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Am J Physiol Heart Circ Physiol 288: H778-H786, 2005. First published October 7, 2004; doi:10.1152/ajpheart.00542.2004
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Frequency-dependent and proarrhythmogenic effects of FK-506 in rat ventricular cells

Jérémy Fauconnier,1 Alain Lacampagne,1 Jean-Michel Rauzier,1 Pierre Fontanaud,2 Jean-Marc Frapier,1 Ole M. Sejersted,3 Guy Vassort,1 and Sylvain Richard1

1Physiopathologie Cardiovasculaire, Institut National de la Santé et de la Recherche Médicale U-637, Université Montpellier 1, and 2Unité Propre de Recherche 9055, Centre National de la Recherche Scientifique, Montpellier, France; and 3Institute for Experimental Medical Research, University of Oslo and Ullevaal University Hospital, Oslo, Norway

Submitted 9 June 2004 ; accepted in final form 30 September 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
FK-506, a widely used immunosuppressant, has caused a few clinical cases with QT prolongation and torsades de pointe at high blood concentration. The proarrhytmogenic potential of FK-506 was investigated in single rat ventricular cells using the whole cell clamp method to record action potentials (APs) and ionic currents. Fluorescence measurements of Ca2+ transients were performed with indo-1 AM using a multiphotonic microscope. FK-506 (25 µmol/l) hyperpolarized the resting membrane potential (RMP; –3 mV) and prolonged APs (AP duration at 90% repolarization increased by 21%) at 0.1 Hz. Prolongation was enhanced by threefold at 3.3 Hz, and early afterdepolarizations (EADs) occurred in 59% of cells. EADs were prevented by stronger intracellular Ca2+ buffering (EGTA: 10 vs. 0.5 mmol/l in the patch pipette) or replacement of extracellular Na+ by Li+, which abolishes Na+/Ca2+ exchange [Na+/Ca2+ exchanger current (INaCa)]. In indo-1-loaded cells, FK-506 generated doublets of Ca2+ transients associated with increased diastolic Ca2+ in one-half of the cells. FK-506 reversibly decreased the L-type Ca2+ current (ICaL) by 25%, although high-frequency-dependent facilitation of ICaL persisted, and decreased three distinct K+ currents: delayed rectifier K+ current (IK; >80%), transient outward K+ current (<20%), and inward rectifier K+ current (IK1; >40%). A shift in the reversal potential of IK1 (–5 mV) accounted for RMP hyperpolarization. Numerical simulations, reproducing all experimental effects of FK-506, and the use of nifedipine showed that frequency-dependent facilitation of ICaL plays a role in the occurrence of EADs. In conclusion, the effects of FK-506 on the cardiac AP are more complex than previously reported and include inhibitions of IK1 and ICaL. Alterations in Ca2+ release and INaCa may contribute to FK-506-induced AP prolongation and EADs in addition to the permissive role of ICaL facilitation at high rates of stimulation.

arrhythmias (mechanisms); long QT syndrome; ion channels; calcium (cellular)

FK-506 (TACROLIMUS) is a potent macrolide widely used for primary immunosuppression or as a rescue therapy for refractory acute rejection after solid organ transplantation. FK-506 binds with high affinity to a class of immunophilins, or protein receptors, referred to as FK-506 binding proteins (FKBPs) (15, 30). The FK-506/FKBP12.6 complex competitively binds to and inhibits the Ca2+- and calmodulin-dependent phosphatase calcineurin (16). This effect results in alterations of intracellular signaling mechanisms central to their immunosuppressive activity (29). The FK-506/FKBP12.6 complex also plays a role in the regulation of the ryanodine receptor (RyR2) and Ca2+-induced Ca2+ release in the heart (18). In addition, FK-506 has the ability to delay cardiac repolarization. This effect is manifested as QT prolongation, which provides a substrate for ventricular "torsades de pointe" (TdP) tachycardia. Several clinical cases of nearly fatal arrhythmias and TdP have been reported after the administration of FK-506 at high blood concentration (3, 13, 14, 25). Moreover, QT dispersion, a marker of risk for arrhythmia and sudden death, is elevated in kidney transplant recipients after they receive oral treatment (Prograf) (11). Quantitative relationships between the concentration of FK-506 and QT prolongation, evaluated in the guinea pig, have shown that delayed ventricular repolarization duration parallels whole blood levels of the drug (22, 23). All these electrical disturbances suggested alterations of ionic currents. Indeed, it has been shown that FK-506 has a high potential to evoke direct inhibition of outward K+ currents [namely, transient outward K+ current (Ito) and delayed rectifier K+ current (IK)] and prolong the action potential (AP) (6–8). In the present study, we show that the effects of FK-506 are more complex and include inhibitions of L-type Ca2+ current (ICaL) and inward rectifier K+ current (IK1). The effect on IK1 has a prominent impact on the AP at low rates of stimulation, resulting in concomitant hyperpolarization of the resting membrane potential (RMP) and prolongation of the late repolarizing phase. High pacing rates dramatically enhance the effect on AP duration (APD) and, thereby, provide conditions for early afterdepolarizations (EADs). Frequency-dependent facilitation of ICaL is involved in this process.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Six- to ten-week-old Wistar-Kyoto rats (Janvier; Le Genest-St-Isle, France) were used. This investigation conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996) and European directives (96/609/EEC).

