were used in this study. and were housrd in cages -11.8 cm wide. by

11.8 cm deep, by 27.7 cm high. with a 12 hour daylnight cycle. and room temperature was maintained at 22 OC. They were fed Rabbit Chow (PMINutrition International. St.

Louis. MO). and had water frerly available to them. Al1 experiments werr approved by the Animal Care Committee of St. Michael's Hospital and conformed to the guiding principles of the Canadian Council on Animal Care.

2.1.1. Langendorff Preparation

Rabbits were anesthetized with sodium pentobarbital(50 mgkg i.v.. MTC

Pharmacrutiçals. Cambridge. ON). and anticoagulatrd with hrpiirin ( 1000 Lu. ix. Lro

Laboratories. Ajax. ON) administered via the right marginal ear vein. Following anesthesia, a midstemal thoracotomy was performed and the ribs retracted to expose the heart. which was rapidly excised by blunt dissection. Al1 hearts weighrd betwren 8.5 and

16.3 g (mean 1 1 .6 + 1.7 g). Each heart \vas mounted in a vertical position by inserting a

cannula into the aorta. above the aonic valve. and held in place with a micro-bulldog

clamp (Harvard Apparatus. Saint Laurent. PQ), and surgical suture. The visceral

pericardium was removed as was any excess tissue. including fat. lungs. and pulrnonary

vesscls. The hem was perfused retrogradely via the aonic cannula on a Langendorff

perfusion apparatus (height 90 cm) with Tyrode's buffer containing the following: NaCl

130 mM. NaHC03 24.2 mM. NaH2P04 1.2 mM. MgCl? 0.6 mM. KC15.6 miM. CaClz 2.2

mM. dextrose 12 mM. and bovine alburnin 40 mM (Sigma Chernicals Co.. St. Louis. MO) dissolved in 2 L of distilled water. The perfusate was filtered with a 0.45 pm membrane (Gelman Sciences. Ann Arbor, MI), and was oxygenated with 95% Oz: 5%

COz at a flow rate of 5.0 Limin, with a pH at 7.4 + 0.05. The temperature of the Tyrode's solution was maintained at 37OC by passing it through a glass spiral warming coi1 within a temperature regulated water bath (Harvard Apparatus. Saint Laurent. PQ). A total volume of 1.72 f 0.06 L of perfusate was recirculated under a head pressure of 68 mmHg. resulting in a flow of 125 mLimin with no han presenr on the Langcndorff apparatus. The effluent was collected in a 1 .j L Pyrex beaker (Coming Lnc.. New York.

NY) and pumped to a 750 mL elevated reservoir by a Veristaltic pump (Jr. Model.

Manostat, New York. NY).

2.1.2. Hemodvnarnic Mrasurements

An air-free. latex balloon was placed in the left ventricle by passing it through an incision in the lefi auricular appendage. The balloon was rxpanded by saline injection resulting in a steady diastolic pressure of 5 2 1 mmHg. A pressure transducer (PX1 D.

Statham. Bionetics. Inc.. Toronto. ON) was comected to the balloon such that bcth systolic and diastolic pressures could be displayrd and rneasured with a pressure amplifier (Hewlett Packard. U.S.A). Systolic pressure was noted before and after each phase of the experîment in mmHg (both baseline and treatment) for each rabbit heart. as

was the rate of coronary flow (measured in mumin). by collecting effluent ejectrd from

the heart .

Heart rate was determined fiom pre- and post-basdine and drug treatment studies

in al1 experiments by averaging three unambiguous successive R-R intervals on the epicardial bipolar electncal signals (recorded from the same channel. either the left or right ventncle), and converted to a value of beats per minute (bpm).

2.1.3. Determination of Electrophvsiolo~icaiParameters

3.1.3.1. Experimental Setup

An oval titanium mesh patch electrode was placed on the rpicardium of the left ventricle. and held in place with a latex tie over the right ventricle (see Figure 8). A contact electrode was also placed in the apical tip of the right ventricular endocardium. guided through the right atrium and the tricuspid valve. These rlectrodes were used to

induce VF as well as to defibrillate the heart during VF episodes. In addition. two pairs of custom-made electrode hooks and a quadripol~contact monophasic action potential

(MAP) 7F cathrter (EP Trchnoiogics Inc.. Sunnyvalr. CA) were placed on the right and

le fi ventricular epicardium. and the right vrntricular rndocardiurn. respecti vel y. One pair

of pins of the M.4P catheter was connected to a Ventrites cardio\,erter/detlbrillator

(Ventritex. Inc.. Sunnyvalr. CA) in order to pace the hart during measurements of

ventricular effective refractory period. and AP duration measurements: the second pair of

pins was co~ectedto a custom-made amplifier (Cartesian Labs. Toronto. ON) to record

left ventricular endocardial MAPs. AI1 cardiac electrical signals were stabilized with a

grounding electrode placed on the metal aortic cannula. Epicardiai and endocardial

electrocardiograms were amplified (Cartesian Labs. Toronto. ON). filtered (low pass 500

Hz; high pass 0.05 Hz). sampled at a rate of ZOO0 Hz. and simultaneously displayed and

recorded using a custorn-made PC compurer software program. ACQUI-2 (Cartesian

Labs. Toronto. ON). Al1 recordings were archived on CD-ROM. and blinded data LV and RV epicardhl bipolar electodes

Figure 8. Schematic representation of the setup of the isolated Langendorff rabbit heart. A defibrillation mesh patch electrode and contact rlectrode (used to induce VF and defibrillate) wrre placed on the lef ventncular rpicardium and right ventricular endocardium. respectiveiy. A monophasic action potential (MAP) catheter placed on the right ventricular endocardium was used to Pace the heart and record MAPs. Bipolar electrodes were placed on the right venticular and lefi ventricular epicardium for acquisition of bipolar epicardial signais. analysis was performed offline by randornizing the treatment phase of each parameter measured.

3. t .3-2. VF Induction and DFT Measurements

Ventricular fibrillation was induced by applying 3 to 5 seconds of 15 V rectified current at 60 Hz via the right ventricular endocardial electrode and the left ventricular epicardial patch. delivered with a custom-made DC fibrillator. A monophasic shock (3 msec pulse duration) was delivered. via the sarne electrodes involved in fibrillation. using an extemal defibrillator (HVS02. Ventritex Inc.. Sunnyvale. CA.) in order to defibrillate the heart following 10 sec of VF. Initial defibrillation shocks of 120 V and 70 V were used for baseline and treatment phases. respectively. If any shock failed to terminate VF, higher voltage shocks were delivered in 10 V increments and at IO second intervals until successful de fibrillation (Figure 9). If. however. a shoçk defibrillaird the heart. the first applied de fibrillation voltage for the next VF episode was decreased by 1O V.

Consecutive fibrillation and defibrillation episodes were separated by 3 minute intervals.

Elrctrocardiogram tracings from both the epicardial and endocardial electrodes. and the hemodynamic state were used as a means to identify the onset of VF and successful defibrillation. When 3 reversals in shock success occurred (failurr followed by success or vice-versa. where each success or failure was counted once). delivery of shocks was ceased. The de fibrillation threshold (DFT)was defined as the lowest voltage required to succrssfully terminate VF. and as such. was defined as the lowest successful shock of such a pair of revcnals. 120 V 130 V sinus I I rhythm

Figure 9. Deti brillation threshold (DFT) determination. Shocks were applied after 10 seconds of ventricular fibrillation (VF). in 10 V increments until successful de fibrillation. In this example I 20 V failed and 1 30 V was successful in terminating VF. In this example 130 V is determined to be the DFT. 2.1.3.3. Measurements of Ventricular Fibrillation Cvcle Length

Ventricular fibrillation cycle length (VFCL) was determined offline by analyzing archived VF episodes including the first defibrillation shock. at a paper speed of 50 mm/sec. The distances betwezn the lowest points of the rlectrocardiogram recorded from the right or lef ventricular epicardiurn dunng VF were manually measured with electronic calipers and reported in msec for the last IO deflections before the first de fibrillation shock for 3 DF episodrs (Figure 10). The same epicardial bipolar channsl was used to measure pre- and post-treatment episodes.

2.1.3.4. Measurements of Ventricular Effective Refractorv Period

The heart was initially paced from the left ventricular endocardial MAP catlirter at a cycle Iength of 400 msec with the Ventritex. using a pulse width of 1.0 msec at 5.0

V. To determine the pacing threshold. the pacing voltage was drcreased in 0.5 V steps

until capture was not consistent. at which point the voltage was increased by 0.1 V

increments to the nearest voltage which consistently captured the ventricles. If sinus

rhythm of the heart \ras too fast for pacing to capture. a mechanical atrioventricular nodal

ablation was performed to reduce the ventricular hcart rate. The Si-SIextrastimulus

technique was applied to determine die ventricular effective refractory period (VERP)

while pacing the heart at twice the diastolic threshold afrrr a 50 beat conditioning train

(Figure 1 1). VERP was defined as the longest interval between SIand Sz beats. where S?

did not capture. An S2 beat was introduced inio the normal pacing of the hart every

righth Si beat. begi~ingat an interval of 120 msec. and incremented in 10 msec strps

until S2 capture was observed. For a more accurate determination of VERP to the closrst

2 msec. the SI-Si interval vas decreased to Figure 10. Ventricular Fibrillation cycle length (VFCL) determination. The activation-activation intervals from the iowest points of the electrocardiogram recorded from the lefi or right ventricular epicardium were manually measured for the last 10 deflections before the fint defibrillation shock. Figure 1 1. Determination of ventricular effective refractory prriod (VERP). An S. beat was introduced into the normal pacing of the heart every eighth SI beat at twico the diastolic thrcshold. The Si stimulus rernained at 400 msec. Initially. the S2 stimulus was delivcred at a non-capture value and incremented until capture was observed. the nearest 10 msec that did not capture. and was subsequently prolonged in 2 msec increments until capture was observed once again. Measurements of pacing threshold and VERP determination were performed during baseline and treatment studiès. 15 minutes after DFT measurements.

2.1.3.5. Measurements of the Action Potential Duration

The heart was paced at a cycle length of 400 msec for a 50 beat train from the right ventricular endocardium with the MAP catheter. 5 minutes following measurements of VERP. Recorded MAPs were printed offline at a paper speed of 50 mdsec and the average MAP duration from beats 46 to 50 were manually measured. The asymptotic end of repolarization renders precise measurernents of the total hlAP duration difftcult. therefore the MAP duration was determined at a repolarization level of 90% (MAPDqo)

with respect to the MAP amplitude (see Figure 12). The MAP amplitude was defined as the distance from the diastolic baseline to the crest of the MAP plateau. not its upstroke

peak. The besinning of the MAP was defined as the instance of fastest rise time of the

MAP upstroke (maximum dV/dt) (Franz. 199 1).

2.1.3.6. Measurement of ORS Duration

The duration of the QRS compiex from the bipolar epicardial electrograms wîs

used as a surrogate measure of ventncular conduction velocity. Stored data was

displayed at a time scale of 100 rnm/sec. and electronic calipers were used to determine

the average of the duration of 3 successive QRS deflections at a cycle length of JO0

msec. Thrse measurements were made pre- and post- drug/saline administration. 1 \ Repolarizatkn

Figure 12. Mrasurements of monophasic action potential duration at 909% repolrirization (M..\PD9,i). The action potrntial amplitude was Jefincd as the crest of the MAP plateau. 2.2. Intact Guinea Pie Heart Ea~eriments

The twenty one male Hartley guinea pigs weighing between 0.9 and 1.1 kg (mean

0.99 k 0.07 kg) used in this study were housed in cages 40.6 cm wide. by 50.8 cm deep, by 2 1.6 cm high, with a 12 hour dayhight cycle, and room temperature was maintained at

23 OC. They were fed Guinea Pig Diet 7006 (Harlan Teklad. Indianapolis. M) and had water freely available to them. Al1 experiments were approved by the Animal Care

Committee of St. Michael's Hospital. and conformed to the guiding principles of the

Canadian Council on Animal Care.

2.2.1. Guinea Pi9 Preparation

Al1 animals were Fasted 12 hours prior to anesthetic induction. Thty were sedated with ketamine i.m. 25 mgkg (Ayerst Veterinary Laboratories. Guelph. ON). and kept on a 37°C water blanket (Micro Temp Pump. Cincinnati Sub-Zero. Cincinnati. OH).

Following sedation. the anterior neck. anterior oblique Irti and right chest walls. and limbs were shaved. Their necks were locally infiltrated with lidocaine (?y6. 0.75 cc sub.q.) (Astra Pharma. Mississauga. ON) ai the site of incision. The guinea pigs were anesthetized with sodium pentobarbital i.p.. 20 mgkg. and maintained in a surgical plane of anesthesia throiighout the study (5 rnbdgihr). The animals were resirained. exposing the neck area for surgery. Platinum needle electrodrs (Grass Inc. Los Angeles. CA) were placed in each of the limbs to obtain a surface ECG (Leads 1.11 and III). A Ag-AdCl reference electrode was placed in a muscle at the site of incision for stabilization of acquired cardiac electrical signais. A11 cardiac rlectricai signais u-rre amplified

(Cartesian Labs. Toronto. ON). filtered (low pass 500 Hz: high pass 0.05 Hz). sampled at a rate of 2000 Hz, and simultaneously displayed and recorded using a custom-made PC computer software program. ACQUI-2 (Cartesian Labs. Toronto. ON). Al1 recordings were archived on CD-ROM. and blinded data analpis [vas perforrned offline.

2.22 Surgical Methods

A 3 cm long incision was made 3 cm below the chin with a size 10 scalpel blade.

Tissue. muscle. and fat were dissected to expose the trachea. A tracheotomy was

performed by inserting a 14 gauge beveled angiocath (4 cm in length) in the trachea. The

lungs were ventilated with room air saturated with 90% Oz at 1 mL/100 g and 85

strokedmin via the canulated trachea with the use of a rodent ventilator (Harvard

Apparatus Canada. Saint Laurent. PQ). The lefi carotid artery and right jugular vein were

isolated by blunt dissection. and canulated with 20 and 16 gaugç angiocaths respectively

(4 cm in length). Blood pressure was monitored by connecting a pressure iransducer

(P23 1 D. Statham. Bionetics. Inc.. Toronto) to the angiocath placed in the lcfi carotid

artery. Systolic and diastoiic pressures were displayed and measured wi th a pressure

amplifier (Hewlett Packard. USA). -4custom-made 3 French quadripolar MAP Ag-

AgICI contact electrode catheter (NuMED. Inc.. N.Y.. U.S.A.) with a 20' bend. 1 cm

from the distal end. was inserted into the right jugular vein. and blindly placed in the right

ventricle. The catheter was guided by displaying electrical signals sensed from the distal

electrode. It was advancrd with the recognition of QRS detlrctions on the endocardial

electrogram. and placed on the right ventricular endocardium. The right jugular vein also

served as a means for i.v. hydration with saline (5 rnlihr). and administration of

anesthetic agents and antiarrhythmic drugs. Titaniurn mesh-patch electrodes were

sutured ont0 the anterior oblique aspect of the le fi and right chest walls for the delivery

DF shocks. 2.3. A Guinea Pie Model Assessine Defibrillation Threshold and Cardiac Refractoriness

2.3.1. Determination of Electroph~sioloeicalParameters

2.3.1.1. VF induction and DFT Measurements

Ventricular fibrillation was induced by apply in9 1 to Z seconds of l 5 V recti fied current at 60 Hz. using a custom-made DC fibrillator. via the two distal electrodes of the right ventncular endocardial MAP catheter. A biphasic shock (10.0 msec pulse duration) was delivered. via the percutaneous mesh patch elrctrodes placed on the chest using an extemal defibrillator ( HVSOL Ventritex Inc.. S unnyvale. CA.) in order to de fibrillate the

heart, following 1 5 seconds of VF. Initial DF shocks of 200 V were used for baseline and treatment phases. If any shock failed to teminate VF. a rescue shock of 30V

(biphasic. 10.0 msec pulse width) was applied. The purpose of this shock was to reinstate

sinus rhythm. and it was not used in the data analysis. When an applied DF shock failed.

the DF voltage was incremented by 20 V for the following VF episode. If. however. a

shock defibrillated the heart. the DF voltage applied for the next VF episode was

decreased by 10 V. Consecutive VF and DF episodes were separated by 2 minutes.

Electrocardiograrn tracings from both the 3 -lead ECG and endocardial electrodes were

used as a means to identify the onset of sustained VF. As electrocardiogram tracings

from both the epicardial and endocardial elrctrodes would not return to baseline

immediately afier an attempt at DF. hemodynamic statr was used as a means to identify

DF success. As in the isolated Langendorff rûbbit hean. when 3 revends in shock

success occurred (failure followed by success or vice-versa. whrre each success or failure

was counted once). delivery of shocks was ceased. The defibrillation threshold (DFT) was defined as the lowest voltage required to successfully terminate ventricular fibrillation. and as such. was defined as the lowest successful shock of such a pair of reversals.

2.3.1.2. Measurements of Ventricuiar Effective Refractory Period

After DFT determination. a period of IO minutes elapsed before ERP measurements were made. The heart was initially paced from the right ventricular endocardial MAP catheter at a cycle length of 250 msec with the Ventritex. usine a pulse width of 2.0 msec at 5.0 V. To determine the pacing threshold, the pacing voltage was decreased in 0.5 V steps until capture was not consistent. nt which point the voltage was incremented by 0.1 V steps to the nrcirest voltage which consistently captured the ventricles. This rate was chosen to ensure 1 : 1 capture for each stimulus delivered. since the sinus rate of the guinea pis heart was nonnally between 300 and 570 rnsec. and 2: 1 block was observed at cycle lengths beiow 130 msec. The SI-S2extrastimulus technique was applied to determine VERP while pacing the heart at twice diastolic threshold after a

50 beat conditioning train (as descnbed for the isolated Langendorff rabbit heart in

section 2.: .M)at each of the following cycle lengths (in msec): 343.220.200. 180. 160.

150. and I JO. An S2 beat was introducrd into the normal pacing of the heart rvery righth

Si beat. beginning at a non-capture coupling interval (e.g. the initial coupling intemal (S?)

was 1 1 O msec at a pacing train (Si)of ?JO msec). The coupling interval was increased at

10 msec increments until Sz capture was observed. For n more accurate determination of

VERP to the ciosest 2 msec. the Si-S- interval was decreased 10 msec to the 1st non-

capture interval. and was subsequently prolonged in 2 msec increments until capture was

observed once again. There was a one minute pause between VERP deteminations at each cycle length. and these measurements were made pre-and post-vehicle

administration.

