Mexiletine-like cellular electrophysiological effects of GS967 in canine ventricular myocardium

Enhancement of the late Na+ current (INaL) increases arrhythmia propensity in the heart, while suppression of the current is antiarrhythmic. GS967 is an agent considered as a selective blocker of INaL. In the present study, effects of GS967 on INaL and action potential (AP) morphology were studied in canine ventricular myocytes by using conventional voltage clamp, action potential voltage clamp and sharp microelectrode techniques. The effects of GS967 (1 µM) were compared to those of the class I/B antiarrhythmic compound mexiletine (40 µM). Under conventional voltage clamp conditions, INaL was significantly suppressed by GS967 and mexiletine, causing 80.4 ± 2.2% and 59.1 ± 1.8% reduction of the densities of INaL measured at 50 ms of depolarization, and 79.0 ± 3.1% and 63.3 ± 2.7% reduction of the corresponding current integrals, respectively. Both drugs shifted the voltage dependence of the steady-state inactivation curve of INaL towards negative potentials. GS967 and mexiletine dissected inward INaL profiles under AP voltage clamp conditions having densities, measured at 50% of AP duration (APD), of −0.37 ± 0.07 and −0.28 ± 0.03 A/F, and current integrals of −56.7 ± 9.1 and −46.6 ± 5.5 mC/F, respectively. Drug effects on peak Na+ current (INaP) were assessed by recording the maximum velocity of AP upstroke (V+max) in multicellular preparations. The offset time constant was threefold faster for GS967 than mexiletine (110 ms versus 289 ms), while the onset of the rate-dependent block was slower in the case of GS967. Effects on beat-to-beat variability of APD was studied in isolated myocytes. Beat-to-beat variability was significantly decreased by both GS967 and mexiletine (reduction of 42.1 ± 6.5% and 24.6 ± 12.8%, respectively) while their shortening effect on APD was comparable. It is concluded that the electrophysiological effects of GS967 are similar to those of mexiletine, but with somewhat faster offset kinetics of V+max block. However, since GS967 depressed V+max and INaL at the same concentration, the current view that GS967 represents a new class of drugs that selectively block INaL has to be questioned and it is suggested that GS967 should be classified as a class I/B antiarrhythmic agent.

Following the large Na + current surge associated with the AP upstroke (called I NaP ) a smaller but sustained current component (called I NaL ) remains active throughout the entire cardiac AP. Enhanced I NaL , due to diseases like heart failure 1-3 , hypertrophic cardiomyopathy 4 or LQT3 5 , is known to increase arrhythmia propensity in the heart 1,6-8 . It has been known for a long time that many of the class I antiarrhythmic drugs inhibit I NaL in addition to blocking I  . This latter effect, however, was considered to enhance proarrhythmic risk and consequently the incidence of sudden cardiac death 15 . As a consequence, development of agents to suppress I NaL selectively was a straightforward strategy in the past decades 2, [16][17][18][19] . One of these agents, 6-(4-(trifluoromethoxy) phenyl)-3-(trifluoromethyl)-[l, 2,4]triazolo [4,3-a]pyridine, known as GS967 or GS458967, was reported to be a particularly selective candidate when tested in rabbit ventricular cells 16 . It was also shown that important speciesdependent variations existed in the electrophysiological properties of myocardial preparations 20,21 , and canine ventricular myocytes are considered a reasonably good model for human ventricular cells [22][23][24] . Furthermore, it was previously shown using the action potential voltage clamp technique that the kinetic properties of I NaL are similar in dogs and humans 20 , while differing from those of other mammals including guinea pigs, rabbits and pigs [25][26][27] . Therefore, we studied the effects of GS967 on I NaL and AP V + max (the indicator of I NaP ), comparing them to similar effects of the class I/B antiarrhythmic agent mexiletine in canine ventricular preparations. In this work, based on experimental evidence, we challenge the present concept that GS967 exerts a selective I NaL blocking effect, since it has very similar class I/B antiarrhythmic properties as mexiletine.

