Is Selective Late Na+ Current Inhibition Different From Class I/B Antiarrhythmic Action? Comparison of The Effects of GS967 to Mexiletine in Canine Ventricular Myocardium

doi:10.21203/rs.3.rs-134972/v1

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Is Selective Late Na+ Current Inhibition Different From Class I/B Antiarrhythmic Action? Comparison of The Effects of GS967 to Mexiletine in Canine Ventricular Myocardium

https://doi.org/10.21203/rs.3.rs-134972/v1

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Enhancement of the late Na⁺ current (I_NaL) increases arrhythmia propensity in the heart, while suppression of the current is antiarrhythmic. GS967 is an agent considered as a selective blocker of I_NaL. In the present study, effects of GS967 on I_NaL, on L-type calcium current (I_Ca), and on action potential (AP) morphology were studied in canine ventricular myocytes by using conventional voltage clamp, action potential voltage clamp and sharp microelectrode techniques. These effects of GS967 were compared to tetrodotoxin (TTX) and to the class I/B antiarrhythmic compound mexiletine. 1 µM GS967, 40 µM mexiletine, and 10 µM TTX dissected largely similarly shaped inward currents under action potential voltage clamp conditions. In case of GS967 and mexiletine, the amplitude and integral of this current was significantly smaller when measured in the presence of 1 µM nisoldipine, while no difference was observed in case of TTX. Under conventional voltage clamp conditions, I_NaLwas significantly decreased by 1 µM GS967 and 40 µM mexiletine (79.0±3.0% and 63.3±2.7% reduction of current integrals, respectively). The integral of I_Ca was moderately but significantly diminished by both drugs (reduction of 9.3±3.3% and 14.1±1.5%, respectively). These changes were associated with acceleration of inactivation of I_Ca. Drug effects on peak Na⁺ current (I_NaP) were also assessed by recording AP upstroke in multicellular preparations. Both GS967 and mexiletine showed fast onset and offset kinetics: 110 ms and 289 ms offset time constants, respectively, as determined from V⁺_max measurements in right ventricular papillary muscles, while the onset kinetics was characterized by 5.3 AP and 2.6 AP, respectively, at 2.5 Hz. Effects on beat-to-beat variability of AP duration (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 I_NaL at the same concentration, the current view that GS967 represents a new class of drugs that selectively block I_NaL has to be questiond and it is suggested that GS967 should be classified as a class I/B (or I/B + IV) antiarrhythmic agent.

Cardiac & Cardiovascular Systems

Drug Discovery, Design, & Development

Physiology

GS967

Mexiletine

Late Na+ current

Ca2+ current

Dog myocytes

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_NaP^[9–14]. 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, 16–19]}. 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 species-dependent variations existed in the electrophysiologic properties of myocardial preparations^{[20, 21]}, and canine ventricular myocytes are considered a reasonably good model for human ventricular cells^[22–24]. 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^[25–27]. Therefore, we studied the effects of GS967 on I_NaL and I_Ca and compared them to those of tetrodotoxin (TTX) and the class I 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, however, it has very similar class I/B antiarrhythmic properties as mexiletine.

2.1. Animals

Adult mongrel dogs of either sex were anesthetized with i.m. injections of 10 mg/kg ketamine hydrochloride (Calypsol, Richter Gedeon, Hungary) + 1 mg/kg xylazine hydrochloride (Sedaxylan, Eurovet Animal Health BV, The Netherlands) according to a protocol approved by the local Animal Care Committees (license N^o: 9/2015/DEMáB at University of Debrecen; and I-74-15-2017, I-74-24-2017 at University of Szeged) and by the Department of Animal Health and Food Control of the Ministry of Agriculture and Rural Development (XIII/3330/2017 and XIII/3331/2017). All animal procedures conform to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and the Guide for the Care and Use of Laboratory Animals (USA NIH publication NO 85 − 23, revised 1996) The study was carried out in compliance with the ARRIVE guidelines.

2.2. 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 Ca²⁺-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 Ca²⁺ for 30 min. Finally, the normal external Ca²⁺ concentration was gradually restored and the cells were stored at 15 ^oC in Minimum Essential Medium Eagle until use. The chemicals used in the experiments were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA).

2.3. 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, CaCl₂ 1.3, MgCl₂ 1, HEPES 10, NaHCO₃ 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.

