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Cardiac safety assessment of NBI-1: Potential role of IKs potassium channel in the observed QT prolongation detected in vivo
240
Abstracts / Journal of Pharmacological and Toxicological Methods 60 (2009) 210–258
written about mouse ECG, some important issues remain unresolved (e.g., which leads, frequency characteristics of the ECG, genesis and normal values of component deflections and intervals, differences among strains, patterns indicating chamber enlargement, myocardial disease and drug toxicity). To address the above issues, orthogonal lead ECGs were recorded from 6 strains, but only 2 (C-57BL, FVB) will be reported here. Recordings were made on isoflurane-anesthetized mice as they lay in a closed Faraday cage with limbs coupled to leads via electrode gel producing a low-impedance interface. In addition to conventional measurements of component deflections, ECGs were signal averaged, Wilson equilateral vectorcardiograms were generated, and frequency components of QRS complexes were quantified by fast Fourier analysis. Vectorcardiographic loops were superimposed on NMF images of the mouse torso/heart, to attempt to identify depolarization of what structures generates components of body surface potentials. Microdeposits of formalin, producing minute localized areas of infarction/fibrosis, were made into various regions of the heart, and the ECG was able to localize the regions of injury. Pulmonic or aortic stenoses were used to produce univentricular hypertrophy, and distributions of body surface potentials demonstrated the hypetrophy but in a manner typical of that for larger mammals. Finally mice were given, IP, isoproterenol that produced subendocardial and papillary muscle necrosis which could be identified, when acute, by J-point deviation, and when chronic by increase in high-frequency components to QRS. A few of the unusual features of the mouse ECG are: frequency components of the QRS complex often reach 900 Hz, median frequency of components exceeds 350 Hz, estimations of vector orientations by scalar electrocardiography is virtually impossible (vectorcardiography is required) due to asynchrony of deflections in scalar leads, it is difficult to predict pathways of cardiac activation with forms of body surface potentials as in larger mammals. Despite profound differences in apparent pathways of ventricular depolarization and in configurations of body surface potentials, the ECG can be highly useful in identifying and quantifying, non-invasively, drug-induced cardiac pathology and/or pathophysiology.
doi:10.1016/j.vascn.2009.04.127
compared to females. This study provided data to validate the rabbit as a GLP model for detecting pharmacological changes in CV parameters for safety pharmacology studies at our facility. doi:10.1016/j.vascn.2009.04.128
The pharmacodynamic/pharmacokinetic relationship of diltiazem in conscious telemetered rats Thomas Vinci⁎, Brian Roche, S. Peter Hong, Brandon Wood, Jeremy Smith, Alecia Peppers, Jessica Wise, LaTonya Mitchell, Jerry Johnson, Steve Graves, Craig Hassler Battelle Memorial Institute, Columbus, OH, United States Introduction: Collecting blood samples from freely moving animals while simultaneously collecting CV or hemodynamic data via telemetry has been discouraged based on the effect the manual (hands on) blood collection procedure has on the CV data. Using the Culex/Empis automated device to collect blood and/or to dose animals in a telemetry study is a model which allows simultaneous collection of CV and PK data in a relatively low stress environment. Objective: To evaluate 3 doses of diltiazem, administered iv by the Empis device, on HR, PR interval and BP via telemetry while simultaneously and without human intervention, collecting post dose blood samples using the automated Culex device in conscious freely moving male rats. Methods: Rats were instrumented with telemetry for measuring ECG and blood pressure as well as a blood collection catheter in the carotid artery and a dosing catheter in the jugular vein. After connection and acclimation to the Culex/Empis device the animals were dosed with 3 doses of diltiazem (3, 5, and 10 mg/kg) with sufficient washout between doses. Results: Low Dose: slight to no change in HR or BP. Mid Dose: decrease in HR and BP, increased PR. PK was variable in extent and duration. High Dose: greater decreases in HR and BP and for longer duration, PR prolongation, AV block. Atypical PK profiles were associated with the significant decreases in HR and BP. Conclusion: The high dose of diltiazem significantly decreased HR and BP resulting in altered pharmacokinetics.