2.4. A Guinea Pie Model as ses sin^ Cardiac Refractoriness: ERP and Dvnamic Restitution

2.3.1. Measurements of Ventricular Effective Refractory Period

Pacing threshold and ERP measurements were deterrnined as described in the

model of defibrillation threshold and cardiac refractoriness (section 2.3.). A more

extensive pacing protocol was employed in this particular modrl. as ERP was dctermined

using the SI-S2extrastimulus technique at the following cycle lengths (in msrc): 300.

280.260,240.220.200. 180. 160. 150 and 1JO msec. There was a one minute pause

between VERP determinations at each cycle length. and these rneasurements were made

pre-and post-vehicle administration.

3.42. Measurements of Activation-Recoverv Intemals and Dvnamic Restitution Kinetics

This pacing protocol was conducted [rom the right ventricular endocardium with

the MAP catheter, 5 minutes proceeding measurements of VERP. The heart was pacrd at

cycle lengths between 300 and 160 msec in 20 msec decrements. between 150 and the

'induced' rastest-follow frrquency (FFF,,) in IO msrc decrements. each for 50 beats. u-iih

a 30 sec pause between each pacing train. The fastest follow frequency (FFF) was

defined as the shonest cycie length of right ventricular endocardial pacing at twice

diastolic threshold which resulted in 1 :1 venvicular capture with continuous pacing. The

FFF,, was detemined by initially pacing the heart at the FFF for 15 beats. and

decrementing the pacing cycle length by 10 msec every 15 beats until the desired pacing

rate. The FFFin was therefore defined as the fastest pacing rate that did not result iri 2: 1 block or VF. In the event of induced VF. a defibrillating shock was delivered at a 300 V shock strength and 10.0 rnsec pulse width. if the heart did not spontaneously revert to sinus rhythm following 20 seconds of VF.

Unipolar MAPs were displayed at a paper speed of 250 mdsec. Activation-

recovery intervals (ARI) were measured as indices of local right ventricular

repolarization as the intervals frorn the mavimurn -dV/dt of the intracardiac QRS to the

mâu + or - dV/dt for inverted or upright T waves, respectively. This has been shown to

correlate with the APD (Gepstein er al.. 1997). However. at the kter cycle lengths

(including 150 msec to the FFF,,). the repolarizing phase of monophasic and biphasic T

waves were interrupted and extinguished by the paced beat which followed. Full

repolarization of the epicardium defines the peak of the T wave. and this was cleariy

discemable on the unipolar MAP signal at ail cycle lengths. The local repolarization

interval from maximum -dVldt of the intracardiac QRS to the peak of the T wave was

defined as the AN(ARlVkr). has been determined to be a surrogate to APD (Yan and

Antzelevitch. 1998. Anyukhovsky et al., 1997). and was therefore ernployed as a rneasure

of cardiac refractoriness in this thesis. The average AMFdT of the last 5 beats of the 50

beat train were manually measured with electronic calipers (Figure 13).

The relationship between AP duration (or in this case. ARIpcdT)and diastolic

interval (DI) descnbes the restitution relation. Each AMvdT was ploned versus its

cortesponding DI (defined as differencc: between pacing cycle length and ARI). ..\II

values were fit using a sigrnoidal Function (sre section 7.6.2.). Figure 13. Measurements of activation-recovery interval (AN) in intact guinea pig hearts. The local repolarization interval from maximum -dVldt of the intracardiac QRS to the peak of the T wave from the unipoiar right ventricular endocardial MAP signal was defrned as the AR1 (.4RIpcak 1. 2.4.3. Measurement of ORS Duration

The duration of the ORS complex from the Mead ECG (typically lead II or III. but the sarne lead used pre- and post-dm@treatment) was used as a surrogate measure of ventncular conduction velocity. Stored data was displayed at a paper speed of 100

rndsec. and electronic calipen were used to determine the average of the duration of 3

successive QRS deflections at a cycle length of 180 msec. These measurements werr:

made pre- and post- drug/vehicle administration.

2.5. Drue Administration

2.5 1. Isolated Langendorff Rabbit Hcart

After determining the percent changes in al1 electrophysiologic and hernodynamic

variables measured were not si gni ficanti y di fferent frorn zero betwren baseline and saline

treatment. animals were nndomly assignai KI each of the druy groups (dofrtilide. sotalol.

barium. combination dofetilide and barium. or combination sotalol and barium).

Measurements of DFT. VFCL, VERP. MAPDqo. QRS duration. heart rate. coron- flow

rate. and intracardiac lefi ventricular pressure were made at baseline and 20 minutes afier

saline or dmg administration. The solution of interest (saline or dmg dissolved in saline)

was administered to the top beaker of the Langendorff apparatus. and the veristaltic purnp

speed was increased 3 fold to hasten drug distribution over 20 minutes. Dmg distribution

and measurernent parameters were measured in an identical fashion in rach group.

Following each experiment, the hearts were removed from the Langendorff apparatus.

and the wet weight was noted. The time coune of al1 expet-iments was between 1 % and

1 % hows. 2.5.1.l. Drug Treatment Groups

2.S.l.l.l. Dofetilide, Sotalol, and Barium Grou~s

Variable doses of dofetilide were administered. and were chosen based on previous Langendorff rabbit heart experiments of the effect of do fet ilide on APD

(Friedrichs et ul.. 1994, Gillis et al.. 1998). QT interval (Barrett et al.. 200 1 ). and its propensity to induce TdP (D'Alonzo et ai.. 1999). The maximum effect of dofetilide on

DFT. VFCL. VERP. and MAPDqowas found at a dose of32 nM. This dose was successively decreased by half until there was a negligible effect on al1 electrophysiologiç parameters. similar to that of controls. The ECjo on VERP. and MAPDq0 was determined to be 8 nM. and was thus chosen as the dose for this study. A total of thirteen rabbit hearts were used for dofetilide treatmrnt. The following doses of dofetilide were administerrd (n=l ) to constnict a preliminary dose-response relationship for each of the electrophysiologic variables measured (in nM): 31. 16. 8.4. 2. .Aller determination of the

ECra on VERP. MAPDqo. and VFCL (see Results). dofetilide was administered to right rabbit he:u-ts at a dose of 8 nM.

The ELoconcentrations of sotalol on DFT and blAPDyoand of barium on DFT and VFCL were previously detrrmined to be J and 3pM respectively (Varma. 1000).

Eight rabbit hearts were also used in each of the sotalol and barium studies.

The stock solution for dofetilide was made by dissolving dofetilide in saline

(0.9% sodium chloride solution. Bavter Corporation. Toronto. ON) to give a final concentration of 25 PM. It was kepr away from light at 4°C. Sotalol and bariurn were made fresh before experimentation. also dissolved in saline. The volume of saline added to the perfusate in controls was i 5 mL. determined to be no larger than any of the volumes of the administered drugs.

2.5.1.1.2. Combination Dofetilide + Barium and Sotalol + Barium Groups

After establishing the concentration-response relationship of the r ffect of dofetilide on DFT, VFCL, VERP, and MAPDqo. combination Ikl and open state Ikr blockade was studied. Eight rabbit hearts were given 8 nM dofetilide and 3 FM barium

(the ECjo of each dru@ in combination. The effect of dmg combinations cm be modified by the concentration-effect relationship of each dmg. If a dmg is administered at a dose producing a close-to-maximal effect. any additional effects of a second dmg in combination would not be observed due to prior saturation of the etfect. Therefore. the aim of administering each of the dnigs at thrir ECrowas to linrarly resolvr additive effects of the drug cornbinations.

To assess the effects of Ikl blockade in combination with a potrntial Ikr closed-

state channel biocker. sotalol and barium were given in combination at doses of 4 pM

and 3 ph.1 (the ECjo of each dru@ respectively. to eight rabbit hearts. Identical

experirnental protocols were followed for the mrasurement of hemodynamic and

electrophysiologic parameters for the drug combination groups as with the control and

individual drug groups.

2.5.2. Intact Guinea Pig Heart

To assess the effectiveness of the nrwly established intact guiiica pig heart modrl

in testing antiarrhythrnic drugs on cardiac refractoriness. control experiments were

conducted. followed by preliminary electrophysiologic studies in the presence of

dofetilide and the chromanol HMR1556. Measurements of VERP. FFF,,. QRS duration, heart rate, systolic and diastolis pressures were made at baseline and 20 minutes after saline. dimethy lsulfoxide (DMS0)ipolyethy lene glycol 400 MW (PEGJOO)/saline combination, or drug administration. The solution of interest (saline or dmg dissolved in organic solvents brought up to 5 mL with saline). was administered i.v. over a 5 minute period. Following each experiment. the animals were heparinized, their hearts were surgically removed by blunt dissection, and weighed. The surgical procedure for al1 experiments was between 35 and 45 minutes in duration. and time course of the electrophysiologic studies was between 1 and 2% hours.

2.5.2.1. Control Group

Saline (0.9% sodium chloride injection, Bmter Corporation. Toronto. ON) was administered in IO guinea pigs. HMRI 556 is not water soluble. therefore varying amounts of DMSO/PEG4OO/saline were used to assess the maximum dose of combination organic solvents producing minimal electrophysiologic effects. As recornmcnded by the supplier (personal communication. Gerlach U. Hoescht Marion

Roussel. Germany). a ratio of 2 : 1 of PEGJ0O:DSMO was necessary to dissolve

HMRl556. Therefore. 0.6 mL PEGJOO + 0.2 mL DMSO brought up to 5 mL with saline

was originally assessed. and successively halved. until no elrctrophysiologic effects were

observed in the modd.

2.5.2.2. Drue Treatment Group

2.52.2.1. Dofetilide Group

As no previous experiments of the rlrctrophysiologic efkcts of do fetilide have

been conducted in guinea pigs in vivo, a pilot dose of 8 pg/kg wvas selected based on

Langendorff guinea pig heart studies assessing epicardial APDs (Williams et al.. 1999). propensity to TdP in the intact rabbit heart (Lu et al.. 2000, Brooks et ai.. 2000). in vivo dog studies of ventricular function (Gout et al.. 1992). and in vivo pig studies of pacing- and ischemia-induced VF (D' Alonzo er al.. 1994, Andersen et al.. 1994). A dose of 4 pgkg was also administered to ensure at ieast one of these doses did not lie at a saturation of effect of the concentration-effect relation. The original dofetilide stock solution described in the Langendorff rabbit heart experiments was also used in this model.

7.5.2.2.2. HMRI 556 Group

In published material. HMR1556 has solely been used in isolated Langendorff guinea pig hearts. These results were used as a guide in selecting a dose of 1 .j mgikg

HMRljj6 in this intact guinea pig heart model (Gogelein et tri.. 2000). Each dose was initially dissolved in 0.05 mL DMSO. diluted in 0.6 mL polyethylcne glycol 400

(PEGJOO), and brought up to a final volume of 5 mL with saline (0.9% saline solution).

A fresh dmg solution was made 2 hours prior to administration and kept at room temperature.

2.5.2.2.3. Combination Dofetilide and HMR1556 Group

The doses which produced between 10 and 15% prolongation in right ventricular

ERP were thought to be close to the EGO. as the maximal effect seen with Ik blockers has been reported to be no greater than 2096. These doses are thought to lie in the linrar portion of the dose-response curve for each dmg. Pilot studies suggested that these wrre

1.5 mgkg HMR1556 and 4 pgkg dofetilide. These were given in combination. each

dissolved in its appropriate solution(s) to a final volume of 2.5 mL. respectively. 2.6. Statistical Analvsis

2.6.1. Isolated Langendorff Rabbit Heart

Differences in DFT, VFCL. VERP. MAPDqO.QRS duration. and hemodynarnic parameters in the control group were determined by cornparhg mean percent changes of each parameter pre- and post-saline with a one-sample t-test analysis. Changes in this group were not found to be significantly different than zero.

Linear dose-response relationships (see Figures I-l to 17) of the mean percent change in MAPDgO.VERP. DFT. and VFCL with dofetilide were plotted. and were each fit to a hyperbolic curve. The drug doses employed in the study of combination antiarrhythmic drug therapy were found to be in the linear portion of the cunes.

The relationship between each dofetilide dose and its respective r ffect on

MAPDW,.VERP. DFT. and VFCL were detetmined by plotting the mean percent change of rach variable from their respective baselinr values. Al1 data was fined to the following non-linear regession one-site binding hyperbolic function:

Where: Y = the drug effect

[XI = the drug concentration

E,,,,, = the maximum dnig effect

ECso = the drug concentration required to reach half-maximal effect

Mean baseline values of al1 hemodynarnic and electrophysiological parameters measured in al1 groups (including saline and drug groups) were compared with a one-way ANOVA. The mean percent change in each variable from baseline in each drug group

(dofetilide, sotalol. barium. combination dofetilide + barium, and combination sotalol + bxiurn) was compared to the mean percent change from baseline in the control group

with a one-way ANOVA. Differences brtween groups were further resolved with a

Bonferonni post-hoc analysis.

Results are presented as mean + standard deviation (SD) in text. and 2s mean k

standard rrror of the mean (SEM) in figures. Values of EC jil and E,, of the pre1imina-y

dofetilide dose-response relationship are reported with their standard error. determined by

computer extrapolation of the curve with the software program GraphPad Prism (version

3.0. GraphPad Software Inc.. San Diego. CA). This program was alsu used to determine

the goodnrss of fit of each concentration-rffect relationship. reportrd with an R~ valus.

and Sy.x values representative of the standard deviation of the vertical distances of the

data points from the fitted curve. For al1 ANOVA and post-hoc analyses. p

considered to be statistically significant.

2.6.2. Intact Guinea Pig Heart

Differences in DFT. VFC L. VERP. ARIPcakr. QRS duration. and hemodynmic

parameters in the control group were drtermined by cornparhg mean percent changes of

rach parameter pre- and post-saline with a one-sample t-test analysis.

The relationship between DI and ARIpcATfor each phase of rach experiment

(baseline and treatrnent/vehicle) was determined by plotting the .4RIVAr versus DI. and

fitted to the following non-linear regression: Where: ARIPkT = the peak activation recovery interval

DI = the diastolic interval corresponding to each .4MpeakT.defined as:

DI, = PCI, - APD,

Where: PCI, = the pacing cycle length intend of the nth beat

APD, = the action potential duration of the nthbeat where:

Dlbiiy = the DI resulting in the shonest ARIWaAr

DI= the DI resulting in the largest ARlpe&r

Dko = the diastolic interval at which ANpcakis halfway between DIhixLyand

DIvm.

Slope = the sterpness of the curve. with a larger value denoting a shallow

curve. and a smaller value denoting a steeper curve.

This sigmoid function has been found to fit experimental restitution data with R' coefficients > 0.98 (Koller rt al.. 1998), and has been found to be superior to previously descrîbed mono- and biexponential fits (Elharrarr et al.. 1984. Elharrar and Surawicz.

1983. Saitoh et al.. 1989).

Ail cuves were fined to this equation with the software program GraphPad Pnsm

(version 3.0. GraphPad Software Inc.. San Diego. CA). The program was used to determine the goodness of fit of each concentration-effect relationship. reponed with an R~ value, and Sy.x values, as well as the steepest slopes of each curve fit. For al1 t-test analyses. p40.05 was considered to be statistically significant. Results are presented as mean k SD in text. and as mean t SEM in figures. 3. RESULTS

3.1. Isolated Laaeendorff Rabbit Heart Mode1

3.1 .1 . Control Experiments

Control experiments involved the administration of saline to the Langendorff rabbit heart to determine the effect of time and the addition of saline (used as a vehicle for the drug studies) on the isolated heart preparation. Particularly. controls were run to ensure the hemodynarnic (coronary flow and left ventricular pressure) and electrophysiologic (DFT. VFCL, VERP. MAPDgOand QRS duration) stability of the

Langendorff heart model. Thus in further experiments the effects of Ikr and Ik 1 blockade could be assessed by anributing any changes in these parameters to the drugs

administered.

3.1.1.1. Electrophvsiolo~icalMeasurements

Mean baseIine and saline values of mesurements of DFT. VFCL. VERP and

MAPDgo are listed in Table I A. A paired t-test analysis of the cight controls indicatrd

that there was no siynificant differencr bctween the mean basdine and mean saline

values of DFT. VFCL. VERP. and MAPDqo+This showrd the stability of this mode1 over

time. and with the addition of 5 mL of saline. The mean percent change from baseline in

DFT. VFCL. VERP. and MAPDq0 was also found not to be different frorn zero. DFT

increased by 0.3 2 5 -4% (from 1 12 i 16 V at baseline to 1 1 -!k 17 V with saline) (p = ns).

VFCL decreased 1.8 t 6.6 % (fiom 85 k 6 msec at baseline to 84 2 7 msec with saline) (p

= ns): VERP at a cycle length of 400 msec decreased 1.1 t 3.3 % (from 150 2 20 msec at

baseline to 149 k 1 9 rnsec with saline) ( p = ns). and MAPDqOincreased 0.5 2 3.6 ?/o ( from

193 k 13 msec at baseline to 194 k 16 msec with saline) (p = ns). 3.1.1.2. Measwements of Cardiac Function and Other Measurements

Mean baseline and saline values of lefi ventricular endocardial systolic and diastolic pressures. coronary flow rate. and heart rate for the control experimcnts are shown in Table 1 B. The mean baseline and saline values of the diastolic and systolic pressures and heart rate were not significantly different from one another. The mean percent change in systolic pressure. diastolic pressure. coronary tlow. and hem rate from baseline to saline treatment were variable. yet not different from zero. Lrfi ventricular endocardial systolic pressure increased slightly by 8.5 f 19.2% (from 20 + 4 mmHg at baseline to 23 2 7 mmHg post saline) (p = ns). Le fi ventricular diastolic pressure increased by 4.4 2 18.8% (from 5 + O mmHg at baseline to 5 2 1 mmHg aftrr saline) (p = ns). Coronary tlow rate did not change. and remained at 2 1 2 7 mLlmin at baseline and afier saline (p = ns). Hem rate decreased by 7.6 2 1 8.0% ( tiom 1 2 1 f 23 bpm at baseline to 121 t 15 bpm post saline) (p = ns).