QT interval measurement. A biocompatible ETA transmitter (Data Sciences; St. Paul, MN) was implanted intraperitoneally in 8-wk-old animals under slight anesthesia. In vivo Holter monitoring was performed by telemetry in untethered rats 1 wk later. FK-506 was injected at 3 mg/kg in the muscle of the left thigh 30 min after the beginning of ECG recordings. The QT interval was normalized into a rate-independent corrected value (QTc). We used Bazett's formula as follows: QTc = QT/, where RR is the interval between two R waves. The blood concentration of FK-506 (in ng/ml) at its maximal effect on the ECG was assessed using a microparticle immunoenzymatical method (University Hospital Lapeyronie, Biochemistry Laboratory).

Cell isolation and cellular electrophysiology. Rats were heparinized and anaesthetized, and ventricular cells were enzymatically isolated as described before (10). Whole cell patch-clamp experiments were performed at room temperature (22–24°C) with an Axopatch 200A (Axon Instruments; Burlingham, CA). Patch pipettes had resistance of 2 M{Omega}. Currents were normalized to the cell membrane capacitance (in pA/pF) measured as before (28). Series resistances were compensated before recordings.

To record APs, the pipette solution contained (in mmol/l) 130 KCl, 25 HEPES, 3 MgATP, 0.4 NaGTP, and 0.5 EGTA (unless otherwise noted); pH was adjusted 7.2 (with KOH). The bath solution contained (in mmol/l ) 135 NaCl, 1 MgCl2, 4 KCl, 11 glucose, 2 HEPES, and 1.8 CaCl2; pH was adjusted to 7.4 (with NaOH). Li+ replaced Na+ in the Na+-free solution. APs were elicited by a 0.2-ms current injection of suprathreshold intensity. During experiments, cells were postrest stimulated by trains of 30 stimuli at 0.1 or 3.3 Hz.

To record ICaL, the bath solution contained (in mmol/l) 1.8 CaCl2, 140 TEA-Cl, 2 MgCl2, 10 glucose, and 10 HEPES; pH was adjusted to 7.4 (with TEAOH) (28). The pipette solution contained (in mmol/l) 140 CsCl, 10 HEPES, 10 EGTA, 0.4 NaGTP, and 3 MgATP; pH was adjusted to 7.2 (with CsOH). ICaL was elicited by test depolarizations (150 ms) from –80 to –10 mV at 0.1 or 3.3 Hz. ICaL amplitude was estimated as the difference between peak ICaL and the current level at the end of the pulse. The decay of ICaL was best fitted by the sum of two exponential components using the following formula: ICaL = Afast x exp(–t/{tau}fast) + Aslow x exp(–t/{tau}slow), where Afast and Aslow are the current amplitudes of the fast and slow components, respectively; t is time; and {tau}fast and {tau}slow are the related time constants of inactivation (8, 28).

