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CARDIOVASCULAR

The Amiodarone Derivative KB130015 [2-Methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran] Induces an Na⁺-Dependent Increase of [Ca²⁺] in Ventricular Myocytes

Laboratory of Experimental Cardiology, Catholic University Leuven, Leuven, Belgium (V.B., D.D., K.R.S.); Interdisciplinary Research Center, Catholic University Leuven Kortrijk, Kortrijk, Belgium (F.V.); and Center for Experimental Surgery and Anesthesiology, Leuven, Leuven, Belgium (K.M.)

Received July 12, 2005; accepted September 14, 2005.

	Abstract

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KB130015 [KB; 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran] is a novel amiodarone derivative designed to retain the antiarrhythmic effects without the side effects. Unlike amiodarone, KB slows Na⁺ current inactivation and could, via an increase in [Na⁺]_i, potentially lead to Ca²⁺ overload. Therefore, we studied the effects of KB on Na⁺ and Ca²⁺ handling in single pig ventricular myocytes using the whole-cell ruptured patch-clamp technique and K₅fluo-3 as [Ca²⁺]_i indicator. KB at 10 µM did not prolong action potential duration but slightly increased the early plateau; spontaneous afterdepolarizations were not observed. The amplitude of the [Ca²⁺]_i transient was larger (434.9 ± 37.2 versus 326.8 ± 39.8 nM at baseline, n = 13, P < 0.05), and the time to peak [Ca²⁺]_i was prolonged. During voltage-clamp pulses, [Ca²⁺]_i transient peak was also larger (578.1 ± 98.9 versus 346.4 ± 52.6 nM at baseline, P < 0.05). Although L-type Ca²⁺ current was reduced (by 21.9% at +10 mV, n = 9, P < 0.05), sarcoplasmic reticulum Ca²⁺ content was significantly enhanced with KB. Forward Na⁺/Ca²⁺ exchange was significantly decreased after KB application, but reverse mode of the Na⁺/Ca²⁺ exchanger was significantly larger, suggesting an increase in [Na⁺]_i with KB. This was confirmed by a 2-fold increase of the [Na⁺]-dependent current generated by the Na/K-ATPase (from 0.17 ± 0.02 to 0.38 ± 0.06 pA/pF, P < 0.05). In conclusion, as predicted from the slowing of I_Na inactivation, KB130015 leads to an increase in [Na⁺]_i and consequently in cellular Ca²⁺ load. This effect is partially offset by a decrease in I_CaL resulting in a mild inotropic effect without the signs of Ca²⁺ overload and related arrhythmias usually associated with Na⁺ channel openers.