There was no difference in the mean absolute value of QRS duration between the baseline study and saline treatment of the control group (see Table I A). The QRS duration increased 3.7 i 4.9% (from 60 2 8 rnsec at baseline to 62 k 9 msec with saline).

3.1.2. Dofetilide Administration in Isolated Rabbit Hearts

3.1 2.1. Effect of Varving Dofetilide Concentration on Electror>hvsiolo~& Measurements

3.1 2.1.1. Monophasic Action Potential Duration at 90% Repolarization

The duration of the MAP was determineci From signals recorded while pacing the isolated rabbit heart at a cycle length of 400 mwc from the right ventricular endocardium with the use of an MAP catheter at baseline. and in the presence of dofetilide (n=I for each do fetilide concentration). There was no signi ficant di fference in mean baseline

MAPDqobetween the control and al1 pooled dofetilide groups (p = ns). Figure 14 displays the dose-response relationship of percent change in MAPDqofrom basrlinr

versus dofetilide concentration with a linear dose scale. The R' and Syx values of the

non-linear regression hyperbolic fit are 0.9776. and 2.7 %. respectively. Each of 2.4. 8.

16. and 32 nM dofetilide produced increases in MAPD90 of 4.2%. 7.0%. 16.106. 2 I .J%.

and 19.0% respectively. A statistical analysis could not be performed as an n=l per

dofetilide concentration was used for determination of the EGO. Quantitativrly. maximal

reduction in MAPDgowas attained at the three hiphrst concentrations studied (8. 16. and

32 IN).Cornputer extrapolation of the E,, and ECso of dofetilide from the hyperbolic

curve yielded values of 26.4 2 4.8%. and 7.2 k 3.6 nM. respectively. [Dofetilide] (nM)

Figure 14. Dose-response curve with a linear dose scale for the effrct of dofetilide on the percent change from baseline (pre-dofetilide) in monophasic action potential duration at 90% repolarization (MAPDqo)at 400 msec pacing cycle length. The non-linear hyperbolic regression fit yielded an R' value of 0.9276 and an Sy.x of 2.7 %. Computer calculation of the E,, yielded a value of 26.4 + 4.8 % and an ECso of72 + 3.6 nM. Dofetilide dose- dependently reduced MAPDqo. n= 1 for rach dose. 3.1.2.1 2. Ventricular Effective Refractory Period

The SI-S2extrastimulus technique was used to determine VERP at a paced cycle length of 400 msec fiom the right ventricular endocardium in the presence of increasing dofetilide concentrations (n=l). There was no significant difference in mean badine

VERP between the control and al1 pooled dofetilide groups (p=ns). Figure 15 shows the hyperbolic fit the of the dose-response relationship of percent change in VERP from baseline with dofetilide. The non-linear regression hyperbolic fit has an R' value of

0.9646. and an Sy.x of 1.8%. Each of 3.4. 8. 16. and 53 nM dofetilide produced

increases in VERP of ?A0'. 5.8%. 14.3?/0. 1 5.8?/,. and 2 1.7% respectively .

Quantitatively. maximal reduction in VERP was attained at the highest concentration

studied (32 nM). Cornputer extrapolation of the E,, and EC so of dofetilide from the

hyperboiic curvr yictlded values of 30.7 2 5.2% and 13.4 k 5.0 nM. resprctivriy. [Dofet ilide] (nM)

Figure 15. Dose-response curve with a linear dose scale for the effect of dofetilide on the percent change From base1 ine (pre-dofetil ide) in ventricular effective refiactory period (VERP) at 400 msec pacing cycle length. The non-linear hyperbolic regcession fit yielded an R' value of 0.96-16 and an Sy.x of 1.8 %. Computer calculation of the E,, yielded a value of 30.7 2 5.1 ?/o and an EC jo of 13 .l k 5.0 nM. Dofeetilide dose-dependently reduced VERP. n=l for each dose. 3.1 2.1 3. Ventricular Fibrillation Cvcle Length

VFCL was measured as an average of the last 10 activation-activation intervals of

3 randomly chosen VF episodes before the first defibnllating shock pre and post-drug treatrnent at each concentration of dofetilide (n=l). There was no significant difference in mean baseline VFCL between control and al1 dofetilide groups (p=ns). Figure 16 displays the dose-response relationship of percent change in VFCL from baseline versus dofetilide concentration with a linear dose scale. The non-linear regression hyperbolic fit has an R' value of0.93 14. and an Sy.x of 3.7 %. Each of 7.4. 8. 16. and 37 nM dofetilide concentrations produced increasrs in VFCL of 4.0%. 12.8%. 246%. 22.4%. and 32.9% respectively. A statistical analysir could not be perfotmed sincr each dose concentration was tested only once. Quantitatively, maximal reduction in VFCL was attained at the three highest concentrations studied (8, 16. and 32 nM). Cornputer extrapolation of the E,, and ECroof dofetilide from the hyperbolic curve yielded values of 40.6 k 7.6% and 8.7 ? 4.2 nM. respectively. [Dofet ilide] ( nM)

Figure 16. Dose-response curve with a linear dose scale for the effect of dofetilidr on the percent change from baseline (pre-dofetilide) in ventncular fibrillation cycle length (VFCL). The non-linear hyperbolic regession fit yielded an R' value of 0.93 14 and an Sy.x of 3.7 ?6. Cornputer calculation of the E,, E,, yielded a value of 10.6 + 7.6 % and an ECroof 8.7 + 4.2 nM. Dofetilide dose-deprndently reduced VFCL. n= 1 for each dose. 3-121.4, Defibrillation Threshold

Defibrillation was measured in triplicate. both at baseline and post-drug administration. There was no significant difference in mean baseline DFT between the control and al1 dofetilide groups. Figure 17 shows the dose-response relationship of dofetilide on percent change in DFT from baseline with a linear dose scale. The non- linear regression hypçrbolic fit has an R' value of 0.8753, and an Sy.x of 6.7%. Each of

2.4. 8. 16, and 32 nM dofetilide produced reductions in DFT of 6.3%. 13.8%. 12.3%.

4 1 .O%. and 38.4% respectively. As the purpose of this dose response study was to determine the ECg, of dofetilide in this model. one animal per dose was used and a statistical anûlysis could not be performed. Quantitatively. maximal reduction in DFT was attained at the two highest concentrations studied (16 and 32 nM). Cornputer extrapolation of the E,, and ECSoof dofetilide from the hyprrbolic cunreyirldrd values of -63.8 k 24.4%and 16.7 k 13.1 nM. respectively. % Change in DFT from Baseline 3.1.2.2. Effect of 8 nM Dofetilide on Electrophysiologic Measurements

Dofetilide was administered to isolated rabbit hearts. A dose of 8 nM dofetilide from preliminary dose-response experirnents was detemined to lie in the linear segment of the dose-response curve. and was determined to be in close approximation to the ECsi1 values on al1 electrophysiologic variables. The mean baseline and treatment (saline and dotètilide) values on measurements of DFT. VFCL, VERP and MAPDgOare listed in

Table II A. No significant difference was found between the mean baseline values of the saline and dofetilide groups with respect to DFT, VFCL. VERP. and MAPDqo(p = ns).

However. after dotètilide administration. DFT decreased by 14.3 f 4.8% ( from 118 k 25

V at base1 ine to 1 09 2 22 V) (p

17.8% (from 10 1 t 1 3 msec at baseline to 126 2 2 1 rnsec) (p< 0.0 1 versus control)

In the absence of the drug combination dofetilide and barium. a highly disorganized and high-frequency pattern of electrical activation was evident on the bipolar epicardial electrogram during VF. Figure 18 shows that afier adrninistering these dmgs in combination. there was a more organized and slower-frequency pattern of VF: the VERP at a cycle length of 400 msec increased by 9.9 ?r 5.6 ( tiom 157 t 19 msrc at baseline to 172 fi 18 mec) (p< 0.0 1 versus control) and the MAPDquincreased 12.7 k

5.0% (from 210 f 14 msec at baseline to 237 I19 msec) (p< 0.01 venus control).

3.1 .U. Effect of Dofetilide on Cardiac Function and Other Measurernents

Table II B shows the mean basdine and treatment values of the lefi ventricular mdocardial systolic and diastolic pressures. coronary Row rate. and hem rate for the control and dofetilide experiments. The mean baseline values of the saline and dofetilide groups of diastolic and systolic pressures and heart rate were not significantly different fkorn one another (p = ns). Afier dofetilide administration. decreases in Iefi ventricular endocardial systolic pressure. coronary flow rate, and heart rate were observed. These decreased by I .l f 36.7% (fiom 18 k 7 mmHg at baselii~eto 17 2 5 rnrnHg) (p = ns versus control), 1.2 2 16.2% (fiom 12 + 6 mL/min at baseline to 22 k 7 mL/min) (p = ns). and 12.7 ?: 2 l .O% (from 1 14 + 23 bpm at baseline to 98 f 24 bpm) (p = ns). respectively.

An increase of 3.5 + 13.1% in left ventricuhr diastolic pressure was observed (from 5 5 1 mmHg at baseline to 5 +- I mmHg) (p = ns).

There was no difference in the mean absolutc value of QRS duration between baseline measurements in the control and dofetilide groups (see Table II A). Although not statistically significant. the QRS duration increased by 3.7 t 4.9% with dofetilide

(from 62 t 7 rnsec at baseline to 64 k 7 mscc) (p = ns).

Dofetilide

Sotalol

Barium

Dofetilide + 8arium Sotalol +

Figure 18. Bipolar electroçrarns recorded Born the lefi ventricular epicardium during fibrillation after treatment with saline or drug(s). Note the more organized. and slower-frequency pattern of VF in the presence of combination dofetilide and barium. compared to al1 other groups. In al1 cases. there was complete loss of lefi venvicular endocardial pressure. 3.1.3. Sotalol Administration in Isolated Rabbit Hearts

The EC jo of sotalol on DFT and MAPDqo in isolated Langendorff rabbit hearts was determined to be 4 pM (Varma 2000). Therefore. this dose was chosen to study the electrophysiologic effects of lkr blockade on eiyht rabbit hearts.

3.1.3.1. Effect of Sotalol on ElectrophvsioIogic Measurements

Mean baseline and study (saline and sotalol) values on measurements of DFT.

VFCL. VERP and MAPD90 are show in Table III A. A cornparison of the mean baseline values of the saline and sotalol groups with respect to DFT. VFCL. VEW. and

MAPDqOshowed no signifiant difference between groups (p = ns). After sotalol administration. DFT decreased by 12.7 2 2.4% (from 174 5 9 V at basrline to 108 i 8 V)

(p< 0.01 versus control). VFCL. VERP and MAPDq(iat a cycle Irngh of 400 msec rach increased by 27.5 k 9.7% (from 92 2 8 msec at baseline to 1 17 I 13 msec) (p< 0.0 I versus control). 8.3 k 1.2 O/O (from 154 + 22 rnstlc at baseline to 167 k 22 msec) (p< 0.01 versus control). and 7.6 + 5.5% (from 190 f 26 msec ût baseline to 205 f 57 msec) (p<

0.0 1 versus control). respectively.

3.1 X.Effect of Sotalol on Cardiac Function and Other Measurements

Table III B shows the mean baseline and treatment values of lefi ventricular endocardial systolic and diastolic pressures. coronary tlow rate. ruid hran rate for the control and sotaiol expenments. The mean baseline values of the saline and sotalol groups of diastolic and systolic pressures and heart rate were not significantly different from one another (p = ns). After sotalol administration. increases in left ventricular endocardial systolic pressure and lefi ventricular diastolic pressure. were observed to be

10.5 I24.7% (fiom 17 i 3 mmHg at baseline to 19 t 4 mmHg) (p = ns versus control), and 9.6 f 34.0% (from 5 ? 1 mmHg at baseline to 5 +- 1 mmHp) (p = ns), respectively.

Coronary flow rate and heart rate decreased by 1.2 2 16.2% (from 22 ?r 6 mLimin at baseline to 22 f 7 mllmin) (p = ns) and 2 1.2 t 12.7% (from 123 f 19 bpm ai basrline to

119 k 22 bpm) (p = ns), respectively.

There was no difference in the mean absolute value of QRS duration between baseline measurenients in the control and sotalol groups (see Table 111 A). .Althou& not statistically significant. the QRS duration increased by 0.4 f 2.4% with sotalol (from 58 k

6 msec at baseline to 58 f 6 msec) (p = ns).

3.1.4. Barium Administration in Isolated Rabbit Wearts

In previous studies, Varma found 3 FM barium to have half-maximal effect on

DFT and VFCL in isolated rabbit hearts (Varrna, 2000). Therefore. this dose was chosen to study the electrophysiologic effects of Ik 1 blockade on right rabbit heans in this study.

3.1.4 1. E ffect of Barium on Electrophvsiolo~icMeasurements

Mean baseline and study (saline and barium) values on measurements of DFT.

VFCL. VERP and M.4PDg0 are listed in Table IV A. There was no significant di fference between the mean baseline values of the saline and barium groups with respect to DFT.

VFCL. VERP. and MAPDgO(p = ns). After barium administration. DFT decreased by

13.9 t 5.0% (from 1 18 k 22 V at baseline to 102 + 22 V) (pi 0.01 venus control): VFCL increased by 25.4 i 15.8% (Irom 95 f I 1 msec at baseline to 1 17 I11 msec) (pc 0.0 1 versus control); VERP at a cycle iength of 400 msec increased by 6.6 + 3.3% (from 153 f

24 msec at baseline to 163 f 25 msec) (pc 0.0 1 versus control); MAPD9o increased 9.4 t

1.9% (from 187 + 12 msec at badine to 204 +_ 25 msec) (p< 0.0 1 versus control).

3.1.4.2. Effect of Bariurn on Cardiac Function and Other Measurements

Mean baseline and treament values of left venuicular endocardial systolic and diastolic pressures, coronary flow rate. and heart rate for the control and barium experiments are indicated in Table IV B. The mean baseline values of the saline and barium groups of diastolic and systolic pressures and kart rate were not significantly different from one another (p = ns). Afier bariurn administration. lefi ventricular endocardial systolic pressure increased by 8.4 f 43.9% (from 20 f 7 mmHg at baseline to

20 t 7 mmHg) (p = ns versus control). Lefi ventrîcular diastolic pressure decreased by 1 1.5 t 2 1.3% (from 5 t 1 mmHg at baseline to 5 + 1 mmHg) (p = ns). Coronary flow rate decreased by 5.6 I1 1.9% (From 12 t 7 mL/min at baselin? to 21 f 7 mL/min) (p = ns). Hem rate decreased by 8.8 + 13.8% ( from 102 + 2 1 bpm at baseline to 93 + 1 3 bpm) (p = ns).

There was no difference in the mean absolute value of QRS duration betwern baseline measurements in the control and barium groups (see Table IV A). Although not statistically significant. the QRS duration increased by 2.2 + 40Y0with barium (frorn 59 f 5 msec at baseline to 60 f 5 msec) (p = ns).

3.1.5. Combination Dofetilide and Barium Experiments In Isolated Rabbit Hearts

Bariurn and dofetilide were given in combination, each at 3 pM and 8 nM respectively. to assess the cardiac eiectrophysiologic effects of simultaneous Ikl and Ikr bloc kade.

3.1 S.1. Effect of the Combination of Dofetilide and Barium on Electrophysioloeic Measurements

Mean baseline and study (saline and combination dofetilide and barium) values on msasurements of DFT. VFCL. VERP and MAPDtlo rire listed in Table V A. Thert.

was no significant difference between the mean baseline values of the saline and

combination dofetilide and barium groups with respect to DFT. VFCL. VERP. and

?vfAPD90(p = ns). Afirr administration of combination dofetilidè and bariurn. DFT

decreased by 3 1 -8 + 1 X?G ( from 1O7 t 1 5 V at baseline to 73 k 14 V) (p< 0.0 1 versus

control. pxO.0 1); VFCL increased by 5 1.4 f 19.8% ( from 96 + 13 msec at baseline to 145

2 24 msec) (pc 0.0 1 versus control): VERP at a cycle length of 400 rnsec increased by

23.7 k 4.0% (from 153 k 18 msec at baseline to 189 f 17 msec) (p<0.0 1 versus control):

MAPDqo increased 20.1 f 5.O0h(from 203 + 20 rnsec at baseline to 243 f II msec)

(p4.01 versus control). Furthemore. al1 electrophysiologic measurements of this group

were found to be si gni ficantl y di fferent from al1 other dmg groups (p<0.01 ).

3.1 .j.I Effect of Combination Dofetilide and Barium on Cardiac Function and Other Measurements

Mean baseline and treatrnent values of lefi ventricular endocardial systolic and

diastolic pressures. coronary flow rate. and heart rate for the contml and combination

dofetilide and barium experiments are indicated in Table V B. The mean baseline values

of the saline and combination dofetilide and barium groups of diastolic and systolic pressures and heart rate were not significantly different fiorn one another (p = ns). After combination dofetilide and baium administration. left ventricular endocardial systolic pressure decreased by 6.8 t 27.0% (from 20 + 8 mmHg at baseline to 18 k 5 mmHg) (p = ns versus control). Lefi ventncular diastolic pressure decreased by 6.7 + 12.8% (from 6 2

1 mmHg at baseline to 5 f 1 mmHg) (p = ns). Coronary tlow rate decreased by 5.2 k

10.7% (from 20 i 3 mL/min at baseline to 19 f 5 mL/min) (p = ns). Heart rate decreased by 18.7 + 12.7% (from 108 2 27 bprn at baseline to 98 f 14 bpm) (p = ns).

There was no difference in the mran absolute value of QRS duration between baseline measurements in the control and combination dofetilide and barium groups (see

Table V A). Although not statistically significant. the QRS duration increased by 1 .O 2

5.0% with combination dofetilide and barium (from 59 t 5 msec at baseline to 59 + 4 msec) (p = ns).

3.1.6. Combination Sotalol and Barium Experiments

3.1.6.1. Effect of Combination Sotalol and Barium on Electro~hvsiolog;ic Measurements

Mean baseline m-d study (saline and combination sotalol and barium) values on measurements of DFT, VFCL, VERP and MAPDqOare listed in Table VI A. There was no significant difference between the mean baseline values of the saline and combination sotalol and barium groups with respect to DFT. VFCL. VERP. and MAPD~U(p = ns).