Validation of the rabbit telemetry model for cardiovascular safety pharmacology studies
doi:10.1016/j.vascn.2009.04.129
Kimberly S. Bynum⁎, Rhea Pittman, Graig Turson, Teresa Gleason, Timothy Edwards, Kasey Landis, Philip Atterson WIL Research Laboratories, LLC., Ashland, OH, United States
Cardiac safety assessment of NBI-1: Potential role of IKs potassium channel in the observed QT prolongation detected in vivo
The rabbit cardiovascular (CV) model presents a Safety Pharmacology paradox: ideal for pre-clinical studies in that they share with humans the ion channels that control ventricular repolarization, yet widely underused in pre-clinical testing. Highly responsive to IKrblocking drugs, rabbits may be particularly sensitive for drug safety screening. Four male and four female rabbits surgically implanted with DSI TL11M2-C50-PXT transmitters were dosed by subcutaneous injection with vehicle, 2.5, 10 or 40 mg/kg dipyridamole using a Latin square dose design. Body temperature, heart rate, arterial blood pressure waveforms and ECG waveforms were collected for 15 s of every 10 min for 1 h pre-dose and for 24 h post-dose. Following a qualitative review, quantitative ECG waveform analysis was performed to determine PR, QRS, RR, and QT values. Administration of dipyridamole resulted in higher heart rate (from 1 up to 14 h), lower blood pressures (from 1 up to 10 h), prolonged RR interval (from 5 up to 14 h), shortened QT interval (from 1 up to 16 h) and lower body temperature (from 1 up to 10 h) as compared to vehicle control, with slightly longer duration of effects in males as
Aida Sacaan⁎, Shirley Lio, Haig Bozigian Neurocrine Biosciences, San Diego, CA, United States NBI-1 is a compound developed for the treatment of CNS diseases. In preparation for first in Man clinical trials, a battery of cardio safety assessment was performed. NBI-1 displayed very weak activity on hERG channel current, inhibiting it by less than 1% at 300 µM. NBI-1 was also tested in the canine Purkinje fiber assay where it did not induce any change in the action potential duration (APD), or in any other parameter at concentrations up to 700 µM. With a clean in vitro profile, NBI-1 was advanced into in vivo testing where it was orally administered (10–200 mg/kg) to telemetered cynomolgus monkeys to assess its effect on ECG and hemodynamic parameters. NBI-1 up to 200 mg/kg had no consistent effect on blood pressure or heart rate. By contrast, administration of NBI-1 resulted in dose-related increases in QTc interval at doses ≥10 mg/kg. This occurred within 1 h and lasted up to 22 h postdose. This unexpected result indicated that either a metabolite has activity at the hERG channel or that the
Abstracts / Journal of Pharmacological and Toxicological Methods 60 (2009) 210–258
parent compound or its metabolite(s) act on non-hERG cardiac channels to produce this QT prolongation. NBI-1 has a diversified metabolic pathway and testing its major metabolite did not reveal any activity on hERG channel. NBI-1 was then tested in HEK cells expressing human IKs or IK1 potassium channels. Results indicated that whereas NBI-1 was inactive against IK1 channels, it produced a dose dependent inhibition (up to 43.1%) of IKs current suggesting that NBI-1 may be an IKs blocker which may explain its QT prolongation in vivo. doi:10.1016/j.vascn.2009.04.130
Simultaneous cardiovascular and pharmacokinetic data collection in freely moving guinea pigs Brian M. Roche⁎, Thomas Vinci, S. Peter Hong, Jeremy Smith, Brandon Wood, Jerry Johnson, Steve Graves, Craig Hassler Battelle Memorial Institute, Columbus, OH, United States The pharmaceutical industry is placing more emphasis on eliminating compounds with deleterious side effects earlier in the development process. Combining methodologies to maximize the desired endpoints (i.e., BP, ECG, PK, etc.) from a single study would be an efficient paradigm for early decision making. Additionally, utilizing a smaller animal with cardiac ion channels similar to humans would provide a more relevant and test article efficient model. Automation allows for dosing and blood collection to occur when appropriate without human presence while minimally affecting cardiovascular endpoints. Combining the