Methods
Animals. Adult  Isolation of cardiomyocytes. Single canine myocytes were obtained by enzymatic dispersion using the segment perfusion technique, as previously described 28 . Briefly, a wedge-shaped section of the ventricular wall supplied by the LAD coronary artery was cannulated, dissected and perfused with a nominally Ca 2+ -free Joklik solution (Minimum Essential Medium Eagle, Joklik Modification, Sigma-Aldrich Co. St. Louis, MO, USA) for a period of 5 min. After this, the tissue was perfused with Joklik solution supplemented with 1 mg/ml collagenase (Type II, Worthington Biochemical Co., Lakewood, NJ, USA; representing final activity of 224 U/ml) and 0.2% bovine serum albumin (Fraction V., Sigma) containing 50 µM Ca 2+ for 30 min. Finally, the normal external Ca 2+ concentration was gradually restored and the cells were stored at 15 °C in Minimum Essential Medium Eagle until use. The chemicals used in the experiments were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA), except for GS967, which was purchased from MedKoo Biosciences, Inc. (Morrisville, NC, USA).
Electrophysiology. Cells were placed in a plexiglass chamber under an inverted microscope, allowing for continuous superfusion with a modified Tyrode solution by gravity flow at a rate of 1-2 ml/min. The modified Tyrode solution contained (in mM): NaCl 121, KCl 4, CaCl 2 1.3, MgCl 2 1, HEPES 10, NaHCO 3 25, glucose 10 at pH = 7.35, which was supplemented according to the actual experimental design. The osmolarity of this solution was 300 ± 3 mOsm, measured with a vapor pressure osmometer. In all experiments, the bath temperature was set to 37 ºC using a temperature controller (Cell MicroControls, Norfolk, VA, USA). Electrical signals were amplified and recorded (MultiClamp 700A or 700B, Molecular Devices, Sunnyvale, CA, USA) under the control of a pClamp 10 software (Molecular Devices) following analogue-digital conversion (Digidata 1440A or 1332, Molecular Devices). Electrodes, having tip resistances of 2-3 MΩ when filled with pipette solution, were manufactured from borosilicate glass. Transmembrane currents were recorded in whole-cell voltage clamp configuration. The series resistance was typically 4-8 MΩ, and the measurement was discarded if it changed substantially during the experiment.
Action potential voltage clamp. Action potential voltage clamp experiments were performed according to the methods described 29,30 . A previously recorded midmyocardial canine ventricular AP was applied as command signal and the current traces were recorded continuously in modified Tyrode solution before and after 5 min superfusion with the Na + channel inhibitor applied. The drug-sensitive current was obtained by subtracting the post-drug trace from the reference pre-drug trace. When measuring the effects of GS967 or mexiletine on I NaL , the experiments were performed in the presence of 1 µM nisoldipine + 1 µM E4031 + 100 µM chromanol 293B, added to the Tyrode solution, in order to eliminate any interference from Ca 2+ and K + currents. The pipette solution contained (in mM): K-aspartate 120, KCl 30, MgATP 3, HEPES 10, Na 2 -phosphocreatine 3, EGTA 0.01, cAMP 0.002, KOH 10 at pH = 7.3 with an osmolarity of 285 mOsm. The amplitude of the dissected I NaL was evaluated at 50% duration of the APD 90 value of the command AP. When determining the current integral, the initial 20 ms after the AP upstroke was excluded from evaluation in order to eliminate the contribution of I NaP . In each experiment 20 consecutive current traces were averaged and analyzed in order to reduce the noise and the trace-to-trace fluctuations of action potential configuration. Ion currents were normalized to cell capacitance, determined in each cell by applying hyperpolarizations from +10 to −10 mV for 15 ms. . Test pulses were clamped to −20 mV for 2 s from the holding potential of −120 mV before and after application of GS967 or mexiletine, while the total amount of I NaL was determined by pharmacological subtraction performed by a final superfusion with 20 µM TTX. The amplitude of I NaL was evaluated at 50 ms after beginning the pulse. For determination of current integral the initial 20 ms was excluded from evaluation in order to minimize the contribution of I NaP . In order to study the voltage-dependence of steady-state inactivation of I NaL , test depolarizations to −20 mV were preceded by a set of 2 s long prepulses clamped to various voltages between −130 and −40 mV for 2 s. Peak currents measured after these prepulses (I test ) were normalized to the peak current measured after the −130 mV prepulse (I max ) and these I test /I max ratios were plotted against the respective prepulse potentials. The data were fitted to the two-state Boltzmann function (y = 100/[1 + exp((x-V1/2)/k)]).