Afier administration of combinarion sotalol and barium. DFT decreased by 12.8 t 1 1 .J%

(from 1 12 f 17 V at baseline to 98 k 19 V) (p<0.01 versus control): VFCL increased by

27.4 t 19.0% (from 1O6 + 19 msec at baseline to 134 f 3 1 msec) (p<00 1 versus control):

VERP at a cycle length of 400 rnsec increased by 8.2 + 3.9% ( from I 59 f. 1 1 msec at badine to 172 t 1 1 msec) (p<0.0 1 versus control): MAPDqOincreased 6.0 + 7.1 % ( from

200 + 2 1 msec at baseline to 2 12 k 23 msec) (pxO.0 1 versus control). In addition. there was no significant difference betwcen the mean effect on each of the electrophysiologic parameten of the sotalol and barium combination group and any other monotherapy dmg group (p = ns).

3.1.6.2. Effect of Combination Sotalot and Barium on Cardiac Function and Other Measurements

Mean baseline and treatment values of le ft ventricular endocardial systolic and diastolic pressures. coron. tlow rate. and hart rate for the control and combination sotalol and bariurn experiments are indicated in Table VI B. The mean baseline values of the saline and ccmbination sotalol and barium groups of diastolic and systolic pressures and heart rate were not significantly different fiom one another (p = ns). After combination sotalol and barium administration, left ventricular endocardial systolic pressure decreased by 1.6 k 32.0% (from 17 f 3 mmHg at baseline to 17 f 8 mmHg) (p = ns versus control). Left ventricular diastolic pressure decreased by 10.8 ? 12.7% (from 6

? O mmHg at baseline to 5 k 1 mmHg) ( p = ns). Coronary tlow rate decreased by 8.0 ?

1 1.6% ( from 1 8 2 2 mL/min at baseline to 16 k 3 mUmin) (p = ns). Hem rate decreased by 17.3 + 32.1% (fiom 1 10 f 3 1 bpm at baseline to 83 + 12 bpm) (p = ns).

There was no difference in the mean absolute value of QRS duration between baseline measurements in the control and combination sotalol and barium groups (see

Table VI A). Although not statistically signifiant, the QRS duration increased by 3.7 +

4.9% tvith combination sotalol and barium (from 60 + 4 msec at baseline to 6 1 k 2 msec)

(p = ns).

3.1.7. Summary of Langendorff Rabbit Heart Experiments

3.1.7.1. Drug effects on DFT and VFCL

Comparing the baseline measurements of the 6 groups showed that there were no statistically significant differences in DFT or VFCL (p = ns), or in changes from baseline to saline treatment (0I5% and -2+7% respectively. p = ns). The mean baseline and post treatment values for DFT and VFCL are listed in Table VI1 for each group. Individual measurements are graphically represented in Figure 19. Significant. and similar decreases in DFT and increases in VFCL were observed afier administration of dofetilide

(-14t5% and 252 18% respectively). sotalol (-1 322% and 27*10%). and barium (-1M% and 75k 16%) compared to control (ail p<0.01). Barium and dofetilide in combination

(Figure 20) had significantly greatrr cffects on DFT and VFCL than cither drug alone (-

32+ 1 5% and 52230% respective1y. pcO.0 I ). whereas the combination of barium and sotalol . although significantly different from control (px0.01). had no greater effect than each dnig alone (- 1 jk 1 1% and 27+ 19% respectively. p = ns).

3.1.7.1. Dm- effects on VERP and MAPDw-

There were no statistically significant differences in VERP or MAPDqObetween the 6 treatrnent groups at baseline (p = ns). or in changes from baseline to control

(vehicle) treatment ( - 1 +2% and Of4 resprctively) (p = ns). The mean baselinr and post treatment values for VERP and MAPDqo are listed in Table VI1 for each group.

Individual measurements are graphically represented in Figure 19. Significant. and similar. increases in VERP and MAPDq0were observed after administration of dofetilide

( 10+6% and 13Whrespectively). sotalol(8S% and 8=6Y0). and barium (7t3% and

9S%)compared to control (al1 p

20).

Figure 19. Individual measurernents of MAPDqo (A). VERP (B). VFCL (C).and DFT (D) in isoiated Langendorfhbbit hearts. Horizontal bars denotr the mean. Baseline measurements from al1 groups were pooled. Dof (A)= 8 nM dofetilide: Sot (a) = 4 pM sotalol; Bar (e) = 3 FM barium; Dof+Bar (A) = combination 8 nM dofetilide and 3 FM bariurn: SotiBar (O) = combination J pM sotalol and 3 FM barium. n=8 per group. Figure 20. Mean percent changes k SEM in MAPDgo(A), VERP (B), VFCL (C). and DFT (D) from baseline to administration of 8 nM dofetilide (Dof). 4 pM sotalol (Sot). 3 barium (Bar). combination 8 nM dofetilide and 3 pM barium (Dof+Bar). and combination 4 PM sotalol and 3 FM barium (Sot+Bar). for each measurement respective1y in isolated rabbit hearts (II=@. tp<0.0 1 versus control '+p<~.~~versus al1 other groups 3.2. Establishinp an Intact Guinea Pig Heart Model

3.2. 1 . Control Exneriments

Control experiments involved the i.v. administration of saline to the intact guinea pig hem in order to determine the efict of time and the addition of saline (used as a vehicle for the drug studies) on this experimentai preparation. Controls were run (n=10) to ensure the stability of this model on hemodynamic measurements (systolic and diastolic blood pressure) and rlectrophysiologic measurements (including DFT. VFCL.

VERP. ARIPar. and QRS duration). Thus. the effects of antiarrhythmic agents on electrophysiologic and hemodynarnic parameters could be assessed by attributing any changes in measurements to the dmgs administered. In order to verify the validity of the model in assessing class III cffects of antiarrliythmic agents. the elrctrophysioiogic effects of dofetilide (8 and 4 pg!kg) were measured. This model was also established in an attempt to explore the rate-dependent and reverse rate-dependent nature of antiarrhythrnic dmgs. both novel and traditional. alone. and in combination. Therrfore. preliminary experirnents with the administration of dofetilide (traditional drug of choice) and HMRI 556 (novel dnig of choice) in combination were conducted.

3.3. A Guinea Pie Model as ses sin^, Defibrillation Threshold and Cardiac Refractoriness

3.3.1. Saline Administration

Mean baseline and saline values on measurements of DFT. VFCL, and QRS duration are listed in Table VI11 Al and measurements of VERP at multiple cycle pacing ienhgths are listed in Table VI11 B. A paired t-test analysis of the ten controls indicated that there was no significant difference between the mean baseline and mean saiine values of DFT, VFCL. and QRS duration (Figure 21), as well as VERP at multiple heart rates (Figure 22). This showed the stability of the model over time. and with the addition of 5 mL of saline. The mean percent change from baseline of al1 electrophysioiogic parameters was also found not to be different from zero: DFT decreased by 0.3 k 6.0 %

(from 189 t 19 V at baseline to 189 + 22 V with saline) (p = ns). and VFCL decreased

1.8 2 5.6 % (from 67 + 6 msec at baseline to 66 I9msec with saline) (p = ns). The right

VERP was measured at 7 different cycle lengths. and was found to decrease as cycle length decreased. At a cycle length of ?JO msec VERP decreased by 0.3 k 0.4 O/O (from

145 2 6 msec at baseline to 145 + 7 mec with saline) (p = ns). decreased by 0.3 t 1.9 % at cycle length 220 msec (frorn 134 5 7 msec at basrline to 133 2 8 msec afier saline) (p

= ns). decreased by 0.2 + 2.6 O/b at cycle Irngth 200 msrc (from 123 + 6 msec at baselinr to 123 k 8 msec after saline) (p = ns). decreased by 0.9 + 1.9 $6 at cycle length 180 msec

(fiom 1 16 t 6 msec at basrline to 1 15 2 7 msec after saline) (p = ns). decreased by 0.4 k

2.8 % at cycle length 160 msec ( from 107 t 5 msec at baseline to 107 2 6 rnsec after saline) (p = ns). decreased by 1.3 k 7.6 % at cycle lrngth 150 msec (from 103 t 5 rnsec at baseiine to 101 k 6 msec dter saline) (p = ns). and decreased by 2.2 f 3.3 % at cycle length 140 msec (from 98 ? 5 msec at badine to 96 k 5 msec afier saline) (p = ns)

(Figure 22). These results reflect the stability of the guinea pig model in measuring both

DFT and VERP.

7 1 J .J .1.2. Hemodvnarnic and Other Measurernents

Mean baseline and saline values of systolic and diastolic pressures. and blood gases @CO2,PO?. and pH) for the control experiments are indicated in Table VI11 D (also see Figure 23). Mean guinea pig weights. heart weights. and changes in hem rate from baseline to saline treatrnent are listed in Table VI11 C. The mean baseline and saline values of the diastolic and systolic pressures. heart rate. and blood gases were not significantly different fiom one another. The mean percent change in systolic pressure. diastolic pressure, heart rate and blood gases from baseline to saline treatment were variable, yet not different from zero. Systolic and diastolic carotid anerial pressure decreased by 6.6 t 17.9 % (from 79 + 9 mmHg at baseline to 72 t 10 mmHg post saline)

(p = ns) and 6.4 + 1 8.1% ( from 5 1 f 8 mmHg at baseline to 47 k 6 mmHg afier saline) (p

= ns). respectively. p02and pC02 increased by 2.5 ? 7.7 % (from 1 11 f 9 mmHg at baseline to 1 14 + 12 mmHg after saline) and 3.2 k 1 1.8 % (from 37 k 3 mmHg at baseline to 38 k 5 mmHg after saline) (al1 p = ns). respectively. However there was no change in hem rate or pH from baseline to saline treatment ( 188 + 10 bpm and 7.39 k

0.04 at baseline to 19 1 + 8 bpm and 7.39 t 0.03 afier saline, respectively) (al1 p = ns).

The mran absolute value of QRS duration also did not change between the baseline study and saline treatment of the control group (50 f 5 msec at baseline at a pacing cycle length of 180 msec. and 50 t 4 msec after saline) (see Table VI11 A). Table Vlll A. Electrophysiologic Mcasurements of DFT, VFCL, and QKS Duration of Intact Guinea Pig f-iearts in the Control Group.

Saline Basel iiic Saline Sali ne

Table VI l l B. Electrophysiologic Measurenieiits of VIW>at Multiple Cycle 1-engths of Intact Guiiica Pig 'fable VI11 C. Physical Measureinrnts of Intact Guinea Pig 1-leurts in the C'ontrol Group. Heürts in the C'oiitrol Group. Daia presented Data prcscnted as itiean k SD. II= 1 O. I ~'ontro~ I 1 ~asclitie 1 Saline -1 VERF, 240 niscc 145 -th 145 _+ 7

VERP 200 nisçc 123 46 123 k 8 VERP 180 msec 11616 115k7

Table VI Il 13. Mcasurements ofs1-fcinodyiianiics and Dlood Güses 01' Intact üuiiiea Piy 1-learts in iIic Control Group. Data presentcd as iiierin i Sl). n= 10. Systolic Blood Diastolic Blooci

--- p = ns btst\vccii Raseline and Seliiic incasiireniciits for al1 paraineters nicüsured. 88.8.. 88.8.. A..

Baseline Saline A Treatrnent

Baseline Saline Treatrnent

Baseline Saline c Treatment

Figure 2 1. Individual measurements of defibrillation threshold (DFT) (A). ventricular fibrillation cycle length (VFCL) (B). and QRS duration (C) in intact guinea pig hearts. Each parameter was obtained in triplicate at baseline (m and afier saline (A)administration in each animai (n=lO). Horizontal lines denote the mean. PCL (msec)

PCL (msec)

Figure 22. Mean values (A) and individual measurements (B)of ventricular effective refractory period (VERP) measured from the right ventricular endocardium at each pacing cycle length at baseline (a) and afier saline (7).Error bars denote SEM and horizontal lines denote the mean (n= 10). 01 Baseline Saline

a Baselme Saline Baseiine Saline

O - Baseline Saline

Figure 23. Individual measurements of systolic and diastolic blood pressure (A). heart rate (B). pC02 (C).p02 (D)and pH (E)obtained at baseline (H) and after saline administration (A)(n=10). Horizontal lines denote the mean. 3.3.2. Combination PEGJOO and DMSO Administration

Baseline and saline values on measurements of DFT. and VERP in the presence of varying concentrations of PEG400 and DMSO combinations are listed in Table IX A

(al1 n=l ).

3.3.2.1. Combination 0.6 mL PEG4OO and 0.2 mL DMSO

3.3.2.1.1. Electrophvsiolorrica1 Measurements

The percent decrease in DFT from baseline to organic solvent treatment was

17.7% (from 170 t 17 V ai baseline to 140 + 17 V afier organic solvent treatment)

(Figure 24). VERP at a cycle length of 2-10 msec increased by 19.7 % ( from 132 rnsec at baseline to 158 rnsec with the organic sol vents). at a cycle length of 220 rnsec increased by 15.9 % (from 126 msec rit baseline to 1 J6 msec with the organic solvents). at a cycle length of 200 msec increased by 1 5.8 O.0 ( frorn 1 14 msec at baseline to 132 msec with the organic solvents). at a cycle length of 180 msec increased by 16.7 9'0 ( from 108 msec at baseline to 126 msec with the organic solvents). at a cycle length of 160 msec increased by 22.9 % (from 96 msec at baseline to 1 18 msec with the organic solvents). at a cycle length of 150 msec increased by 2 1.3 % (from 94 msec at baseline to 1 14 msec with the organic solvents). and at a cycle length of 1 JO msec increased by 17.8 % (from 90 rnsec at baseline to 106 msec with the organic solvents) (Figure 25). This was the greatest dose of combination organic solvents adrninistrred in this thesis. and it was found to dramatically decrease DFT and increase VERP at al1 paced heart rates.

3 3 2.2. Hernodvnamic and Other Measurements

Baseline and post-organic solvent treatment values of systolic and diastolic pressures, heart rate and blood gases @CO?, p02, and pH) for the controi expenments are listed in Tables IX B and C. Systolic and diastolic carotid arterial pressure decreased by

24.5 % (from 49 mmHg at baseline to 37 mmHg post organic solvent administration) and

26.9 % (from 26 mmHg at baseline to 19 mmHg after organic solvents). respectively.

Heart rate also decreased by 6.6 % (1 82 bpm at baseline toi 70 bpm afier organic solvents), as did p02and pH by 5.8 % (from 98 mmHg at baseline to IO4 mmHg afier organic solvents). and 3.0 % (7.41 at baseline to 7.38 after organic solvents). respectively. pCQ2 increased by 1 1.4 % (from 35 mmHg at baseline to 39 mmHg aftcr organic solvents).

QRS duration between the baseline study and organic solvcnt treatment of this control group increased by 5.5 % (55 + 3 msec at baseline at a pacing cycle length of 180 msec to 58 k 4 msec after organic solvents) (see Table IX C).

2.2Combination 0.3 mL PEG300 and 0.1 rnL DMSO

3.3 2.2.1. Electrophvsiolo~icalMeasurements

There was an 8.7 % percent decrease in DFT from baseline to organic solvent treatment (from 330 f 17 V at baseline to 2 10 + 17 V afier organic solvent combination)

(Figure 24). VERP at a cycle lrngth of 240 msec increased by 1 .J ?' (from 140 msec at baseline to 1 J2 msec with the organic solvents). at a cycle length of 220 msec increased by 3.0 941 (hm134 msec at baseline to 138 msec with the organic solvents). at a cycle length of 200 msec increased by 1.6 % (from 126 msec at baseline to 128 mec with the organic solvents). did not change at a cycle length of 180 mec (1 18 msec at baseline and with the organic solvents). at a cycle length of 160 msec decreased by 3.6 % (from 1 12 msec at baseline to 108 msec with the organic solvents). at a cycle length of 150 msec decreased by 3.8 % (From 106 mec at baseline to 102 msec with the organic solvents), and did not change at a cycle length of 140 msec (98 msec at baseline and afier organic solvent administration) (Figure 25). As with the higher dose of combination organic solvents (section 3.3 2.1.1 .). this medium dose was also found to mimic class III effects of decreasing DFT and increasing VERP.

3.3 22.2. Hemodvnamic and Other Measurements

Values of systolic and diastolic pressures. hrart rate and blood gases (pCOz. p02. and pH) for these control experirnents (pre- and post-organic solvent treatment) are indicated in Table IX A and C. Systolic carotid arterial pressure increased by 4.9 Oh

(from 61 mmHg at baseline to 64 mmHg post organic solvent administration). Diastolic pressure decreased by 5.0 % (from 42 mmHg at baseline to 40 mmHg after organic solvents). Heart rate also decreased by 1 .O % (19 1 bpm at basdine to 189 bpm after organic solvents). pOz and pC02 increased by l2.J ?/O (from 9? mmHg at baseline to 109 mrnHg after organic solvents) and 8.1 O'O ( from 34 mmHg at baseline to 37 mmHg after organic solvents). respectivelp. pH decreased by 0.5 % (frorn 7.43 at baseiine to 7.39 after organic solvents).

QRS duration between the baseline study and organic solvent treatment of this control group decreased by 5.1 % (59 + 4 msec at baseline at a pacing cycle length of 1 80 msec to 56 ?r 3 msec after organic solvents) (see Table IX C).

3.3.3.3. Combination 0.15 mL PEG400 and 0.05 mL DMSO

3.3 2.3.1. Electrophvsiological Measurements

There was no change in DFT from baseline to organic solvrnt treatment (190 k 30

V at baseline to 190 + O V after solvent combination) (Figure 24). VERP at cycle lengths of 240. 180. 160. 150, and 140 msec did not change (140. 1 14.106, 102. and 98 msec respectively at baseline and after organic solvent treatment). However. VERP at cycle lengths of 220 and 200 msec decreased by 1 .j % (from 134 msec at baseline to 132 msec with the organic solvents), and 1.6 % (frorn 122 msec at baseline to 120 msec with the organic solvents). respectively (Figure 25). The lowest dose of solvents yielded results very similar to that seen in the control group receiving saline(section 3.3.1.I .) by not altering DFT or VERP.

3.3.2.3 .?. Hemodvnarnic and Other Measurements

Baseline and organic solvent treatment values of systolic and diastolic pressures. heart rate and blood gases (pCO?.pOz. and pH) for the control experiments are indicated in Tables IX B and C. Systolic carotid arterial pressure decreased by 5.3 % (from 57 mmHg at baseline to 54 mmHg post organic solvent administration). Diastolic pressure decreased by 1.9 O/o (from 35 mmHg at baseline to 34 mmHg afier organic solvents).