K+ currents were recorded with the solution used for APs but with 10 µmol/l tetrodotoxin and 2 mmol/l Co2+ added extracellularly to block Na+ current and ICaL, respectively. They were elicited from a holding potential of –80 mV by test depolarizations (1 s) varying between –50 and +70 mV at 0.1 Hz. The amplitude of Ito was calculated as the difference between the peak of the fast Ito and steady-state current at the end of depolarizing pulses. Amplitudes of IK were measured between the holding current and the steady-state current at the end of depolarizations. Quasi-steady-state current-voltage (I-V) relations of IK1 were also obtained by applying voltage ramps from –120 to +50 mV with a slope of 0.04 V/s.

Measurements of Ca2+ transients. Cells were loaded for 30 min at room temperature with indo-1 AM (10 µmol/l, Molecular Probes). Cells were field stimulated at 0.2 and 1.0 Hz. Fluorescence was measured using a multiphotonic microscope (LSM510-LNO, Zeiss; Le Pecq, France; coupled to a 5-mW laser, TiSa Mira-Verdi, Coherent; Orsay, France). The fluorescences, emitted at 405 and 480 nm after a femtosecond laser pulse excitation at 740 nm, were simultaneously recorded in the line-scan mode (1.9 ms/line). Their ratio (F405/F480) was used to determine the qualitative variation of intracellular Ca2+ concentration ([Ca2+]i)

Analyses and statistics. ECGs were analyzed using automatized software (Ecg Auto 1.4 rev2, Emka Technologies; Paris, France). Electrophysiological data acquisition and analyses were performed using pCLAMP (version 8.1, Axon Instruments). RMP, AP amplitude, and APDs at 20%, 50%, and 90% repolarization were measured. Ca2+ transients were analyzed by subroutines of IDL software (RSI; Paris, France). Ca2+ influx during activation of Ca2+ channels was measured as the integrated surface area during the depolarization. All averaged data are presented as means ± SE. Statistics were performed using Student's t-test (for paired or unpaired samples). Differences were considered significant with *P < 0.05, highly significant with **P < 0.01, and extremely significant with ***P < 0.001.

Solutions. FK-506 was prepared as 25-mmol/l stock solutions in DMSO. Nifedipine (Sigma) was prepared as a 10-mmol/l stock solution in ethanol. On the day of experiments, aliquots were diluted to the desired concentrations in the perfusion solution. Control and test solutions were applied as described (1). For in vivo experiments, Prograf injectable solution was prepared daily at the desired concentration in an isotonic saline solution. FK-506 and Prograf were generous gifs from Fujisawa Pharmaceutical (Osaka, Japan).

Simulation. We used the model of Pandit et al. (26) in conjunction with a mechanistic representation of the frequency-dependent facilitation of ICaL, which we have described previously (17, 28). Differential equations derived from the structure of the complete model were solved using a fourth-order Runge-Kutta numerical integration routine (31) with the WinPP version of XPPAUT (obtainable at http://www.math.pitt.edu/~bard/xpp/xpp.html) (9). Step size was sufficiently small (dt = 2.10–5 s) to ensure accurate numerical integration for all state variables. The effect of FK-506 on a given ionic current was assessed by changing its maximal conductance, consistently with the values obtained experimentally. To modelize the effect of a shift in the I-V curve of IK1, we changed the expression of IK1 by adding a constant shift value in the following equation (26):

where EK is the K+ Nernst potential, gK1 is IK1 conductance, F is Faraday's constant, R is the gas constant, T is temperature, and [K+]o is extracellular K+ concentration.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of FK-506 on the QT interval. Holter recordings were performed during 12 h overnight, corresponding to the active period of animals. Figure 1 A shows a representative evolution of the ECG after the injection of 3 mg/kg FK-506 (see METHODS). Prolongation of the QT/QTc interval started 40 min after the injection and lasted for several hours (Fig. 1B). Similar effects were observed in four different animals. Measurements of the blood concentration of FK-506 during its maximal effect in other animals demonstrated the presence of circulating FK-506 at concentrations varying between 40 and 220 ng/ml (i.e., range 50–250 nmol/l). We concluded, therefore, that FK-506 could prolong the QTc interval also in the rat, as described previously in humans (see Introduction) and in an experimental model such as the guinea pig (22, 23). We did not detect arrhythmic events in these experiments.