The major drawback of current antiarrhythmics is the earlier experiences of increased mortality in the CAST studies (CAST Investigators, 1989). The newer class III agents with a pure K⁺ channel blocking action have also been associated with proarrhythmia, notably a high incidence of Torsade-de-Pointes in the SWORD study (Waldo et al., 1996). They are not a first choice for ventricular arrhythmias occurring in the setting of ischemic cardiomyopathy where there is often already prolongation of the action potential at baseline. The multiaction drug amiodarone has not been reported to have the proarrhythmic effect of other class III agents (Amiodarone Trials Meta-Analysis Investigators, 1997). It has specific effects on multiple cardiac ion channels that differ in acute and chronic treatment (Kodama et al., 1997). After myocardial infarction, treatment with amiodarone has been reported to prevent arrhythmic death (Cairns et al., 1997; Julian et al., 1997), but a recent trial in heart failure patients confirmed superiority of ICD (Bardy et al., 2005). Because of its low proarrhythmic potential, amiodarone remains a widely used and efficient drug. However, its long-term clinical use is limited by its significant extracardiac toxicity. Because of close structural similarity with thyroid hormones, symptoms of hypothyroidism can occur. Corneal deposits and development of lung fibrosis can be major reasons for discontinuation of amiodarone treatment (Martin, 1990). Therefore, several compounds have been developed based on the amiodarone structure, aiming to retain the efficiency and cardiac safety of amiodarone, with less of the extra-cardiac side effects. KB130015 (KB) is one such new drug (Carlsson et al., 2002). Preliminary data in guinea pig suggest that KB has a toxicity profile more advantageous that amiodarone. In the same study in guinea pig papillary muscle, KB has been shown to prolong action potential duration suggesting a potential antiarrhythmic effect. Like amiodarone, it acts on many ion channels, including Ca²⁺, Na⁺, and K⁺ channels (for review, see Mubagwa et al., 2003). Unlike amiodarone, KB slows the inactivation of voltage-dependent Na⁺ channels (Macianskiene et al., 2003b). This effect is expected to increase intracellular [Na⁺] and thus the cellular Ca²⁺ load through an increased influx via Na⁺/Ca²⁺ exchange, a potentially positive inotropic effect. However, KB also decreases L-type Ca²⁺ current, which would lower the cellular Ca²⁺ load (Macianskiene et al., 2003a). The net effect of these opposite actions is yet unknown and is of considerable interest given the earlier experience of increased mortality during chronic treatment in patients with heart failure with agents that increased cellular Ca²⁺ (Packer, 1993). Preliminary data indicated that KB increased cell shortening, especially at high concentrations (Mubagwa et al., 2003). Therefore, in the present study, we examined the effect of KB on cellular Ca²⁺ load and Na⁺/Ca²⁺ exchange function associated with alterations in increased Na⁺ influx. We used pig ventricular myocytes that have action potential duration and frequency behavior close to that of humans.

	Materials and Methods

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Cell Isolation. Single left ventricular myocytes were enzymatically isolated from the hearts of domestic pigs of either sex (body weight, 41 ± 4 kg; n = 10) as previously described (Stankovicova et al., 2000). We used only cells isolated from the midmyocardial layer, mostly from the left ventricular anterior free wall, but in a number of experiments also from the left ventricular posterior wall. Cells were stored at room temperature and used within 24 h.

Solutions and Drugs. All experiments were performed in normal Tyrode's solution 137 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl₂, 1.8 mM CaCl₂, 11.8 mM Na-Hepes, and 10 mM glucose 10, pH 7.40. The pipette solution for whole-cell patch clamp contained 120 mM K-aspartate, 10 mM NaCl, 20 mM KCl, 10 mM K-Hepes, 5 mM MgATP, and 0.05 mM K₅fluo-3, pH 7.2.

A stock solution of KB130015 (a gift from Karo-Bio, Huddinge, Sweden) was prepared in dimethyl sulfoxide at a concentration of 10 mM. The final concentration to be used was obtained by diluting 1:1000 (v/v) in normal Tyrode's solution. A 1:1000 dilution of the solvent in normal Tyrode's solution had no effects by itself (n_cells = 9, data not shown). NiCl₂ (Sigma-Aldrich, St. Louis, MO) was dissolved directly in the Tyrode's solution at 2.5 mM. Dihydroouabain (DHO; Sigma-Aldrich) was prepared as a 1 mM stock in distilled water and diluted 1:100 in the Tyrode's solution before use.

Experimental Setup. [Ca²⁺]_i transients were measured with fluo-3 as a [Ca²⁺]_i indicator. The setup for fluorescence and membrane currents recording was as described before (Antoons et al., 2002). The fluorescence signals were corrected for the background recorded after the seal formation. This signal was further calibrated to [Ca²⁺]_i values, using an F_max reading obtained at the end of the experiment (Trafford et al., 1999; Antoons et al., 2002).

Experimental Protocols. We recorded action potentials and [Ca²⁺]_i transients in current clamp mode, applying 5-ms current injections to trigger action potentials. During voltage-clamp experiments, the holding potential was –70 mV. For measuring L-type Ca²⁺ current, I_CaL, the Na⁺ current was inactivated by a prepulse to –50 mV. I_CaL was taken as the difference between the peak inward current and the current at the end of the pulse during depolarizations to –40 up to +50 mV. The Ca²⁺ content of the sarcoplasmic reticulum (SR) was measured during a fast application of 10 mM caffeine at a holding potential of –50 mV following a train of 10 conditioning pulses from –70 to +10 mV at 1 Hz.