Heart rate also decreased by 3.7 % ( 191 bpm at baseline to 1 87 bpm afier organic solvents). p02 increased by 6.7 % (from IO5 mmHg at baseline to 1 12 mmHg after organic solvents). pCOz increased by 17.2 % (from 39 mmHg at baseline to 34 mmHg afier organic solvents). pH decreased by 1.1 % (7.44 at baseline to 7.36 afier organic solvents).

QRS duration between the baseline study and organic solvent treatment of this control group increased by 5.7 % (53 k 2 msec at baseline at a pacing cycle length of 180 msec to 56 r 1 msec after organic solvents) (see Table iX C).

3.3 .hl.Surnmm of Combination PEG4OO and DMSO Experiments

Between the three combinations administered. the lowest amount of organic solvents (0.15 mL PEGJOO and 0.05 mL DMSO in combination) has the least effect on electrophysiologic parameters. by not altering DFT. nor prolonging VERP. Furthemiore. this amount of combination organic solvents can be used to completely dissolve the chromano1 HMR1556. the dmg of interest which is insoluble in saline.

1reatment

Figure 34. The effect of high (combination 0.6 mL PEG4OO and 0.2 mL DMSO). medium (combination 0.3 mL PEGJOO and 0.1 mL DMSO). and low (combination O. 15 mL PEG4OO and 0.05 mL DMSO)doses of organic solvents on DFT in the intact guinea pig heart, measured at baseline and post solvent treatment. 0.6 ml PEGQOO + 0.2 ml DMSO 50.3 mi PEG4OO + 0.1 ml DMSO

PCL (rnsec)

8 Baseline ,/ 0.6 ml PEG + 0.2 ml DMSO t 0.3 ml PEG + 0.1 ml OMS0 a 0.15 ml PEG + 0.05 ml DMSO

?CL (msec)

Figure 25. The effect of varying combinations of PEG400 and DMSO on the ventricular effective refractory penod (VERP) (B). and on the percent change in VERP of the intact guinea pig hem (A) from baseline to post soivent treatment. Baseline values of VERP at multiple cycle lengths frorn three experirnents are displayed as the mean k SEM. Minimal changes in VERP are observed with the lowest dose of combination organic solvents administered (0.15 mL PEG400 and 0.05 mL DMSO). n= 1 per group. 3.3.3. Administration of 8 and 4 &kg Dofetilide

3.3.3.1. Effects of 8 ug/kg Dofetilide on Electrophvsiological Measurements

Treatment of the guinea pig with 8 pgAg dofetilide decreased the DFT from a baseline of 207 2 12 V to 147 + 3 1 V. a difference of -29.07 % (n=l) (Table X A). After administration of dofetilide post DFT rneasurements, VERP was decreased at every paced cycle length: at 210 rnsec VERP was decreased by 10.4 % (from 154 msec at baseline to 138 msec with dofetilide). by 8.3 % at a cycle lengh of220 rnsec ( from 144 msec at baseline to 1 32 msec with do fet ilide). by 1 1.6 % at a cycle length of 200 msec

(from 138 msec at baseline to 122 msec with dofetilide), by 10.9 % at a cycle length of

180 msec (from 118 msec at baseline to 1 14 msec with dofetilide), by 1 1.9 % at a cycle

length of 160 msec ( from 1 18 msec at baseline to 1 O-l msec with dofrtilide). by 14.3 % at a cycle length of 1 50 msec ( from 1 12 msec at baseline to 96 msec with dofetilide). and by

13.2 % at a cycle length of 140 msec (from 106 rnsrc at baseline to 92 rnsec with dofetilide) (Table X A). Although this dose of dofetilide decreased DFT. its effects on

VERP were not comparable. In fact. this same dmg dose increased VERP to a greater

extent at al1 paced cycle lengths in the absence of DF shocks (see section 3.4.2.1.1 .).

3.3.3.2. Effect of 4 pdke Dofetilide on Electroohysioloeical Measurernents

The dose of dofetilide was reduced to 4 pg/kg, (n=2) resulting in a decrease of

25.4 20.6 % in DFT from baseline (from 183 + 33 V at baseline to 137 + 24 V afier

dofetilide) (Table X A). At each paced cycle length. the VERP also decreased: VERP at

a cycle length of 240 msec decreased by 0.6 f 2.7 %(from 156 + O msec at baseline to

155 f 4 msec with dofetilide), at a cycle length of 220 rnsec decreased by 2.7 f 0.1 % (from 149 k I msec at baseline to 145 f 1 msec with dofetilide), at a cycle length of 200 msec decreased by 2.1 f 2.9 % (from 140 t 6 msec at baseline to 1 3 7 + 1 msec with dofetilide). at a cycle length of 180 msec decreased by 3.0 k 4.2 % (from 130 k 6 msec at baseline to 126 k O msec with dofetilide). at a cycle length of 160 rnsec decreased by 2.5 k 1.1 % (fiom 120 k 6 msec at baseline to 117 k 4 msec with dofetilide). at a cycle length of 150 msec decreased by 0.8 t 1.2 % (from 1 12 I8 msec at baseline to 1 1 1 k 7 msec with dofetilide). and at a cycle length of 140 msec decreased by 1.9 + 0.2 % (from 107 2

10 msec at baseline to 105 + 10 msec with dofetilide) (Table X A). These results are qualitatively sirnilar to those obtained with 8 pg/kg dofetilide (section 3.3.3.1 .). The magnitude of VERP prolongation obtained with dofetilide while applying defibrillation shocks is less than that obtained without DFT testing (see section 3.4.7.2.1 .).

3.3.3.3. Effects of 8 ~gi'kpDoktilide on Hemodvnamic and Other Measurements

Baseline and dofetilide treaûnent values of systolic and diastolic pressures. heart rate and blood gases (pCOz. pOz, and pH) for the 8 pgkg dofetilide experiments are indicated in Tables X B and C. Systolic and diastolic pressure decreased by 6.9 % (frorn

58 mmHg at baseline to 54 mmHg post dofetilide administration) and 5.0 ?' (from 40 rnrnHg at baseline to 38 mmHg after dofetilide). respectively. Hem rate and blood pH ais0 decreased by 4.3 % ( 194 bpm at baseline to 186 bprn afier dofetilide) and 3 .O % (7.4 1 at baseline to 7.38 alter dofetilide). respectively. POr and pCOz increased by 10.5 %

(from 86 mmHg at baseline to 95 mmHg after dofetilide) and 19.4 % (fiom 3 1 mmHg at baseline to 37 rnrniig after dofetilide), respectively. QRS duration between the baseline study and dofetilide treatment group decreased by 3.3

% (60 rnsec at baseline at a pacing cycle length of 180 msec to 58 msec after dofetilide)

(see Table X C).

33.34. Effects of 4 pdke Dofetilidr on Hemodvnamic and Other Measurements

Tables X B and C list baseline and dofetilide treatrnent values of systolic and diastolic pressures. heart rate and blood gases (pCO,. p02, and pH) for the 4 pgkg dofetilide experirnents. Systolic and diastolic pressure decreased by 4.9 2 4.5 % (from 60 k 4 mmHg at baseline to 57 k 1 mmHg post dofetilide administration) and 9.8 k 0.7 %

(fiom 41 f 3 mmHg at baseline to 37 + 3 mmHg after dofetilide) respectively. Hem rate also decreased by 3.6 I0.6 % ( from 1 96 k 1 bpm at baseline to 1 89 + 3 bpm after dofetilide). p02 increased by 2.7 k 5.4 % ( from 1 12 k 5 mmHg at baseline to 1 1 5 2 7 mmHg after dofetilidc). pC02 increased by 13.9 i 1.80/0 (from 56 k 2 mmHg at basdine to 41 f 5 mmHg after dofetilide). pH decreased by 0.3 2 5.8 % (from 7.37 k 0.15 at baseline to 7.35 I0.2 1 after dofetilide).

QRS duration between the baseline study and do fetilide treatment group increasrd by 1.7 I1.5 % (58 t 1 msec at baseline at 3 pacing cycle Irngth of 180 msec to 59 I7 msec afier dofetilide) (see Table X C).

Table X C. Physicül Measurements of Intact Giiinca Pig Hearts in ihe Control and Dofetilide-Treated Groups. Data presriited as mean f SI>. n=10 for control group. n=l for 8 !@kg dofetilide group. n=2 for 4 pgkg dofetilide group.

Guinea Pig

-- I Control 0.99 + 0.07 50 + 5 50 t 5

Post = post-treûtniciit [saline in control expcriments, 8 or 4 pglkg dofetilide in drug treainient expcriments]. 3.1. A Guinea Pip Mode1 Assessinn Cardiac Refractoriness: ERP and Dvnamic Restitution

3.41. Control Experiments

3.4.1.1. Electro~hvsiologicaIMeasurements

Mean baseline and saline values of VERP and ARIF&T measurements are listed in

Tables XI A and XI B respectively. A paired t-test analysis of the four saline controls indicated that there was no significant difference between the mean baseline and saline values of VERP and ARIpcakTat each pacing cycle length (Figure 26 and 27). This showed the stability of this mode1 ovrr time. and with the addition of 5 mL of saline. The mean percent change from baseline in VERP and ARI,~T.was also detemined not to be different from zero. VERP at a cycle length of 300 msrc decreased by 1.5 + 4.1 % (from

159 t 9 msec at baselinr to 156 k 10 msrc after saline) (p = ns). at a cycle length of 280 msec decreased by 0.6t 3.8 % (from 154 k 7 msec at baseline to 153 + 7 msec afier saline) (p = ns). at a cycle length of 260 msec decreased by I .O t 2.7 % (from 149 f 6 msec at baseline to 147 f 5 msec afier saline) (p = ns). at a c yclr length of 240 msec decreased by 0.3 t 0.7 % (from 142 k 5 msec at baseline to 1-12 i 5 msec after saline) (p

= ns). at a cycle length of 220 msec increased by 0.4 f 2.1 % (from 136 f 2 msec at baseline to 137 + 4 msec after saline) (p = ns). at a cycle lcngth of 200 msec decreased by

0.4 k 1 .j % (frorn 130 t 3 msec at baseline to 130 i 4 msec afier saline) (p = ns). at a cycle length of 1!i0 msec decreased by 1.2 + 1.6 % (from 123 + 3 msec at baseline to 122 f 4 msec after saline) (p = ns), at a cycle length of 160 msec decreased by 0.4 + 1.6 %

(from 1 15 + 5 msec at baseline to 1 15 i 6 msec afier saline) (p = ns). at a cycle length of

150 msec decreased by 0.9 + 1.O % (Frorn 1 10 f 7 msec at baseline to 109 It 6 msec after saline) (p = ns), and at a cycle length of 140 rnsec decreased by 0.5 k 1.9 % (from 105 t

7 msec at baseline to 104 f 7 msec afier saline) (p = ns) (Figure 26).

ARIpcakTat a cycle length of 300 msec decreased by 0.1 f 1.9 % (from 174 + 1 O msec at baseline to 174 t 7 msec with saline) (p = ns), at a cycle length of Z8O msec increased by 0.9 t 3.2 % (from 169 + 4 msec at baseline to 169 + 6 msec with saline) (p

= ns). did not change at a cycle length of 260 msec (from 163 f 8 msec at baseline ro 163

+ 7 msec with saline) (p = ns). at a cycle length of 210 msec decreased by 0.2 f 2.2 %

(from 160 k 7 msec at baseline to 159 + 6 msec with saline) (p = ns), did not change at a cycle length of 220 msec (from 153 2 8 msec at baseline to 153 f 5 msec with saline) (p

= ns). at a cycle length of 200 mec increased by 0.8 f 4.3 % (from 146 2 7 msec at baseline to 147 f 3 msec with saline) (p = ns). at a cycle length of 180 msec increased by

0.3 + 2.3 46 ( from i 3 8 + 7 msec at baseline to 139 k 5 msec with saline) (p = ns). did not change at a cycle length of 160 msec (from 13 1 + 7 msec at baseline to 13 1 f 4 msec with saline) (p = ns). at a cycle length of 150 msec decreased by 0.6 k 3.7 % ( from 126 k

8 msec at baseline to 175 I4 msec with saline) (p = ns). at a cycle length of 140 msec decreased by 0.8 f 2.8 % (from 1 19 + 10 msec at baseline to 1 17 k 7 msec with saline)

(n=3. p = ns). at a cycle length of 130 msec decreased by 0.4 2 2.1 % (from 1 12 t 4 msec at baseline to 112 k 1 msec with saline) (n=3. p = ns). at a cycle leiigth of 120 msec decreased by 2.4 f 2.4 % (from 1 10 f 4 msec at baseline to 107 I1 rnsec with saiine)

(n=3, p = ns). and at a cycle length of 110 msec increased by 2.8 + 2.5 % (frorn 99 + 6 msec at baseline to 102 r 6 msec with saline) (n=2. p = ns) (Figure 37). Both VERP and

ANWakTwere found to decrease with increasing heart rate (i.e. decreasing pacing cycle length), similar to the results obtained in the control experiments assessing DFT (section

3.3.1.1 .).

3.4.1.2. Dvnarnic Restitution Kinetics of Control Experiments

The dynamic restitution relation was determined for both baseline and saline treatment groups (Figure 28). At baseline. the non-linear regression sigrnoidal fits have an R' value of 0.9645,0.9782.0.9926.and 0.9853 t'or each experiment. respectively.

Sx.y values were determined to be j.=l0/0 2.8%. 5.4%. and 4.7*/0 respectively. The steepest slopes of each cuwe are as follows: 1.47,0.38, 1.30. and 0.55 respectively (mean k SD = 0.93 + 0.54). After saline treatrnenr. the sigrnoidal regression fits have R' values of 0.9724. 0.9698. 0.9824 and 0.9736 respectively. The steepest slopes of each cunc are

1.69.0.40. 1-59. and 0.55 respectively.(mean + SD = 1 .O6 t 0.68). This resultrd in an overall increase of 10.6 t 9.9 % in the steepest siope of the dynarnic restitution curve.

3 .A. 1 2. Hemodvnmic and Other Measurements

Mean baseline and saline values of systolic and diastolic pressures. hem rate and blood gases (pCOz. pOz. and pH) for the control experiments are indicated in Table XI C and D. The mean baseline md saline values of the diastolic and systolic pressures. heart rate. and blood gases were not significantly different from one another. The mean percent change in systolic pressure. diastolic pressure. heart rate and blood gases from baseline to saline treatment were variable. yet riot different from zero. Systolic pressure decreased by 3.9 k 9.1 % (fiom 57 ?r 1 1 mmHg at baseline to 58 f 6 mmHg post saline)

(p = ns). Diastolic arterial pressure decreased by 0.7 I8.2% (from 37 k 10 mmHg at baseline to 36 t 7 mmHg after saline) (p = ns). Heart rate increased by 1.3 I3.6 % (frorn

192 k 5 bpm at bascline to 194 I5 bpm after saline) (p = ns). p02 increased by 1.9 + 5.8 % (fiom 101 t 10 mmHg at baseline to 102 t 4 rnrnHg after saline). pC02 decreased by

2.3 2 18.8 % (from 35 t 6 mmHg at baseline to 34 f 4 mmHg after saline) (p = ns). pH decreased by 0.1 f 0.7 % (from 7.40 i 0.02 at baseline to 7.39 2 0.03 afier saline) (p = ns).

QRS duration increased by 0.1 k 3.9 % (from 46 k 4 msec at a pacing cycle length of 180 msec at baseline. to 46 k 3 msec after saline) (Table X1 D).

Baseline Saline

A ?CL (msec) Baseline Sali ne

B PCL (m~e~)

Figure 26. (A) The mean ventricular effective refractory period (VERP) measured at each pacing cycle length at baseline (m) and afier saline administration (a). Error bars denote SEM. (B)Individual measurements of VERP obtained at each pacing cycle length at baseline (m) and after saline (a). Horizontal lines denote the mean. All p = ns between baseline and saline measurements at each pacing cycle length. n=4 Baseline Saline

120 140 160 180 200 220 240 260 280 300 320 PCL (mec)

Baseline Saline

Figure 27. (A) The mean activation recovery intervals measured from maximum - dV/dt of the QRS deflection to the peak of the T wave (ARIPeaT)for each pacing cycle length at baseline (m) and after saline administration (a). Error bars denote SEM. (B) Individual measurements of ARIpd obtained at each pacing cycle length. Horizontal lines denote the mean. Al1 p = ns between baseline and saline measurements of AMpcdTat each respective cycle length. n 4. Baseline Saline

O 25 50 75 IO0 125 150 DI (msec)

Figure 28. Dynamic restitution kinetics at baseline (W) and after saline administration (0)of a control experirnent. Al1 non linear regressions were fit to sigmoidal curves with R' values >0.96. and Sy.x 4.4%. The steepest slopes at baseline and afier saline were determined to be 0.93 ?r 0.54 and 1 .O6 r 0.68 respectively. 3 A.2. Do fetilide Experiments

3 .M.1. Administration of 8 udke Dofetilide

3 A.2.l.1. Effects of 8 p.&e Do fetilide on Electrophvsiolo~icalMeasurements

Mean baseline and dofetilide values on measurements of VERP and ARIprakTare listed in Tables XII A and B, respectively. VERP at a cycle length of 300 msec increased by 19.7 % (fiom 142 msec at baseline to 170 msec after dofetilide), at a cycle length of

180 msec increased by 1 8.6 % (from 140 msec at baseline to 166 rnsec after do fetilide). at a cycle length of 760 msec increased by 16.2 % (from 136 msrc at baseline to 158 msec afier dofetilide), at a cycle length of 340 rnsec increased by 15.2 % (from 132 msec at baseline to 1 52 msec after do fetilide). at a cycle length of 120 msec increased by 10.9

% ( from 128 msec at baseline to 1 42 msec afer do ktilide). at a cycle length of 200 msec increased by 9.8 96 ( from 122 msec at baseline to 134 msec afier dofctilide). at a cycle length of 180 msrc increased by 10.7 % ( frorn 1 11 msec at basrline to 124 msec after dofetilide), at a cycle length of 160 mec increased by 7.5 % (from 1 O6 msec at baseline to 1 14 msec after dofetilide). at a cycle length of 150 msec increased by 3.9 06 (from 102 msec at baseline to 1 O6 msec after dofetilide). and did not change at a cycle length of 1-10 msec ( 1 00 msçc both at baselinr and after dofetilide) (Figures 29 and 30). Tliese results differed from the previous study with the same drug dose in that the effect of dofetilide on prolonging VERP was more pronounced in the absence of defibrillation shocks (see

7 7 1 section J.J.J. 1 .).