use of the Culex/Empis automated device to collect blood and/or to dose guinea pigs with a cardiovascular telemetry study is a model that enhances the amount of data (knowledge) about a potential new drug from a single study. We evaluated the use of guinea pigs, instrumented with a telemetry device capable of collecting ECG, blood pressure and body temperature, in conjunction with the Culex/Empis automated device to establish the PK/PD relationship of several known compounds: QT prolonging agent, terfenadine; hypotensive agent, nitroprusside; hypertensive agent, phenylephrine; chronotropic agent, zatebridine; and negative QT control enalapril. doi:10.1016/j.vascn.2009.04.131
Effect of pacing rate and beta blockade on cardiac refractory periods and his-bundle conduction Jinbao Huang CorDynamics, Inc., Chicago, IL, United States Stimulation of the sympathetic nervous system releases norepinephrine, activating beta receptors and triggering increases in heart rate (HR) and atrioventricular (AV) conduction velocity. Metoprolol is a selective beta receptor blocker, which causes a decrease in sinus HR and slows AV conduction. The purpose of this study was to assess the effects of pacing and beta blockade on cardiac electrophysiologic parameters in the anesthetized dog. Two bipolar plunge electrodes were used to determine atrial and ventricular refractory periods (AERP and VERP), as well as to record atrial electrograms. A multipolar electrophysiology catheter positioned distal to the bicuspid valve was also used to record His bundle electrograms to determine atria to His (AH) and His to ventricle (HV) interval measurements. The AV nodal refractory period (AVNERP) was also calculated. In nontreated dogs (n = 11),
241
AVNERP and AH intervals were prolonged at 180 bpm compared to 120 bpm (AVNERP: 201 ±17 vs. 192 ± 18 msec and AH: 128 ± 6 vs. 119 ± 5 ms, respectively). Atrial ERP, VERP and HV intervals were similar when hearts were paced at 120 and 180 bpm. Treatment with 0.1, 0.3 and 1.0 mg/kg metoprolol dose-dependently decreased sinus HR (10 to 20% from baseline) and increased AVNERP (10 to 25% from baseline) with 180 or 120 bpm pacing. Metoprolol also prolonged AH conduction time. In conclusion, increasing pacing rate prolongs AVNERP and AH conduction time. In contrast, beta blockade decreases HR, but also increases AH interval, suggesting slowing of supraventricular conduction.
doi:10.1016/j.vascn.2009.04.132
Induction of Torsades de Pointes by FPL64176, DPI-201106, dofetilide, and chromanol 293B in isolated guinea pig and rabbit hearts Hsien C. Cheng⁎, Josephine Incardona Sanofi-Aventis, Bridgewater, NJ, United States Introduction: For predicting torsades de pointes (TdP) liability of a compound, most of previous studies have used surrogate markers such as hERG inhibition or QT prolongation which may or may not lead to TdP. In this study, we have used isolated hearts for testing induction of TdP. Method: Spontaneously beating isolated guinea pig and rabbit hearts were perfused according to the Langendorff method in hypokalemic (2.1 mM) solution. Lead II ECG and incidence of TdP were monitored for 1 h. Results: FPL 64176, a calcium channel activator, and DPI-201106, a sodium channel modulator, produced TdP in isolated guinea pig and rabbit hearts in a concentration dependent manner; guinea pig hearts were more sensitive than rabbit hearts. In contrast, dofetilide, an IKr inhibitor, gave a low incidence of TdP in guinea pig hearts (1/24), and no TdP at all in rabbit hearts. Chromanol 293B, an IKs inhibitor, elicited TdP concentration dependently in guinea pig but not in rabbit hearts. Conclusion: Thus IKs inhibition, rather than IKr inhibition, appears to be critical in inducing TdP in isolated guinea pig hearts. Rabbit heart did not produce TdP by IKs inhibitor presumably due to a low level of IKs channels in the heart. TdP produced in this study are consistent with the notion that their production may be a consequence of reduced repolarization reserve, thereby causing ventricular abnormalities (Roden, J internal Med., 2006; Jost et al., A.N.E., 2007). Thus, these isolated guinea pig and rabbit hearts may be useful for predicting TdP liability of compounds in drug development.
