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CARDIOVASCULAR

Mechanisms Underlying the Effects of the Pyrethroid Tefluthrin on Action Potential Duration in Isolated Rat Ventricular Myocytes

Division of Pulmonary and Critical Care Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland

Received February 11, 2005; accepted June 20, 2005.

	Abstract

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Due to increased global use, acute exposures to pyrethroid insecticides in humans are of clinical concern. Pyrethroids have a primary mode of action that involves interference with the inactivation of Na⁺ currents (I_Na) in excitable cells, which may include cardiac myocytes. To investigate the possible cardiac toxicity of these agents, we have examined the effects of a type-1 pyrethroid, tefluthrin, on isolated rat ventricular myocytes. Under whole-cell current-clamp, tefluthrin prolonged the mean action potential duration at 90% repolarization (APD₉₀) by 216 ± 34% in 19 myocytes isolated from 14 hearts. About one-third of this prolongation was apparently due to persistent I_Na, with the balance associated with spontaneous cytosolic Ca²⁺ waves, and Na⁺-Ca²⁺ exchange. In some action potentials, tefluthrin also activated early after-depolarizations (EADs). Using a selected EAD-containing action potential clamp, we observed that EADs could evoke a Cd²⁺-sensitive membrane current (I_EAD) that triggered secondary sarcoplasmic reticulum (SR) Ca²⁺ release. The notion that EADs could stimulate Ca²⁺ current was strengthened by the persistence of I_EAD in myocytes exposed to extracellular Li⁺ and Sr²⁺ ions, used to minimize Na⁺-Ca²⁺ exchange and SR Ca²⁺ release, respectively. Tefluthrin inhibited I_EAD by approximately 10%. Together, our results support an arrhythmogenic model whereby tefluthrin exposure stimulated Na⁺ influx, provoking cellular Ca²⁺ overload by reverse Na⁺-Ca²⁺ exchange. During Ca²⁺ waves, forward Na⁺-Ca²⁺ exchange prolonged the action potential markedly and kindled EADs by permitting the reactivation of Ca²⁺ current. Similar mechanisms may be involved in pyrethroid toxicity in vivo, and also in type 3 long QT syndrome, wherein Na⁺ channel mutations prolong I_Na.

Based on structural differences, pyrethroids are divided into two types that have distinct neurotoxic profiles (Tabarean and Narahashi, 1998; Vais et al., 2000; Soderlund et al., 2002). In cardiac myocytes, both classes of pyrethroids can prolong the action potential duration (APD) and provoke sporadic early after-depolarizations (EADs) (Spencer et al., 2001). EADs are significant for exacerbating the dispersion of myocardial action potential duration, with a possible direct link to arrhythmias (Belardinelli et al., 2003; Restivo et al., 2004). For pyrethroids, this arrhythmogenic effect could be explained by increased I_Na persistence because these agents shift Na⁺ channel activation to more negative potentials and can slow the rate of voltage-dependent inactivation and/or deactivation (Honerjager, 1982; Denac et al., 2000; Soderlund et al., 2002). As such, these effects mimic, at least qualitatively, Na⁺ channel mutations associated with type 3 long QT syndrome (LQT3) (Bennett et al., 1995; Nuyens et al., 2001) and toxins, including pyrethroids, provide a valuable experimental model of this disease (Priori et al., 1996; Restivo et al., 2004).

Despite intensive research into the effects of both toxins and LQT3 mutations on I_Na, it is not yet clear whether persistent I_Na prolongs the cardiac action potential directly or indirectly, due to the effect of Na⁺ entry on cellular Ca²⁺ balance, mediated by Na⁺-Ca²⁺ exchange (Ravens et al., 1991; Hoey et al., 1994). Accordingly, in the present investigation, we determined the relative effects of prolonged I_Na, modified by the type I pyrethroid tefluthrin, and Na⁺-Ca²⁺ exchange current (I_NaCa) on APD in rat ventricular myocytes. Our results suggest that direct depolarization by I_Na accounted for about one-third of tefluthrin-induced APD prolongation, with the remaining two-thirds due to alterations in intracellular [Ca²⁺] ([Ca²⁺]_i) transients. Moreover, EADs seemed to be associated with reactivating Ca²⁺ current (I_Ca), suggesting that multiple inward currents contribute synergistically to APD prolongation, EADs, and arrhythmogenesis. Similar complex interactions between conductances could play a prominent role in pyrethroid toxicity in vivo and may underlie some of the arrhythmias provoked by LQT3 mutations.