AMFtT at a cycle length of 300 rnsec increased by 9.0 % (from 163 k 1 msec at baseline to 177 f 1 mec after dofetilide), at a cycle length of 280 msec increased by 9.3

(frorn 157 + 2 msec at baseline to 172 + 1 msec afier dofetilide). at a cycle length of 260 msec increased by 8.7 % ( from 153 + 2 msec at baseline to 166 f 1 msec after dofetilide), at a cycle length of 240 msec increased by 10.0 % (fiom 147 f 2 msec at baseline to 16 1 t 2 msec after dofetilide). at a cycle length of 220 msec increased by 9.1

% (from 142 k 2 msec at baseline to 155 + 1 msec afier doktilide). at a cycle length of

200 msec increased by 7.8 % (from 136 + 1 msec ai baseline to 147 + O msec after dofetilide), at a cycle length of 180 msec increased by 6.6 % (from 13 1 k 1 msec at baseline to 139 + 3 msec afier dofetilide). at a cycle length of 160 msec increased by 10.6

% (from 120 t 7 msec at baseline to 133 k 2 msec after dofetilide). at a cycle length of

150 msec increased by 6.5 % (from 1 19 f 2 msec at baseline to 126 k 2 msec after dofetilide), at a cycle length of 140 msec increased by 5.9 % (from 1 13 2 3 msec at baseiinr: to 1 30 k 1 msec after dofetilide). at a cycle length of 130 msec increased by 4.6

% (from 109 + 2 msec at baseline to I 14 t 1 msec afier dofetilide). at a cycle length of

120 msec increased by 4.1 % (from 106 f 7 msec at baseline to 1 10 k 1 msec after dofetilide), and at a cycle length of 1 10 msec increased by 6.8 % (from 98 ? 2 msec at baseline to 104 r 1 msec after dofetilide) (Figure 29B). Refractoriness tended to shorten with increasing pacing rate.

3.4.2. t -3. Dvnamic Restitution Kinetics of the 8 udkg Dofetilide Ex~eriment

The dynamic restitution relation was deterrnined for both the baseline and

dofetilide treatment phases of the expenment (Figure 3 1). At baseline. the non-linear

regression sigrnoidal fit has an R' value of 0.9879. an Sx.y value of 1.8%. and a steepest

slope of 0.91. Afier dofetilide treatment. the sigrnoidal regression fit haan R' value of

0.9957, an Sx.y value of 1.3%, and a steepest dope of 1.13, resulting in a 23.6 % increase in the slope fiom baseline to dofetilide administration. This did not differ greatly from the results obtained with 4 pgkg dofetilide (section 3.4.2.2.2.) or from control experirnents (section 3.3 A.?.).

3 .M.l.3. Effects of 8 @kg Dofetilide on Hemodynamic and Other Measwements

Baseline and dofetilide treatment values of systolic and diastolic pressures. heart rate and blood gases (pC02, pOz, and pH) for the 8 pgkg dofetilide expenments are indicated in Tables XII C and D. Systolic and diastolic pressures decreased by 7.7 */O

(from 52 mml-ig at baselinr to 48 mmHg post dofetilide administration) and 7.9 % (from

38 mmHg at baseline to 35 mmHg after dofetilide). respectively. Heart rate also decreased by 4.9 % ( 193 bpm at baxline to 1 83 bpm afler do fetilide). pOz increased by

1 1 .O % (frorn 1 10 mmHg at baseline io 123 mmHg afier dofetilide). pCO? increased by

18.1 % ( from 32 mmHg at baseline to 38 mmHg afier dofetilide). pH decreased by 0.7 %

(7.44 at baseline to 7.39 after dofetilide).

QRS duration between the baseline study and do fetilide treatment group increased by 5.8 % (from 52 t I msec at baseline at a pacing cycle length of 1 80 msec to 55 2 3 mec afier dofetilide) (sec Table XII D). C +I a 'a C

Cl1 +I rCi IA c.

b +I C'i \O C -

Od +I m \O C -

'3 9 e'Ji C C Q PJ; z2 < -

180 Baseline Dofetilide

PCL (msec)

200 Baseline 8 pglkg Dofetilide

80 l I 1 1 I I I I 80 120 160 200 240 280 320 B PCL (msec)

Figure 79. The ventncular effective refractory period (VERP) (A), and mean activation-recovery intervals (ANvtT)(B) at each respective paced cycle length measured at baseline (H). and after administration of 8 pg/kg doktilide (A). Figure 30. The effect of 8 @cg Dofetilide on the percent change in right VERP meisured from badine in the intact guinea pig heart. Note the magnitude of effect diminish as the pacing cycle length is decreased. Baseline Dofetilide 8 pglkg

Figure 3 1. Dynamic restitution kinetics at baselinr (H) and afier administration of 8 pdkg do fetilide (A)(n= 1). The non linear regressions pre- and post-drug treatment wrre fit to sigrnoidai curves with R- values >0.98. and Sy.x < 1.8 %. The steepest dope increased 23.6% from 0.9 1 at baseline to 1.13 after treatment wi th do fetilide. 3.4.2.2. Administration of 4 pgkg Dofetilide

j.4.2.2.l.Effects of 4 u_g/kgDofetilide on Electrophvsiological Measurernents

Mean baseline and dofetilide values on measurements of VERP and AMpeakTare listed in Table XII1 A and Xi11 B respectively. VERP at a cycle length of 300 msec increased by 14.7 % (from 150 msec at baseline to 172 msec after dofetilide). at a cycle length of 280 msec increased by 10.7 ( from 1 50 msec at baseline to 1 66 msec afier dofetilide). ai a cycle length of 260 msec increased by 10.8 % (from 148 msec at baseline to 164 msec afier dofetilide). at a cycle length of NOmsec increased by 12.9 % (from

140 msec ai baseline to 158 msec after dofetilide). at a cycle length of 220 msec increased by 1 1.8 ?/o ( from 1 36 msec at baseline to 153 msec after dofetilide ). at a cycle length of 200 msec increased by 1 0.8 ( from 130 msec at badine to 144 msec afier dofetilide). at a cycle length of 180 msèc increased by 8.3 ?/O (From 122 msec at baseline to 132 msec afier dofetilide). at a cycle length of 160 msec increased by 7.0 % ( from 1 14 msec at baseline to 122 msec after dofetilide). at a cycle length of 150 msec increased by

5.6 % (from 108 msec at baseline to 1 14 msec afier dofetilide). and at a cycle lrngth of

140 msec increased 5.9 % ( from 102 msec at baseline to 1 O8 msec afier dofetilide)

(Figures 32 and 33). The increases in VERP with this dose of dofetilide are qualitatively comparable to those seen with 8 pglkg dofetilide in the absence of high voltage shocks

(see section 3.4.2.1.1. ).

AMvAT at a cycle length of 300 msec increased by 8.6 % ( from 163 + 2 msec at

baseline to 177 t 1 msec afier dofetilide). at a cycle length of 280 msec increased by 1 1.4

% (from 157 ?r 2 msec at baseline to 175 k 1 rnsec after dofetilide). at a cycle Iength of

260 msec increased by 12.1 % (fiorn 154 k 4 rnsec at baseline to 172 k 2 mec afier dofetilide), at a cycle length of 240 msec increased by 8.3 % (from 152 f 2 msec at

baseline to 165 f 2 msec after dofetilide), at a cycle length of 220 msec increased by 8.1

% (frorn 147 + 3 rnsec at baseline to 159 + 2 msec after dofetilide). at a cycle length of

200 msec increased by 6.9 % (from 14 1 + 1 msec at baseline to 1 50 k 1 msec afier

dofetilide), at a cycle length of 180 msec increased by 6.5 % (frorn 135 i 2 msec at

baseline to 143 & 2 msec afier dofetilide). at a cycle length of 160 msec increased by 7.5

% (from 125 t 2 msec at baseline to 134 ? 3 msec after dofetilide). at a cycle length of

150 msec increased by 3.2 % (from 124 k 1 msec at baseline to 128 + 1 msec after

dofetilide). at a cycle length of 1JO msec increased by 1.9 % (frorn 1 17 2 3 msec at

baseline to 120 ir 2 msec after dofetilide). at a cycle length of 130 msec increased by 5.7

% (from 1 1 1 f 2 msrc at baseline to 1 1 7 k 1 msec afier do fet ilide). at a cycle length of

120 msec increased by 0.3 % (from 106 + 1 msec at baseline to 107 k 3 msec afier

dofetilide). and at a cycle length of 1 10 msec decreased by 1 .O ?/o ( from 100 c 1 msec at

baseline to 99 t 1 msec a%rrdofetilide) (Figure 32 B).

3.4.2.2.2. Dvnamic Restitution Kinetics of the 4 ~g/kgDofetilide Experiment

The dynamic restitution relation was determined for both the baseline and

dofetilide treatment phase (Figure 34). At baseline. the non-linear regression sigmoidal

fit has an R' value of O.99j7. an Sx.y value of 1.2%. and a steepest slopè of: 1.14. Afier

dofetilide treatment. the sigmoidal regression fit has an R' value of 0.9829. an Sx.y value

of 3.5%. and a steepest slope of 1.54. resulting in an increase of 34.6 % fiom the slope

detemined at baseline. The trend of increasing dope was also seen with this drug dose.

compared to the 8 pg/kg dofetilide expenment (see section 3.4.2.1.2.) 3.4.2.2.3. Effects of 4 pe/ke. Dofetilide on Hemodynamic and Other Measurements

Baseline and dofetilide treatment values of systolic and diastolic pressures. heart rate and blood gases (pCOz. pOz. and pH) for the 4 pgikg dofetilide experirnents are indicated in Table XIII C and D. Systolic pressure decreased by 7.9 % (from 63 mmHg at badine to 58 mmHg post dofetilide administration). Diastolic pressure decreased by

6.7 % (from 45 mmHg at baseline to 42 mmHg after dofetilide). Hrart rate also decreased by 8.4 % (from 196 bpm at baseline toi 80 bprn after dofetilide). p02

increased by 6.3 % ( from 99 mmHg at baseline to 105 mmHg after dofetilide). pC02

increased by 7.4 % ( from 4 1 mmHg at baseline to 44 mrnHg aher dofetilide). pH

decreased by 0.5 % ( from 7.42 at bascline to 7.38 after do fer ilide).

QRS dwation between the baseline study and doktilide treatment group increased

by 2.1 % (from 48 + 3 msec at baseline at a pacing cycle length of 180 msec to 49 k 2

msec after dofetilide) (see Tabie XIII D).

Baseline Dofetilide 4uglkg

PCL (msec)

Baseiine 4 pglkg Dofetilide

I I I I I I 80 120 160 200 240 280 320 B PCL (msec)

Figure 32. The ventricular effective refractory period (VERP) (A) and mean activation-recovery intervals (AMpcJLT)(B) at each respective paced cycle length measured at baseline (m), and afier administration of 4 pgkg dofetilide (A). % Change in VERP from Baseline Baseline Dofetilide 4pgfkg

DI (msec)

Figure 34. Dynamic restitution kinetics at baseline (m) and after administration of 4 pg/kg dofetilide (A)(n=l). The non linear regressions pre- and post-drug treatment were fit to sigrnoidal curves with R- values > 0.98. and Sy.x < 3.5 %. The steepest dope increased 34.6% from I .l4 at baseline to 1 54 after dmg. 3.4.3. Administration of 1 -5 melkg HMRlSS6

3.4.3.1. Effects of 1.5 m&e HMR1556 on Electrophysiolo~icalMeasurements

Mean baseline and dofetilide values on measurements of VERP and ARIw&Tare listed in Table XIV .4 and B, respectively. VERP at a cycle length of 300 msec increased by 9.5 % ( from 148 msec at baseline to 1 62 msec afier HMRl556). at a cycle length of

280 msec increased by 9.6 % (from 146 msec at baseline to 160 msec after HMRl556). at a cycle length of 260 msec increased by 8.3 % (from 144 msec at baseline to 156 msec after HMRl556). at a cycle length of 2-10 rnsec increased by 7.2 % (from 138 msec at baseline to I J8 msec after HMRI 556). at a cycle length of220 msec increased by 9.0 O6

(from 134 rnsec at baseline to 146 msec after HMRl556). at a cycle length of 100 msec

increased by 6.3 % (from 126 msec at baseline to 134 msec afier HMRl556). at a cycle

Iength of 180 msec increased by 1.6 % (frorn 124 msec at baseline to 126 msec afier

HMR 1 556). at a cycle length of 160 msec increased by 5.2 % (frorn 1 16 msec at basclinc

to 122 msec after HMR 1556). at a cycle length of 150 msec increased by 7.5 % ( from

106 msec at baseline to 1 14 msec after HMR1556). and did not change at a cycle length

of 140 msec (102 msec at baseline and atirr HMRl556) (Figures 35.4 and 36).

Prolongation of VERP with HMR1556 was more pronounced at faster than at slower

paced rates. compared to the effects of dofetilide (see sections 3.4.2.1 .l. and 3 .4.2.2.l.).

MUvAr at a cycle length of 300 msec increased by 2.3 % (from 186 f 1 mec at

baseline to 191 f 1 msec after HMR1556). at a cycle lengih of 180 msec increased by 5.5

% (fiom 1752 1 msec at baseline to 185 + 1 msec afier HMR 1 556). at a cycle length of

260 msec increased by 4.6 % (from 172 t il mec at baseline to 180 t 7 msec after

HMR 1556). at a cycle length of 240 msec increased by 0.4 % (fiom 169 + 2 msec at baseline to 170 t 1 mec afier HMR1556). at a cycle length of 220 msec increased by 4.1

% (fiorn 161 k 2 msec at badine to 168 I 1 rnsec after HMR1556), at a cycle length of

200 msec increased by 3.0 % (from 153 t 1 msec at baseline to 158 f 1 msec after

HMR1556). at a cycle length of 180 msec decreased by 1.1 % ( from 119 k 2 msec at baseline to 148 + 2 mec afrer HMRl556). at a cycle length of 160 msec decreased by 0.5

% ( from 1 39 t 1 msec at baseline to 138 ?: 2 msec aRer HMR 1 556). at a cycle length of

150 msec increased by 1.7 % ( from 134 k 1 msec at baseline to 1 36 r 1 msec after

HMR 1 556). at a cycle length of 140 msec increased by 1.8 % ( from 1 28 + 2 rnsec at baseline to 13 1 k 2 msec afirr HMR 1556). and ai a cycle length of 1 30 msec increased by

0.5 % ( from 1 26 k 3 msec at baseline to 126 + 3 msec after HMR1556) (Figure 368).

3.4.3.2. Dvnamic Restitution Kinetics of the 1.5 mg /kg- HMR1556 Ex~eriment

The dynarnic restitution relation was detrnnined for both the baseline and

HMR 1 5 56 treatment phases ( Figure 3 7). At baseline. the non-linear regression sigmoidal fit has an R' value of 0.9724. an Sx.y value of 4.1%. and a steepest siope of 1.17. After

HMRlS6 treatment. the sigmoidal regression fit has an R' value of 0.9875. an Sx.y value of 1.8 %. and a steepest slope of 0.99. resulting in an decrease of 15.7 % from the slope detemincd at baseline.

3.43.3. Effects of 1.5 m.dg HMR 1 556 on Hemodvnamic and Other Measurements

Baseline and HMR1556 treatment values of systolic and diastolic pressures. heart rate and blood gases (pCO,. PO,.and pH) for the 1.5 mg/kg HMRI 556 rxperiments are indicated in Tables XIV C and D. Systolic pressure decreased by 7.1 % (fiom 57 mmHg at baseline to 53 mmHg post HMRlS6 administration). Diastolic pressure decreased by 5.0 % (hm40 mmHg at baseline to 38 mmHg afier HMRlSj6). Heart rate also decreased by 1.6 % (from 194 bpm at baseline to190 bprn after HMRlj56). p02 increased by 15.4 Oh (from 99 mmHg at baseline to 1 14 rnrnHg after HMRljj6). pCOz increased by 18.1 % (from 29 mmHg at baseline to 34 mmHg after HMRljj6). pH decreased by 0.5 % (from 7.43 at baseline to 7.39 afier HMRl556).

QRS duration between the baseline study and HMRl556 treatment group increased by 4.3 % (from 47 k 4 msec at baseline at a pacing cycle length of 180 msec to

49 k 3 msec afier HMRlj56) (see Table XIV D). NOTE TO USERS

Page(s) not included in the original manuscript and are unavailable from the author or university. The manuscript was microfilmed as received.

This reproduction is the best copy available. 9 -

ICI +i chi iA œ -

- a in LI

d tf L - iCI +I m d a -

\O +I 3 d œ - O i aO t-4 &- upi: > -

Baseline HMR1556 1.5 mg/ kg

A PCL (mec) Baseline

A 1.5 mglkg HMR1556

Y" 1 80 120 160 200 240 280 320 PCL (msec)

Figure 35. The ventricular effective refractory period (VERP) (A)and mean activation-recovery intervals (ARIw&T)(6) at each respective paced cycle length measured at baseline (m), and after administration of 1.5 m_&g HMRljS6 (A). PCC (msec)

Figure 36. The effect of 1.5 mb& HMR1556 on the percent change in right VERP measured from baseline in the intact guinea pig hem. Note the magnitude of effect does not diminish at the fastest pacing cycle lengths (i.e. 160 and 1 50 msec). Baseline

Figure 37. Dynamic restitution kinetics at baseline (U) and after administration of 1.5 mgkg HMR1556 (A) (n=l). The non linear regressions pre- and post- dnig treatment were fit to sigmoidal curves with R' values > 0.98. and Sy.x < 4.1 %. The steepest dope decreased by 15.7 % from 1.17 at baseline to 0.99 after dnig. 3.4.4. Administration of Combination 4 pelkg Dofetilide and 1 .j mg/kp,- HMRl556

3.4.4.1. Effects of Combination 4 udkg Dofetilide and 1.5 mgl/kg HMR 1556 on Electrophvsioloaical Measuremenis

Mean baseline and combination dofetilide and HMRlS56 values on measurements of VERP and ARlvaT are listed in Table XV A and B, respectively.

