Short- and Long-Term Effects of Amiodarone on the Two Components of Cardiac Delayed Rectifier K⁺ Current

Rabbit Ventricular Myocytes

Japanese white rabbits of either sex weighing 1.5 to 2.2 kg were killed under anesthesia with thyamiral sodium (30 mg/kg IV), and the hearts were removed. Single myocytes were isolated enzymatically from the apical region of the left ventricle (approximately one third in the apex/base axis) as described previously.¹⁴ To observe the long-term effects of amiodarone, the rabbits were administered oral amiodarone (100 mg · kg⁻¹ · d⁻¹) for 4 weeks before cell isolation.¹¹

Electrophysiological Recording

The single-pipette, whole-cell, voltage-clamp technique was used for recording membrane currents. Cell capacitance was measured by integrating the capacitive transient evoked by applying a hyperpolarizing pulse to −5 mV from a holding potential of −50 mV. The cell capacitance and series resistance were electrically compensated by 60% to 90%. Command potential generation and data acquisition were controlled by pCLAMP software (version 6.0.3, Axon Instruments) and an IBM-compatible computer. Current signals were filtered at 1 kHz and digitized at a sampling frequency of 2 kHz. Current measurement was started after 40-minute superfusion with normal Tyrode’s solution to avoid possible retention of amiodarone and its active metabolite (desethylamiodarone)³ on the cell surface in rabbits with oral treatment.

cRNA Injection and Voltage-Clamp of Oocytes

Isolation and maintenance of Xenopus oocytes and injection with HERG cRNA were performed as described previously.⁷ Stage V and VI oocytes were injected with 10 ng HERG cRNA. KVLQT1 cRNA (5 ng) and minK cRNA (1 ng) were coinjected to induce I_Ks. Currents were recorded at room temperature (22°C to 24°C) by standard 2-microelectrode voltage-clamp techniques¹⁵ 2 to 4 days after cRNA injection.

Ribonuclease Protection Assay

For the ribonuclease protection assay, rabbit ERG,KVLQT1, and minK subunit cDNA fragments were amplified by reverse-transcriptase polymerase chain reaction. The nucleotide sequences of the primers and the amplified regions are listed in Table 1⇓. Gene bank accession numbers are U75212 (nucleotides187 to 456) for ERG, U70068 (nucleotides 502 to 1133) for KVLQT1, and L41659 (nucleotides 239 to 477) for minK. A HindIII site (AAGCTT) was introduced into the 5′ end of the sense primers of the rabbit ERG,KVLQT1, and minK. The amplified cDNA was cloned into pGEM-T vector with the TA cloning system (Promega). The ribonuclease protection assay was performed as described previously.¹⁶

Solution and Drugs

The Tyrode’s solution used for cell isolation and the single myocyte experiments was composed of (mmol/L) NaCl 143, KCl 5.4, CaCl₂ 1.8, MgCl₂ 0.5, NaH₂PO₄ 0.25, HEPES 5.0, and glucose 5.6; pH was adjusted to 7.4 with NaOH. The glass pipette had a resistance of 3 to 5 MΩ after filling with the internal pipette solution containing (mmol/L) KOH 60, KCl 80, aspartate 40, HEPES 5, EGTA 10, MgATP 5, sodium creatinine phosphate 5, and CaCl₂ 0.65 (Pca 8.0) at pH 7.2. The external solution used to measure K⁺ currents was maintained at 34°C and was composed of the following (mmol/L): N-methyl-d-glucamine 149, MgCl₂ 5, HEPES 5, and nisoldipine 0.003. I_Ks and I_Kr were separated by applying 2 specific blockers, chromanol 293B (30 μmol/L)¹⁷ and E-4031 (10 μmol/L),¹⁴ respectively.

Oocytes were bathed in a modified ND96 solution containing (mmol/L) NaCl 94, KCl 4, MgCl₂ 2, CaCl₂ 0.1, and HEPES 5 (pH 7.6). CaCl₂ was reduced to 0.1 mmol/L to suppress endogenous Ca²⁺-activated Cl⁻ current.

Amiodarone hydrochloride (Sigma Chemical Co) was dissolved in dimethyl sulfoxide to prepare a stock solution of 300 mmol/L. On the day of experiments, aliquots of the stock solution were diluted with the bath solution. Dimethyl sulfoxide at 0.1% had no significant effect on outward currents in rabbit ventricular myocytes or Xenopus oocytes. E-4031 was kindly provided by Eisai Pharmaceuticals (Tokyo, Japan); chromanol 293B, by Aventis Pharma (Frankfurt, Germany).