	Materials and Methods

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Isolation of Ventricular Myocytes. Adult male Wistar rats (250 g) were completely anesthetized by intraperitoneal injection of pentobarbitone sodium (100 mg kg^–1) and were euthanized by removal of the heart in accordance with the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences, 1996) and Johns Hopkins University Animal Care and Use Committee. Rats were selected for study due to their relevance, via similarities between action potentials, to transgenic models of LQT3 (Nuyens et al., 2001), and to elucidate further upon our previous report (Spencer et al., 2001). To isolate ventricular myocytes, the heart was perfused retrogradely at 38°C for 5 min with nominally Ca²⁺-free HEPES-buffered Tyrode's solution. This was followed by recirculation of the same solution containing (units ml^–1) collagenase (type I) and one protease (type XIV) for 10 min and perfusion with enzyme-free Tyrode's solution containing 0.2 mM CaCl₂ for a further 5 min to stop enzymatic digestion. Ventricles were cut and myocytes dispersed in the same solution at room temperature for experiments within 8 h of cell isolation.

Electrophysiology. Signals were recorded using Axopatch amplifiers in whole-cell current- or voltage-clamp mode (Axon Instruments Inc., Union City, CA). Patch pipettes had resistances of 2.5 to 5 M when filled with intracellular solutions. For adequate physical access to the cytoplasm, the settling time-constant of the voltage-clamp was maintained at 300 to 700 µs. All experiments were conducted at room temperature (approximately 25°C) to maximize the electrophysiological stability of myocytes and to facilitate cellular retention of fluorescent indicators. The ruptured-patch method avoids the accumulation of indicators in intracellular compartments. Action potentials were stimulated (0.05 to 0.1 Hz) at zero holding current by 5-ms depolarizing pulses (<1.5 nA) through the pipette. For voltage-clamp experiments, the voltage command waveform consisted of a prolonged action potential (AP clamp) containing an EAD, previously recorded from a rat ventricular myocyte exposed to tefluthrin. To identify the ionic currents potentially flowing during such EADs, various known blockers and pharmacological agents were used. Block of ionic currents was confirmed using voltage ramps from –90 to +50 mV at 0.32 V s^–1 (not shown), as described previously (Spencer et al., 2001). Cs⁺-based patch pipette solutions served to avoid contamination of voltage-clamp recordings by K⁺ currents.

Intracellular Ca²⁺ Measurements. The Ca²⁺-sensitive fluorescent indicator fluo-4 was included in the patch pipette solutions (200 µM) to measure localized [Ca²⁺]_i by laser scanning confocal microscopy. Confocal images were acquired using a Zeiss LSM 510 microscope (Carl Zeiss Microimaging Inc., Thornwood, NY) with a Zeiss Plan-Neofluor 40x oil immersion objective (numerical aperture 1.3). Fluo-4 was excited at 488 nm, and fluorescence was recorded at wavelengths >505 nm. Images were acquired in line-scan mode (pinhole 200 µm) with the scan-line oriented parallel to the long axis of the cell, and 512 pixels/line (0.15 µm/pixel) were scanned repeatedly at 2-ms intervals for 2 to 3 s. Photobleaching and laser damage to the cells were minimized by attenuating the laser power to 0.25 W. Image acquisition was synchronized to electrophysiological protocols such that line-scans were initiated 100 ms before the stimulation of an action potential or application of a voltage-clamp pulse. The precise timing of events was marked by LED flashes into the microscope's transmitted light channel. After background subtraction, fluo-4 fluorescence was expressed relative to its resting value (F₀), as F/F₀. A pseudoratio [Ca²⁺]_i calibration for fluo-4 (K_d = 1 µM; Molecular Probes, Eugene, OR) was used as necessary (Cheng et al., 1993). In other experiments, the high-affinity Ca²⁺ indicator, indo-1 (1 mM) was used to buffer [Ca²⁺]_i, preventing [Ca²⁺]_i transients from activating inward I_NaCa. To verify this, indo-1 fluorescence was excited at 360 ± 8 nm, and emission was recorded simultaneously at 405 ± 15 and 485 ± 12.5 nm by conventional fluorescence microscopy. After background subtraction, the emission ratio F₄₀₅/F₄₈₅, which reflects [Ca²⁺]_i, was calculated.