2.3.1. 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. These measurements were performed either in the presence or absence of 1 µM nisoldipine added to the Tyrode solution. The pipette solution contained (in mM): K-aspartate 120, KCl 30, MgATP 3, HEPES 10, Na₂-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 current (I_NaL in the presence of nisoldipine) was evaluated at half-duration 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.

2.3.2. Conventional voltage clamp

Conventional voltage clamp experiments, using rectangular command pulses, were performed to study the effects of GS967 and mexiletine on I_NaL and I_Ca at stable test potentials.

When measuring I_NaL the external solution was a HEPES-buffered Tyrode solution containing (in mM): NaCl 144, NaH₂PO₄ 0.4, KCl 4.0, CaCl₂ 1.8, MgSO₄ 0.53, glucose 5.5 and HEPES 5.0, at pH = 7.4) supplemented with 1 µM nisoldipine, 0.5 µM HMR-1556 and 0.1 µM dofetilide in order to block Ca²⁺ and K⁺ currents. The composition of the pipette solution was (in mM): CsCl 125, TEACl 20, MgATP 5, EGTA 10, HEPES 10, at a pH = 7.2). 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 the case of I_Ca measurements the bath solution was modified Tyrode solution supplemented with 3 mM 4-aminopyridine to suppress K⁺ currents. The pipette solution contained (in mM): K-aspartate 120, KCl 30, MgATP 3, HEPES 10, Na₂-phosphocreatine 3, EGTA 0.01, cAMP 0.002, KOH 10 at pH = 7.3. Test pulses to + 5 mV, lasting for 200 ms, arose from the holding potential of -80 mV, while a prepulse to − 40 mV for 15 ms was interposed between the holding potential and the test pulse to inactivate Na⁺ channels. In this case the current integral contained the total amount of current carried by I_Ca from the beginning to the end of the test pulse.

2.3.3. 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 it was 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 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 APD₉₀ 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.

2.3.4. 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:

SV = Σ (│APD_i+1 minus APD_i│) / [n_beats * √2]

where SV is short term variability, APD_n and APD_n+1 indicate the durations of the i^th and i + 1^th 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.

2.4. 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.

3.1. Effects of GS967, mexiletine and TTX under action potential voltage clamp conditions

Under action potential voltage clamp conditions, 10 µM TTX, 1 µM GS967 and 40 µM mexiletine dissected similarly shaped inward current profiles dominated by I_NaL (Fig. 1). These concentrations were chosen because the densities, measured at half-duration of the AP (50% of APD₉₀), and integrals of the dissected currents were largely comparable in size. TTX was applied as a reference, since 10 µM TTX was shown to suppress more than 80% of I_NaL in canine ventricular cells^[20]. Importantly, in all action potential voltage clamp experiments performed with mexiletine, the bath solution contained 1 µM E4031 and 100 µM chromanol 293B in order to eliminate any interference from K⁺ currents^[33]. Since I_Ca was not suppressed in these experiments, the measurements were repeated in the presence of 1 µM nisoldipine (NISO) and the results were compared to those obtained without nisoldipine (NO NISO) as presented in Fig. 2. In the case of GS967 and mexiletine, the half-duration current densities were significantly smaller in the presence (− 0.27 ± 0.02 and − 0.25 ± 0.02 A/F) than in the absence (− 0.42 ± 0.03 and − 0.45 ± 0.04 A/F) of nisoldipine. Similar differences were found when comparing the current integrals: −48 ± 4 and − 43 ± 4 mC/F in the absence, while − 68 ± 5 and − 74 ± 10 mC/F in the presence of nisoldipine, respectively. Importantly, there was no significant difference between the NISO and NO NISO data in the case of TTX. These results suggest that both GS967 and mexiletine inhibit I_Ca in addition to I_NaL blockade.

3.2. Effects of GS967 and mexiletine on I_NaL under conventional voltage clamp conditions

The effects of 1 µM GS967 and 40 µM mexiletine on I_NaL were also studied under conventional voltage clamp conditions by applying 2 s duration depolarizations to − 20 mV from the holding potential of − 120 mV (Fig. 3). 1 µM GS967 significantly reduced 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 on the average of 6 myocytes). 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 1 µM GS967 and from − 76.4 ± 7.6 to − 26.7 ± 2.6 mC/F (63.3 ± 2.7% reduction) by 40 µM mexiletine.

