Introduction

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The focus of heart failure therapy has recently evolved from direct support of cardiac inotropy to the prevention of "maladaptive" responses underlying the evolution of myocardial damage. Nonetheless, direct inotropic support remains a primary need in the management of patients with overt heart failure (Eichhorn and Gheorghiade, 2002) and may even be a prerequisite to establish therapies (e.g., -blockers and angiotensin converting enzyme inhibitors) potentially associated with initial hemodynamic deterioration. Inotropic interventions based on positive modulation of cAMP concentration, albeit suitable for acute treatment, cannot be used chronically because they increase the risk of sudden death (Cohn et al., 1998). This explains why, in spite of their narrow therapeutic range, cardiac glycosides remain a valuable tool in the chronic treatment of symptomatic heart failure, even when sinus rhythm is preserved (The Task Force of the Working Group on Heart Failure of the European Society of Cardiology, 1997). In particular, the use of digoxin is justified by its ability to reduce hospitalization for relapses and improve patients' functional state (The Digitalis Investigation Group, 1997). Although suggestive of hemodynamic amelioration, these effects are not associated with a decrease in mortality rate; conceivably, this reflects the proarrhythmic effect of cardiac glycosides.

Inhibition of the Na⁺/K⁺ pump and the resulting increase in intracellular Ca²⁺ are commonly held to underlie both inotropy and proarrhythmia, thus making the two actions apparently inseparable (Schwartz and Noble, 2001; Bers, 2002). This view is challenged by the observation that the ratio between inotropic and toxic effect may vary widely among different Na⁺/K⁺ pump inhibitors (Wasserstrom et al., 1991).

A sharp dissociation between inotropy and proarrhythmia has been recently observed with a novel nonglycoside Na⁺/K⁺ pump inhibitor, (E,Z)-3-((2-aminoethoxy)imino)androstane-6,17-dione hydrochloride (PST2744). In single cell, tissue, and whole animal studies the incidence of arrhythmias, observed at similar inotropy, was markedly lower for PST2744 than for the reference compound digoxin (Micheletti et al., 2002).

The present study compares the effects of PST2744 and digoxin on the electrical activity and relevant membrane currents of guinea pig ventricular myocytes. The aim of such evaluation is to detect differences in the actions of the two agents that might account for their widely distinct therapeutic index. To this end, except when dose-response curves were obtained, drug effects were compared at concentrations of the two agents exerting similar inotropic effects (equi-inotropic concentrations) as determined by previous experiments on single guinea pig myocytes (Micheletti et al., 2002).

	Materials and Methods

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Myocyte Preparation

Guinea pig ventricular myocytes were isolated by using a retrograde coronary perfusion method previously published except for minor modifications (Zaza et al., 1998). The investigation conforms to the Guide of the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication 85-23, revised 1996); all experiments were carried out according to the guidelines issued by the Animal Care Committee of the University of Milan. In brief, female guinea pigs weighing 200 to 300 g were anesthetized with a xylazine (1.5 mg/kg b.wt.) and ketamine (20 mg/kg b.wt.) mix, killed through cervical dislocation, and exsanguinated. Hearts were quickly removed and perfused in sequence with 1) modified Tyrode's solution (37°C) containing 130 mM NaCl, 4.5 mM KCl, 0.75 mM CaCl₂, 5 mM MgCl₂, 23 mM HEPES-NaOH, 21 mM D-glucose, 1 mM NaH₂PO₄, 20 mM taurine, 5 mM creatine, and 5 mM pyruvate, adjusted to pH 7.3, and equilibrated with 100% O₂; 2) a nominally Ca²⁺-free Tyrode's solution containing 3.3 µM EGTA and adjusted to pH 7.0; 3) a 0.075 mM CaCl₂ Tyrode's solution containing 140 U/ml collagenase (type 2; Worthington Biochemicals, Freehold, NJ), 0.17 U/ml protease (type XIV; Sigma-Aldrich, St. Louis, MO), and bovine serum albumin (1 mg/ml). The ventricles were then chopped at 37°C and triturated in nominal Ca²⁺-free solution adjusted to pH 7.3. The supernatant was collected every 5 min, filtered through a nylon mesh, and centrifuged at 500 rpm for 3 min. To separate myocyte and nonmyocyte cells, the pellets were resuspended (50%, v/v) in a continuous Percoll gradient containing 0.9% NaCl and centrifuged at 500 rpm for 15 min. Finally, isolated myocytes were resuspended in 1 mM CaCl₂ Tyrode's solution containing gentamycin (10 µg/ml) and stored at room temperature until use. Rod-shaped, Ca²⁺-tolerant myocytes, obtained with this procedure, were used within 12 h from dissociation. Measurements were performed only in quiescent myocytes with clear-cut striations.