Solutions. Unless indicated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO). HEPES-buffered Tyrode's solution contained 137 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 10 mM D-glucose, and 10 mM Na-HEPES (HEPES neutralized to pH 7.4 with NaOH). For cell superfusion, 2 mM CaCl₂ was added. The pyrethroid tefluthrin (Reidel-de Haën/Sigma-Aldrich) was dissolved in dimethyl sulfoxide at 50 mM and was diluted with Tyrode's solutions for experiments. Because pyrethroids seem to dissolve in cell membrane lipids before association with their Na⁺ channel target (Tabarean and Narahashi, 1998), a rapid rate of tefluthrin action was ensured by superfusing cells with final concentrations between 10 and 25 µM. The amount of dimethyl sulfoxide vehicle (0.05% or less) did not affect APD in the absence of the pyrethroid (not shown). Sr²⁺ and Li⁺ Tyrode's solutions were prepared by replacing CaCl₂ with equimolar SrCl₂ and/or by substituting NaCl with equimolar LiCl. For the latter, the pH of HEPES was also adjusted to 7.4 using LiOH instead of NaOH. In some experiments, CdCl₂ (0.3–0.5 mM) was added directly to the Tyrode's solution from 100 mM aqueous stock to block L-type I_Ca. Throughout this study, external solutions around the myocyte of interest were changed rapidly (<1 s) using a computer-controlled concentration-clamp system, assuming a dead time of 750 ms, which was determined in separate experiments in which CdCl₂ abolished I_Ca.

For recording action potentials, patch pipettes were filled with K⁺-based intracellular solution containing 85 mM K-glutamate, 20 mM KCl, 10 mM NaCl, 0.5 mM MgCl₂, 10 mM MgATP, 5 mM pyruvic acid, and 30 mM K-HEPES (HEPES neutralized to pH 7.2 with KOH). As necessary, both forward and reverse Na⁺-Ca²⁺ exchange were minimized by including 1 mM indo-1 and 20 mM LiCl in the same K⁺-based intracellular solution, after omitting NaCl. For voltage-clamp studies, the K⁺ salts were replaced in the intracellular solution by Cs-glutamate, CsCl, and Cs-HEPES at the same concentrations as noted above, and NaCl was omitted. The K⁺ salts of fluo-4 and indo-1 were purchased from Molecular Probes.

Data Handling. Electrophysiological signals were recorded and analyzed using pCLAMP software (version 8.1). Currents, [Ca²⁺]_i transients, and action potentials were low-pass filtered at 500 Hz and digitized at 1 kHz, and action potential duration at 90% repolarization (APD₉₀) was determined. Measurements were made by cursor. Results are expressed as mean ± S.E., and comparisons used Student's paired or unpaired t tests, assuming each myocyte was an independent sample. A P value of <0.05 is taken to indicate a statistically significant difference. The parameters "n" and "A" represent the numbers, respectively, of myocytes studied and hearts used for isolating these cells. To verify the statistical power in our results, t tests were repeated after averaging the data obtained from all cells isolated from each individual heart. The values were similar using either approach. To serve the purposes of illustration, capacitance transients are truncated in figures, and fluorescence line-scans were transformed into pseudocolor using custom software (IDL; Research Systems, Boulder, CO). Ionic currents were normalized to cell capacitance to correct for variations in cell volume.

	Results

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Effects of Tefluthrin on Evoked Action Potentials. Our previous work showed that several pyrethroids, including tefluthrin, prolonged I_Na in rat ventricular myocytes, causing APD prolongation and EADs (Spencer et al., 2001). As shown in Fig. 1, in the present studies also, APD₉₀ was increased significantly from 282 ± 24 to 881 ± 117 ms (n = 19; A = 14), a mean increase of 216 ± 34%, after 1 min of superfusion with tefluthrin (10–25 µM). During these long action potentials, the time to peak of [Ca²⁺]_i transients was also significantly prolonged from 112 ± 17 to 405 ± 92 ms (n = 16; A = 14). At their greatest prolongation, the [Ca²⁺]_i transients incorporated discrete, delayed cytosolic Ca²⁺ waves (Fig. 1, bottom). The membrane potential changes associated with these Ca²⁺ waves varied between APD prolongation at relatively constant voltage (Fig. 1a), slow depolarizations (Fig. 1b), and some with EADs (Fig. 1c). In such action potentials, there was a delay of 132 ± 16 ms between the stimulus and Ca²⁺ wave upstroke, at which time, the action potentials had repolarized to 7 ± 5 mV (n = 10; A = 10). Ca²⁺ waves also exceeded the amplitude of stimulated [Ca²⁺]_i transients by 1.6 ± 0.3 times, and sometimes originated within the imaged cytoplasmic volume as a "V-shaped" fluorescence change (Fig. 1, b and c). Although membrane potential changes and Ca²⁺ waves most often did not align exactly in register, very markedly prolonged depolarizations were not observed in the absence of Ca²⁺ waves, signifying the presence of an underlying Ca²⁺ wave-dependent, depolarizing conductance.