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  		Fig. 1. FK-506 prolongs the QT interval in the rat. A: representative effect of FK-506 (3 mg/kg) on the ECG. B: averaged (means ± SE) corrected QT (QTc) in unthetered rats. Each trace is the average of 1,000 ECGs recorded for 5 min every 15 min.

 
Effect of FK-506 on single cells. Prolongation of the QT/QTc interval is considered to be a hallmark of increased susceptibility to arrhythmias. At the cellular level, this is related to AP prolongation. Figure 2A,a shows the effect of FK-506 (25 µmol/l) on the AP of a cardiomyocyte recorded at 0.1 Hz. FK-506 induced both a lengthening of the AP and a hyperpolarization. Steady state was achieved after 8 min. APD90 was increased from 25.6 ± 3.8 to 35.5 ± 4.5 ms (Fig. 2A,b) with a mean prolongation of 20.9 ± 5.0%. RMP hyperpolarized from –80.1 ± 0.3 (control) to –83.9 ± 0.6 mV (***P < 0.001). Increasing the pacing rate from 0.1 to 3.3 Hz in the presence of FK-506 caused further prolongation of the AP, considerably exceeding that observed in control cells (Fig. 2B,a and b). This effect was accompanied by a depolarization of the RMP to –78.8 ± 0.4 mV (***P < 0.001). Furthermore, high pacing rates triggered EADs (Fig. 2B,a) in 59% of cells (n = 17). EADs developed at plateau potentials. All these effects were reversible upon washout of FK-506 for at least 10 min. They were also observed at 5 µmol/l, but they were less pronounced than at 25 µmol/l. Hence, most of the study was conducted using this latter concentration (see also Refs. 6–8).



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  		Fig. 2. FK-506 prolongs the action potential (AP) and hyperpolarizes the resting membrane potential in single cells. A,a: effect of FK-506 (25 µmol/l) on APs recorded at 0.1 Hz. A,b: averaged AP durations [AP durations at 20, 30, 50, and 90% repolarization (APD20, APD30, APD50, and APD90, respectively); means ± SE, n = 17] at 0.1 Hz. B,a: effects of an increase in the pacing rate from 0.1 to 3.3 Hz on the AP before and after FK-506. The control shown is steady-state AP after the change in frequency [stimulation (Stim) 5]; FK-506 shows two stimulations, stimulations 1 and 5, after the change in frequency. B,b: averaged percentage (means ± SE, n = 17) of the steady-state increase in APD90 induced at 3.3 Hz. ***P < 0.001.

 
Effect of FK-506 on ICaL. An increase in the APD is expected to reflect an increase in an inward current or/and a decrease in an outward current. We (10) previously related frequency-dependent changes of rat ventricular AP duration to changes in ICaL. We reasoned that ICaL could be involved in the pacing-dependent effects and in the EADs induced by FK-506. This hypothesis was further supported by the fact that the Ca2+ channel blocker nifedipine (2 µmol/l) prevented the occurrence of EADs in all four cells tested. This is illustrated in Fig. 3A. Because nifedipine abolishes the whole cardiac excitation-contraction coupling process, i.e., Ca2+ entry via ICaL, subsequent sarcoplasmic reticulum Ca2+ release, and Ca2+-activated currents, we investigated the effect of FK-506 directly on ICaL recorded in voltage-clamp conditions. Surprisingly, FK-506 did not increase but rather decreased ICaL peak amplitude (Fig. 3B) with no change in the I-V relationship (Fig. 3C) and ICaL decay kinetics (Fig. 3D,a) and with no use dependence. The decrease was unrelated to spontaneous rundown of ICaL peak amplitude because recovery was observed after washout of the drug (Fig. 3E). Detailed analysis showed that Afast (see METHODS), the Ca2+-dependent fast inactivating component (1), was decreased with no change in the slow component Aslow (Fig. 3C,b), which resulted in a decrease of the [Afast/(Afast + Aslow)] ratio. This decrease of Afast, therefore, accounted for the decrease in global peak ICaL (Fig. 3D,b). Importantly, the frequency-dependent facilitation of ICaL, which we routinely assessed by changing the rate from 0.1 to 3.3 Hz (10), persisted consistently (Fig. 3E). In the examples shown in Fig. 3E, Ca2+ entry, measured as integrated surface area during depolarizations, was increased by 55% after the change from 0.1 to 3.3 Hz in control conditions (top). In the presence of FK-506 (Fig. 3E, middle), the increase was still important (40%) and exceeded largely the reduction induced by FK-506 (–6% of Ca2+ entry during the depolarization) that occurred mainly on current peak amplitude. Therefore, we concluded that the slowing of ICaL decay kinetics can clearly be a major factor in the triggering of EADs.