Statistical Analysis

Data are presented as mean±SEM unless otherwise specified. When relative densities of I_Kr and I_Ks and effects of long-term amiodarone administration on the ratio were determined, current amplitudes from 2 to 3 myocytes per each rabbit heart were averaged and served as 1 datum point. Statistical comparisons between the different experimental groups were obtained by ANOVA. Comparisons between multiple group means were performed with a Bonferroni-corrected t test for all group comparisons. Differences were considered significant at P<0.05. Concentration-response relationships were fit to the Hill equation to determine the concentration of drug required for 50% inhibition (IC₅₀). A nonlinear least-squares curve-fitting program (Calmpfit 6.0) was used to analyze deactivation kinetics.

I_Kr and I_Ks of Rabbit Ventricular Myocytes

The relative contributions of chromanol 293B–resistant and –sensitive components of I_K in rabbit isolated ventricular myocytes are illustrated in Figure 1⇓. Depolarizing pulses for 2 seconds from −40 to 70 mV were applied from the holding potential at −75 mV at 0.03 Hz. The tail current (I_{K, tail}) was measured on repolarization back to −50 mV (Figure 1A⇓). Application of 30 μmol/L chromanol 293B decreased the time-dependent outward current during depolarization and the tail current on repolarization. Additional application of 10 μmol/L E-4031 eliminated most time-dependent current. Figure 1B⇓ illustrates the chromanol 293B–sensitive component obtained by subtraction of the current traces. The chromanol 293B–resistant and –sensitive components of I_K were used in this study to define I_Kr and I_Ks, respectively.

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Figure 1.

Delayed rectifier K⁺ currents in rabbit ventricular myocytes. A, Representative current traces for I_K. Bath application of 30 μmol/L chromanol 293B caused partial inhibition of I_K. The 293B-resistant component (I_Kr) was eliminated after additional application of 10 μmol/L E-4031. B, The 293B-sensitive component (I_Ks) obtained by digital subtraction. C, Tail current-voltage (I_t-V) relationship for total I_K, I_Kr, and I_Ks. Density of peak tail current (mean±SE, n=10) was plotted vs test potentials. D, Averaged activation curves of total I_K, I_Kr, and I_Ks. Data points in C were normalized and fitted to Boltzmann equation. Half-activation voltages (V_1/2) were −5.8 (total I_K), −8.8 (I_Kr), and 9.6 (I_Ks) mV. Slope factors were 14.4 (total I_K), 11.3 (I_Kr), and 13.9 (I_Ks) mV.

Figure 1C⇑ shows a tail current-voltage (I_t-V) relationship for total I_K, I_Kr, and I_Ks in control rabbit ventricular myocytes (n=10, 22 cells). Based on sensitivity to chromanol 293B, 71% of the tail current amplitude after depolarization to 50 mV was attributable to I_Kr and 29% to I_Ks. Tail current amplitudes were normalized to peak values, and the resulting I-V relation was fitted to a Boltzmann equation to construct isochronal activation curves (Figure 1D⇑).

The time courses of activation at 50 mV and deactivation at −50 mV were determined by exponential fitting. The time constants of I_Kr and I_Ks in control myocytes are summarized in Table 2⇓. There were no significant differences between the 2 components of I_K at these potentials.

Short-Term Effects of Amiodarone on I_Kr and I_Ks

The short-term effects of amiodarone on I_Kr were examined in ventricular myocytes pretreated with 30 μmol/L 293B (Figure 2⇓). Application of amiodarone (0.1 to 10 μmol/L) caused a concentration-dependent inhibition of both time-dependent outward currents and tail currents (Figure 2A⇓). The residual tail currents after application of 10 μmol/L amiodarone were abolished after additional application of 10 μmol/L E-4031. Figure 2B⇓ shows averaged I-V relationships for I_Kr (n=8). The tail current density after depolarization to 50 mV was reduced by amiodarone at 1 and 10 μmol/L by 29% and 75%, respectively, from baseline (0.53±0.12 pA/pF). The IC₅₀ for block of I_Kr was 2.8 μmol/L (Figure 2C⇓).

