Rabbit Left Ventricular Wedge Preparation Research Articles

Intracellular uptake of agents that block the hERG channel can confound the assessment of QT interval prolongation and arrhythmic risk.

Oliceridine is a biased ligand at the μ-opioid receptor recently approved for the treatment of acute pain. In a thorough QT study, corrected QT (QTc) prolongation displayed peaks at 2.5 and 60 minutes after a supratherapeutic dose. The mean plasma concentration peaked at 5 minutes, declining rapidly thereafter. The purpose of this study was to examine the basis for the delayed effect of oliceridine to prolong the QTc interval. Repolarization parameters and tissue accumulation of oliceridine were evaluated in rabbit left ventricular wedge preparations over a period of 5 hours. The effects of oliceridine on ion channel currents were evaluated in human embryonic kidney and Chinese hamster ovary cells. Quinidine was used as a control. Oliceridine and quinidine produced a progressive prolongation of the QTc interval and action potential duration over a period of 5 hours, paralleling slow progressive tissue uptake of the drugs. Oliceridine caused modest prolongation of these parameters, whereas quinidine produced a prominent prolongation of action potential duration and QTc interval as well as development of early afterdepolarization (after 2 hours), resulting in a high torsades de pointes score. The 50% inhibitory concentration values for the oliceridine inhibition of the rapidly activating delayed rectifier current (human ether a-go-go current) and late sodium channel current were 2.2 and 3.45 μM when assessed after traditional acute exposure but much lower after 3 hours of drug exposure. Our findings suggest that a gradual increase of intracellular access of drugs to the hERG channels as a result of their intracellular uptake and accumulation can significantly delay effects on repolarization, thus confounding the assessment of QT interval prolongation and arrhythmic risk when studied acutely. The multi-ion channel effects of oliceridine, late sodium channel current inhibition in particular, point to a low risk of devloping torsades de pointes.

Assessment of drug-induced proarrhythmia: The importance of study design in the rabbit left ventricular wedge model

In the present study, we investigated an impact of the stimulation rate on the detection of the proarrhythmic potential of 10 reference compounds with effects on different cardiac ion channels in the isolated arterially-perfused rabbit left ventricular wedge preparation. The compounds were tested in the wedge model using two distinct protocols; including baseline stimulation at 1-Hz followed by a brief period at 0.5-Hz, either without an additional brief period of 2-Hz stimulation (i.e. Protocol 1) or with 2-Hz stimulation (i.e. Protocol 2). As expected, QT-prolonging drugs (ibutilide and quinidine) prolonged the QT interval, similarly increased the Torsades de Pointes (TdP) score, and elicited early afterdepolarizations (EADs) in both protocols. HMR1556 and JNJ-303 (IKs blockers) also prolonged the QT interval up to 1μM similarly in both protocols. Nifedipine (Ca2+ antagonist) shortened the QT interval, and reduced force of contraction similarly in both protocols. However, Na+ channel blockers (Ia, Ib, Ic) widened the QRS duration more in Protocol 2 than in Protocol 1. Furthermore, it was only possible to detect non-TdP-like ventricular tachycardia/fibrillation (VT/VF) induced by Na+ blockers and by QT-shortening drugs (levcromakalim and mallotoxin) using the 2-Hz stimulation (Protocol 2).Our data suggest that the inclusion of a brief period of fast stimulation at 2Hz is critical for detecting drug-induced slowing of conduction (QRS widening), QT shortening and associated (non-TdP-like) VT/VF, which are distinct from the QT prolongation/TdP proarrhythmia in isolated, arterially-perfused rabbit left ventricular wedges.