View larger version (41K): [in this window] [in a new window]   Fig. 1. Membrane potential and [Ca2+]i changes induced by tefluthrin. a, top, superimposed action potentials (Em) evoked in control Tyrode's solution (solid trace) before and during superfusion with 10 µM tefluthrin (dotted trace). Middle, superimposed spatially averaged fluorescence profiles corresponding to the action potentials shown above and normalized to the control resting fluorescence (tefluthrin, dotted trace). Bottom, fluorescence line-scan images, in pseudocolor, for the profiles depicted in the middle panel. b, membrane potential (Em), spatial mean fluorescence, and line-scans, as in a, from another myocyte. c, as in b from a further myocyte. Note that Ca2+ waves traversing the scanned line during tefluthrin exposure have a characteristic slanted appearance due to their nonsynchronous activation. An EAD is indicated by an arrow in c. Time scale bar applies to all panels.

Ca²⁺-Dependent Sarcolemmal Currents during Action Potential Prolongation. In rodent hearts, SR Ca²⁺ release has been shown to cause APD prolongation by activating forward Na⁺-Ca²⁺ exchange, generating inward I_NaCa (Knollmann et al., 2003; Spencer and Sham, 2003). Therefore, to examine the involvement of this conductance in tefluthrin-induced APD₉₀ prolongation, myocytes were exposed briefly to extracellular Li⁺ ions, which permeate Na⁺ channels but fail to participate in Li⁺-Ca²⁺ exchange, specifically abolishing inward I_NaCa. As illustrated in Fig. 2a, mean APD₉₀ was significantly prolonged from 267 ± 48 to 715 ± 120 ms after 1 min of superfusion with 10 µM tefluthrin (n = 7; A = 4). In the same myocytes, briefly switching to Li⁺ Tyrode's solution, for a single (10-s) stimulation interval, immediately abbreviated APD₉₀ to 303 ± 24 ms, and the difference between this value and control was nonsignificant (Fig. 2b). The action potential rise times and overshoot potentials were unaltered by Li⁺, suggesting that Na⁺ channel current was similar when carried by either Na⁺ or Li⁺. In agreement with this, during voltage ramps, the observed amplitude of Na⁺ channel current was unaltered after substituting Li⁺ for extracellular Na⁺ (data not shown). Interestingly, when both forward and reverse Na⁺-Ca²⁺ exchange were minimized simultaneously, using intracellular solution containing 1 mM indo-1 plus 20 mM LiCl (Fig. 2, c and d), APD₉₀ was only prolonged by 60 ± 11% (n = 14; A = 5) after 1 min of exposure to 20 µM tefluthrin (Fig. 2, c and d). No EADs occurred under these conditions, and [Ca²⁺]_i transients were negligible. Therefore, when all Na⁺-Ca²⁺ exchange was effectively abolished, tefluthrin-induced APD prolongation was significantly reduced (by about 3-fold).

