A new mechanism preventing proarrhythmia in chronic heart failure: rapid phase-III repolarization explains the low proarrhythmic potential of amiodarone in contrast to sotalol in a model of pacing-induced heart failure

Induction of heart failure

Heart failure was induced via 3 to 4 weeks of ventricular pacing at 400 bpm. Adult female New Zealand white rabbits were anaesthetized with ketamine (75 mg/kg) and xylazine (5.8 mg/kg), with additional doses applied as needed. A pacemaker lead for transient pacing in the clinical setting was implanted into the right ventricle via the right jugular vein under fluoroscopic guidance. After testing the pacing threshold, the wounds were closed. One week after the operation, an external pacemaker was connected and pacing started at 400 bpm at twice the diastolic threshold. Over a period of 3 to 4 weeks, the rabbits were monitored by clinical examination and ECG to observe whether they developed CHF.14 In addition, sham-operated rabbits served as controls.

Preparation of hearts for perfusion

Thirty-seven white female New Zealand rabbits (weighing 3.5–4.0 kg) received oral amiodarone treatment (50 mg/kg body weight by mouth per day) for 6 weeks. It has previously been shown by our group in the same model that this dosage prevents ventricular tachycardias and ventricular fibrillation, and that achieved tissue levels correspond to human tissue levels under amiodarone treatment (6.5 ± 0.9 to 11.9 ± 2.3 μg in this study).15 The drug was mixed into the normal food and application started 3 weeks prior to surgery. Then, 17 of these 37 rabbits (randomly assigned) received a pacemaker as described above while the remaining 20 rabbits were sham-operated and observed. Amiodarone was fed continuously until the Langendorff experiment was performed. In addition, 18 untreated rabbits of comparable weight also received a pacemaker while 14 untreated rabbits were sham-operated. These animals served as ‘baseline’-controls for the amiodarone group before their hearts were infused with sotalol later on.

The method of preparing the hearts has previously been described in detail.16 In short, female New Zealand white rabbits were anaesthetized with sodium thiopental (200–300 mg intravenously). The complete hearts were removed and immediately placed in an ice-cold Krebs–Henseleit solution (composition in mM: CaCl₂ 1.80, KCl 4.70, KH₂PO₄ 1.18, MgSO₄ 0.83, NaCl 118, NaHCO₃ 24.88, Na-pyruvate 2.0, and d-glucose 5.55). The spontaneously beating hearts were perfused at constant flow (52 mL/min) with warm (36.8–37.2°C) Krebs–Henseleit solution. Perfusion pressure was kept stable at 100 mmHg. The perfusate was equilibrated with 95% O₂ and 5% CO₂ (pH 7.35; 37°C). The cannulated and perfused hearts were attached to a vertical Langendorff apparatus (Hugo Sachs Electronic, Medical Research Instrumentation, March-Hugstetten, Germany). A deflated latex balloon was inserted into the left ventricle and connected to a pressure transducer to control haemodynamic stability. The atrioventricular (AV) node was ablated. This resulted in complete AV-dissociation with a ventricular escape rate below 60 bpm.

Electrocardiographic and electrophysiological measurements

Signals from a simulated ‘Einthoven’ configuration were amplified by a standard ECG amplifier (filter settings: 0.1–300 Hz), and QT-interval was measured. Monophasic action potential (MAP) recordings and stimulation were accomplished simultaneously using contact MAP-pacing catheters (EP Technologies, Mountain View, CA, USA). The MAP electrograms were amplified and filtered (low pass 0.1 Hz, high pass 300 Hz). Monophasic action potentials were analysed using specifically designed software, permitting precise definition of the amplitude and duration of the digitized signals. The recordings were considered reproducible and, therefore, acceptable for analysis only if they had a stable baseline amplitude with a variation of less than 20% and a stable duration measured at 90% repolarization (APD₉₀). Seven MAPs were evenly spread in a circular pattern around both ventricles; one MAP was recorded from the left endocardium (Figure 2). Pacing at twice diastolic threshold was performed for 1 min at each cycle length (CL) from 900 to 300 ms (Universal Programmable Stimulator, UHS 20, Biotronik, Germany). All data were digitized at a rate of 1 kHz with 12-bit resolution and subsequently stored on a removable hard disk (BARD LabSystem, Bard Electrophysiology, Murray Hill, MA, USA).