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Figure 2.

Short-term effects of amiodarone on I_Kr. I_K was elicited by same voltage-clamp pulse protocol as in Figure 1⇑. A, Representative current traces in ventricular myocyte pretreated with 30 μmol/L chromanol 293B. Bath application of 10 μmol/L amiodarone caused marked inhibition of 293B-resistant component of I_K (I_Kr). Additional application of 10 μmol/L E-4031 resulted in complete elimination of time-dependent currents. B, I_t-V relationship for I_Kr before and after application of 1 and 10 μmol/L amiodarone. Density of peak tail current (mean±SE, n=8) was plotted vs test potentials (*P<0.05 vs control). C, Concentration-response relationship for block of tail current after pulse to 50 mV by amiodarone. IC₅₀ and Hill coefficient were 2.8 μmol/L and 0.91, respectively (n=8).

Short-term effects of amiodarone on I_Ks were examined in myocytes pretreated with 10 μmol/L E-4031 (Figure 3⇓). Application of 10 μmol/L amiodarone to these myocytes caused only a slight inhibition of currents. The residual time-dependent currents in the presence of 10 μmol/L amiodarone were abolished after additional application of 30 μmol/L 293B. To estimate the short-term effects of amiodarone on I_Ks more precisely, 293B-sensitive components were obtained by subtraction of current traces in the absence and presence of 30 μmol/L 293B (Figure 3B⇓, a-c and b-c). A minimal reduction in both time-dependent outward currents and tail currents by 10 μmol/L amiodarone was recognized more clearly in such subtracted current traces. Figure 3B⇓ shows the I_t-V relationship of I_Ks, in which the tail current amplitude in the subtracted current was plotted against test potential (n=9). Amiodarone (10 μmol/L) caused a slight reduction in currents that was associated with a 15-mV shift in the I_t-V relationship.

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Figure 3.

Short-term effects of amiodarone on I_Ks. Current was elicited by same voltage-clamp pulse protocol as in Figure 1⇑. A, Representative current traces in ventricular myocyte pretreated with 10 μmol/L E-4031. Bath application of 10 μmol/L amiodarone caused minimal inhibition of E-4031–resistant component of I_K (a, b). Additional application of 30 μmol/L 293B resulted in complete elimination of the time-dependent currents (c). Changes in 293B-sensitive component of I_K (I_Kr) after application of 10 μmol/L amiodarone are shown by digital subtraction (a-c, b-c). B, I_t-V relationship for I_Ks before and after 10 μmol/L amiodarone (n=10; *P<0.05 vs control).

Effects of Amiodarone on HERG and KvLQT1/minK Currents

HERG currents were measured with a 2-step pulse protocol at a frequency of 0.03 Hz. From a holding potential at −90 mV, a 2-second depolarization (−80 to 50 mV) was applied to activate outward currents, followed by return of the membrane potential to −70 mV to evoke tail currents (Figure 4A⇓). The amplitude of outward tail currents exceeded the amplitude of the activating currents, characteristic of HERG channel behavior.^{11 18} Bath application of amiodarone (1 to 100 μmol/L) for 30 minutes resulted in a concentration-dependent decrease in outward currents during depolarization and tail currents. Figure 4B⇓ and 4C⇓ shows the I-V relationship for currents at the end of the depolarization step and at the peak of the tail after repolarization, respectively (n=5). The IC₅₀ for amiodarone block of the HERG channel tail current was 37.9 μmol/L (Hill coefficient=0.61; n=5).

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Figure 4.

Short-term effects of amiodarone on HERG and KvLQT1/minK currents in Xenopus oocytes. A, Representative HERG current traces recorded from oocyte before and after application of 30 μmol/L amiodarone. Voltage-clamp pulse protocol is shown above current traces. B, I-V relationship for currents measured at end of 2-second depolarization step (n=5). Data were obtained before (○) and 30 minutes after amiodarone at 3 μmol/L (▵), 30 μmol/L (□), and 100 μmol/L (•). C, I_t-V relationship for peak tail currents. D, Representative current traces recorded from oocyte expressing KvLQT1 and minK subunits before and after application of 300 μmol/L amiodarone. E, I-V relationship for currents measured at end of 7.5-second depolarization step (n=5). Data were obtained before (○) and 30 minutes after (•) 300 μmol/L amiodarone. F, I_t-V relationship for peak tail currents (n=5).