The amplitude of the reverse mode of Na⁺/Ca²⁺ exchange current was measured as the nickel-sensitive current during a depolarizing step to +40 mV. The current generated by the Na/K-ATPase was measured as DHO-sensitive current at a holding potential of –50 mV following a train of 10 conditioning pulses from –70 to +10 mV at 1 Hz. All the experiments were done at 37°C.

Statistics. All data are shown as mean ± S.E.M. As statistical test, a paired Student's t test was used for single measurements, and ANOVA was used for multiple measurements.

	Results

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KB Effects on the [Ca²⁺]_i Transient during Action Potentials. Figure 1A illustrates the effect of KB on the action potential and the pooled data from 13 myocytes. On average, KB did not prolong the action potential duration at 90% of repolarization [318.8 ± 25.9 versus 352.5 ± 23 ms in control (CTRL)]. Resting membrane potential was unchanged, but the early repolarization tended to be less with an increase of V_m at 15 ms in 10 of 13 cells. Figure 1B illustrates the corresponding effect on the [Ca²⁺]_i transient with mean data for [Ca²⁺]_i at rest and at peak. Resting [Ca²⁺] was significantly enhanced with KB (182.2 ± 18.9 versus 121.8 ± 12.7 nM in CTRL, n_cells = 25, P < 0.01), and the amplitude of the [Ca²⁺]_i transient was increased (peak [Ca²⁺]_i = 434.9 ± 37.2 versus 326.8 ± 39.8 nM in CTRL, P < 0.05). The kinetics of the [Ca²⁺]_i transient are given in Fig. 1C. Time to peak was significantly prolonged, and the time from onset of depolarization to half-relaxation was also prolonged, although the rate of relaxation itself was not altered.

View larger version (38K): [in this window] [in a new window]   Fig. 1. KB increases [Ca2+]i transients evoked by action potentials. A, typical example of an action potential recorded before and after 10 µM KB application with a plot of individual and averaged values of the action potential duration at 90% of repolarization (APD90) and membrane potential (Vm) at 15 ms after depolarization. B, [Ca2+]i transient recorded before and after 10 µM KB application with a plot of averaged values for [Ca2+] at rest and peak values of [Ca2+]i (ncells = 25; **, P < 0.01). C, kinetics of the [Ca2+]i transient with time to peak (TTP), time to 50% decline of [Ca2+]i, measured from the onset of depolarization (time to Ca50) and time constant of the rate of decline of [Ca2+]i. For the mean data, ncells = 13; *, P < 0.05; **, P < 0.01.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Effect of KB on the [Ca2+]i transient during square voltage-clamp steps. Example of the effect of KB application during single steps of 250 ms from –70 to +10 mV on the [Ca2+]i transient (left panel) with pooled data for the peak values of the [Ca2+]i transient, the time to peak (TTP), and the time constant of the rate of decline of the [Ca2+]i transient. ncells = 10; *, P < 0.05; **, P < 0.01.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Effects of KB on [Ca2+]i transients with sequential activation of INa and ICaL. A, example of the effect of KB washing on the membrane current and the associated [Ca2+]i transient during a pulse from –70 to –50 mV followed by a step to +10 mV. B, pooled data for the [Ca2+]i transients activated by ICaL during the second step: peak, TTP, and the time constant for the rate of decline of [Ca2+]i before and after KB. ncells = 11; *, P < 0.05.

In a similar protocol as in Fig. 3A, we studied further the effect of KB on the amplitude and the voltage dependence of I_CaL by varying the amplitude of the second depolarizing step. KB decreased significantly the amplitude of I_CaL (by 21% at +10 mV, n_cells = 9, P < 0.05) without affecting its voltage dependence (Fig. 4A). Despite the reduction in I_CaL, KB significantly increased the amplitude of the associated [Ca²⁺]_i transient (at +10 mV, peak [Ca²⁺]_i transient 419.9 ± 46.4 versus 302.6 ± 58.6 nM in CTRL, P < 0.05) (Fig. 4B). Note that this effect is smaller than what is seen during a single step from –70 to +10 mV (Fig. 3B).