doi:10.1016/j.vascn.2009.04.133
Possible role of the ultra-rapid delayed rectifier potassium current (IKur) in action potential repolarization in rabbit heart Carol Wilson⁎, Samantha Robinson, Sara Graham, Nick McMahon, Bronagh Heath GlaxoSmithKline, The Frythe, Welwyn, Hertfordshire, United Kingdom The ultra-rapid delayed rectifier K+ current (IKur) plays an important role in early cardiac action potential repolarization in a number of species. Although the rabbit heart has proven useful for
Abstracts / Journal of Pharmacological and Toxicological Methods 60 (2009) 210–258
written about mouse ECG, some important issues remain unresolved (e.g., which leads, frequency characteristics of the ECG, genesis and normal values of component deflections and intervals, differences among strains, patterns indicating chamber enlargement, myocardial disease and drug toxicity). To address the above issues, orthogonal lead ECGs were recorded from 6 strains, but only 2 (C-57BL, FVB) will be reported here. Recordings were made on isoflurane-anesthetized mice as they lay in a closed Faraday cage with limbs coupled to leads via electrode gel producing a low-impedance interface. In addition to conventional measurements of component deflections, ECGs were signal averaged, Wilson equilateral vectorcardiograms were generated, and frequency components of QRS complexes were quantified by fast Fourier analysis. Vectorcardiographic loops were superimposed on NMF images of the mouse torso/heart, to attempt to identify depolarization of what structures generates components of body surface potentials. Microdeposits of formalin, producing minute localized areas of infarction/fibrosis, were made into various regions of the heart, and the ECG was able to localize the regions of injury. Pulmonic or aortic stenoses were used to produce univentricular hypertrophy, and distributions of body surface potentials demonstrated the hypetrophy but in a manner typical of that for larger mammals. Finally mice were given, IP, isoproterenol that produced subendocardial and papillary muscle necrosis which could be identified, when acute, by J-point deviation, and when chronic by increase in high-frequency components to QRS. A few of the unusual features of the mouse ECG are: frequency components of the QRS complex often reach 900 Hz, median frequency of components exceeds 350 Hz, estimations of vector orientations by scalar electrocardiography is virtually impossible (vectorcardiography is required) due to asynchrony of deflections in scalar leads, it is difficult to predict pathways of cardiac activation with forms of body surface potentials as in larger mammals. Despite profound differences in apparent pathways of ventricular depolarization and in configurations of body surface potentials, the ECG can be highly useful in identifying and quantifying, non-invasively, drug-induced cardiac pathology and/or pathophysiology.
doi:10.1016/j.vascn.2009.04.127
compared to females. This study provided data to validate the rabbit as a GLP model for detecting pharmacological changes in CV parameters for safety pharmacology studies at our facility. doi:10.1016/j.vascn.2009.04.128
The pharmacodynamic/pharmacokinetic relationship of diltiazem in conscious telemetered rats Thomas Vinci⁎, Brian Roche, S. Peter Hong, Brandon Wood, Jeremy Smith, Alecia Peppers, Jessica Wise, LaTonya Mitchell, Jerry Johnson, Steve Graves, Craig Hassler Battelle Memorial Institute, Columbus, OH, United States Introduction: Collecting blood samples from freely moving animals while simultaneously collecting CV or hemodynamic data via telemetry has been discouraged based on the effect the manual (hands on) blood collection procedure has on the CV data. Using the Culex/Empis automated device to collect blood and/or to dose animals in a telemetry study is a model which allows simultaneous collection of CV and PK data in a relatively low stress environment. Objective: To evaluate 3 doses of diltiazem, administered iv by the Empis device, on HR, PR interval and BP via telemetry while simultaneously and without human intervention, collecting post dose blood samples using the automated Culex device in conscious freely moving male rats. Methods: Rats were instrumented with telemetry for measuring ECG and blood pressure as well as a blood collection catheter in the carotid artery and a dosing catheter in the jugular vein. After connection and acclimation to the Culex/Empis device the animals were dosed with 3 doses of diltiazem (3, 5, and 10 mg/kg) with sufficient washout between doses. Results: Low Dose: slight to no change in HR or BP. Mid Dose: decrease in HR and BP, increased PR. PK was variable in extent and duration. High Dose: greater decreases in HR and BP and for longer duration, PR prolongation, AV block. Atypical PK profiles were associated with the significant decreases in HR and BP. Conclusion: The high dose of diltiazem significantly decreased HR and BP resulting in altered pharmacokinetics.