View larger version (14K): [in this window] [in a new window]   Fig. 2. Inhibition of Na+-Ca2+ exchange during tefluthrin superfusion. A, top, shown are superimposed action potentials stimulated in control Tyrode's solution before (solid trace) and after 1 min of superfusion of a representative myocyte with 10 µM tefluthrin (dotted trace). Tefluthrin exposure prolonged the APD90 from 183 to 689 ms. Bottom, superimposed spatially averaged fluorescence profiles from the same cell, normalized to control resting fluorescence, in control (solid trace) and tefluthrin-containing Tyrode's solution (dotted trace). b, panels identical to panel a for the same cell during superfusion with Li+ Tyrode's solution containing 10 µM tefluthrin (solid traces) and after returning to tefluthrin-containing Tyrode's solution (dotted traces). In the continued presence of tefluthrin, the brief switch to Li+ Tyrode's solution, for a single (10-s) stimulation interval, abbreviated APD90 back to 261 ms and prompted a slight increase in resting [Ca2+]i. Subsequent return to Li+ -free Na+-containing solution allowed APD90 to relengthen immediately to 853 ms. c, action potentials recorded in two different myocytes (top and bottom panels) dialyzed by intracellular solutions including 1 mM indo-1 plus 20 mM LiCl to minimize Na+-Ca2+ exchange. Shown are superimposed action potentials recorded in control Tyrodes' solution (solid traces) and after 1 minute in Tyrode's solution containing 20 µM tefluthrin (dotted traces). Time scale bar applies to all panels.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Action potential voltage-clamp with an EAD-containing command. a, membrane voltage command (Em) with APD90 of 690 ms, an EAD take-off potential of –39 mV, and peak at –3 mV. b, membrane current (Im) recorded during superfusion with control Tyrode's solution using the command shown in a. The current response to the EAD (IEAD) is marked by an arrow. c, spatially averaged fluorescence changes during the action potential clamp of b from the same myocyte. d, membrane current in the same myocyte after adding 300 µM CdCl2 to the superfusion solution. e, Cd2+-sensitive difference current calculated by subtracting record d from record b. f, results from a different myocyte in which a rapid CdCl2 puff was timed to occur just before the voltage-clamped EAD. The control current (dotted) is superimposed on the current recorded with the rapid Cd2+ application (solid line), and the effect of this maneuver on IEAD is shown (for the same cell) at greater resolution in the inset. Time scale bar applies to all panels.

The Ionic Basis of I_EAD. To determine the relative contributions of different known currents to I_EAD, we used selective modification of I_Ca, I_NaCa, and I_Na. Figure 4b shows that when extracellular Ca²⁺ was replaced by Sr²⁺ ions, which permeate Ca²⁺ channels without triggering SR Ca²⁺ release, [Ca²⁺]_i transients immediately disappeared (Spencer and Berlin, 1997). Sr²⁺-containing solution also caused a significant outward shift in holding current from –7.2 ± 0.4 to –2.9 ± 0.5 A F^–1 (n = 16; A = 8) by inhibiting inward rectifier K⁺ channels (Fig. 4b). This effect was confirmed using voltage ramps (not shown). When carried by Sr²⁺, the amplitude of I_EAD was 92 ± 6% (n = 10; A = 6) of its value in Ca²⁺-containing solution, and the membrane potential of peak I_EAD was not significantly altered, supporting the identity of I_EAD as predominantly Ca²⁺ current. The half-time for I_EAD decay was also slightly, but significantly, increased from 21 ± 2 to 31 ± 2 ms (n = 10; A = 6) in cells exposed to Sr²⁺. In five other myocytes from two hearts, when cellular Ca²⁺ overload was provoked by replacing 50% of extracellular Na⁺ by Li⁺, late-occurring Ca²⁺ waves abolished I_EAD (Fig. 4, c and d). This effect, which is consistent with Ca²⁺-induced inactivation of I_Ca, also suggests that inward I_NaCa was not a major contributor to I_EAD, as was confirmed by the persistence of I_EAD in myocytes dialyzed with 1 mM indo-1 plus 20 mM LiCl (data not shown) to abolish Na⁺-Ca²⁺ exchange completely (n = 15; A = 3). Moreover, Fig. 5, a and b, shows that I_EAD also persisted during simultaneous extracellular replacement of Na⁺ by Li⁺, and Ca²⁺ by Sr²⁺, preventing both inward I_NaCa and SR cation release. (The delayed Ca²⁺ wave in Fig. 6c confirms that the SR cation load was not released by I_Sr.)

View larger version (31K): [in this window] [in a new window]   Fig. 4. The influences of SR divalent cation release on IEAD. a, top, membrane voltage command (Em) as in Fig. 3a. Middle, membrane current (Im) recorded during superfusion with control Tyrode's solution using the command shown in a; IEAD is marked by arrow. Bottom, spatially averaged fluorescence and confocal line-scan image. b, panels identical to a in the same cell during superfusion with Sr2+ Tyrode's solution. Note the inhibition of phasic fluorescence transients, but persistence of IEAD. c, panels and axes identical to a in a cell superfused with a 50:50 mixture of Li+ Tyrode's and control Tyrode's solutions ([Na+] 74 mM; see Materials and Methods). d, results from a different myocyte, axes as in a, also during low [Na+] superfusion as in c. In d, a spontaneous Ca2+ wave abolished IEAD. Time scale bar applies to all panels.