Injection of oocytes with cRNA encoding KVLQT1 and minK subunits induced I_Ks, characterized by a linear I-V relationship (Figure 4D⇑).^{8 9} Currents were measured during a 7.5-second depolarizing pulse to potentials ranging from −80 to 40 mV, with tail currents measured at −80 mV. Pulses were applied at a frequency of 0.03 Hz. Bath application of amiodarone did not affect the time-dependent currents even at the highest concentration tested (300 μmol/L). Figure 4E⇑ and 4F⇑ shows the I-V relationships for currents during the test pulse (step) and for tail currents. Unlike what was observed for I_Ks in rabbit myocytes, the averaged I-V curve was not shifted by 300 μmol/L amiodarone.

Long-Term Effects of Amiodarone on I_Kr and I_Ks

Figure 5⇓ shows I_Kr and I_Ks in ventricular myocytes isolated from rabbits treated with oral amiodarone (100 mg · kg⁻¹ · d⁻¹) for 4 weeks. Averaged current densities of total I_K in these myocytes were significantly less than total I_K measured in cells isolated from control rabbits. The tail current density after depolarization to 50 mV was 0.81±0.13 pA/pF in controls (n=10; 22 cells) and 0.45±0.04 pA/pF in cells isolated from rabbits treated long term with amiodarone (n=9; 26 cells). Application of 30 μmol/L 293B caused no substantial change in both time-dependent outward currents and tail currents (Figure 5A⇓). Additional application of 10 μmol/L E-4031 resulted in complete elimination of the time-dependent currents. These findings indicate that I_Ks was very small in cells isolated from rabbits treated long term with amiodarone.

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Figure 5.

Delayed rectifier K⁺ currents recorded from ventricular myocytes isolated from rabbits treated long term with amiodarone. I_K was elicited by same voltage-clamp pulse protocol as in Figure 1⇑. A, Representative traces of I_K before and after application of 30 μmol/L chromanol 293B, then again after addition of 10 μmol/L E-4031. B, I_t-V relationships (n=10) for total I_K, I_Kr, and I_Ks. C, Averaged isochronal activation curves for total I_K, I_Kr, and I_Ks. Data points in B were normalized and fitted to Boltzmann equation. V_1/2 values were −6.2 (total I_K), and −9.0 (I_Kr) mV. Slope factors were 14.1 (total I_K) and 15.3 (I_Kr) mV.

Figure 5B⇑ shows I_t-V relationships for total I_K, I_Kr, and I_Ks in myocytes treated with long-term amiodarone (n=9; 26 cells). After depolarization to 50 mV, 92% of the tail current was 293B resistant (I_Kr), and only 8% was chromanol 293B sensitive (I_Ks). The tail current density of I_Kr in the amiodarone-treated myocytes (0.41±0.04 pA/pF at 50 mV) was reduced by 29% (P<0.05) compared with control myocytes (0.58±0.11 pA/pF at 50 mV). In contrast, the tail current density of I_Ks in the amiodarone-treated myocytes (0.04±0.02 pA/pF at 50 mV) was reduced by 83% (P<0.05) compared with control (0.23±0.02 pA/pF at 50 mV). Figure 5C⇑ shows isochronal activation curves for total I_K and its 2 components. V_1/2 of total I_K and I_Kr did not differ significantly from the corresponding values in control myocytes. The amplitude of I_Ks was too small to analyze precisely, but its activation curve (Figure 5C⇑, dotted line) was apparently shifted toward more positive potentials compared with those of I_K and I_Kr. There were no significant differences between control and amiodarone-treated myocytes in the activation and deactivation time constants of I_Kr (Table 2⇑).

Channel Subunit Expression

The effects of long-term treatment of amiodarone on mRNA encoding ERG, KVLQT1, and minK subunits were measured with the ribonuclease protection assay using hearts obtained from control and amiodarone-treated rabbits killed after 28 days. Cyclophilin mRNA expression levels were used for the internal control. As shown in Figure 6⇓, the levels of rabbit ERG,KVLQT1, and minK mRNAs did not exhibit a significant difference between control and amiodarone-treated rabbits.

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Figure 6.

Rabbit ERG, KVLQT1, and minK mRNA expression after 28 days of oral amiodarone administration. From left to right, levels of rabbit ERG, KVLQT1, and minK mRNA were measured by ribonuclease protection assay from RNA extracted from apical part of left ventricles. Cyclophilin mRNA (Cyclo) served as internal control.