Preclinical assessment of drug-induced proarrhythmia in arterially perfused rabbit left ventricular wedge preparations: An important role for stimulation rates

Preclinical assessment of drug-induced proarrhythmia in arterially perfused rabbit left ventricular wedge preparations: An important role for stimulation rates

LYSOPHOSPHATIDIC ACID PROLONGS ACTION POTENTIAL DURATION AND INCREASES ELECTROPHYSIOLOGICAL INSTABILITY OF ADULT RABBIT VENTRICULAR MYOCARDIUM BY AUGMENTING L-TYPE CALCIUM CURRENT

ObjectivesLysophosphatidic acid (LPA) has various actions on cardiovascular system and is widely reported to modulate multiple ion currents in non-myocardiocytes, but little is known about its electrophysiological effects on cardiac...

Lysophosphatidic Acid Increases the Electrophysiological Instability of Adult Rabbit Ventricular Myocardium by Augmenting L-Type Calcium Current

Lysophosphatidic acid (LPA) has diverse actions on the cardiovascular system and is widely reported to modulate multiple ion currents in some cell types. However, little is known about its electrophysiological effects on cardiac myocytes. This study investigated whether LPA has electrophysiological effects on isolated rabbit myocardial preparations. The results indicate that LPA prolongs action potential duration at 90% repolarization (APD90) in a concentration- and frequency-dependent manner in isolated rabbit ventricular myocytes. The application of extracellular LPA significantly increases the coefficient of APD90 variability. LPA increased L-type calcium current (ICa,L) density without altering its activation or deactivation properties. In contrast, LPA has no effect on two other ventricular repolarizing currents, the transient outward potassium current (Ito) and the delayed rectifier potassium current (IK). In arterially perfused rabbit left ventricular wedge preparations, the monophasic action potential duration, QT interval, and Tpeak-end are prolonged by LPA. LPA treatment also significantly increases the incidence of ventricular tachycardia induced by S1S2 stimulation. Notably, the effects of LPA on action potentials and ICa,L are PTX-sensitive, suggesting LPA action requires a Gi-type G protein. In conclusion, LPA prolongs APD and increases electrophysiological instability in isolated rabbit myocardial preparations by increasing ICa,L in a Gi protein-dependent manner.

Role of mixed ion channel effects in the cardiovascular safety assessment of the novel anti‐MRSA fluoroquinolone JNJ‐Q2

JNJ-Q2, a novel broad-spectrum fluoroquinolone with anti-methicillin-resistant Staphylococcus aureus activity, was evaluated in a comprehensive set of non-clinical and clinical cardiovascular safety studies. The effect of JNJ-Q2 on different cardiovascular parameters was compared with that of moxifloxacin, sparfloxacin and ofloxacin. Through comparisons with these well-known fluoroquinolones, the importance of effects on compensatory ion channels to the cardiovascular safety of JNJ-Q2 was investigated. JNJ-Q2 and comparator fluoroquinolones were evaluated in the following models/test systems: hERG-transfected HEK293 cells sodium channel-transfected CHO cells, guinea pig right atria, arterially perfused rabbit left ventricular wedge preparations and in vivo studies in anaesthetized guinea pigs, anaesthetized and conscious telemetered dogs, and a thorough QT study in humans. The trend for effects of JNJ-Q2 on Tp-Te, QT, QRS and PR intervals in the non-clinical models and the plateau in QTc with increasing plasma concentration in humans are consistent with offsetting sodium and calcium channel activities that were observed in the non-clinical studies. These mixed ion channel activities result in the less pronounced or comparable increase in QTc interval for JNJ-Q2 compared with moxifloxacin and sparfloxacin despite its greater in vitro inhibition of I(Kr). Based on the non-clinical and clinical cardiovascular safety assessment, JNJ-Q2 has a safe cardiovascular profile for administration in humans with comparable or reduced potential to prolong QT intervals, compared with moxifloxacin. The results demonstrate the importance of compensatory sodium and calcium channel activity in offsetting potassium channel activity for compounds with a fluoroquinolone core.