View larger version (32K): [in this window] [in a new window]   Fig. 5. Simultaneous inhibition of SR divalent cation release and Na+-Ca2+ exchange does not eliminate IEAD. a, top, membrane voltage command (Em) as in Fig. 3a. Middle, membrane current (Im) recorded during superfusion with control Tyrode's solution using the command shown in a; IEAD is marked by arrow. Bottom, spatially averaged fluorescence changes during the action potential clamp and corresponding confocal line-scan image. b, panels and axes identical to a in the same cell during the superfusion with Sr2+ Tyrode's solution in which all Na+ was replaced by Li+. Note that despite the inhibition of phasic fluorescence transients and inward INaCa, IEAD persisted. c, panels, axes, and solution identical to that in b, in a different myocyte. The dotted line indicates that a spontaneous SR divalent cation release occurred after IEAD had decayed. Each time scale bar applies to panels above and/or to the left of it.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Influences of tefluthrin on IEAD. a, membrane voltage command (Em) as in Fig. 3a. b, membrane current (Im) recorded during superfusion with control Tyrode's solution using the command shown in a; IEAD is marked by arrow. c, Im recorded in the same cell during superfusion with control Tyrode's solution containing 20 µM tefluthrin. d, tefluthrin-sensitive difference current calculated by subtracting record c from record b. Note the large initial current, the small persistent current, and slight inhibition of IEAD (a positive deflection) indicated by the asterisk. e, spatially averaged fluorescence transients during the action potential clamp currents of b. f, spatially averaged fluorescence transients during the action potential clamp currents of c. Time scale bar applies to all panels.

	Discussion

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This investigation examined the electrophysiological effects of the type 1 pyrethroid tefluthrin in isolated rat ventricular myocytes. Our goal was to elucidate the membrane potential changes induced by this agent, which modifies I_Na, causing APD prolongation and EADs, since such changes could be involved in cardiotoxic effects. Our novel findings include 1) that tefluthrin-induced APD prolongation and EADs were linked directly to both I_Na, and I_NaCa, and 2) that an additional Ca²⁺ current (I_EAD) was activated specifically during EADs. By analogy, the chain of events revealed by our results may be typical for agents that hinder Na⁺ channel inactivation gating, including LQT3 mutations.

Pyrethroid-Induced APD Prolongation. In early studies, cardiac inotropic effects produced by pyrethroids were attributed to Na⁺ influx, independent of actions on intracardiac nerves (Forshaw and Bradbury, 1983; Berlin et al., 1984). Subsequently, several pyrethroids were demonstrated to slow the inactivation of cardiac I_Na, similar to the effects of these agents on neurones (Grant et al., 1993; Spencer et al., 2001). This activity, which is shared by other toxins, generates persistent I_Na that may prolong APD by direct sarcolemmal depolarization, and/or by indirectly enhancing forward Na⁺-Ca²⁺ exchange (Ravens et al., 1991; Hoey et al., 1994; Bennett et al., 1995; Nuyens et al., 2001; Spencer et al., 2001). However, the relative contributions of these two mechanisms have not been determined. In the present study, we demonstrated that APD₉₀ was exquisitely sensitive to the abolition of inward I_NaCa by extracellular Li⁺ ions, such that tefluthrin-induced APD prolongation was reversed during superfusion by Li⁺-containing solution (Fig. 2b). It follows that inward I_NaCa dominated the control of APD during Ca²⁺ overload induced by tefluthrin-stimulated Na⁺ influx (Spencer and Sham, 2003). Furthermore, I_Na and I_Li induced by voltage ramps were similar (not shown), consistent with the known permeability of Na⁺ channels to these two ions (Lipp and Niggli, 1994; Sakmann et al., 2000). Thus, in the continued presence of tefluthrin, less APD shortening may have been expected to follow Li⁺ exposure, due to the presence of persistent I_Li. This current was therefore most likely masked by outward I_NaCa (reverse exchange) for which the driving force would be essentially infinite in zero extracellular [Na⁺]. To investigate this possibility further, the effects of persistent I_Na on APD were also studied after abolishing forward and reverse Na⁺-Ca²⁺ exchange simultaneously, without removing extracellular Na⁺ (Fig. 2, c and d). In myocytes dialyzed by 1 mM indo-1 plus 20 mM LiCl, tefluthrin-induced APD prolongation was only about 60%, compared with roughly 200% prolongation when Na⁺-Ca²⁺ exchange was fully active. This lesser degree of APD prolongation may indicate the true effect of persistent I_Na, which alone caused only limited sarcolemmal depolarization.