View larger version (16K): [in this window] [in a new window]   Fig. 4. Voltage dependence of ICaL and the associated [Ca2+]i transient. A, voltage dependence of L-type Ca2+ current measured as the peak-minusend of pulse current before and after adding KB. B, voltage dependence of the peak [Ca2+]i transient before and after KB. ncells = 9; *, P < 0.05.

View larger version (32K): [in this window] [in a new window]   Fig. 5. SR Ca2+ content and Ca2+ removal by the Na+/Ca2+ exchanger. A, membrane currents and [Ca2+]i transients evoked by 10 mM caffeine application for 12 s at a holding potential of –50 mV following a train of pulses to +10 mV. The traces are the average of 10 cells. B, pooled data of the amplitude of the evoked [Ca2+]i transient, before and after KB. C, integral of the inward Na+/Ca2+ exchange current. D, time constant of the rate of decline of the caffeine-induced [Ca2+]i transient. E, peak inward Na+/Ca2+ exchange current induced by caffeine. F, time constant of the rate of decline of the Na+/Ca2+ exchange current. ncells = 10; *, P < 0.05; **, P < 0.01.

Increased [Na⁺]_i and incomplete removal of Ca²⁺ are also expected to lead to fast reloading of the SR after removal of caffeine. This was tested by applying a second pulse of caffeine 10 s after washout of the first application, with the membrane potential held constant at –50 mV. As illustrated in Fig. 6A, a second caffeine application in CTRL conditions did not evoke a second Ca²⁺ release (no transient increase of [Ca²⁺]_i, only an increase of baseline [Ca²⁺]_i by less than 100 nM). However, with KB, a second release of Ca²⁺ from the SR was observed as a [Ca²⁺]_i transient during the second caffeine pulse, consistent with incomplete removal during the first application, and fast reloading of the SR (peak of the [Ca²⁺]_i transient during the second pulse of caffeine was 316.6 ± 49.7 versus 156.15 ± 25.7 nM for the maximal value of [Ca²⁺]_i during caffeine application in CTRL, n_cells = 10, P < 0.01, Fig. 6B).

View larger version (20K): [in this window] [in a new window]   Fig. 6. Faster SR Ca2+ reloading in unstimulated cells. A, example illustrating the probing SR reloading in unstimulated cells at –50 mV; 10 mM caffeine was applied for 12 s, and a second caffeine application was given 8 to 10 s later (each application indicated by a horizontal line). B, pooled data of the peak [Ca2+]i values during the second caffeine application. ncells = 10; *, P < 0.05; **, P < 0.01.

View larger version (14K): [in this window] [in a new window]   Fig. 7. KB increases reverse-mode Na+/Ca2+ exchange through increased [Na+]i. A, individual and averaged data of the amplitude of nickel-sensitive outward current during a step to +40 mV before and after KB superfusion. ncells = 7; **, P < 0.01. B, pooled data of the amplitude of the outward DHO-sensitive current at –50 mV, recorded after a series of depolarizing pulses from –70 to +10 mV. ncells = 5; *, P < 0.05.

	Discussion

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KB130015, at a dose of 10 µM that significantly slows I_Na inactivation, increases the amplitude of the Ca²⁺ transient during action potentials but without inducing spontaneous Ca²⁺ release; an increase of [Ca²⁺]_i is also seen during square voltage steps. The SR Ca²⁺ content is increased, but the L-type Ca²⁺ current is reduced. The enhanced cellular Ca²⁺ load may result from a reduced forward mode Na⁺/Ca²⁺ exchange and increased reverse mode of Na⁺/Ca²⁺ exchange. These changes in Na⁺/Ca²⁺ exchange activity can be related to an increase in [Na⁺]_i.