Differentiating electrophysiological effects and cardiac safety of drugs based on the electrocardiogram: A blinded validation

The ventricular components (QRS and QT) on the electrocardiogram (ECG) depend on the properties of ventricular action potentials that can be modulated by drugs via specific ion channels. However, the correlation of ECG ventricular waveforms with underlying ion actions is not well established and has been extensively debated. To conduct a blinded in vitro assessment of the ionic mechanisms for drug-induced ECG changes. Fourteen cardiac and noncardiac drugs with known effects on cardiac ion channels were selected by the study sponsor, and were tested in the rabbit left ventricular wedge preparation with recording of the ECG and contractility. The investigators who performed the experiments and analyzed the data were blinded to names, concentrations, and molecular weights of the drugs. The compounds were prepared by the sponsor and sent to the investigators as 56 stock solutions. The effects of I(Kr), I(Ks), I(Ca,L), I(Na) blocker, and I(KATP) opener on QRS, QT, and T(p-e), were evaluated. Disclosure of the names and concentrations after completion of the study revealed that there were highly correlated ECG changes with underlying ionic mechanisms and proarrhythmic potential of drugs that, respectively, target I(Kr), I(Ks), I(Ca,L), I(Na), and I(KATP). Among ECG parameters, T(p-e) was more useful in differentiating drugs' actions. Specific electrophysiological action and the consequent proarrhythmic potential of a drug can be accurately determined by analysis of drug-induced changes in ECG in the rabbit left ventricular wedge preparation. Change in T(p-e) provides the most relevant information.

Modulation of the late sodium current by ATX-II and ranolazine affects the reverse use-dependence and proarrhythmic liability of IKrblockade

Drug-induced torsades de pointes (TdP) often occurs during bradycardia due to reverse use-dependence. We tested the hypothesis that inhibition or enhancement of late sodium current (I(Na,L) ) could modulate the drug-induced reverse use-dependence in QT and T(p-e) (an index of dispersion of repolarization), and therefore the liability for TdP. Arterially perfused rabbit left ventricular wedge preparations were used. Action potentials from the endocardium were recorded simultaneously with a transmural ECG. The effects of Anemonia sulcata toxin (ATX-II) (an I(Na,L) enhancer), d,l-sotalol, clarithromycin and ranolazine (an I(Na,L) blocker) on rate-dependent changes in QT, T(p-e) and proarrhythmic events were tested, either alone or in combination. Rate-dependent QT and T(p-e) slopes and TdP score (a combined index of TdP liability) were calculated at control and during drug infusion. ATX-II (30 nM) and sotalol (300 µM) caused a marked increase in QT and T(p-e) intervals, steeper QT-basic cycle length (BCL) and T(p-e) -BCL slopes (i.e. reverse use-dependence), and TdP. Addition of ranolazine (15 µM) to ATX-II or sotalol significantly attenuated QT-BCL, T(p-e) -BCL slopes and the increased TdP scores. In contrast, clarithromycin (100 µM) moderately prolonged QT and T(p-e) without causing R-on-T extrasystole or TdP, but addition of ATX-II (1 nM) to clarithromycin markedly amplified the QT-BCL and T(p-e) -BCL slopes and further increased TdP score. Modulation of I(Na,L) altered drug-induced reverse use-dependence related to QT as well as T(p-e) , indicating that inhibition of I(Na,L) can markedly reduce the TdP liability of agents that prolong QT intervals.

Vulnerability to Unidirectional Conduction Block and Reentry in Rabbit Left Ventricular Wedge Preparation: Effects of Stimulation Sequence and Location

Many factors influence the initiation of unidirectional conduction block and reentry. To explore the influential factors on the temporal vulnerable window of the unidirectional conduction block, we investigated the effect of stimulation sequence and location on the temporal vulnerability in epicardial and endocardial sites in an arterially perfused rabbit left ventricular wedge preparation at three basic cycle lengths (BCL) of 2,000, 1,000 and 500 ms. An extrastimulus (S2) was introduced at coupling intervals incremented by 1 ms to scan the entire diastolic interval. The results showed that the vulnerable window increased with the lengthening of BCL, which ranged from 48.55 +/- 18.04 ms at BCL of 500 ms to 92.50 +/- 25.59 ms at BCL of 2,000 ms for endocardial, and is a similar style for epicardial from 21.00 +/- 14.02 ms at BCL of 500 ms to 75.71 +/- 16.34 ms at BCL of 2,000 ms (P < 0.05). The vulnerable window of endocardial was wider than that of epicardial (P < 0.05). Furthermore, the window size for reentry was about 39% smaller than that for unidirectional conduction block. Meanwhile, we found that increasing the number of premature beats enlarged the vulnerable window markedly due to enhanced transmural dispersion of repolarization (TDR) of ventricle. The vulnerable window is an effective index to evaluate the increased risk for unidirectional conduction block. Therefore, the factors that increase the vulnerable window are associated with the higher incidence of ventricular arrhythmias.