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  		Fig. 3. FK-506 decreases L-type Ca2+ current (ICaL) but frequency-dependent facilitation remains. A: effect of nifedipine (2 µmol/l) on early afterdepolarizations (EADs) induced by FK-506 at 3.3 Hz. B: typical effect of FK-506 (25 µmol/l) on ICaL peak amplitude with best fits of inactivation. ICaL was evoked at –10 mV from a holding potential (HP) of –80 mV. The ratio [Af/(Af + As)] decreased from 0.83 to 0.74, where Af and As are the current amplitudes of the fast and slow components. C: current-voltage (I-V) curves of ICaL (means ± SE, n = 18). D: fast ({tau}f) and slow time constants of inactivation ({tau}s) of ICaL evoked at –10 mV from a HP of –80 mV (a) and their related amplitudes, Af and As, respectively (b; means ± SE, n = 27). E: steady-state effect of FK-506 and recovery after washout on ICaL recorded at 0.1 and 3.3 Hz.

 
Effect of FK-506 on Ito and IK. FK-506 decreased both Ito and IK (Fig. 4A,a). Steady-state inhibition of IK was obtained within <2 min, whereas steady-state inhibition of Ito needed >10 min (Fig. 4A,b). FK-506 blocked IK nearly completely at all voltages, whereas Ito was much less sensitive (Fig. 4B,a and b). Inhibition of Ito was slightly enhanced by higher pacing rates (<10% of the remaining peak current, data not shown), as reported previously (7).



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  		Fig. 4. FK-506 decreases transient outward K+ current (Ito) and blocks delayed rectifier K+ current (IK). A: decreasing effect of FK-506 (25 µmol/l) on both Ito and IK recorded at +50 mV from a HP of –80 mV at 0.1 Hz. A,a: original data traces with arrows showing how the transient Ito and sustained IK were measured. A,b: time course of effects. B: I-V curve of K+ currents recorded between –50 and +70 mV (protocol in inset). B,a: original data traces. B,b: averaged data (n = 12). Ito and IK were measured as shown in A,a.

 
Effect of FK-506 on IK1. FK-506 also blocked IK1. We used 1 mmol/l Ba2+ to block, and thereby isolate, IK1 (39). Quasi-steady-state I-V relations were studied by applying voltage ramps from –120 to +50 mV with a slope of 0.04 V/s. Figure 5A shows the traces obtained before and after perfusion of Ba2+ and the subtracted current reflecting pure Ba2+-sensitive IK1 (IK1-Ba). Figure 5B summarizes the effect of FK-506 on IK1-Ba. FK-506 reduced both the inward and outward components of IK1, and a leftward shift was observed (–4.8 ± 0.9 mV, n = 6). This shift may partly account for the RMP hyperpolarization induced by FK-506 (Fig. 2A,a) Block of the outward current occurred at voltages corresponding to AP repolarization (between –40 mV and RMP).



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  		Fig. 5. FK-506 decreases inward rectifier K+ current (IK1). A: quasi-steady-state I-V relations of pure Ba2+-sensitive IK1 (IK1-Ba) before (control, Icont) and after the addition of 1 mmol/l Ba2+ (IBa) in the bath; the difference current (Icont – IBa) determines IK1-Ba. B: effect of FK-506 (25 µmol/l) on IK1-Ba between –120 and 0 mV. Reversal potential (Erev) was shifted from –83 to –90 mV in this cell (same as in A).