Mechanisms Underlying the Increase in [Ca²⁺]_i with KB. In the presence of a reduction of I_CaL, the first explanation for the increase in the amplitude of the [Ca²⁺]_i transient with KB is the increase in SR Ca²⁺ content resulting from the higher intracellular [Na⁺]. We also have to consider, however, that with the increase in [Na⁺]_i, enhanced influx of Ca²⁺ via reverse-mode Na⁺/Ca²⁺ exchange during depolarization can contribute to this increase. We have found previously that reverse mode per se is a very inefficient trigger for Ca²⁺ release and that the Ca²⁺ release seen with the Na⁺ current most likely represents spurious activation of Ca²⁺ channels rather than activation of release channels via reverse mode (Sipido et al., 1995, 1997). This view has been supported by others (for review, see Bers, 2002), although there have been reports that Ca²⁺ release triggered by reverse-mode Na⁺/Ca²⁺ exchange following Na⁺ influx via the channel had a specific profile (Lipp and Niggli, 1994). Others have argued that Ca²⁺ influx via Na⁺/Ca²⁺ exchange can modulate and reinforce the trigger function of I_CaL (Litwin et al., 1998). In Fig. 3B, we saw that the increase in amplitude of the [Ca²⁺]_i transient activated by I_CaL alone during the step from –50 to +10 mV is less than the increase in the [Ca²⁺]_i transient during the action potential or the voltage-clamp step from –70 to +10 mV that activated both I_Na and I_CaL. This can be taken to support the idea that Ca²⁺ influx associated with the Na⁺ current contributes to the enhanced [Ca²⁺]_i transient with KB. It is not absolute proof, however, because the release with the step from –70 to –50 mV has partially depleted the sarcoplasmic reticulum. Another argument in favor of a role for reverse mode is the prolongation of the time to peak [Ca²⁺]_i during action potentials and single square voltage steps from –70 to +10 mV but not in the steps from –50 to +10 mV. This is compatible with the presence of a slower process triggering Ca²⁺ release or contributing direct Ca²⁺ influx in the presence of Na⁺ current only. This could be reverse-mode Na⁺/Ca²⁺ exchange, in particular in the presence of a reduced I_CaL.

Another argument in favor of increased Ca²⁺ influx via reverse-mode Na⁺/Ca²⁺ exchange during depolarization comes from examining the Ca²⁺ flux balance. With a depolarizing step, Ca²⁺ extrusion via Na⁺/Ca²⁺ exchange on repolarization should match the Ca²⁺ influx during the step (Bridge et al., 1990; Trafford et al., 1997; Bers, 2002). We calculated the integral of the Na⁺/Ca²⁺ exchange forward mode on repolarization after the single depolarizing pulse and found that it was unchanged with KB (data not shown). However, because Ca²⁺ influx via I_CaL is reduced, we expect to find a reduced value; thus, this observation can support the concept that there has been additional Ca²⁺ influx via reverse-mode Na⁺/Ca²⁺ exchange.

Is KB a Useful Inotropic and/or Antiarrhythmic Agent? The finding of a moderate increase in Ca²⁺ release without spontaneous Ca²⁺ oscillations can be taken as favorable in comparison with other Na⁺ channel openers such as DPI 201-106, BDF 9148, and BDF 9198 (for review, see Flesch and Erdmann, 2001). To some extent, this must be seen as a dose effect. Indeed, in a few experiments, we used KB at 50 µM and saw a larger increase in [Ca²⁺]_i and [Na⁺]_i, with arrhythmogenic effects (data not shown). On the other hand, there is a genuine difference between KB and these substances, namely the presence of Ca²⁺ channel blockade that limits total Ca²⁺ influx and protects against excessive Ca²⁺ loading. Caution remains, however, in the failing heart because [Na⁺]_i is already elevated, and a further increase in [Na⁺]_i might more rapidly lead to Ca²⁺ overload.