Recording Techniques

Myocytes suspension was placed in a 30-mm Petri dish, with a plastic ring to reduce total volume to 1 ml. The dish was perfused at 2 ml/min with standard external Tyrode's solution, containing 154 mM NaCl, 4 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES-NaOH, and 5.5 mM D-glucose, adjusted to pH 7.35. The cell under study was held within 300 µm from the tip (1 mm) of a thermostated multiline pipette allowing for rapid switch between solutions. The solution temperature was monitored at the pipette tip with a fast-response digital thermometer (BAT-12; Physitemp, Clifton, NJ) and kept at 35 ± 0.5°C except when otherwise specified (see Results).

Membrane potential and current were measured in the whole cell configuration (Axopatch 200-A; Axon Instruments, Inc., Foster City, CA) by borosilicate glass pipettes with a tip resistance between 2.5 and 4 M. The pipette solution contained 110 mM K⁺-aspartate, 23 mM KCl, 0.4 mM CaCl₂ (calculated free-Ca²⁺ = 10⁷ M), 3 mM MgCl₂, 5 mM HEPES-KOH, 1 mM EGTA-KOH, 0.4 mM GTP-Na⁺ salt, 5 mM ATP-Na⁺ salt, and 5 mM creatine phosphate, pH 7.3. Pipette solutions were modified in specific cases, as detailed in the relevant sections of Results. Membrane capacitance and series resistance (<5 M) were measured in every cell; the latter was compensated by approx. 70% of its initial value. An average junction potential of about 5 mV, measured upon moving the electrode tip from Tyrode to "intracellular" (K⁺-aspartate), was left uncompensated. Potential and current signals were filtered at 2 KHz, digitized through a 12-bit A/D converter (Digidata 1200, sampling rate 5 KHz; Axon Instruments, Inc.) and simultaneously recorded by an adapted VCR system. Trace acquisition and analysis was controlled by dedicated software (pClamp 8.0; Axon Instruments, Inc.).

Experimental Protocols

Different ionic currents have been studied, each requiring specific conditions detailed here.

Na⁺/K⁺ Pump Current (I_NaK) Recordings. I_NaK was measured as the holding current recorded at 40 mV in the presence of Ni²⁺ (5 mM), nifedipine (5 µM), and Ba²⁺ (1 mM) to minimize contamination by changes in Na⁺/Ca²⁺ exchanger, Ca²⁺ and K⁺ currents, respectively. Tetraethylammonium-Cl (20 mM) and EGTA (5 mM) were added to the pipette solution and intracellular K⁺ was replaced by Cs⁺. To optimize the recording conditions, I_NaK was enhanced by increasing intracellular Na⁺ (to 10 mM) and extracellular K⁺ (to 5.4 mM). In each cell, the current was recorded at steady state during exposure to increasing concentrations of the drug under test and, finally, to a saturating concentration of ouabain (20 µM) (Fig. 2a). Because I_NaK inhibition was expressed as percentage of ouabain effect, the latter was used as the asymptote for the estimation of EC₅₀ values. The rate of onset of I_NaK inhibition was measured by linear interpolation of the early phase of the change in current.

High-Threshold Ca²⁺ Current (I_CaL) Recordings. I_CaL was recorded during depolarizing pulses to 0 mV from a holding potential of 40 mV as the difference between currents recorded in the absence and presence of 10 µM nifedipine, respectively (nifedipine-sensitive current, I_nife). To prevent contamination by K⁺ currents, intracellular K⁺ was replaced by equimolar Cs⁺, the latter was also added to the superfusing solution at a concentration of 10 mM. A low EGTA concentration (0.5 mM) was used to control basal Ca²⁺ levels without suppressing Ca²⁺ transients.