Recording of APs from multicellular preparations. Multicellular preparations (right ventricular papillary muscles) were selected to prevent the limitations of isolated myocyte studies, like the absence of intercellular clefts or potential damage to channel proteins, allowing better representation of in vivo conditions. The experiments were performed as previously described 31 . Briefly, transmembrane potentials were recorded using 3 M KCl filled sharp glass microelectrodes having tip resistance between 10 and 20 MΩ. These electrodes were connected to the input of a high impedance electrometer (MDE GmbH, Heidelberg, Germany). Preparations were paced by a pair of platinum electrodes using 1 ms wide rectangular current pulses with twice the threshold amplitude at 37 °C. The pacing cycle length was set to 1 s for at least 60 min allowing the preparations to equilibrate before starting the experiment. Following equilibration at 1 s cycle length, the cycle length was sequentially varied between 0.3 and 5 s. At each cycle length the 25th AP was recorded, and the cycle length was then changed. Under these conditions a quasi steady-state rate-dependence could rapidly be obtained. APs were digitized at 100 kHz using an ADA 3300 data acquisition board (Real Time Devices Inc., State Collage, PA, USA) and stored for later analysis. After taking control records at each cycle length the preparations were superfused with either GS967 or mexiletine for 20 min and then the protocol was repeated. Efforts were made to maintain the same impalement throughout each experiment. If, however, an impalement became dislodged, adjustment was attempted, but when the parameters of the re-established impalement deviated by more than 5% from the previous record, the experiment was discarded.
Restitution kinetics of the maximum rate of depolarization (V + max ) is considered as the indicator of the offset time constant. To determine the restitution time constant for V + max the preparations were paced using a train of 20 basic stimuli delivered at a basic cycle length of 1 s. Each train was followed by a single extra stimulus applied with successively longer coupling intervals. The train of basic stimuli was reinitiated following the delivery of the extra stimulus. In this way, each 20th basic AP was followed by a single extra AP occurring at gradually increasing diastolic intervals. The diastolic interval was defined as the time from APD 90 of the last basic member of the train to the upstroke of the extra AP. Recovery curves were generated by plotting the V + max of each extra AP as a function of the respective diastolic interval and data were fitted to a single exponential function.
Onset kinetics of drug action on V + max were determined by stimulating the preparation at a cycle length of 0.4 s following a few min period of rest and the initial 40 APs were recorded and data were plotted against the number of the analyzed AP within the train. The rate of development of block was obtained by monoexponential fitting of the V + max values.

Determination of beat-to-beat variability of APD in isolated myocytes.
Since beat-to-beat variability of APD is relatively small in multicellular preparations due to the balancing effect of the neighboring cells, these experiments were performed in isolated myocytes. Series of 50 consecutive action potentials were analyzed to estimate the beat-to-beat variability of APD, defined as short term variability (SV), according to the following formula: where SV is short term variability, APD i and APD i+1 indicate the durations of the ith and i + 1th APs, respectively, at 90% level of repolarization and n beats denotes the number of consecutive beats analyzed 32 . Changes in SV were presented as Poincaré plots where 50 consecutive APD values are plotted, each against the duration of the previous AP.

Statistics.
Results are expressed as mean ± SEM values, n denotes the number of myocytes or multicellular preparations studied. Statistical significance of differences was evaluated using one-way ANOVA followed by Student's t-test for paired or unpaired data as pertinent. Differences were considered significant when p was less than 0.05. Data were processed, analyzed and figures were created in Excel (Microsoft Corp., Redmond, WA, USA), and Origin 2015 (Originlab Corp., Northampton, MA, USA).

Results
Effects of GS967 and mexiletine on I NaL using conventional voltage clamp. First, the effects of GS967 and mexiletine on I NaL were studied under conventional voltage clamp conditions by applying 2 s duration depolarizations to −20 mV from the holding potential of −120 mV (Fig. 1). 1 µM GS967 significantly reduced www.nature.com/scientificreports/ the density of I NaL , measured at 50 ms after the beginning of the pulse (from −0.313 ± 0.05 to −0.062 ± 0.01 A/F, corresponding to an 80.4 ± 2.2% reduction in the 6 myocytes studied). For comparison, this parameter was also significantly decreased by 40 µM mexiletine from −0.385 ± 0.036 to −0.156 ± 0.014 A/F (reduction of 59.1 ± 1.8%, n = 12). Similar results were obtained when comparing the current integrals, i.e. the charge carried by the current with the exclusion of the initial 20 ms. As determined from the same experiments, the current integrals were significantly decreased from −69.1 ± 7.9 to −15.4 ± 3.9 mC/F (79.0 ± 3.1% inhibition) by GS967 and from −76.4 ± 7.6 to −26.7 ± 2.6 mC/F (63.3 ± 2.7% reduction) by mexiletine. Since class I antiarrhythmics are known to modify inactivation kinetics of I NaP , the effects of 1 µM GS967 and 40 µM mexiletine on the voltage dependence of steady-state inactivation of I NaL was also examined using a conventional prepulse protocol. The amplitude of I NaL was determined at 50 ms after beginning of the test-pulse. Both GS967 and mexiletine caused a significant leftward shift in the steady-state inactivation curve (Fig. 2). The V 0.5 value was shifted by −17.2 and −13.5 mV in the presence of GS967 and mexiletine, respectively. The slope factors of the curves were not influenced by GS967 or mexiletine.