Calcium-activated chloride current contributes to action potential alternations in left ventricular hypertrophy rabbit

T-wave alternans, characterized by a beat-to-beat change in T-wave morphology, amplitude, and/or polarity on the ECG, often heralds the development of lethal ventricular arrhythmias in patients with left ventricular hypertrophy (LVH). The aim of our study was to examine the ionic basis for a beat-to-beat change in ventricular repolarization in the setting of LVH. Transmembrane action potentials (APs) from epicardium and endocardium were recorded simultaneously, together with transmural ECG and contraction force, in arterially perfused rabbit left ventricular wedge preparation. APs and Ca(2+)-activated chloride current (I(Cl,Ca)) were recorded from left ventricular myocytes isolated from normal rabbits and those with renovascular LVH using the standard microelectrode and whole cell patch-clamping techniques, respectively. In the LVH rabbits, a significant beat-to-beat change in endocardial AP duration (APD) created beat-to-beat alteration in transmural voltage gradient that manifested as T-wave alternans on the ECG. Interestingly, contraction force alternated in an opposite phase ("out of phase") with APD. In the single myocytes of LVH rabbits, a significant beat-to-beat change in APD was also observed in both left ventricular endocardial and epicardial myocytes at various pacing rates. APD alternans was suppressed by adding 1 microM ryanodine, 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and 100 microM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). The density of the Ca(2+)-activated chloride currents (I(Cl,Ca)) in left ventricular myocytes was significantly greater in the LVH rabbits than in the normal group. Our data indicate that abnormal intracellular Ca(2+) fluctuation may exert a strong feedback on the membrane I(Cl,Ca), leading to a beat-to-beat change in the net repolarizing current that manifests as T-wave alternans on the ECG.

Ventricular repolarization in an acute canine model of long QT syndrome

In the present study, we investigated an impact of the stimulation rate on the detection of the proarrhythmic potential of 10 reference compounds with effects on different cardiac ion channels in the isolated arterially-perfused rabbit left ventricular wedge preparation. The compounds were tested in the wedge model using two distinct protocols; including baseline stimulation at 1-Hz followed by a brief period at 0.5-Hz, either without an additional brief period of 2-Hz stimulation (i.e. Protocol 1) or with 2-Hz stimulation (i.e. Protocol 2). As expected, QT-prolonging drugs (ibutilide and quinidine) prolonged the QT interval, similarly increased the Torsades de Pointes (TdP) score, and elicited early afterdepolarizations (EADs) in both protocols. HMR1556 and JNJ-303 (IKs blockers) also prolonged the QT interval up to 1 μM similarly in both protocols. Nifedipine (Ca2 + antagonist) shortened the QT interval, and reduced force of contraction similarly in both protocols. However, Na+ channel blockers (Ia, Ib, Ic) widened the QRS duration more in Protocol 2 than in Protocol 1. Furthermore, it was only possible to detect non-TdP-like ventricular tachycardia/fibrillation (VT/VF) induced by Na+ blockers and by QT-shortening drugs (levcromakalim and mallotoxin) using the 2-Hz stimulation (Protocol 2).Our data suggest that the inclusion of a brief period of fast stimulation at 2 Hz is critical for detecting drug-induced slowing of conduction (QRS widening), QT shortening and associated (non-TdP-like) VT/VF, which are distinct from the QT prolongation/TdP proarrhythmia in isolated, arterially-perfused rabbit left ventricular wedges.