Transient Inward Current (I_TI) Recordings. I_TI was elicited by repolarization during a Ca²⁺ transient. Because the aim was to test the effects of PST2744 and digoxin independently of the inhibition of the Na⁺/K⁺ pump, the latter was blocked during all measurements by exposure to a K⁺-free solution containing 20 µM ouabain. To minimize contamination by components independent of the Ca²⁺ transient, I_TI was obtained as the subtraction of traces recorded, respectively, under loading (Fig. 4, inset 1) and depletion (Fig. 4, inset 2) of the sarcoplasmic reticulum. Under such conditions, I_TI should be mostly carried by the Na⁺/Ca²⁺ exchanger (NaCaX) (Fedida et al., 1987; Zygmunt et al., 2000), whose driving force is transiently increased upon repolarization. I_TI was measured at room temperature under ruptured-patch conditions with low intracellular EGTA concentration (1 mM). To prevent contamination by K⁺ currents, intracellular K⁺ was replaced by equimolar Cs⁺ and the extracellular solution contained Ba²⁺ (1 mM) and Cs⁺ (2 mM). Because the contour of I_TI was also affected by digoxin, effects were more accurately quantified by measuring "average I_TI" as the ratio of I_TI time integral over the integration interval (avg I_TI).

Na⁺/Ca²⁺ Exchanger Current (I_NaCa) Recordings. I_NaCa was measured as the current sensitive to 5 mM Ni²⁺ (Kimura et al., 1987) by the voltage protocol shown in the inset of Fig. 5. As for I_TI, measurements were performed in the presence of Na⁺/K⁺ pump blockade (see described above), and Na⁺ concentration in the pipette solution was increased to 20 mM to improve measurement of outward I_NaCa. Contamination by I_CaL and cytosolic Ca²⁺ oscillations were minimized by adding 0.2 µM nisoldipine and 5 mM EGTA to the extra- and intracellular solutions, respectively. Current/voltage relations of I_NaCa were obtained by applying repolarizing voltage ramps (dV/dt = 85 mV/s), which allowed measurement of I_NaCa reversal potential (E_rev) (Hobai et al., 1997). Drug effects on forward and backward NaCaX operation modes were separately quantified by measuring I_NaCa at potentials symmetrical to E_rev (±60 mV from E_rev). This type of measurement provides an estimate of inward and outward NaCaX "conductances".

Delayed Rectifier Current (I_Kr and I_Ks) Recordings. Delayed rectifier currents were measured as the amplitude of the "tail" elicited upon returning to the holding potential after an activating pulse (protocols at the top of Figs. 6 and 7). To isolate I_Ks, I_Kr was blocked by 5 µM E-4031, and tail contamination by drug-induced changes in I_CaL and I_NaCa were prevented by ouabain (20 µM), Ni²⁺ (5 mM), and nisoldipine (0.2 µM). Under such conditions, the time-dependent outward current was completely suppressed by 10 µM chromanol 293B, thus confirming its identity with I_Ks. I_Kr was isolated by subtracting currents recorded in the absence and presence of 5 µM E-4031 (Sanguinetti and Jurkiewicz, 1990).

Substances

Nifedipine (Sigma-Aldrich) and nisoldipine, E-4031, and chromanol 293B stock solutions were prepared by dissolving the substances in ethanol, water, and dimethyl sulfoxide, respectively. PST2744 and ouabain (Sigma-Aldrich) were dissolved in water, and digoxin (Sigma-Aldrich) was dissolved in dimethyl sulfoxide; E-4031, chromanol 293B, and nisoldipine were generous gifts from Sanofi Recherche (Montpellier, France), Roche Diagnostics, and Bayer Pharmaceuticals (Milano, Italy), respectively.

Statistical Analysis

Means were compared by Student's t test or analysis of variance for paired or unpaired observations as appropriate. A probability level P < 0.05 was used to define significance throughout the study (N.S., not significant). In the text and figures, values are presented as mean ± standard error.