Effects of GS967 and mexiletine under action potential voltage clamp conditions. Based on the non-equilibrium gating theory of Clancy et al., gating of the Na + channel is intimately influenced by the shape of the voltage protocol applied 33 . Therefore, the effects of GS967 and mexiletine on I NaL were studied also under action potential voltage clamp conditions using canonic mid-myocardial APs as command signals. 1 µM GS967 and 40 µM mexiletine dissected inward I NaL profiles having current densities (measured at 50% of APD 90 ) of −0.37 ± 0.07 and −0.28 ± 0.03 A/F, and current integrals of −56.7 ± 9.1 and −46.6 ± 5.5 mC/F, respectively. These differences, however, were not significant statistically. Since the Ca 2+ and K + currents were previously blocked in these experiments, the recorded current profiles are considered as I NaL inhibited by GS967 and mexiletine during a regular ventricular AP (Fig. 3A-C).
The total density and integral of I NaL in canine myocytes were estimated using concentration-response curves obtained with TTX, which was applied as a reference compound. The maximum value of the Hill-fit gave the total magnitude of I NaL (Fig. 3D,E). Comparing the respective current densities and integrals dissected by 1 µM GS967 and 40 µM mexiletine to these maximal values obtained with TTX, the inhibiting effects of GS967 and mexiletine could be expressed in a percentage form. Accordingly, GS967 and mexiletine blocked 88% and 67% www.nature.com/scientificreports/ of total I NaL density, and 78% and 64% of total I NaL integral, respectively. More importantly, the current profiles dissected by GS967 and mexiletine were different in shape. The GS967-sensitive current decreased monotonically during the AP, similarly to the TTX-sensitive current (not shown), while the mexiletine-sensitive current displayed a saddle-like profile. This difference is likely attributable to the threefold faster dissociation of GS967 than mexiletine from the Na + channel (see later).
Effects of GS967 and mexiletine on action potential upstroke. After determining the effects on I NaL , the actions of GS967 and mexiletine on I NaP were studied and compared. Since direct measurement of cardiac I NaP is difficult at 37 °C, the maximum velocity of depolarization during the action potential upstroke (V + max ) was used as an approximate, although not linear, measure of I NaP [34][35][36] . Due to the more physiological situation (e.g. higher stability) in multicellular cardiac preparations, these experiments were performed in right ventricular papillary muscles using sharp microelectrodes. As demonstrated in Fig. 4A,B, 40 µM mexiletine significantly reduced V + max in the entire frequency range applied under steady-state conditions, while this effect of GS967 was significant only at the shortest cycle lengths of 0.3 and 0.4 s. This difference can well be explained by the faster offset kinetics of GS967 (Fig. 4C,D). The time constant of recovery of V + max , determined following a constant 1 Hz stimulation was 110 ms for GS967, while almost three times longer, 289 ms for mexiletine. The onset kinetics of V + max block was studied by application of a constant stimulation rate at 2.5 Hz and the initial 40 APs were recorded. The onset rate constant was 5.3 AP for 1 µM GS967 and 2.6 AP for 40 µM mexiletine (Fig. 4E,F).

Effects of GS967 and mexiletine on APD.
Although APD in right ventricular trabeculae was shortened by both GS967 and mexiletine in a reverse rate-dependent manner, this effect was statistically significant only at the longest constant cycle lengths above 1.5 s (Fig. 5).