Biventricular pacing and transmural dispersion of the repolarization

In 2003, Medina-Ravell et al .1 published a study that suggested that biventricular pacing could be arrhythmogenic in a subset of patients because of the reversal of the normal endocardial-to-epicardial activation sequence by left-ventricular epicardial pacing. As epicardial action potentials are briefer than endocardial action potentials, transmural dispersion of the repolarization (TDR) is larger with an epicardial-to-endocardial activation sequence than with an endocardial-to-epicardial activation sequence. Medina-Ravell et al . demonstrated this in an isolated arterially perfused rabbit left-ventricular wedge preparation. They also showed, in a quasi-ECG derived from this preparation, that TDR was faithfully reflected by the T peak-end interval and that T peak-end was larger with epicardial pacing than with endocardial pacing of the preparation. Their conclusion that a similar effect occurs in intact hearts in humans was substantiated by …

In-vitro experimental models for the risk assessment of antibiotic-induced QT prolongation

The prolongation of the ventricular repolarization and proarrhythmic effects (Torsade de Pointes: TdP) of five reference antibiotics were compared in four in-vitro models. 1. Using the patch clamp technique on the human ether-a-gogo-related gene (HERG) current, the rank order for blockade of the HERG-current (IC 50) was: sparfloxacin (44 μM) > telithromycin = moxifloxacin = erythromycin (± 100 μM). 2. Assessing their effects on action potential duration (APD 90) and incidence of early afterdepolarizations in isolated rabbit Purkinje fibers, the rank order was: sparfloxacin > moxifloxacin > telithromycin > erythromycin (prolongation of APD 90 at 100 μM: 83%, 48%, 33% and 17% from baseline compared to + 5% with solvent, P < 0.05, respectively). 3. Assessing the drug effects on the APD 60, triangulation, reverse use-dependency, and instability in isolated Langendorff-perfused rabbit hearts, the rank order was: moxifloxacin > erythromycin > sparfloxacin > telithromycin. 4. Assessing their torsadogenic potentials (scores of effects on QT-interval, peak of the T wave to end of T wave: T p–e, T p–e/QT ratio, R wave on T wave (R on T) and TdP in isolated rabbit left ventricular wedge preparations, the rank order for their TdP risk score was: sparfloxacin > erythromycin > moxifloxacin > telithromycin. Additional experiments with grepafloxacin indicate that the rank order to detect grepafloxacin-induced long QT was the wedge preparation > the Purkinje fiber > HERG > the isolated heart, where the isolated heart was unable to detect grepafloxacin-induced APD prolongation. The present study demonstrates that the first three in-vitro models can be used to assess the ability of antibiotic compounds to delay ventricular repolarization. However, with respect to their known clinical effects on QT and TdP incidence, the wedge preparation appears to be more predictive and suitable for detecting torsadogenic action of antibiotics.

Use of arterially perfused rabbit ventricular wedge in predicting arrhythmogenic potentials of drugs

Introduction A growing number of drugs have reportedly been associated with delayed ventricular repolarization and a potentially fatal but rare arrhythmia, torsade de pointes (TdP). There is obviously a call for a validated proarrhythmia model that distinguishes proarrhythmic drugs from nonarrhythmogenic drugs. Methods In this article, we validated the arterially perfused rabbit left ventricular wedge preparation model and examined its use in predicting proarrhythmic potentials of drugs. A fairly detailed methodological description about this technically challenging model was given, aiming to help others establish the assay successfully. Parameters commonly used in the action potential studies were verified and critical experimental conditions (e.g. stability and reproducibility of recordings) were examined. Six commercially available compounds with various proarrhythmic potentials were administered in the model to evaluate their correlations with individual clinical outcomes. Results Our study indicated that, in a successful experiment, the action potential duration (APD) can be stably maintained for several hours without intervention. Dofetilide, dl-sotalol, cisapride, risperidone and moxifloxacin increased endo- and epicardial APD 90, QT interval and T P–E (peak-to-end time of the T wave) in a reverse use-dependent manner within clinically relevant concentration ranges. Phase 2 early afterdepolarizations (EADs) were observed at 1.6, 2.3, 16.7, 37.5 and 7.9 fold, respectively, their corresponding unbound therapeutic concentrations. In contrast, fluoxetine at up to 3 μM (∼ 35 fold unbound therapeutic mean plasma concentration after 60 mg/day, p.o. for 5 weeks) had only a mild prolonging effect on APD 90 and QT with essentially no effect on T P–E. Discussion Our results strongly support the usefulness of this model in predicting a compound's arrhythmogenic potential in humans within clinically relevant concentration ranges, and the experimental results with this model need to be interpreted in light of each drug's pharmacokinetic and pharmacodynamic behavior in clinic.