 
Role of extracellular Na+ and intracellular Ca2+ in the genesis of EADs. In the presence of FK-506, high pacing rates triggered EADs in 59% of single cells dialyzed with 0.5 mmol/l EGTA in the patch pipette (Fig. 2B,a). Interestingly, EADs occurred in only 25% of cells (n = 12) dialyzed with 10 mmol/l EGTA and never in cells (n = 7) dialyzed with 20 mmol/l BAPTA (data not shown). Hence, the occurrence of EADs was related to [Ca2+]i. We further assessed this hypothesis in intact cells loaded with indo-1 and stimulated at 1.0 Hz. The results revealed two populations of cells. In one-half of the cells, FK-506 had no effect on [Ca2+]i. In the other one-half, FK-506 generated doublets of Ca2+ transients (Fig. 6A,a) that were associated with increased diastolic (Fig. 6A,b) and systolic Ca2+ (Fig. 6A,c). Therefore, increased [Ca2+]i caused by FK-506 may contribute to the genesis of EADs. Because Ca2+ overload will favor Na+/Ca2+ exchanger activity and, thereby, induce inward Na+/Ca2+ exchanger current (INaCa), we replaced Na+ by equimolar Li+ to block INaCa. EADs were abolished in this condition, although rapid pacing-induced prolongation of the AP was still observed (Fig. 6B).



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  		Fig. 6. Intracellular Ca2+, extracellular Na+, and EADs. A: effect of FK-506 (25 µmol/l) on diastolic and systolic intracellular Ca2+ concentration measured with indo-1 in single cells. A,a: example of a doublet of Ca2+ transients observed at 1 Hz in one-half of the cells. A,b: averaged basal Ca2+ and peak transient Ca2+ in the absence (control, n = 23) and presence of FK-506 (n = 14); cells exhibiting doublets of Ca2+ transients (n = 7), corresponding to EADs, were discriminated (white bars vs. gray bars). B: APs recorded during trains of stimulation at 2 Hz before and after FK-506. EADs were induced by FK-506 (1). EADs could last in the absence of any stimulation and spontaneous oscillations were observed (2). EADs were not observed when Li+ replaced extracellular Na+. All experiments were performed in the same cell. AU, arbitrary units.

 
AP simulation. We assessed the mechanistic details of the pacing-dependent effect of FK-506 on the AP using a theoretical model of rat ventricular cells (see METHODS). To mimic the effect of FK-506, we decreased all conductances inhibited by FK-506 consistently with experimental observations: 25% for ICaL, 20% for Ito, 80% for IK, and 40% for IK1. We also shifted the reversal potential of IK1 (–3 mV). At a pacing rate of 0.1 Hz, simulations resulted in RMP hyperpolarization and prolongation of the repolarizing phase of the AP (Fig. 7A,a). These effects were quite consistent with the experimental effects of FK-506 (Fig. 2A,a). The shift of IK1 was required to produce the hyperpolarization but not sufficient per se to induce the AP prolongation (data not shown). Simulation with only a decrease in IK1 showed that block of this current by FK-506 accounts for most of the effect of the drug on the AP at low pacing rates (data not shown). An increase in the pacing rate from 0.1 to 3.3 Hz immediately promoted a depolarization of the RMP, a marked prolongation of the AP, and occurrence of EADs, reproducing all the effects of FK-506 (Fig. 7B,a and b). These effects were antagonized by suppressing facilitation of ICaL (i.e., by suppressing frequency-dependent slowing of the decay of ICaL in the model; Fig. 7C), which indicated that this current has a central role. However, the extent of block of Ito is also potentially critical (Fig. 7D). Finally, when INaCa was set to 0, EADs were prevented, although a substantial prolongation of the AP remained (Fig. 7E), as observed experimentally (Fig. 6B).