Effects of GS967 and mexiletine on beat-to-beat variability of APD. Elevated beat-to-beat QT
interval variability is a good predictor of ventricular arrhythmia in a wide variety of patients, while at a single cell level it translates into beat-to-beat variability of APD (also called short term variability, SV) -both parameters are considered as indicators of proarrhythmic risk [37][38][39][40] . In contrast to multicellular preparations, where the neighboring cells may effectively balance the individual differences in APD, SV is more pronounced in single cells, therefore the effects of GS967 and mexiletine were studied on SV in isolated ventricular cells, paced at a constant cycle length of 1 s. Under these conditions SV was significantly decreased by 1 µM GS967 and 40 µM mexiletine (reduction of 1.01 ± 0.19 and 0.63 ± 0.02 ms, respectively, corresponding to 42.1 ± 6.5% and 24.6 ± 12.8% decrease), while APD was moderately shortened by 32.5 ± 6.9 and 41.4 ± 4.1 ms, respectively, inactivation of I NaL , determined using a conventional prepulse-protocol presented in the insets. 2 s long prepulses, ranging from -130 to −40 mV were followed by a test pulse clamped to −20 mV for 2 s. Each I NaL amplitude (I test ) was normalized to the peak current measured after the −130 mV prepulse (I max ) and these I test /I max ratios were plotted as a function of the corresponding prepulse-potential. Finally, data were fitted to a Boltzmann function and the results are given below the stead-state inactivation curves. Symbols and bars are mean ± SEM values, numbers in parentheses indicate the number of myocytes studied, asterisks denote significant differences between data of the pre-drug control and the GS967-or mexiletine-treated groups. www.nature.com/scientificreports/ (all p < 0.05 and n = 8). All these effects were largely reversible upon washout (Fig. 6). Since APD itself is also known to affect the magnitude of SV, the term of relative SV (RSV) was introduced 31,41 . Accordingly, RSV = dSV / dAPD, i.e. the change in SV is normalized to that of APD. The value of RSV was significantly higher for GS967 than mexiletine (0.039 ± 0.007 versus 0.015 ± 0.007) predicting a better antiarrhythmic effectivity of GS967 than mexiletine.

Discussion
We studied and compared the effects of GS967 (1 µM) to those of the class I/B antiarrhythmic drug mexiletine (40 µM) on I NaL and I NaP in canine ventricular myocardium by combining the conventional microelectrode, voltage clamp and action potential voltage clamp techniques. It was found that GS967, which is generally considered as a selective blocker of I NaL 16 , inhibits I NaP as well, similarly to the class I/B antiarrhythmic drug mexiletine, but with higher potency. Based on the hypothetically selective I NaL blocker nature of GS967, the drug was previously mentioned as a novel class VI antiarrhythmic agent 42 . However, without questioning the theoretical possibility of the concept for selective I NaL inhibition, an alternative approach should also be considered, since the concept whether I NaL can be blocked selectively is an interesting and still unresolved issue. There is evidence that Na + current in cardiac tissues may also be conducted by Na + channels other than the cardiac specific Na v 1.5 channels, which were suggested to contribute to both I NaP and I NaL . However, proper functional evidence for their role is still lacking while considerable interspecies differences were observed [43][44][45][46][47][48][49][50][51] . If these channels play a role in I NaL but not in I NaP , their pharmacological inhibition may result in selective I NaL blockade. However, GS967 has not been shown to inhibit these types of sodium channels but it was reported to inhibit cardiac type Na v 1.5 channels 9,10 . Therefore‚ it is difficult to estimate the relative contribution of neuronal type Na + channels to the GS967-sensitive current.