Validation of the rabbit telemetry model for cardiovascular safety pharmacology studies
doi:10.1016/j.vascn.2009.04.129
Kimberly S. Bynum⁎, Rhea Pittman, Graig Turson, Teresa Gleason, Timothy Edwards, Kasey Landis, Philip Atterson WIL Research Laboratories, LLC., Ashland, OH, United States
Cardiac safety assessment of NBI-1: Potential role of IKs potassium channel in the observed QT prolongation detected in vivo
The rabbit cardiovascular (CV) model presents a Safety Pharmacology paradox: ideal for pre-clinical studies in that they share with humans the ion channels that control ventricular repolarization, yet widely underused in pre-clinical testing. Highly responsive to IKrblocking drugs, rabbits may be particularly sensitive for drug safety screening. Four male and four female rabbits surgically implanted with DSI TL11M2-C50-PXT transmitters were dosed by subcutaneous injection with vehicle, 2.5, 10 or 40 mg/kg dipyridamole using a Latin square dose design. Body temperature, heart rate, arterial blood pressure waveforms and ECG waveforms were collected for 15 s of every 10 min for 1 h pre-dose and for 24 h post-dose. Following a qualitative review, quantitative ECG waveform analysis was performed to determine PR, QRS, RR, and QT values. Administration of dipyridamole resulted in higher heart rate (from 1 up to 14 h), lower blood pressures (from 1 up to 10 h), prolonged RR interval (from 5 up to 14 h), shortened QT interval (from 1 up to 16 h) and lower body temperature (from 1 up to 10 h) as compared to vehicle control, with slightly longer duration of effects in males as
Aida Sacaan⁎, Shirley Lio, Haig Bozigian Neurocrine Biosciences, San Diego, CA, United States NBI-1 is a compound developed for the treatment of CNS diseases. In preparation for first in Man clinical trials, a battery of cardio safety assessment was performed. NBI-1 displayed very weak activity on hERG channel current, inhibiting it by less than 1% at 300 µM. NBI-1 was also tested in the canine Purkinje fiber assay where it did not induce any change in the action potential duration (APD), or in any other parameter at concentrations up to 700 µM. With a clean in vitro profile, NBI-1 was advanced into in vivo testing where it was orally administered (10–200 mg/kg) to telemetered cynomolgus monkeys to assess its effect on ECG and hemodynamic parameters. NBI-1 up to 200 mg/kg had no consistent effect on blood pressure or heart rate. By contrast, administration of NBI-1 resulted in dose-related increases in QTc interval at doses ≥10 mg/kg. This occurred within 1 h and lasted up to 22 h postdose. This unexpected result indicated that either a metabolite has activity at the hERG channel or that the
Abstracts / Journal of Pharmacological and Toxicological Methods 60 (2009) 210–258
parent compound or its metabolite(s) act on non-hERG cardiac channels to produce this QT prolongation. NBI-1 has a diversified metabolic pathway and testing its major metabolite did not reveal any activity on hERG channel. NBI-1 was then tested in HEK cells expressing human IKs or IK1 potassium channels. Results indicated that whereas NBI-1 was inactive against IK1 channels, it produced a dose dependent inhibition (up to 43.1%) of IKs current suggesting that NBI-1 may be an IKs blocker which may explain its QT prolongation in vivo. doi:10.1016/j.vascn.2009.04.130
Simultaneous cardiovascular and pharmacokinetic data collection in freely moving guinea pigs Brian M. Roche⁎, Thomas Vinci, S. Peter Hong, Jeremy Smith, Brandon Wood, Jerry Johnson, Steve Graves, Craig Hassler Battelle Memorial Institute, Columbus, OH, United States The pharmaceutical industry is placing more emphasis on eliminating compounds with deleterious side effects earlier in the development process. Combining methodologies to maximize the desired endpoints (i.e., BP, ECG, PK, etc.) from a single study would be an efficient paradigm for early decision making. Additionally, utilizing a smaller animal with cardiac ion channels similar to humans would provide a more relevant and test article efficient model. Automation allows for dosing and blood collection to occur when appropriate without human presence while minimally affecting cardiovascular endpoints. Combining the use of the Culex/Empis automated device to collect blood and/or to dose guinea pigs with a cardiovascular telemetry study is a model that enhances the amount of data (knowledge) about a potential new drug from a single study. We evaluated the use of guinea pigs, instrumented with a telemetry device capable of collecting ECG, blood pressure and body temperature, in conjunction with the Culex/Empis automated device to establish the PK/PD relationship of several known compounds: QT prolonging agent, terfenadine; hypotensive agent, nitroprusside; hypertensive agent, phenylephrine; chronotropic agent, zatebridine; and negative QT control enalapril. doi:10.1016/j.vascn.2009.04.131