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  		Fig. 7. Computer simulations of FK-506 effects on rat ventricular APs. A: APs at 0.1 Hz after reducing various conductances consistently with the experimental effects of FK-506 on ionic currents (see text). B: APs at 0.1 and 3.3 Hz before (a) and after (b) simulating the effect of FK-506. C: consequence of suppressing facilitation of ICaL in the model. No EADs occurred at 3.3 Hz. D: effects of different degrees of block of Ito (0, 17, and 23%) on EADs in the model. Block of Ito favors the occurrence of EADs at 3.3 Hz. E: role of the Na+/Ca2+ exchanger. When the Na+/Ca2+ exchanger current was set to 0, pacing-dependent prolongation of AP occurred, but EADs were suppressed.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
FK-506 is a promising immunosuppressant agent (27). FK-506 acts primarily as an inhibitor of the T cell response by preventing dephosphorylation and translocation of the nuclear factor of activated T cells. FK-506 binds to its cellular target, the FKBPs, among which FKBP12.6 is tightly associated to the RyR2, and plays a role in the regulation of Ca2+-induced Ca2+ release in the heart (18). However, large QT dispersion along with prolonged QT interval and cases of ventricular TdP tachycardia have been reported in organ recipients with high FK-506 blood levels (3, 11, 13, 14, 25). Various effects of FK-506 have also been reported before in rat ventricular cells, including AP prolongation, increased intracellular Ca2+ transient, Ca2+ release from the sarcoplasmic reticulum, effects on electrogenic INaCa, and direct effects on Ito and IK (6, 8, 20, 34, 38). The novel findings here are as follows: 1) FK-506 also inhibits ICaL and IK1, and 2) its effects on the AP are highly frequency dependent. The effect on IK1 has major electrophysiological consequences such as RMP hyperpolarization and AP prolongation. Frequency-dependent facilitation of ICaL plays a permissive role in the frequency-dependent prolongation of the AP and triggering of EADs.

FK-506 inhibits ICaL. Our detailed analysis shows that most of the moderate effect of FK-506 on ICaL reflects preferential inhibition of the magnitude of the fast inactivating component (Afast), suggesting a link with Ca2+-dependent inactivation of Ca2+ channels (1, 5, 32, 33). Inactivation may result from local Ca2+ elevation from enhanced activity of the RyR2 by FK-506, thereby decreasing the number of channels available for opening. Previous reports have concluded that FK-506 has no effect on ICaL peak amplitude (8, 20, 34). A possible explanation for this discrepancy with our results is that, in these studies, ICaL was evoked from a depolarized holding potential (–40 mV). Depolarizing the holding potential to this level indeed decreases Afast preferentially (1, 28), which might further prevent theeffect of FK-506 on ICaL. Importantly, despite partial inhibition of peak ICaL by FK-506, high pacing-induced facilitation of ICaL persisted. Facilitation compensated largely the decreasing effect of FK-506 on current peak amplitude and provided the triggering event for EADs here. Large increases in Ca2+ entry were indeed still promoted by high rates in the presence of FK-506. Both the nifedipine effect (Fig. 3A) and numerical simulation (Fig. 7C) demonstrated that the high rate-induced increase in Ca2+ entry via Ca2+ channels provide the depolarization required for the genesis of EADs in the presence of FK-506.

FK-506 inhibits IK1. IK1 has a major role in maintaining the RMP near the high negative K+ equilibrium potential in ventricular cells (24). Moreover, between the RMP and –30 mV as during the AP plateau, the outward current will contribute to phase III repolarization (21). In contrast to its effects on Ito and IK, the inhibition of IK1 by FK-506 was diminished in the presence of intracellular Ca2+ buffers (unpublished results), which may be interpreted as reflecting Ca2+-dependent block of the outward flow due to increased rectification of IK1 channels (19, 39). The consequences of this block were primarily a prolongation of the AP late phase and a hyperpolarization of the RMP due to a leftward shift in the IK1-V curve. Block of the outward current is indeed expected to shift the K+ Nernst potential from its resting value to more hyperpolarized potentials (4). From a physiological point of view, inhibition of IK1 might be, at least partially, involved in the prolongation of the QTc interval observed experimentally (Fig. 1). Selective block of IK1 by Ba2+ has been associated with prolonged QTc in isolated rabbit hearts (37). Similarly, in guinea pig hearts probed with Kir2.1 overexpression and dominant negative suppression, the QTc interval is prolonged by IK1 suppression (21).