Assessment of the Proarrhythmic Potential of the Novel Antiarrhythmic Agent AZD7009 and Dofetilide in Experimental Models of Torsades De Pointes

This study examined the proarrhythmic potential of the novel antiarrhythmic agent AZD7009 and dofetilide. The electrophysiological and proarrhythmic effects of AZD7009 and dofetilide were assessed in the arterially perfused canine and rabbit left ventricular wedge preparation. The proarrhythmic potential of AZD7009, dofetilide, and azimilide was further assessed in the methoxamine-sensitized rabbit model of torsades de pointes (TdP) in vivo. AZD7009 lengthened the action potential duration (APD) and the QT interval in a bell-shaped manner (15.9 +/- 1.3% in canine wedge and 46.1 +/- 2.9% in rabbit wedge) occurring at 3 and 1 microM. In contrast, dofetilide did not show the bell-shaped concentration response and the QT interval was lengthened more extensively (27.7 +/- 1.6% and 100.8 +/- 10.0%). Furthermore, whereas dofetilide prolonged the midmyocardial and endocardial APD predominantly, resulting in an increased transmural dispersion of repolarization (TDR), AZD7009 prolonged the APD more homogenously in all cell layers. At 1 microM, AZD7009 produced phase 2 early afterdepolarizations (EADs) in 1/4 rabbit preparations but without ventricular R-on-T extrasystoles or TdP. In contrast, starting at 0.03 microM, dofetilide-induced EADs, R-on-T extrasystoles and TdP in 6/6, 5/6, and 4/6 preparations. Following intravenous infusion of AZD7009 (210 nmol/kg/minute), dofetilide (2 nmol/kg/minute) or azimilide (3.33 micromol/kg/minute), TdP was induced in 0/8, 5/8, and 5/8 rabbits (P = 0.026 vs AZD7009), respectively. In 5/5 rabbits, AZD7009 promptly suppressed TdP induced by dofetilide. In animal models of TdP, AZD7009 delays ventricular repolarization in a self-limited way associated with a low risk of repolarization-related proarrhythmia.

Effect of epicardial or biventricular pacing to prolong QT interval and increase transmural dispersion of repolarization

Background— The present study examined pacing site–dependent changes in QT interval and transmural dispersion of repolarization (TDR) and their potential role in the development of torsade de pointes (TdP). Methods and Results— In humans, the QT interval, JT interval, and TDR were measured in 29 patients with heart failure during right ventricular endocardial pacing (RVEndoP), biventricular pacing (BiVP), and left ventricular epicardial pacing (LVEpiP). In animal experiments, pacing site–dependent changes in ventricular repolarization were examined with a rabbit left ventricular wedge preparation in which action potentials from endocardium and epicardium could be simultaneously recorded with a transmural ECG. In humans, LVEpiP and BiVP led to significant QT and JT prolongation. LVEpiP also enhanced TDR. Frequent R-on-T extrasystoles generated by BiVP and LVEpiP but completely inhibited by RVEndoP occurred in 4 patients, of whom 1 developed multiple episodes of nonsustained polymorphic ventricular tachycar...