	Results

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Effect on Membrane Potential. Myocytes were studied in the ruptured patch mode, during stimulation by 2-ms current pulses delivered through the patch pipette at a cycle length of 0.5 s (Fig. 1, a-c).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Drug effects on the action potential. a, APD90 measured in an individual cell during stimulation at 2 Hz is plotted as a function of time. Exposure to PST2744 (PST) and digoxin (digo) is marked by the bars. The insets show action potentials recorded at the times marked on the APD graph by the labels a-f. b and c, summary of Ediast and APD90 changes observed during exposure to the specified equi-inotropic drug concentration pairs (n = 5, 7, and 9 for both agents). *, P < 0.05 versus control; #, P < 0.05 PST versus digo.

Effect on I_NaK. This set of experiments was designed to compare directly the concentration dependence of Na⁺/K⁺ pump inhibition by PST2744 and digoxin (Fig. 2, a-c). The effect of each drug concentration on I_NaK was expressed as percentage of the ouabain-induced change (see Materials and Methods). As occurred for inotropic effects (Micheletti et al., 2002), digoxin (EC₅₀ = 2.66 µM) was more potent than PST2744 (EC₅₀ = 6.75 µM) (Fig. 2b) in inhibiting I_NaK. However, the extent of I_NaK inhibition associated to a given inotropic effect was slightly greater for PST2744 than for digoxin (Fig. 2c). Thus, digoxin may require less Na⁺/K⁺ pump inhibition than PST2744 to induce the same inotropic response. When measured at the same concentrations, the onset of I_NaK inhibition was faster for PST2744 than for digoxin (e.g., at 2.5 µM, 4.9 ± 0.9 versus 2.1 ± 0.9 pA/s; P < 0.05).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Drug effects on INaK. a) membrane current recorded at 40 mV from an individual cell during exposure to increasing concentrations (micromolar) of PST2744 (PST) followed by 20 µM ouabain (ouab). The effect of a single digoxin (digo) concentration (micromolar) applied after ouabain washout is shown for comparison. B, dose dependence of INaK inhibition by PST2744 (open circles, n = 9, 14, 17, and 6) and digoxin (filled circles, n = 7, 9, 13, and 7). c, myocyte twitch amplitude is plotted as a function of INaK inhibition at various PST2744 (0.5, 2.5, 10, and 20 µM) and digoxin (0.5, 0.8, 2.5, and 5 µM) concentrations. Data of twitch amplitude are from Micheletti et al. (2002).

Effect on I_CaL. The purpose of this set of experiments (Fig. 3, a-d) was to test whether inhibition of I_CaL might contribute to drug-induced changes in the action potential contour. Equi-inotropic drug concentrations corresponding to the mid-portion of the inotropy dose-response curve (Micheletti et al., 2002) were used. Neither PST2744 (4 µM; Fig. 3a) nor digoxin (1 µM; Fig. 3b) significantly affected the peak amplitude of I_CaL (PST2744, 0.52 ± 5.5%, N.S.; digoxin, 6.23 ± 4.2%, N.S.). The current decay was best fitted by a biexponential function. The faster exponential component, reflecting Ca²⁺-dependent I_CaL inactivation, was significantly accelerated by both PST2744 (_fast, 17.05 ± 3.2%, P < 0.05) and digoxin (_fast, 13.53 ± 2.8%, P < 0.05) (Fig. 3d). The slow component was unchanged by PST2744 and slightly slowed by digoxin (_slow, 5.4 ± 1.5%, P < 0.05).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Drug effects on ICaL. a and b, nifedipine-sensitive currents (10 µM, Inife) in control and during drugs superfusion at equi-inotropic concentrations (PST2744 4 µM, n = 4 and digoxin 1 µM, n = 6). Current traces were recorded during steps to 0 mV from a holding potential of 40 mV every 2 s; to emphasize changes in ICaL inactivation, each trace was normalized to the peak amplitude. c and d, average results on peak Inife and fast of ICaL inactivation. *, P < 0.05.