Experimental ex vivo protocol

Cycle length dependence of MAPs was investigated under baseline conditions in amiodarone-treated hearts and in the control group by pacing the hearts at CLs between 900 and 300 ms. Subsequently, programmed stimulation was performed with up to two extrastimuli (increment of 2 ms) to determine the refractory period. Thereafter, the potassium concentration was lowered to 1.5 mM to provoke EADs and TdP in spontaneously beating hearts with their slow ventricular escape rate. Five minutes later, the potassium concentration was set back to 5.8 mM and the control hearts were perfused with d,l-sotalol in increasing concentrations (50–100 µM) over a period of 20 minutes. The experimental set-up was designed to reproduce conditions and circumstances that are clinically known to be associated with an increased propensity to the development of TdP. Pacing, MAP recording, and measurement of ECG parameters were started 20 min after drug infusion. The protocol steps were repeated for each drug concentration. In summary, data were obtained from CHF-hearts and control hearts, both chronically treated with amiodarone, and CHF-hearts and control hearts at baseline and after perfusion with 50 and 100 µM sotalol.

APD₉₀ was measured as the average interval between the fastest MAP upstroke and 90% repolarization. Dispersion of APD₉₀ was expressed as the difference between the maximum and the minimum of APD₉₀, simultaneously recorded in eight endocardial and epicardial catheters. Monophasic action potential morphology was assessed by calculating the ratio of APD_90/50, thereby quantifying a triangular or rectangular action potential shape. Early afterdepolarizations were defined as a positive voltage deflection that interrupted the smooth contour of phase-II or III repolarization of the action potential. Torsade de pointes was defined as a polymorphic ventricular tachyarrhythmia of more than five beats with a changing ventricular axis and spontaneous termination.

Data acquisition and statistical analysis

The observed data were entered into a computerized database (Microsoft Excel 2003) and statistically evaluated using SPSS Software Release 15.0.1 (November 2006, Chicago). Categorical variables were expressed as frequency and percentage, whereas continuous variables were presented as mean ± SD. The effect of sotalol and amiodarone on CL dependence QT-interval, action potential duration, and dispersion of repolarization were assessed using general linear model for repeated measurements. After confirming significant group differences over CL, post hoc comparisons with Tukey's procedure were applied to determine differences between groups. McNemar tests were used to compare the appearance of EADs and TdP. The χ² test and Fisher's test were used to compare TdP incidence. T-tests were used to compare the difference between two independent study groups, and the paired-samples t-test between dependent study groups. Differences were considered significant at P< 0.05.

Assessment of heart failure

During the period of fast ventricular pacing, rabbits became lethargic, fatigued, showed loss of appetite, and developed respiratory distress. Moreover, the paced rabbits developed ascites, pleural and pericardial effusion, and peripheral oedema. To monitor the progress of left ventricular (LV)-function impairment, two-dimensional ECG was carried out periodically and demonstrated a significant decrease in ejection fraction from 74±6–22±8% in the presence of a dilated LV after 3 to 4 weeks. After removal, the failing hearts were found to have a significantly greater LV-diameter (mean—left ventricular enddiastolic diameter: 1.86 ± 0.18 cm in heart failure as compared with 1.64 ± 0.14 cm in control hearts, P< 0.05), and increased weight (mean 19.3 ± 2.7 g in heart failure as compared with 16.5 ± 2.1 g in controls; P<0.05). Histological examination showed an increase of perivascular and interstitial fibrosis. The comparison of baseline electrophysiologic values of intact and failing hearts revealed a significant prolongation of QT-interval and action potential duration (Figure 1A).

Effect of sotalol on QT-interval, action potential duration, and dispersion of repolarization in sham and failing hearts

After a stabilization period of 10 min, MAP recordings and pacing thresholds (mean threshold 1.6 ± 1.2 mA) remained highly reproducible throughout the experimental protocol. The MAP amplitude did not change by more than 20% for the subsequent investigation period. Sotalol (50 and 100 µM) in sham-operated (n= 14) and failing (n= 18) hearts led to a significant increase in QT-interval and monophasic action potential duration (APD₉₀; P< 0.05), thereby mimicking LQT syndrome. Figure 1B and C illustrate the effects of sotalol on QT-interval. In the presence of 100 µM sotalol, the QT-interval increase ranged between 15% at a CL of 300 ms and 29% at a CL of 900 ms (P< 0.05) in sham hearts. This so-called reverse-frequency dependence17 was even more marked in failing hearts (17–72%; P< 0.05) representing a reduced repolarization reserve in heart failure (Figure 1D). The increase in QT-interval by sotalol was paralleled by a significant increase in APD₉₀. Up to eight simultaneous epi- and endocardial MAPs demonstrated a significant increase in dispersion of repolarization in sotalol-treated hearts from baseline values of 41–70 ms in sham hearts. Again, the increase in dispersion of repolarization was more marked in failing hearts due to a reduced repolarization reserve (35–102 ms in failing hearts after drug infusion; Figure 2). Dispersion of repolarization was markedly increased at slow heart rates (Table 1) and resulted from an increase in interventricular (recorded between right epicardium and left epicardium (basal and apical MAP recordings) and transmural (recorded between left endocardium and left epicardium) dispersion.