Short-Term Effects of Amiodarone on I_Kr and I_Ks

We used myocytes isolated from the apical region of the left ventricle to minimize cell-to-cell variation of I_Kr and I_Ks resulting from their regionally different distribution.¹⁴ The activation-deactivation kinetics of I_Kr (chromanol 293B–resistant component) and I_Ks (chromanol 293B–sensitive component) in control rabbits were similar to those in our previous reports,^{14 19} in which I_Kr and I_Ks were estimated as E-4031–sensitive and –insensitive components, respectively. Unlike other animal species, activation-deactivation kinetics of I_Kr and I_Ks in rabbits are similar (Table 2⇑). Therefore, it was difficult to discriminate between the 2 components on the basis of kinetics, necessitating a pharmacological approach to distinguishing between I_Kr and I_Ks.

Short-term application of amiodarone to rabbit isolated ventricular myocytes resulted in a concentration-dependent decrease in the 293B-resistant component of I_K (I_Kr) with minimal change in the 293B-sensitive component (I_Ks). This confirms our previous findings, also in rabbit ventricular myocytes, that short-term amiodarone (1 to 10 μmol/L) inhibited the E-4031 (10 μmol/L)–sensitive component of I_K (I_Kr) without affecting the E-4031–resistant component (I_Ks).¹¹ However, these results are at odds with a previous report on guinea pig ventricular myocytes by Balser et al.¹⁰ Differences in animal species and experimental conditions might explain the discrepancy.

The study of heterologously expressed human channels in Xenopus oocytes confirmed the short-term effects of amiodarone observed in isolated rabbit cardiac myocytes. We confirmed the findings of a recent study by Kiehn et al¹⁸ that HERG channels can be blocked by short-term amiodarone (IC₅₀, 9.8 μmol/L) and demonstrated a lack of effect of the drug on KvLQT1/minK current.

Long-Term Effects of Amiodarone on I_Kr and I_Ks

The most prominent effect of long-term amiodarone on cardiac muscles is a moderate and frequency-independent prolongation of action potential duration.⁵ Information available to explain the mechanism underlying the action potential duration prolongation is limited. It was shown in our previous study in ventricular myocytes isolated from rabbits treated with oral amiodarone (100 mg · kg⁻¹ · d⁻¹ for 4 weeks) that the current densities of I_{K, tail} and I_to (transient outward current) were decreased significantly compared with control rabbits (by ≈50% and ≈30%, respectively), without any appreciable changes in their voltage dependence.^{5 11} Qualitatively similar findings have been reported by Varró et al.¹² In these reports, I_K was not separated into the 2 components I_Kr and I_Ks. In the present study, the tail current density of total I_Kr in amiodarone-treated rabbits was decreased by 29%, whereas the tail current density of I_Ks was reduced by 83% from controls. This indicates that I_K inhibition by long-term amiodarone is predominantly due to a reduction in I_Ks.

Recently, Bosch et al¹³ reported that long-term treatment of guinea pigs with intraperitoneal amiodarone (80 mg · kg⁻¹ · d⁻¹ for 7days) caused a substantial reduction in the current density of I_Kr and I_Ks of ventricular myocytes to a similar extent (≈60% reduction) without affecting their voltage-dependence or kinetics. Different periods of amiodarone administration (4 versus 1 week), different experimental protocols, and different animal species (rabbits versus guinea pigs) may underlie the discrepancy between our data and their observations.

Study Limitations

We focused our investigation on the short- and long-term effects of amiodarone on I_Kr and I_Ks because block of these currents is an obvious candidate mechanism for the class III properties of this drug. However, the antiarrhythmic action of amiodarone likely results from inhibition of multiple channels and receptors.^{3 5} In addition, long-term effects of amiodarone on the heart are modulated by plasma and tissue accumulation of the parent drug and its active metabolite desethylamiodarone, which would impose their direct effects.^{3 5}

mRNA levels of ERG, KVLQT1, and minK potassium channel subunits were not affected by long-term amiodarone, indicating that the reduction in I_Ks density cannot be ascribed to reduced transcription of mRNA. The mechanism of reduced I_Ks is unknown but may be due to an effect on the posttranscriptional processes of channel protein synthesis.