Effect on I_TI. Representative examples of the effects of PST2744 and digoxin on I_TI are shown in Fig. 4, a and b, respectively. PST2744 (4 µM) consistently reduced avg I_TI (42.8 ± 7.9%, P < 0.05; Fig. 4c). Although digoxin (1 µM) also caused a small decrease in I_TI in part of the cells, its effect was inconsistent and failed to achieve significance (8.8 ± 11.5%, N.S.; Fig. 4d).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Drug effects on ITI in the presence of Na+/K+ pump blockade. a and b, INaCa measured as ITI elicited by repolarization during a Ca2+ transient in the presence of 20 µM ouabain. The voltage protocols (inset) included a conditioning train (1 Hz) followed by a test pulse, after which ITI was measured (shown by the box). The duty cycle of the conditioning train was such as to favor cell Ca2+ loading in protocol 1 and Ca2+ depletion in protocol 2. Protocols 1 and 2 were applied in each cell, and ITI was obtained by subtraction between the respective current traces. c and d, effects of PST2744 (PST 4 µM, n = 14) and digoxin (digo 1 µM, n = 12) on avg ITI. *, P < 0.05.

Effect on I_NaCa. Representative examples of the effects of PST2744 and digoxin on I_NaCa are shown in Fig. 5, a and b, respectively. Baseline I_NaCa current and its E_rev were similar between PST2744 and digoxin groups. PST2744 (4 µM; Fig. 5a) decreased inward (Fig. 5c) and outward (Fig. 5d) I_NaCa conductance similarly (inward, 14.9 ± 4.8%, P < 0.05; outward, 13.5 ± 4.1%, P < 0.05) and slightly shifted E_rev in the negative direction (26.3 ± 3 versus 23.6 ± 3.2 mV, P < 0.05). Digoxin (1 µM; Fig. 5b) affected neither I_NaCa nor E_rev (18.5 ± 2.1 versus 17.1 ± 1.9 mV; N.S.)

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Drug effects on INaCa in the presence of Na+/K+ pump blockade. a, INaCa measured as the current sensitive to 5 mM Ni2+ (see text for details). Current/voltage curves were obtained by applying slow hyperpolarizing V ramps (0.085 V/s, inset); inward and outward INaCa were measured at ±60 mV from the current reversal potential, respectively. a and b, examples of the effects of PST2744 (PST 4 µM) and digoxin (digo 1 µM) on INaCa current/voltage curves. Drug effects on inward and outward INaCa are summarized in c and d, respectively (n = 10 for PST and n = 11 for digo). *, P < 0.05.

Effect on Delayed Rectifier Currents. Evaluation of PST2744 and digoxin effects on delayed rectifier currents was prompted by the arrhythmogenic potential of their inhibition and by the observation of a difference in transient APD changes upon drug washin and washout (see above). PST2744 (4 µM) and digoxin (1 µM) were applied during separate measurement of I_Ks (Fig. 6, a-c) and I_Kr (Fig. 7, a-c) (Sanguinetti and Jurkiewicz, 1990) (see Materials and Methods). I_Kr was marginally reduced by PST2744 (5.99 ± 1.5%, P < 0.05) and unaffected by digoxin (2.99 ± 5.3%; N.S.). I_Ks was slightly but significantly reduced by both PST2744 and digoxin to a similar extent (21.5 ± 4.0 versus 14.6 ± 2.6%; N.S.). Thus, concentrations of PST2744 and digoxin exerting similar inotropic effect slightly reduced I_Ks. PST2744 also inhibited I_Kr but to a very small extent.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Drug effects on IKs. The voltage protocol used to measure IKs is shown at the top (see text for details). Examples of PST2744 (PST 4 µM) and digoxin (digo 1 µM) effects are shown in a and b, respectively. Drug-induced changes in the amplitude of IKs tail current density are summarized in c (n = 5 for PST and n = 4 for digo). *, P < 0.05.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Drug effects on IKr. The voltage protocol used to measure IKr (current sensitive to 5 µM E-4031, IE-4031) is shown at the top (see text for details). Examples of PST2744 (PST 4 µM) and digoxin (digo 1 µM) effect are shown in a and b, respectively; because IKr flowing during the activating step was very small, only IKr tail currents are displayed at high magnification. Drug-induced changes in the amplitude of IKr tail current amplitude are summarized in c (n = 6 for PST and n = 8 for digo). *, P < 0.05.

	Discussion

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Effects on Membrane Potential. The aim of this set of experiments was to look for peculiarities in the modulation of action potential contour that might suggest a mechanism for the different proarrhythmic effects of PST2744 and digoxin.