Effect of amiodarone on QT-interval, action potential duration, and dispersion of repolarization

In sham hearts, chronic administration of amiodarone led to a prolonged QT-interval as compared with untreated control hearts (271 –238 ms). The prolonged QT-interval was accompanied by a significant increase of APD₉₀. In contrast to sotalol, no reverse-frequency dependence (14% increase at a CL of 300 ms, 10% at 900 ms, Figure 1C) and no significant increase in dispersion of repolarization could be observed (Figure 2 top). In failing amiodarone-treated hearts, only a non-significant rise in QT-interval (271–280 ms; Figure 1D) and APD₉₀ was observed. Again, there was no increase in dispersion of repolarization (Figure 2, bottom) compared with failing baseline hearts.

Refractoriness and action potential configuration

A comparison between sham and failing hearts at baseline revealed an increase in the refractory period from 173 to 199 ms. The administration of both sotalol and amiodarone augmented the refractory period in control hearts from 173 to 199 ms (amiodarone) and from 173 to 222 ms (100 µM sotalol). In failing hearts, sotalol increased the refractory period from 199 to 291 ms. Comparable with APD₉₀, amiodarone hearts did not show a significant increase.

Sotalol led to a triangular action potential configuration (ratio APD₉₀/APD₅₀ sham: 1.29 at baseline, 1.44 at 100 µM sotalol; CHF: 1.11 at baseline, 1.35 at 100 µM sotalol; Figures 3A and 3B), whereas amiodarone did not cause triangularization but caused a more marked phase-II prolongation (APD₉₀/APD₅₀ sham: 1.29 at baseline in controls, 1.30 after treatment with amiodarone; CHF: 1.11 at baseline in controls, 1.15 after treatment with amiodarone; Figures 3A and 3B).

Early afterdepolarizations and torsade de pointes

In the presence of sotalol, EADs and triggered activity were a frequent finding. After complete AV-block and lowering of potassium concentration from 5.88 to 1.5 mM/l, 7 of 14 sotalol-treated sham hearts showed MAP recordings with EADs and TdP (50%, 35 single episodes (Figure 4). A typical example during sotalol 100 µM in a CHF heart is shown in Figure 5. Torsade de pointes were always associated with EADs [sotalol: P< 0.05 (Fisher-test)]. In failing sotalol-treated hearts, TdP could reproducibly be observed in 16 out of 18 hearts (Figure 4; 89%, 154 single episodes). In amiodarone-treated sham and failing hearts, no EADs or TdP could be observed.

Extent of myocardial repolarization

In failing hearts, there was a significant prolongation of QT-interval and action potential duration as compared with non-failing hearts. Action potential prolongation, mainly due to deceleration of phase-III-repolarization, served as the key electrophysiological factor for the formation of triggered activity and potentially life-threatening arrhythmias. In heart failure, this results from a reduction in outward, repolarizing ion currents, especially due to down-regulation of potassium channels.18 Thus, CHF leads to a reduced ‘repolarization reserve’, which means a patient-specific response to risk factors that influence the repolarization process.19 Additional administration of agents that prolong repolarization may be harmful. The major antiarrhythmic mechanism of class-III drugs is prolongation of action potential duration, which unfortunately is also the mechanism underlying its proarrhythmic potential.6 However, despite comparable QT-prolongation, different cardiovascular and non-cardiovascular drugs, including the antiarrhythmic agents sotalol and amiodarone have a different proarrhythmic potential. Thus, prolongation of repolarization may act as a ‘conditio sine qua non’ for the occurrence of this type of proarrhythmia, but the correlation between the extent of QT-prolongation and the incidence of TdP is poor.20