VERP at a cycle length of 300 msec increased by 19.2 Oh (from 156 msec at baseline to

186 msec after the combination of dofetilide and HMRI556). at a cycle length of 280 msec increased by 20.0 % (from 150 msec at baseline to 180 msec after the dnig combination), at a cycle length of 260 msec increased by 19.2 % (from 144 msec at baseline to 174 msec afier the combination). at a cycle length of XOmsec increased by

15.5 % (frorn 142 msec at baseline to 164 msec afier the combination). at a cycle lrngth of 210 msec increased by 13.2 ?O (from 136 msec at baseline to 154 rnsec afier the combination). at a cycle length of 200 msec increased by 14.1 % (from 138 msec at baseline to 146 msec after the combination), at a cycle length of 180 msec increased by

17.2 % (frorn 1 16 rnsec at baseline to 136 msrc aftrr the combination). at a cycle length of 160 msec increased by 12.5 % (from 1 12 msec at baseline to 126 msec after the combination). at a cycle length of 150 msec increased by 1 1 .j % (fiom I O4 msec at

baseline to 1 16 msec afier the combination). and at a cycle length of 140 msec increased

by 10.2 % (from 98 msrc at baseline to 1O8 msec after the combination) (Figure 38A and

39). Prolongation in VERP was greater with the drug cornbination than with either dnig

alone, particularly at the shortest paced cycle lengths (see sections 3 -4.2.2.1. and

3.4.3.1.). AMFdT at a cycle length of 300 msec increased by 12.0 % (fiom 173 t 1 msec at baseline to 193 + 1 msec after the combination of dofetilide and HMRl556). at a cycle length of 280 msec increased by 10.7 % (from 169 k 3 msec at baseline to !87 f 2 msec after the combination). at a cycle length of 260 msec increased by 7.9 % (from 163 +- 3 msec at badine to 178 k 2 mec aAer the combination). at a cycle length of 210 rnsec increased by 6.0 % (fiom 16 1 t 1 msec at baseline to 17 1 + 2 msec afier the combination). at a cycle length of 220 msec increased by 4.7 % (from 156 f 1 msec at baseline to 164 2 1 msec afirr the combination). at a cycle length of 200 msec increased by 7.2 % (from 147 t 2 msec at baseline to 158 t O msec aFter the combination). at a cycle length of 180 msec increased by 4.5 % (from 142 t 2 msec at baseline to 149 t 1 msec after the combination). ai a cycle length of 160 msec increased by 5.5 % (from 132 f 1 msec at baseline to 1 JO c 1 rnsec afier the combination). and did not change at a cycle length of 1 j0 msec (from 128 f 1 msec at baseline to 178 t 2 after the combination)

(Figure 388).

3.4.4.2. Dvnarnic Restitution Kinetics of the Combination 4 udkg Dofetilide and 1.5 mg /kg HMRljj6 Experirnent

The dynarnic restitution relation was determined for both the baseline and combination treatment phases (Figure 40). At baseline. the non-linear regression sigrnoidal fit has an R' value of 0.98% an Sx.y value of 2.8%. and a steepest dope of

1.17. After treatment of combination HMR1556 and dofetilide, the sigmoidal regression fit has an R' value of 0.9862. an Sx-y value of 1.4 %, and a steepest slope of 0.84. resulting in an decrease of 26.4 % from the dope determined at baseline. 3.4.4.3. Effects of Combination 4 pe/kg Dofetilide and 1.5 mg/ke HMRl556 on Hemodvnamic and Other Measurements

Baseline and post-treatment values olsystolic and diastolic pressures. hart rate and blood gases (pC02. PO,. and pH) for the 4 pgikg dofetilide and 1 .j mgkg HMR1556 combination experiment are indicated in Tables XV C and D. Systolic pressure decreased by 5.1 % (from 59 mmHg ai baseline to 56 mmHg post dofetilide and

HMR 1 556 administration). Diastolic pressure decreased by 7.0 % ( from 43 mmHg at baseline to 40 mmHg afier the combination). Heart rate also decreased by 2.8 % (from

192 bpm at baseline to 187 bpm aher the combination). pOz increased by 12.4 % (from

97 mmHg at baseline to 1 IO mmHg afièr the combination). pCOz increased by 18.2 %

(from 33 mmHg at baseline to 38 mmHg aftrr the combination). pH decreased by 0.7 O'O

(from 7-46 at baseline to 7.41 aAer the combination).

QRS duration between the baseline study and combination dofetilide and

HMRl556 treatment group increased by 2.2 % (from 46 + 3 msec at baseline at a pacing cycle length of 180 msec to 47 k 3msec afier the combination) (see Table XV D).

200 --, Baseline

A PCL (msec)

Baseline HMR7 556+Dofetilide

B PCL (msec)

Figure 38. The ventricular effective refractory period (VERP) (A) and mean activation-recovery intervals (ARIvdT) (B) at each respective paced cycle length measured at baseline (H). and after administration of combination 4 p_gkg dofetilide and 1-5 mgkg HMR 1556 (A). % Change in VERP Baseline HMRl556+Dofetilide

DI (msec)

Figure JO. Dynamic restitution kinetics at baseline (B) and after administration of combination 4 pg/kg do fetilide and 1.5 mgkg HMR 1 556 (A)(n= 1). The non linear regressions pre- and post-drug combination were fit to sigrnoidal curves with R' values > 0.98, and Sy.x values< 2.8 %. The steepest slope decreased by 26.1 % from 1.17 at baseline to 0.81 after drug treatment. 4. DISCUSSION

4.1. Isolated Lanpendorff Rabbit Hearts

The original hypothesis set fonh in this thesis was not correct. Ikr block with dofetilide was found to be additive to Ikl block with barium with respect to VERP,

MAPDgO.VFCL, and DFT. However, the effects of barium and sotalol (another Ikr blocker) in combination were not additive on any of the electrophysioiogic variables measured. In fact. the latter combination produces effects no greaiêr than rach dnig alone. To the author's knowledge, this is the fint study to show that different Ikr blockers can have differing effecrs when combined with another potassium channel blocker. These results suggest that in virro studies of the potential statc-dependence of

Ikr block have in vivo correlates (Waldo er cil., 1996. Kaber. 19%). and also suggest that combinations of Ik block may have differing effects. perhaps depending on the state of the Ikr channel being blocked (Carmcliet. 1993).

4.1.1 . Dofetilide Dose-Response Determination

Although the dose-response detemination of do fetilide on re fractoriness and DFT was based on single observations (n= 1 for each concentration), these were prelirninary experiments never previously conducted in an isolated heart modrl. The purpose of

performing a preliminary drug study in a concentration-dependent manner was to limit

the variability due to dose by approximating the dose of dofetilide that would produce

close to half-maximal effect (EC jo) on electrophysiological variables of cardiac

refractoriness and DFT. At the least. these studies provided a dose range within the dose-

response relation in which the effects of drug combinations could be linearly resolved.

The effect of drug combinations cm be modified by the concentration-effect relationship of each drug. If a drug is administered at a dose producing a close-to-maximal effect. any additional effects of a second drug in combination would not be observed due to prior saturation of the effect. Since the endpoint of this study was to determine the effects of combination dmgs, the additive effects (if any) could be resolved by administenng dmgs near. if not at their EC jo.

4. t 2. Selectivitv of Ikr Block with Dofetilide. Sotalol. and Bariurn

The Ikr channel is a time- and voltage-dependent potassium channel that opens rapidly within a few hundred milliseconds (Courtney et ul.. 1992). It possesses different states. including open, closed. and inactive (Kiehn et al.. 1999. Wang et al.. 1997).

Dofetilide has been show to be a potent and selective blocker of the open state of the Ikr channel (Kiehn rf ol.. 1996. Snyders and Chaudh.;. 1996. Spector et rd.. 1 996). and was therefore used as one of the probes in studying the effects of Ikr blockade on electrophysiologic parameters. However. there are conflicting interpretations in the literature regarding the state-dependence of Ikr block with sotalol. Numaguchi rf

(2000) found that d-sotalol block was not reduced in an inactivation-mutation of the human ether-a-gogo (HERG) K' potassium channel (whic h encodes a potassium channel believed to be the basis for the Ikr current). suggesting that sotalol does noi prirnariirily bind to the inactivated state. These investigators also postulated that d-sotalol accesses its target receptor in the open pore of HERG channels. Ficker and colleagues ( 1998) suggested that methanesuifonanilide binding (such as with do fetilide) is localized to the pore region of the HERG channel. However. studies in isolated guinea pig ventricular myocytes found that with the administration of sotalol. the amplitude of the tail current of

Ikr was already reduced fiom the Fust applied depolarkation and did not change with repetition of the clamp (Carrneliet. 1993). This points to the potential of sotalol blocking the rested (closed) state ot' Ikr. as activation of the Ikr channel was not required for channel block in the presence of drug. If sotalol were to preferentially block the open

state of the channel (such as dofetilide or E-403 1), its effects on prolonging repolarization

by Ikr block would not be apparent in the depolarization afirr a quiescent ceil state. the

tail current would gradually decrease with repetitive depolarizations. and block would

accumulate with time (in a use-dependrnt mamer). Hoaever. given the limited

experimental evidence of the state-selrctivity of Ikr block with sotalol. its state-

dependence of bloc k still remains unclear.

The Ikl channel is voltage-dependent and displays strong inward rectification at

potentials positive to the equilibrium potential for potassium (Courtney et al.. 1992).

Barium has long been considered a relatively selective blocker of the Iklchannel at the

concentrations reported in this thesis. and may have antiarrhythmic properties (Dorian rr

al.. 199Jb. Kavanagh er cil.. 1998). However. recent evidence suggests that barium also

blocks HERG channels (Weerapura er d..2000). Weenpura and colleagues reported

concentration-dependent inhibition of HERG tail current. with minimal effects at

concentrations of 1 and 10 ph.1 barium. and increased inhibition with 500 pM and 2 tnbl

barium concentrations. Although barium rnay bloc k other potassium currents at very

high concentrations. block of Ikr is minimal at the ECso of barium on refractoriness in

this thesis (3 FM) (Varma. 2000).

4.1 3. Effect of Ikr or Ik 1 Block on Elrctrophvsiological Variables

Each of dofetilide, sotalol. and bariurn prolong APD. VERP. and decrcase DFT in

the mammalian heart. In this study. both drugs (when used alone) had similar effects on prolonging refractoriness. measured by EKP, MAPD90. and VFCL. which were not found to be statistically significant from one another. but different from the saline-treated control group. This signified a prolongation of myocardial refractoriness, which was expected given that these drugs are known to possess class III antiarrhythrnic properties.

The class III effect of soialol in prolonging APD in this thesis is near half the

maximal effect seen in humans (tppically brtween 15 and 20 % APD prolongation)

(Edvardsson et al., 1980. Neuvonen et al., 198 1). In addition. the class III effects of

sotalol in humans are very similar to those of dofetilide. by prolonging refractoriness

(rneasured by APD and ERP) by close to equal arnounts (Montero and Schmitt. 1996).

consistent with the results of this thesis. The dose of bariurn administered also prolonged

APD by approximately half the maximal effect of APD prolongation seen in open-chest

anesthetized dogs (Dorian et tri.. 1994h 1. Thrrefore. the doses of cach drue in this study

produced effects that were near half-maximal effect on cardiac refractoriness. as

rneasured by ventricular ERP and MAPDqU.

At the approximate ECroof each drug on prolonging cardiac refractoriness. DFT

was found to decrease significantly. The decrease in DFT found with each drug was not

found to be significantly different From one another. but was different from control.

Prolongation in cardiac refractoriness may account for the obsemed decreases in DFT.

4.1 .J. Effect of Ikr or Ikl Block on Hemodvnamics and ORS duration

The range of baseline measurements of systolic and diastolic left ventricular

endocardial pressures were found to be similar to oiher studies of Langendorff-perfused

rabbit hearts (Dhein et ai.. 1993. GilIis et cd.. 1998). Barium. sotalol. dofetilide. and the

drug combinations did not significantly alter the force of cardiac contraction (as evidenced by a lack of change in lefi systolic pressure), heart rate. or coronary flow rate

fiom their baseline measurements.

These results are consistent wirh other studies of barium in both Langendorff

isolated rabbit hearts (Gillis et ai., 1998). which reported no change in coronary flow. as well as open-chest anesthetized dogs (Dorian et al., 1994b). which showed no change in heart rate. The effect of dofetilide on measures of cardiac function arc in agreement with those found in the literature. as this drug has not been show to alter blood pressure in dogs (Davis ci ai.. 1999) or man (Yuan ef al.. 1994. Bashir et ai.. 1995). Although

sotalol has not been shown to alter hean rate or rate of coronary flow in the same

eaperimental model. the lack of drue efkcts on hemodynarnics drtermined in this study

are in conflict with the results of Dhein and colleagues (1993). They found a significant

reduction in left ventricular systolic pressure aHer administration of sotalol in isolated

rabbit hearts. However. drug effects were assessed following a prolonged equilibration

period of one hour. after which timr viability of the myocardium is often compromised.

In both man and baboon. sotalol has been show to significantly decrease heart rate and

contractility (Mahmarian ef al.. 1990. Rogers et al.. 1982). In contrast. in vitro studies of

cat papillary muscle (Kauman and Olson. 1968) and rat atrium (Tandr and Rrfsum.

1988) have show an association between prolongation of APD and increased

contractility. Yet, the findings of this thesis, demonstrated a significant increase in .4PD

without an accompanying increase in conuactility. Therefore. it is uncertain whether

sotalol has no effect on the force of contraction in the isolated rabbit hem. or a type II

error occurred whereby contractility is indeed affected. although observed to be undtered

under the conditions of this study. 4.1 S. Combination Ikr and Ikl Blockade

Dofetilide and barium in combination yielded additive effects on cardiac refractoriness

(measured by APD. ERP. and VFCL) and DFT, whereas the combination of sotalol and barium did not. The former combination not only showed a substantial increase in

VFCL, but also displayed more organized homogeneous local activation patterns dunng fibrillation than either drug alone or the soialol and barium combination. Apart from producing additive effects on measures of refractoriness during paced rhythm. this phenomenon may be partly responsible for the decreased DFT (Dorian er al.. 1996).

Greater homogeneity of fibrillating wavefronts would increase the probability that a larger volume of the myocardium will be in a state of refractoriness. and therrfore electrically inexcitable. As a result. there will be greaier homopneity in temporal. and perhaps spatial refractoriness (a consequence of decreased temporal and/or spatial dispersion). facilitating the success of a drfibrillating shock. This has previously bern demonstrated in both the clinical and èxperimrntal srttings. Dop studies have show a close correlation between drug-induced refractory period extension (with dofetilide) and decreases in DFT (Davis er al., 1999). Dorian and colleagues also found a correlation between increases in activation intervals during fibrillation and decreases in defibrillation energy requirements in patients receiving sotalol (Dorian cr al.. 1994a).

4.1.6.Mechanisms of Combination Drug Effects

Previous studies have shown that two drugs acting on the same ion channel given in combination will produce additive (if not synergistic) effects if rach of the drugs binds to an independent site. or the sarne site on that channel. provided there has not been pior saturation of drug binding to the receptor. For example, the class 1 antiarrhythmic combination of propafenone and mexilctine (each binding to srparate sites on Na' channels) were found to produce effects greater than each drug alone on the percent suppression of fiequent premature ventricular contractions (Takanaka et ai,. 1992).

Another exarnple is the administration of two Ikr-selective blockers in combination studied in guinea pig ventricular myocytes (Sanguineni and lurkiewicz.

1990). These investigators found that the effects of combination d-sotalol and E-403 1 were no greater than each drug alone on reducing Ikr tail currents. However. each drug was administered at its maximal effective concentration ( IO0 pM d-sotalol ad 5 pM E-

403 l). Thus. additive effects would not be resolved in such a manner even if the. were to exist. as the effect of each drug on Ikr channels would be saturated with such high drug concentrations. Provided that the mechanism of closed-state Ikr block by sotalol is correct. each of the drugs may bind to different sites on the Ikr channel. but they would do so at different phases of the cardiac cycle: E-103 1 would bind to the open-state of Ikr during the AP (Basin and Lynch. 1994). and sotalol wouid bind to the closed-state. primarily between APs (Carmeliet. 1993). Therefore. ion channel binding site as well as the state of channel block are both factors to be considered in determining the nature of combination channel block.

4.1.7. Possible Mechanisms of Combination Ikr and Ik1 Block

The results of combination K' channel block in this thesis could by explained by three possible mechanisms: a) binding site cornpetition, b) allosteric interactions. and c) compensatory current densities. 4.1.7.1. Binding Site Com~etition

Although HERG block with barium rnay provide a potential explanation for the observed additive effects with dofetilide. it does not explain why the effects of sotalol and bariurn are not additive. One possible mechanism rnay be that barium and sotalol (but not dofetilide) compete for the same binding site on the Ikr channel. The maximal effect of bariurn on cardiac refractoriness (measured by APD and VERP) in this mode1 has been determined to be less than that of sotalol (Varma. 2000). Therefore, although the potencies of barium and sotalol rnay be very similar (as their EC on MAPDso are 3 and

4 PM. respectively). their efficacy (measurcd by maximal ERP and APD prolongation with maximal concentrations) for the Ikr channel rnay be very different. As such. barium rnay be acting as a partial agonist (a ligand that cannot elicit a mêuimal response even at very high concentrations) on the Ikr channel. Wlien given in combination with sotalol. barium would not produce additive effects as the particular binding site for barium and sotalol on the Ikr channel rnay have a greater affinity for one drug over the other.

However. this explanation is purely speculative as the findings of barium and HERG interactions is recent (Weerapura rf ab. 2000). and further research in particular binding sites of barium and sotaiol on the Ikr channel need to be explored.

Furthemore. 'chemical antagonisrn' rnay be mechanism involved in the reduced efficacy of the barium-sotalol combination. in that bariurn and sotalol rnay form a chemical complex. which would not allow one of the agents to interact with the Ikr charnel. Thus. when given in combination only one of the agents would rxert its effects.

Although this rnay be a possible explanation for the observed resultsl it is not a direct Ikr channel effect. It should also be noted that the experirnental system containing the receptor cm

influence the pattern of binding site competition, therefore these observed patterns may not necessarily denote molecular mechanism.