Effect of pacing rate and beta blockade on cardiac refractory periods and his-bundle conduction Jinbao Huang CorDynamics, Inc., Chicago, IL, United States Stimulation of the sympathetic nervous system releases norepinephrine, activating beta receptors and triggering increases in heart rate (HR) and atrioventricular (AV) conduction velocity. Metoprolol is a selective beta receptor blocker, which causes a decrease in sinus HR and slows AV conduction. The purpose of this study was to assess the effects of pacing and beta blockade on cardiac electrophysiologic parameters in the anesthetized dog. Two bipolar plunge electrodes were used to determine atrial and ventricular refractory periods (AERP and VERP), as well as to record atrial electrograms. A multipolar electrophysiology catheter positioned distal to the bicuspid valve was also used to record His bundle electrograms to determine atria to His (AH) and His to ventricle (HV) interval measurements. The AV nodal refractory period (AVNERP) was also calculated. In nontreated dogs (n = 11),
241
AVNERP and AH intervals were prolonged at 180 bpm compared to 120 bpm (AVNERP: 201 ±17 vs. 192 ± 18 msec and AH: 128 ± 6 vs. 119 ± 5 ms, respectively). Atrial ERP, VERP and HV intervals were similar when hearts were paced at 120 and 180 bpm. Treatment with 0.1, 0.3 and 1.0 mg/kg metoprolol dose-dependently decreased sinus HR (10 to 20% from baseline) and increased AVNERP (10 to 25% from baseline) with 180 or 120 bpm pacing. Metoprolol also prolonged AH conduction time. In conclusion, increasing pacing rate prolongs AVNERP and AH conduction time. In contrast, beta blockade decreases HR, but also increases AH interval, suggesting slowing of supraventricular conduction.
doi:10.1016/j.vascn.2009.04.132
Induction of Torsades de Pointes by FPL64176, DPI-201106, dofetilide, and chromanol 293B in isolated guinea pig and rabbit hearts Hsien C. Cheng⁎, Josephine Incardona Sanofi-Aventis, Bridgewater, NJ, United States Introduction: For predicting torsades de pointes (TdP) liability of a compound, most of previous studies have used surrogate markers such as hERG inhibition or QT prolongation which may or may not lead to TdP. In this study, we have used isolated hearts for testing induction of TdP. Method: Spontaneously beating isolated guinea pig and rabbit hearts were perfused according to the Langendorff method in hypokalemic (2.1 mM) solution. Lead II ECG and incidence of TdP were monitored for 1 h. Results: FPL 64176, a calcium channel activator, and DPI-201106, a sodium channel modulator, produced TdP in isolated guinea pig and rabbit hearts in a concentration dependent manner; guinea pig hearts were more sensitive than rabbit hearts. In contrast, dofetilide, an IKr inhibitor, gave a low incidence of TdP in guinea pig hearts (1/24), and no TdP at all in rabbit hearts. Chromanol 293B, an IKs inhibitor, elicited TdP concentration dependently in guinea pig but not in rabbit hearts. Conclusion: Thus IKs inhibition, rather than IKr inhibition, appears to be critical in inducing TdP in isolated guinea pig hearts. Rabbit heart did not produce TdP by IKs inhibitor presumably due to a low level of IKs channels in the heart. TdP produced in this study are consistent with the notion that their production may be a consequence of reduced repolarization reserve, thereby causing ventricular abnormalities (Roden, J internal Med., 2006; Jost et al., A.N.E., 2007). Thus, these isolated guinea pig and rabbit hearts may be useful for predicting TdP liability of compounds in drug development.
doi:10.1016/j.vascn.2009.04.133
Possible role of the ultra-rapid delayed rectifier potassium current (IKur) in action potential repolarization in rabbit heart Carol Wilson⁎, Samantha Robinson, Sara Graham, Nick McMahon, Bronagh Heath GlaxoSmithKline, The Frythe, Welwyn, Hertfordshire, United Kingdom The ultra-rapid delayed rectifier K+ current (IKur) plays an important role in early cardiac action potential repolarization in a number of species. Although the rabbit heart has proven useful for