In Fig. 1, Ca²⁺ waves and membrane potential changes were out of register, which is explained largely by the recording of membrane potential over the whole sarcolemma, whereas confocal line-scans sampled only a small cytosolic volume. Interestingly, the first Ca²⁺ wave during highly prolonged action potentials was delayed by over 100 ms with respect to stimulation, coinciding with early repolarization (to about +10 mV). Moreover, the amplitude of such Ca²⁺ waves most often exceeded the stimulated [Ca²⁺]_i transient (Figs. 1, b and c, and 4d), suggesting that the SR Ca²⁺ load available for Ca²⁺ wave propagation was greater than at stimulation. This slightly surprising observation may have arisen because reverse Na⁺-Ca²⁺ exchange, although being a poor trigger of SR Ca²⁺ release, can preferentially load the SR with Ca²⁺ after an evoked [Ca²⁺]_i transient (Sham, 2000). Therefore, the recovery of refractory Ca²⁺ release channels during SR refilling may have permitted the threshold Ca²⁺ load for spontaneous Ca²⁺ release to be attained 100 to 200 ms after a stimulation (Han et al., 1994; Spencer and Berlin, 1995; Sham et al., 1998). Pseudoratio calibrations suggested a maximal [Ca²⁺]_i of about 1.6 µM at the peak of Ca²⁺ waves; and assuming that peak subsarcolemmal [Ca²⁺]_i was similar, such a concentration could represent a lower limit for subsarcolemmal [Ca²⁺]_i at the time of tefluthrin-modified I_Na (Weber et al., 2002). The reversal potential for Na⁺-Ca²⁺ exchange might then have been determined by changes in subsarcolemmal [Na⁺]_i, as such, being steeply dependent on the decay of pyrethroid-induced Na⁺ influx (Weber et al., 2003). Simple calculations suggest that exchanger reversal potentials could range from +26 mV, if subsarcolemmal [Na⁺]_i equalled the pipette [Na⁺], to –30 mV if it was double this (20 mM). Therefore, increases subsarcolemmal [Na⁺]_i mediated by I_Na, by favoring outward Na⁺-Ca²⁺ exchange (Ca²⁺ influx) during early repolarization, could be expected to progressively Ca²⁺ load the SR, possibly synchronizing the first Ca²⁺ waves (Bers, 2002). The dependence of I_NaCa on both subsarcolemmal Na⁺ and Ca²⁺ gradients could also contribute to APD variability which is a consistent observation in myocytes exposed to pyrethroids (Spencer et al., 2001).

Ionic Basis for Tefluthrin-Induced EADs. When used as a voltage command, a tefluthrin-induced EAD evoked prominent inward current (I_EAD) analogous to currents previously implied indirectly (January and Riddle, 1989). Evidence that this current was conducted predominantly, if not entirely, by sarcolemmal Ca²⁺ channels can be summarized as follows: 1) I_EAD was associated with rapid, synchronous secondary [Ca²⁺]_i transients (Fig. 3c), indicating Ca²⁺-induced Ca²⁺ release, which disappeared when Ca²⁺ channels were blocked by Cd²⁺ (Cheng et al., 1993; Bouchard et al., 1995). Cd²⁺ sensitivity was unchanged whether the blocking ion was present in the bulk superfusion solution, or when CdCl₂ was rapidly applied as a "puff" during the plateau of the APC, showing that I_EAD was insensitive to the level of SR Ca²⁺ loading. 2) I_EAD was abolished by Ca²⁺ waves, consistent with Ca²⁺-induced inactivation of Ca²⁺ channels (Fig. 4d). 3) I_EAD persisted after replacing extracellular Ca²⁺ with Sr²⁺ to prevent [Ca²⁺]_i transients (Spencer and Berlin, 1997) and after the simultaneous replacement of extracellular Ca²⁺ with Sr²⁺ and Na⁺ with Li⁺, eliminating the possibility that Na⁺-Ca²⁺ exchange was responsible for this current (Fig. 5). 4) I_EAD was not stimulated, but it was slightly reduced by exposure to tefluthrin-containing solution (Fig. 6d), indicating that Na⁺ channels were not involved in this current. In itself, this also implies that persistent tefluthrin-sensitive current was carried by Na⁺, because other available conductances are inhibited by pyrethroids (Soderlund et al., 2002).