3.1.7.2. AlIosteric Interactions

Another mode of dnig interactions betwern barium and sotalol at the receptor

level can involve separate binding sites of these two agents on the Ikr channel. Under

such circurnstances. occupancy of barium on the Ikr channel does not precludr binding of

sotalol and vice-versa. The binding of bariurn or sotalol to the Ikr channel may produce conformational changes in the channel. rendering the second drug's binding

unfavourable. Such 'allosteric' interactions with barium would hinder the binding of

sotalol to the Ikr channel (and vice-versa). resulting in non-additive effects in the

presence of this dnig combination.

4.1 -7.3. Conipensaton: Crirrcnt Densitiss

An explanation of these novel findings may involve the voltage kinetics of the Ikr

channel. Block of Ikl channels by barium may sliphtly depolarize cardiac cells. Such a

decrease in membrane potential to a lrss nrgative potential may drive Ikr channels into

the open state. resulting in greüter outward K- condiictance through the Ikr channel at any

given voltage. In other words. slight depolarization of the resting membrane potential by

Ikl block with barium will allow for accumulation of Ikr channels in the open state.

producing a greater overall net Ikr current. If more Ikr channels are in the open state in

the presence of barium, thrre would be less closed Ikr channels to which sotalol cm bind.

resulting in a diminished sotalol effect (Strauss et al., 1970, Hondeghem and Snyders.

1990). This postulated mechanism is supported by studies showing a reduced sotalol effect in the presence of high extracellular K' concentration (Cobbe et a/.. 1985. Cobbe.

1988, Baskin and Lynch, 1994). High extracellular K' has been shown to slightly depolarize the resting membrane potential in dogs (Babbs et al., 1980). and may account for decreased effectiveness of sotalol. as this agent will bind to less available closed-state

Ikr channels (Carmeliet. 1993). If combined administration of an Ikl channel blocker and a closed-state Ikr channel blocker is not additive, then the combination of dofetilide

(an open-state Ikr blocker) (Cmeliet. 1991. Yang et al.. 1994) and barium would be

expected to produce additive (if not synergistic) e ffects on prolongation of cardiac

repolarization. and perhaps reduction in DFT. This would be tme since more Ikr channels would be 'forced' to the open state by a slightly depolarized membrane

potential with Ikl block. and more of the Ikr blocker would be able to exert its class III

actions.

Furthemore. in the presence of Ikl block by bûrium. Ikr currents may increase in

magnitude as a tùnction of' their voltage- and time-dependent properties, and as a

consequence of the delay in repolarization caused by Ikl block. Mechanistically. open-

state Ikr block with dofetilide rnay reduce thrse "compensatory" increasrs in Ikr (inducrd

by Ikl block) which rnay occur during the late repolarization phase of the AP. The result

would be additive class III effects of dofetilide and barium. On the other hmd. if sotalol

blocks Ikr (in part) in the closrd state. sotalol may not diminish recruitrnent of additional

Ikr current Iatr in the AP. thus allowing relative increases in Ikr to cornpensate for

reduced Ikl current (resulting fiom ikl block with barium).

In summary, regardless of the blocking properties of bariurn on repolarizing

potassium currents, this study suggests that dofetilide and sotalol act differently on the Ikr charnel (or have non-Ikr related effects), and that this difference can have important consequences under conditions resulting in a diminished Ikl repolarizing potassium current.

4.1.8. Relevance of Ikr block in the Presence of Diminished Ikl current

IkI currents have been show to be diminished in conditions of terminal heart failure in humans (Beuckelmann et al.. 1993) dogs (O'Rourke es ai.. 1999). and rabbits

(Tsuji et ul., 2000). The results of this thesis suggest that dofetilide and sotalol differ in their effectiveness in prolonging cardiac refractoriness in cardiac diseased States. To the knowledge of the author. little esperimental evidence exists conceming the actions of class III antiarrhythrnics. such as dofetilide or sotalol. in models of induced hart failure.

Studies in rabbit and cat hearts have shocvn that dofetilide had less of an effect on APD prolongation in hypertrophic compared to control hearts (Kowey er ol. . 199 1. Gillis et al..

1998). However. these studies involved dofetilide doses in the millimolar range. where dmg toxicity is more likely to occur. However, a few clinical trials provide insight to the rfficacy of these two Ikr blockers in patients with diseased hearts. The effect of dofetilide on mortality and morbidity in patients with congestive heart failure was studied in the DIAMOND trial (DIAMOND Investigators. 1977. Kaber. 1998). Prelirninary results showed a neutral effect on mortality. and a relatively Iow incidence of TdP. This contrasts with the SWORD (Survival with Oral D-Sotalol) study. in which there was increased total mortality in post-myocardial infarciion patients treated with d-sotalol

(Waldo el d..1 996). However. sotalol has been shown to be more effective than many class 1 antiarrhythmic agents with respeci to total mortality, sudden death, cardiac death, and VT recurrence (Manson. 1993b). The lack of effectiveness of selective Ikr blockade with d-sotalol in patients of the SWORD trial could be explained in part with the results of this thesis. Dofetilide may bave been effective in reducing mortaiity and maintaining patients in sinus rhythm since its effects of prolonging cardiac refiactoriness persist in the presence of diminished Ikl cunents. On the contras. blockade with d-sotalol (the racemate responsible for class III actions) rnay have been clinically ineffective since it was administered to patients with diseased hearts. possibly possessing downregulated Ikl channels. resulting in diminished Ikl current density. However. the effectiveness of sotalol compared to its d-isomer rnay be due primarily to its P-blocking properties. although not many studies have been performed with sotalol. and the mechanism of its effectiveness stili remains unclear.

4.2. Intact Guinea Pip. Hearts

This study validated thc guinea pig ris a feasible mode1 for rlectrophysiologic experimrnts of cardiac refractoriness. It is stable over time and with the addition of drug vehicles (saline or organic solvents). Although the results of the drug study in intact guinea pig hearts were obtained in single observations (n=l per dmg concentration). the data suggests that the hypothesis postulated in this thesis regarding Ikr and Iks blockade may be correct. Ikr block displayed reverse rate-dependence with a more pronounced prolongation of VERP at longer than at shortcr paced cycle lengths. while Iks block was found to exhibit less reverse rate-dependence than Ikr block. Combination Ikr and Iks block resulted in a greater prolongation of ERP at each paced cycle length than selective blockade of either channel alone. and displayed less reverse rate-dependence than with

Ikr block alone. 4.2.1. Measures of Cardiac Refiactoriness in Control Expenments

To the knowledge of the author, an in vivo guinea pig model has not been employed to determine the effects of class 111 antiarrhphrnic agents on cardiac refiactoriness measured by ERP. Furthemore. this is the first study (although results are preliminary) to assess the effects of a phmacological intervention on dynamic

restitution kinetics in an in vivo model.

4.2.3. Seiectivitv of Agents for Potassium Channel block

Dofetilide has been shown to be a potent. and selective blocker of Ikr channels in

the open state (as discussed earlier in %olated Langendorff Rabbit Hearts"). HMR1556

has recently been developed, and has been found potently and selectively block Iks

currents with an Goof 34 nM in guinea pig ventricular myocytrs (Gogelein et al..

2000). .4t a concentration approximately 30 fold grrater ( IO pM). it was found to block

Ikr and Ikl only slightly. and moderately drcrease Ito. and the L-type 1Ca currents by no

more than 25%.

4.2.3. Effect of Ikr andior Iks Block on DFT. ERP. and FFF

Dofetilidr ai doses of 8 and J pp'kg were found to decrease DFT in each

experiment by 29 and 25%. respectively. consistent with results of in vivo studies in open

chest dogs (Davis et al.. 1999). However, in the mode1 of testing DFT and cardiac

refiactoriness. dofetilide at each dose either decreased or did not change VERP. contrary

to its known class III effects. Experiments in which the dofetilide effect on DFT was not

evaluated. and thus defibrillation shocks were not delivered. were valid in assessing the

e ffect of do feti lide on prolonging cardiac re fractoriness. Shock-induced catecholarnine

release may be responsible for the ineffectiveness of dofetilide in prolonging ERP in these prelirninary experiments. This is consistent with the results of Marschang and colleagues (2000), who showed that adrenergic stimulation (with isoproterenoi) antagonized the class III action of do fetilide in isolated canine cardiomyocytes.

The intact guinea pig hem was paced at multiple cycle lengths to detemine the effect of Ikr or Iks block on cardiac refiactoriness measured by VERP and FFF,,. At a doses of 8 and 4 pg/kg, dofetilide was found to proiong VERP by 20 and 15% at the slowest paced hem rate. and by O and 6% at the tàstest paced heart rate. respectively.

HMRl556 at a dose of 1.5 mgkg was found to prolong VERP between approximately 1O and 6 % at near!y al1 paced cycle lengths. Therefore, dofetilide and HMR1556 were each found to prolong VERP in a reverse and non-reverse rate-dependent manner. respectively. These results are consistent with the experimental findings that dofetilide acts in a reverse rate-dependent fashion in dogs (Bauer rr trl.. 1999) and hurnans

(Derakhchan et aL .200 1. Yang et di.. 1Wa). In addition. Bauer and colleagues found positive rate-dependence on cardiac refractoriness with Chromanol739b. an Iks blocker with less selectivity and potency than HMRl556. in guinea pig ventricular myoc'es

(Bauer et ai.. 1999. Gogelein et al.. 2000). Furthermore. HMR1556 and dofetilide given

in combination (at 1.5 mgkg and 4 pg/kg respectively). prolonged VERP to a greater

degree than either drug alone. Although the effect was not additive at slower heart rates.

the dmgs displayed rate-dependent. and perhaps additive effects. in combination at faster

rates. This is consistent with the concept that two agents acting at two pharmacologically

distinct targets would each produce an effect. The lack of additive effects at slower rates

may be due to a dose-effect, whereby dofetilide may have been given at a dose producing near maximal effect, and thus any prolongation in VERP by another agent would not be resolved due to prior saturation of the effect.

Each dose of each drug increased the shonest cycle length at which 1 :1 capture was maintained (FFFln)by 10 msec. which was slightly greater than that found in the control (saline) group (3 + 5 msec). However. the combinntion of HMRl556 and dofetilide (at the doses stated previously) was found to prolong FFFlnby 30 msec frorn baseline measurements. The measurement of FFF,, represents ventricular functional refractoriness. and the increase in this parameter suggests that the dmgs in combination have a greater effect on limiting the fastest possible ventricular rates and thus rnight be more effective in preventing the establishment of a tachycardia which requires adaptation of refractory periods for tachycardia maintenance (Vinet rr tri.. 1996).

4.2.4. Effect of Ikr and/or Iks Block on Dynamic Restitution Kinetics

The kinetics of dynamic restitution were assessed by plotting values of ARIp,~r

versus their corresponding Dis. and drtemining the steepest slope of the fitted cun7e

pnerated by a Boltzmann sigmoidal equation. Dofrtilide at 8 and 4 pg/kg increased the

sreepest dope of the restitution relation to 1 .l3 and 1 54 respective1y. Although one

would expect a class III antiarrhythmic agent (whose principal mechanism of action is to

prolong APD) to flatten the dope of the DR relation to a value less than unity, these

results are somewhat intuitive. Dofetilide is well-know-n to display reverse rate-

dependence. as show by other investigators as well as in this thesis. It is also known to

be a proarrhythmic agent at bradycardic rates by mechanisms arising from EADs and

triggered activity, increasing the propensity to TdP. The dynamic restitution relation thus characterizes dofetilide as an agent that may promote wavefront splitting during a tachycardia, possibly due to its reverse rate-dependent effects.

However, Hh!R 15% decreased the sterpest slope of the DR relation to 0.99. In combination with dofetilide (4 pg/kg) the slope was evcn more sliallow. at 0.84. Given that drugs exerting reverse rate-dependent effects will increase the slope. a drug exhibiting less reverse rate-dependence would be expected to decrease the slope. as its effects persist at fast. as well as slow hem rates. The drugs given in combination flatten the restitution relation more than each dmg alone. suggesting that the propensity to VF induction is low as APD altemans would not persist. This could be explained by the additive effects of the drugs in combination on cardiac refractoriness (at fast rates). as measured by ERP. Prelirninq resdts suggest that a greater prolongation of refractoriness may occur rit both slow and fast rates with combination Ikr and Iks block.

Dmg effects on DR have been studied in perfused tissue, where DAM and cytochalasin D (uncoupling agents) were found to fiatten (Garfinkel et ci/. . 2000). and

increase (Banville and Gray. 2000) the restitution slope. respectively. Garfinkel and

colleayues (2000) alsn found high concentrations of bretylium to organize the

morphoiogy of the VF pattern. as well as decrease the slope of the DR relation less than

unity.

With respect to Krchannel blockrrs and their effects in this study. when APD

under the influence of a drug shortens faster as rate increases than at baseline

measurements (reverse rate-dependence) the steepest DR dope wiil be 2 1. However. if

APD (prolonged by a class III antiarrhythmic) shortens to the same extent as in the dnig

free state. the DR slope will be less than unity. 4.3. Limitations

The studies in this thesis (in part) involved healthy rabbit hearts, and it is

important to exercise caution when extrapolating conclusions cf such an experirnental setting to diseased heart states in a physiologic environment. The rabbit hearts in this dicsis were continuously perfused throughout the entire experimental protocol. and VF

induction in shon duration may not have permitted myocardial ischemia. However, the

in vivo heart becomes ischemic during fibrillation. resulting from decreased cardiac

output. Many antiarrhythmic agents. such as sotalol and dofetilide. lose their class III

effects of APD prolongation in the presence of high extracellular K' concentrations

(Cobbe et al., 1985. Baskin and Lynch. 1994). as seen in ischemic cardiac tissue.

However. ischemia does not alter DFT. or drug-induced changes in DFT (Cames and

Mehdirad. 2000. Echt et al.. 1989b. Yakaitis et al.. 1975). Even though ischemia was not

a parameter measured in this thesis. its effects would have been apparent in abolishing

drug-induced prolongation of re frac toriness.

The isolated, denervated rabbit heart used for the Langendorff experiments did

not have the influence of the auionomic nervous system on drug effects. Ikr blockers

have been show to be less effective in prolonging repolarization with increased

sympathetic activity (Vanoli et al., 1995). Furthemore, the decreased tissue viability

after prolonged pacing trains does not allow expenmentation of the rate- or reverse rate-

dependent effects of antiarrhythmic drugs.

Sotalol used in this study has beta blocking as well as 'pure class III' effects.

However, P-blocken have no significant effects on DFT or APD in isolated hearts (Sharma et al., 1983), and thus the P-blocking moiety in sotaloi is unlikely to account for the differential interaction with barium.

In addition, sotalol, aithough suggested to bind to the closed state of the Ikr channel, has not been clearly defined as a state-selective Ikr blocker as there is limited patch-clamp data on its state-dependent blockade. There may also exist other mechanisms underlying the interaction that have not yet been explored. and the differential effect of sotalol and dofetilide may not only be due to state-dependent Ikr channel block.

Although the guinea pig mode1 has been show to be very stable ovrr time and with the addition of drug vehicle in measuring DFT. VERP and DR. definitive conclusions on the effects of Ikr and/or Iks block in the intact guinea pig hean cannot be made. since cach of the drug doses ber<:srudied in one animal.

The ineffectiveness of class III antiarrhythrnic Ik.block on cardiac refractoriness cannot be assessed in expenments where DF shocks have been delivereù (either for DFT determination or reinitiation of sinus rhythm after VF-induced rapid pacing). Shock- induced catecholarnine release rnay be responsible for the lack of ERP prolongation. previously determilied in rxperimental preparations under adrenergic stimulation

(Marschang et al.. 2000).

The only DR studies performed have been in isolated perfused preparations. DR kinrtics have never been tested in an intact beating heart where electrophysiology may be influenced in part by neurohumoral and autonomic effects. as vie11 as hemodynamic and anatomical differences. Furthennore, although both models employed in this thesis represent a Iess than ideal complex systern. the hearts of guinea pigs and rabbits do exhibit similarities to the human heart, and in particular, possess similar repolarizing K+ channels. although their current densities may differ (Giles and imaizumi. 198 8). 4.4. Conclusions

The results of this thesis confirrn (in part) the hypothesis that different selective

Ikr blocken cmhave different effects on cardiac refractoriness (measured by VERP.

MAPD9a,and VFCL) and DFT in the mammalian heart in the presence of reduced Ikl current.

Administration of dofetilide and barium in combination has additive effects on

VERP. MAPDgO,VFCL and DFT. whereas the effect of sotalol and bariurn in combination is no different on these parameters than with eithrr drug alone.

Furthemore. in this thesis the guinea pig intact heart has been validated as a rnodel for the study of class III antiarrh'hmic agents. their rate-dependence, and their effects on dynamic restitution kinrtics. Prelirnin.; results suggest that Ikr and Iks block in combination can be more effective in prevtntinp the grnesis of reentrant arrhythrnias. perhaps by an additive effect on prolonging ERP at multiple cycle lengths. eliminating reverse rate-dependent effects of Ikr block alone. 4.5. Recommendations for Future Research

To confirm the postulated mechanism that sotalol in part blocks the Ikr channel in a closed state, the combination of sotalol and an open channel blockrr (such as dofetilide or E-403 1) could be administered in ventricular rnyocytes each at their ECjo concentrations and the degree of current block could be measured. If sotalol is in fact a closed-state Ikr channel blocker, the combination of dofetilide and sotalol would be

additive in prolonging cardiac refractoriness as measured by intracellular APD and

VERP. Although previous studies have shown sotalol and E-403 1 to have no additive

effects. this is mainly due to a dose effect (Le. drugs were given each ai their maximal

effective concentration. resulting in a saturation of rffect) (Sanguinetti and Jurkiewicz.

1 990).

It is also essentiaI to detemine if the additive effects of Ikr and Ik1 block found in

this thesis are altered by autonornic reflexes by assessing their effects in an in vivo animal

model. Such data wiil providc a better prediction of the rffect of (possible state-

dependent) Ikr in combination with lkl block in humans.

The class III effect of dofetilide on prolonging VERP was shown to be abolished

in the presence of DF shocks. prrhaps in part due to catecholarnine release. In vitro

studies have shown that in the presence of adrenergic stimulation with isoproterenol.

HMRl556 displays positive rate-dependence. in that its class III action of prolonging

cardiac retiactonness is niuch greater at faster than at slower heart rates (Gogelein er al..

2000). Such an action would allow for the assessment of DR as well as inducibility of

VF in in vivo studies. within the same animal. This would confirrn DR in the intact heart

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