Action potential morphology

Our group has previously shown in healthy rabbit hearts that differences in action potential morphology, different effects on dispersion of repolarization, and differences in reverse-frequency dependence contribute to the low torsadogenic potential of amiodarone.13 The present study showed for the first time, that comparable mechanisms play a role in non-ischaemic heart failure. Differences in the morphology of the action potential (rectangular vs. triangular) are associated with a divergent potential of a great variety of repolarization-prolonging drugs to provoke proarrhythmia. The proarrhythmic potential of a triangular action potential configuration was first presented by Hondeghem et al.21 Our group and others have also reported that the configuration of a prolonged action potential is a relevant factor for the proarrhythmogenic potential of QT-prolonging drugs.20 Moreover, we recently showed in healthy rabbit hearts ex vivo, that a rapid phase-III repolarization caused by amiodarone, leading to a rectangular action potential, is associated with a low proarrhythmic potential.13 In contrast, sotalol was found to prolong phase-III of the repolarization process. This marked prolongation enhanced the origination of EADs by creating more time in the voltage window for calcium channel reactivation as compared to amiodarone22 in the present study. By prolonging action potential duration within the L-type Ca²⁺ ‘window’ voltage range, more calcium can enter the cell and the Na⁺/Ca²⁺ exchanger is activated. This leads to an increased inward current that could redepolarize the sarcolemmal membrane to a potential from where I_Na and/or I_Ca becomes reactivated.23 The resulting inward current may depolarize the sarcolemmal membrane to a potential from where a new and premature action potential can be triggered.24 Besides, triangulation is capable of prolonging the recovery of the sodium current, which lengthens the vulnerable period.17 In contrast, in our model of normal and failing hearts, amiodarone did not cause triangulation but a rectangular action potential configuration due to a more marked phase-II prolongation by taking advantage of its effects on multiple ion channels, in this case especially inhibition of I_Cal. Compared with sotalol, in the setting of the failing rabbit heart amiodarone enhanced a fast and better synchronized repolarization that was less vulnerable to proarrhythmia.

Trigger and substrate for proarrhythmia

In non-ischaemic heart failure, triggered activity is a crucial mechanism for the origination of arrhythmias in the experimental25 and the clinical setting.26 In our study, the appearance of proarrhythmia was always associated with the occurrence of EADs and an increased dispersion of repolarization. The importance of EADs, especially in CHF, is underlined by the work of Pogwizd et al.26 in human failing hearts prior to explantation. They reported that focal initiation of ventricular tachycardia in heart failure was related to triggered activity initiated in the subendocardium of non-ischaemic dilated cardiomyopathy. In addition, an increased dispersion of repolarization is considered as the responsible substrate for the perpetuation of arrhythmias in the setting of proarrhythmia. It has been shown in experimental models and in humans that differences in the degree of increasing dispersion of repolarization contribute to a divergent proarrhythmic potential of many drugs.16,27 In the present study, the importance of dispersion of repolarization as the underlying substrate for proarrhythmia in CHF was measured by several simultaneously recorded MAPs. Using epicardial and endocardial MAP recordings, differences in action potentials recorded from different layers of myocardium were evident. The observed minor increase of dispersion by amiodarone compared with sotalol contributes to the lack of proarrhythmia. Shimizu et al.28 used MAP recordings in humans to support the hypothesis that proarrhythmia is maintained by a reentrant mechanism due to increased dispersion of repolarization. Following comparable results in different studies, dispersion has been added to the relevant factors for the initiation of proarrhythmia.29 One underlying mechanism for the increase in dispersion of repolarization is a heterogenous connexin-43-expression that has been identified as a relevant factor that increases vulnerability to ventricular tachyarrhythmias in CHF.30 Moreover, a significant decrease of connexin-43-expression has been reported in an animal model of tachycardiomyopathy.31 The decrease of the major gap junction protein leads to an uncoupling between muscle layers resulting in an increase of dispersion of repolarization that may facilitate the occurrence of ventricular tachyarrhythmias. In addition, in the context of CHF, many positive inotropic agents, used in treatment of end-stage heart failure, increase dispersion of repolarization.32 This leads to proarrhythmia, thus increasing mortality.33 Amiodarone has been shown to attenuate the proarrhythmic effects of dobutamine in patients with advanced heart failure,34 probably by creating a homogeneous repolarization.

Reverse-frequency dependence is regarded as a risk factor for the development of TdP.35 In sham hearts and even more pronounced in failing hearts, treated with sotalol, we observed a more marked prolongation of action potential duration at slower than at faster heart rates. This finding contributes to proarrhythmia occurring in CHF, because the higher increase of prolongation at slow rates leads to a marked increase of dispersion of repolarization (Table 1), thus unmasking a reduced repolarization reserve in CHF. In addition, the increased dispersion of repolarization at slower heart rates explains why bradycardia is a contributing factor for the occurrence of proarrhythmia. Neither failing nor intact amiodarone hearts showed reverse-frequency dependence. The lack of a remarkable effect on dispersion of repolarization during bradycardia contributes to the understanding of the low proarrhythmic potential of amiodarone.