Na v 1.5 channels have complex and multiple open and closed states 52 with different drug binding properties governing active, inactive and resting channel states. Accordingly, drugs interacting with the binding sites of the channels depending on their actual open or closed channel states may produce variable effects on I NaP and I NaL . For example, when a drug binds rapidly and with high affinity to open and inactivated channel states, and it dissociates rapidly from closed resting channel states, it would not inhibit I NaP but I NaL . This would be due to complete drug dissociation from its binding site in the resting closed states unless frequency was very high or at least the cycle length would be shorter than its dissociation from the channel. Consequently, whether a drug inhibits I NaP or I NaL selectively or both of them, largely depends on the stimulation protocol but not on existing www.nature.com/scientificreports/ specific I NaP or I NaL binding sites. Present results and other recently published data 9,10 are consistent with this suggestion and do not support a mechanism which is based on specific I NaL inhibition that is distinctly different from class I/B antiarrhythmic actions described for drugs such as mexiletine 14,52 , lidocaine 9,10,14 , amiodarone 11 and ranolazine 53 . This approach is also in line with the reported high (38-fold) selectivity of ranolazine on I NaL over I NaP 2 , which is intermediate (13-fold) for amiodarone 3 and much lower (only threefold) for flecainide 54 . In our experiments, both GS967 and mexiletine significantly depressed V + max at high stimulation rates. It was previously established that changes of V + max and I NaP are not linear and a relatively modest decrease of V + max can represent robust depression of I NaP 35,36 . Accordingly, the 20-30% reduction of V + max measured in the present study in papillary muscle preparations (at pacing cycle lengths of 0.3-0.4 s) following GS967 and mexiletine application can represent a similar degree of I NaP depression as the measured 60-80% reduction of I NaL obtained in ventricular myocytes. Therefore, in theory, neither GS967 nor mexiletine can be considered as "selective" I NaL inhibitors. However, when therapy is concerned -assuming that decreased I NaP is proarrhythmic, while reduced I NaL is antiarrhythmic -GS967, which has about threefold faster offset kinetics, can be more beneficial than mexiletine since I NaP would be affected in a lesser extent than I NaL by GS967 at normal or moderately enhanced heart rates.
On the basis of present and other results it is clear that GS967 affects I NaP in a strongly rate-and moderately species-dependent fashion. Similarly to our results, V + max was reduced by 0.3 µM GS967 in murine myocytes 55 , while the same concentration of the drug failed to modify V + max in canine Purkinje strands 56 . On the other hand, GS967 shortened APD in a variety of preparations, including rat 57,58 , murine 55 , rabbit 16,59 and human 60 ventricular  The respective protocols are shown in the insets -for further details see Methods section. Solid lines were obtained by monoexponential fitting. Symbols and bars are mean ± SEM, numbers in parentheses indicate the number of preparations studied, asterisks denote significant differences between data of the pre-drug control and the GS967-or mexiletine-treated groups. www.nature.com/scientificreports/ cells within a wide concentration range (0.1-1 µM) -similarly to the present observations in isolated canine ventricular cells. In our multicellular preparations, however, a significant APD shortening effect appeared only at cycle lengths longer than 1.5 s. This can be explained by the well-known higher drug-sensitivity of single cells comparing to multicellular preparations. The reduction of I NaL by GS967 may result the GS967-induced shortening of the elongated APD, reduction of the enhanced dispersion and short term variability of repolarization, changes often preceding torsade de pointes arrhythmias [38][39][40] . Taken together the present results and the literature, it is likely that a compound having kinetic properties similar to GS967 would be a very promising new antiarrhythmic agent, since several in vitro [16][17][18][19][55][56][57][58][59][60] and in vivo 57,61 studies support the potent antiarrhythmic activity of GS967. Its kinetic properties are better than those of mexiletine, as shown in this study, and also than those of ranolazine 9 , agents known to suppress I NaL . GS967 had high brain penetration 62 , which can be utilized as a potential new antiepileptic compound 63 , and caused a profound use-dependent block on all sodium channel isoforms studied, making the compound prone for possible central nervous system side effects. It is unclear at present whether the effects of GS967 on the nervous system preclude the use of this drug as an antiarrhythmic therapeutic, however, a new agent exhibiting the same kinetic properties of GS967 without CNS side effects would represent a promising candidate for future development.
In summary, GS967 -similarly to mexiletine-inhibited both the peak and late components of Na + current and decreased beat-to-beat variability of APD. Based on its kinetic properties, GS967 should be classified as a new potent class I/B antiarrhythmic agent. The results of the present study also suggest that investigations of "selective" I NaL inhibitors should be carried out through a wide range of stimulation rates since the effect of drugs like GS967 or mexiletine, that possess fast offset kinetics of I NaP inhibition, can be misinterpreted.

Control
Washout Mexiletine * * * * Figure 6. Reversible effects of GS967 (A-C) and mexiletine (D-F) on beat-to-beat variability of APD in isolated myocytes. Superimposed sets of action potentials (A,D) and Poincaré plots (B,E) obtained in control, following exposure to 1 µM GS967 or 40 µM mexiletine, and after washout of drugs. (C) and (F) show the average effects on beat-to-beat variability (SV) and APD. Symbols and bars are mean ± SEM, small dots represent individual data, numbers in parentheses indicate the number of myocytes studied, asterisks denote significant differences between data of the pre-drug control and the GS967-or mexiletine-treated groups.