FK-506-induced AP prolongation at low pacing rate. FK-506 inhibits various currents involved in the repolarizing phase of the AP. These currents have two opposite roles: depolarization (ICaL) and repolarization (Ito, IK1, and IK). In theory, inhibition of ICaL shortens the AP plateau, whereas inhibition of Ito, IK1, and/or IK delays repolarization. At a low pacing rate (0.1 Hz), FK-506 has a stronger effect on the late phase of the AP. In addition, it hyperpolarizes the RMP. The opposite effects on Ito and ICaL are probably more or less counterbalancing each other during the early phase. Because IK has very little contribution in rat ventricular cells (4, 12), we suggest therefore that most of the prolongation of the AP at the low pacing rate reflects inhibition of IK1. This hypothesis was confirmed by computer simulation, which reproduced the IK1-mediated effect of FK-506 on both APD and RMP.

High pacing rates and EADs: mechanisms. APD is an important arrhythmogenic determinant. AP lengthening can result in the occurrence of EADs (36). In normal conditions, high pacing rates enhance APD in rat ventricular cells. This is a highly dynamic process that involves facilitation of ICaL and occurs through beat-to-beat adaptation (10, 35). We show here that the rapid pacing-induced increase in APD is enhanced by FK-506. Both the plateau phase and late repolarization are markedly prolonged, and EADs eventually develop at plateau potentials. Despite partial inhibition of ICaL by FK-506, high pacing-induced facilitation of ICaL persists and plays a key role (Fig. 7C). Although facilitation of ICaL is not enhanced per se, persistence of the slowing of ICaL decay kinetics after pacing acceleration provides sufficient net depolarizing current during the AP plateau (Fig. 7C) and contributes to or possibly amplifies intracellular Ca2+ overload to generate EADs. In addition, inhibition of Ito and IK1 by FK-506 contributes synergistically to this process in two different ways: 1) it slows AP repolarization within the "window" voltage range of Ca2+ channels, thereby allowing reactivation and enhancement of ICaL; and 2) the use-dependent block of Ito (see Refs. 7 and 8 and present results) causes further depolarization (Fig. 7D).

The EADs are also linked to extracellular Na+ concentration and elevated [Ca2+]i. Manipulations of the Ca2+ buffer in patch-clamped cells and direct measurements of [Ca2+]i in intact cells (Fig. 6A) both highlighted the contribution of [Ca2+]i. FK-506 increases both diastolic [Ca2+]i and the amplitude of [Ca2+]i transients, as reported by others in rat and mouse ventricular myocytes (8, 20, 34). Arrhythmia is associated with these increases (Fig. 6A). FK-506 has probably no direct effect on the Na+/Ca2+ exchanger (20, 34). However, our data suggest that forward INaCa is important (Fig. 6B) in conjunction with Ca2+ overload. Depolarizing INaCa is enhanced by AP prolongation, causing reactivation of ICaL and, thereby, EADs (36), which was confirmed by numerical simulations: setting INaCa to zero prevents EADs (Fig. 7E). Therefore, we conclude that enhancement of INaCa also contributes to acceleration-induced EADs (2) in the presence of FK-506. However, the fact that changes in Ca2+ release function caused by FK-506 were not included in our computational modeling might limit the interpretation concerning the precise contribution of Ca2+-dependent mechanisms.

In conclusion, this study provides explanations for the proarrhythmogenic potential of FK-506. Inhibition of repolarizing K+ currents contributes to AP prolongation and disordered QT. The effect on IK1, responsible for prolonged terminal repolarization of the AP, is the main effect at low pacing rates, and it has probably a significant role in the QT prolongation. We demonstrated that a use-dependent increase in Ca2+ entry, initiated by frequency-dependent facilitation of ICaL, has a permissive effect for the occurrence of EADs with a contribution of electrogenic INaCa. These currents act in combination with elevated intracellular Ca2+ induced by FK-506. This model of drug-induced long QT syndrome provides interesting insights in the cellular mechanisms involved in acceleration-induced EADs.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the Fondation pour la Recherche Médicale (to S. Richards), Région Languedoc-Roussillon, Association Française contre les Myopathies, and Aurora programme (to S. Richard and O. M. Sejersted).


   ACKNOWLEDGMENTS
 
We thank Fujisawa Pharmaceutical (Osaka, Japan) for providing us with FK-506.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Richard, Physiolpatholgie Cardiovascularie, Institut National de la Santé et de la Recherche Médicale U-637, Université Montpellier 1, CHU Arnaud de Villeneuve, 35295 Montpellier cedex 5, France (E-mail: srichard{at}montp.inserm.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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