The rapid phase of I_EAD, which activated at –15 mV, was preceded by a slower inward creep of current, suggesting that different gating processes underpinned the fast and slow phases (Fig. 3e). One possibility is that the current creep reflected the slow reactivation of window I_Ca (Hirano et al., 1992). It is also notable that Ca²⁺ waves could both prolong the action potential, facilitating the development of I_EAD and also inactivate this current (Fig. 5d). Therefore, the relationship between Ca²⁺ waves and late-occurring EADs may be complex, possibly explaining why EADs are not associated with every action potential during a given stimulation train. At present, it seems unlikely that persistent I_Na supplied the necessary depolarization for initiating I_EAD, because this Na⁺ current seemed largely invariant, and because tefluthrin-induced EADs were not observed after minimization of Na⁺-Ca²⁺ exchange (as in Fig. 2, c and d). Although the role for Na⁺-Ca²⁺ exchange in EADs continues to be debated (Marban et al., 1986; January and Riddle, 1989; Volders et al., 2000), our results suggest that this mechanism mediated both SR Ca²⁺ loading and the inward current underlying APD prolongation, even if the EAD upstroke was ultimately attributable to I_Ca.

Toxicology and Clinical Implications. The effects of tefluthrin on action potentials resemble those of the anemone toxin ATXII (Hoey et al., 1994) and of several type I and type II pyrethroids studied in our previous investigation (Spencer et al., 2001). Therefore, many agents interfering with Na⁺ channel inactivation could have the potential for both direct depolarization of cardiac cells and indirect effects attributable to Ca²⁺ overloading. In previous work, however, tetramethrin proved ineffective against cardiac I_Na, so our findings may not apply automatically to all pyrethroids (Spencer et al., 2001). Due to high lipophilicity, pyrethroids effect a rather uniform bodily distribution after oral ingestion (Soderlund et al., 2002), and if the volume distribution is close to that of other hydrophobic drugs such as phenobarbitone (0.7), the LD₅₀ of 25 mg kg^–1 for tefluthrin (in rats) corresponds to a tissue concentration approaching 100 µM. Although continuous superfusion of myocytes cannot mimic a one-time oral pyrethroid dose, our present findings suggest the potential for arrhythmogenic effects in acute pyrethroid intoxication, especially since 20 µM tefluthrin was able to modify I_Na comprehensively within just 20 s of superfusion. In addition, pyrethroids have lesser effects on other conductances, which may be manifest at higher concentrations (Soderlund et al., 2002; Hildebrand et al., 2004). Considering LQT3 models, the present results suggest that indirect potentiation of Na⁺-Ca²⁺ exchange by persistent I_Na could be a cryptic factor involved in LQT3 arrhythmias (Nuyens et al., 2001). Moreover, since substantial increases in [Na⁺]_i may occur in heart failure (Pogwizd et al., 2003), and about 50% of heart failure patients die of arrhythmia, the present findings may also suggest that Ca²⁺ overload during the action potential could be a novel arrhythmogenic risk factor in this disease as well.

	Footnotes

 
This work was funded by American Heart Association Mid-Atlantic Affiliate Beginning Grant-in-aid Award 0465502U (to C.I.S.) and by National Institutes of Health Awards HL-071835 and HL-63813 (to J.S.K.S.).

doi:10.1124/jpet.105.084822.

ABBREVIATIONS: APD, action potential duration; EAD, early after-depolarization; LQT3, type 3 long QT syndrome; [Ca²⁺]_i, intracellular calcium concentration; AP, action potential; APD₉₀, action potential duration measured at 90% of repolarization; SR, sarcoplasmic reticulum.

¹ Current address: CV Therapeutics, Inc., Palo Alto, CA.

Address correspondence to: Dr. C. Ian Spencer, CV Therapeutics, Inc., 3172 Porter Drive, Palo Alto, CA 94304. E-mail: ian.spencer{at}cvt.com

	References

Top Abstract Materials and Methods Results Discussion References

 

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