Effects on Na⁺/K⁺ Pump Current. According to the classical interpretation of glycosides action, inotropy should be directly related to Na⁺/K⁺ pump blockade. However, other mechanisms may contribute to their inotropic effect (Tian et al., 2001; Sagawa et al., 2002) and affect the relationship between Na⁺/K⁺ pump inhibition and inotropy (Wasserstrom et al., 1991; Wasserstrom, 2002). This set of experiments was designed to assess whether differences might exist in such a relationship between PST2744 and digoxin. Data from a previous study were used for the concentration dependence of the inotropic effect (Micheletti et al., 2002). Comparison of such data with the present results shows that the extent of Na⁺/K⁺ pump inhibition required to produce a given inotropic effect was slightly smaller for digoxin than for PST2744. The ratio between Na⁺/K⁺ pump inhibition and inotropy for various digitalis compounds may correlate with the ability to facilitate opening of Ca²⁺ release channels of the sarcoplasmic reticulum (Wasserstrom et al., 1991). Such an action has been demonstrated for digoxin (Sarkozi et al., 1996; Sagawa et al., 2002) and, if not shared by PST2744, might justify the difference observed between the two agents. The onset of Na⁺/K⁺ pump inhibition was faster for PST2744 than for digoxin. This is consistent with the previous observation that association and dissociation rate constants of digitalis compounds to the Na⁺/K⁺ pump are decreased by the glycosidic group (aglycones bind faster than the respective glycoside) (Yoda, 1974), which is not present in PST2744 structure.

Effects on I_TI and Na⁺/Ca²⁺ Exchanger Current. This group of experiments was aimed at testing the primary effects (i.e., independent of Na⁺/K⁺ pump inhibition) of PST2744 and digoxin on I_TI and its charge carrier NaCaX.

Effects on Delayed Rectifier and Ca²⁺ Currents. Due to its proarrhythmic potential, inhibition of delayed rectifier currents has recently become a major concern in drug safety; this motivated an analysis of PST and digoxin effects on I_Kr and I_Ks. While both agents exerted some effects, their functional relevance is questionable for the following reasons. Although major inhibition of delayed rectifier currents is generally required to modulate repolarization (Bosch et al., 1998), the effect on I_Ks were relatively small and those on I_Kr almost negligible. Second, I_Ks inhibition is generally associated with a relatively low proarrhythmic risk, because of the lack of reverse use dependence of its effect on APD (Bosch et al., 1998). Finally, although induction of early after-depolarizations and arrhythmias related to I_K inhibition may be secondary to APD prolongation (Brachmann et al., 1983; January et al., 1988; Antzelevitch and Sicouri, 1994), the net steady-state effect of PST2744 and digoxin was a shortening of APD (see above). Nonetheless, it is fair to stress that some APD prolongation might theoretically occur in other cardiac tissues expressing drug-sensitive currents in a different proportion (e.g., Purkinje fibers and M cells).

Relevance to Arrhythmogenic Effects. The cell targets of drug action evaluated in this study encompass those most likely to be involved in arrhythmogenic effects based on electrophysiological alterations. Among the actions observed, the one not shared by digoxin and most likely to account for the lower arrhythmogenic effect of PST2744 is inhibition of I_TI. Indeed, I_TI is directly involved in the genesis of DADs, the main mechanism of digitalis-induced arrhythmias (Bers, 2002). The extent of I_TI reduction was large compared with direct inhibition of I_NaCa, thus suggesting that PST2744 might modulate the amplitude and/or kinetics of intracellular Ca²⁺ transients, a hypothesis that requires further investigation. Although in guinea pig and human ventricular myocytes I_TI is mostly carried by NaCaX (Fedida et al., 1987; Koster et al., 1999), other Ca²⁺-sensitive conductances may contribute to it in other species (Zygmunt et al., 1998). On the other hand, Ca²⁺-sensitive conductances would still be affected by drug-induced changes in cytosolic Ca²⁺ dynamics. Although other differences have been disclosed between the two agents, they concern quantitatively small effects, which, as discussed above, are unlikely to contribute to the arrhythmogenic profile.