Thus, although the effects on systolic Ca²⁺ can be overall considered rather favorable, the effects on diastolic Ca²⁺ are less favorable. Indeed, the total duration of the [Ca²⁺]_i transient is somewhat prolonged and diastolic [Ca²⁺]_i levels are slightly increased, factors that both will likely reduce diastolic filling in the intact heart. This is particularly relevant for patients with a compromised diastolic function as often observed in heart failure (Kass et al., 2004).

In contrast to classical Na⁺ channel openers, KB does not markedly prolong the action potential; thus, its potential antiarrhythmic activity cannot be related to a typical class III effect. However, there are some unique properties that may help to explain the observed protection (Mubagwa et al., 2003). Triggered early and delayed afterdepolarizations are important mechanisms underlying ventricular arrhythmias in heart failure; KB may partially reduce the likelihood of both these mechanisms.

Early afterdepolarizations have been ascribed to reactivation of I_CaL (January and Riddle, 1989; Zeng and Rudy, 1995); thus, a partial block of I_CaL may be a favorable property of KB. It could be argued that Na⁺ window currents could provide EADs with KB, as seen with anemone toxin II (Boutjdir et al., 1994) or veratridine (Verdonck et al., 1991). A difference with these substances is, however, that they induce a large persistent current that is again not seen with KB. Inward Na⁺/Ca²⁺ exchange current in the setting of enhanced Ca²⁺ loading can provide the conditioning current to allow the EAD (Zeng and Rudy, 1995; Volders et al., 1997; Wehrens et al., 2000). As we have seen, the inward Na⁺/Ca²⁺ exchange current is not increased but rather reduced with KB, probably because of the higher intracellular Na⁺ and leftward shift of the reversal potential. This same mechanism, i.e., a reduced forward Na⁺/Ca²⁺ exchange current, can also help to reduce the likelihood and amplitude of delayed afterdepolarizations if spontaneous Ca²⁺ release would occur. Lastly, the higher amplitude of the outward sodium/potassium pump current may exert a stabilizing influence on the resting membrane potential. As elegantly demonstrated by Pogwizd et al. (2001), a reduction in outward I_K1 at the resting membrane potential, as seen in the rabbit with heart failure, is an important factor in facilitating delayed afterdepolarizations. KB would counteract enhanced excitability by providing additional outward current at the resting membrane potential.

Despite its delaying of Na⁺ channel inactivation, KB does not prolong the action potential. The reduction of I_CaL is one element that contributes, as discussed before (Mubagwa et al., 2003). In the present study, we identify another, namely the larger sodium/potassium pump current. The lack of an increase in action potential duration is favorable to reduce EADs and may also be favorable for the diastolic function.

Slowing of the Na⁺ channel inactivation by KB130015 leads to an increase in [Na⁺]_i and consequently in cellular Ca²⁺ load. This effect is partially offset by a decrease in I_CaL, resulting in a mild inotropic effect without signs of Ca²⁺ overload and related arrhythmias. Activation of the sodium/potassium pump current by the increased [Na⁺]_i may contribute to an antiarrhythmic effect.

	Footnotes

 
This work was supported by grants from the FWO, the Flanders Fund for Scientific Research (to K.R.S. and K.M.).

doi:10.1124/jpet.105.092221.

ABBREVIATIONS: ICD, implantable cardiac defibrillator; KB130015 (KB), 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran; DHO, dihydroouabain; SR, sarcoplasmic reticulum; CTRL, control; DPI 201-106, (±)-4-[3-(4-diphenylmethyl-1-piperazinyl)-2-hydroxy propoxy]-1H-indole-2-carbonitrile; BDF 9148, (±)-4-[3-[[1-(diphenylmethyl)-3-azetidinyl]oxy]-2-hydroxypropoxy]-1H-indole-2-carbonitrile; EAD, early after-depolarization.

Address correspondence to: Dr. Karin R. Sipido, Laboratory of Experimental Cardiology, KUL, Campus Gasthuisberg O/N 7th Floor, Herestraat 49, B-3000 Leuven, Belgium. E-mail: karin.sipido{at}med.kuleuven.be

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