3.3. Effects of GS967 and mexiletine on I_Ca under conventional voltage clamp conditions

Since action potential clamp experiments suggested some inhibition of I_Ca by both GS967 and mexiletine, this was clarified using conventional voltage clamp, as shown in Fig. 4. Although peak I_Ca was not altered significantly by 1 µM GS967 (− 4.92 ± 0.16 and − 4.76 ± 0.04 A/F), the current density, measured at 50 ms after the beginning of the pulse, was significantly reduced (from − 0.66 ± 0.05 to − 0.62 ± 0.05 A/F). Accordingly, the current integral (measured from the beginning to the end of pulse) was also significantly decreased by GS967 in the 7 myocytes studied (from − 111.9 ± 5.0 to − 100.7 ± 3.1 mC/F, reduction of 9.3 ± 3.3%). The suppressive effect of 40 µM mexiletine on I_Ca was somewhat more pronounced. It was significant for the reduction of peak current (from − 4.9 ± 0.4 to − 4.4 ± 0.4 A/F), and for the current measured at 50 ms (from − 0.86 ± 0.08 to − 0.70 ± 0.07 A/F), as well as the current integral (from − 114 ± 11 to − 98 ± 9 mC/F, corresponding to 14.1 ± 1.5% reduction on average of 5 experiments). Both drugs accelerated the decay time of I_Ca, determined between 90% and 10% of current amplitudes, significantly: from 48.8 ± 2.5 to 44.2 ± 2.9 ms by GS967, and from 50.8 ± 5.4 to 44.9 ± 5.0 ms by mexiletine. This acceleration of current decay may account for the decreased current integrals.

3.4. Effects of GS967 and mexiletine on action potential upstroke

After determining the effects on I_NaL and I_Ca, the actions of GS967 and mexiletine on I_NaP were studied and compared. Since direct measurement of cardiac I_NaP is difficult at 37 ^oC, the maximum velocity of depolarization during the action potential upstroke (V⁺ _max) was used as an approximate, although not linear, measure of I_NaP^[34–36]. 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. 5.A,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. 5.C,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. 5.E,F).

3.5. 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. 6).

3.6. 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^[37–40]. 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, (all p < 0.05 and n = 8). All these effects were largely reversible upon washout (Fig. 7). Since APD itself is also known to affect the magnitude of SV, the term of relative SV (RSV) was introduced^{[41, 42]}. 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.

We studied and compared the effects of GS967 (1 µM) to those of the class I/B antiarrhythmic drug mexiletine (40 µM) on I_Ca, 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_Ca and I_NaP as well, similarly to the class I/B antiarrhythmic drug mexiletine^[33] but with higher potency. The I_Ca blocking effects were more prominent under action potential voltage clamp than under conventional voltage clamp conditions in the case of both drugs (compare Figs. 2 and 4). The reason for this difference is unknown at present, however, it highlights the advantage of the action potential voltage clamp technique in cardiac cellular electrophysiology and pharmacology.

Based on the hypothetically selective I_NaL blocker nature of GS967, the drug was previously mentioned as a novel class VI antiarrhythmic agent^[43]. 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 sodium current in cardiac tissues may also be conducted by sodium channels other than the cardiac specific Na_v1.5 channels, such as Na_v1.8^[44]. Also, relatively high mRNA expression levels of Na_v2.1 were reported in human ventricle^[45] but proper functional evidence for its role is still lacking. If these channels play a role in I_NaL but not in I_NaP, pharmacological inhibition of these channels may result in selective I_NaL blockade. However, GS967 has not been shown to inhibit these latter types of sodium channels but it was reported to inhibit cardiac type Na_v1.5 channels^{[9, 10]}. Na_v1.5 channels have complex and multiple open and closed states^[46] 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 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, 46]}, lidocaine^{[9, 10, 14]}, amiodarone^[11] and ranolazine^[47]. 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 3-fold) for flecainide^[48].

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 3-fold 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^[49], while the same concentration of the drug failed to modify V⁺ _max in canine Purkinje strands^[50]. On the other hand, GS967 shortened APD in a variety of preparations, including rat^{[51, 52]}, murine^[49], rabbit^{[16, 53]} and human^[54] ventricular 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 moderate reduction of I_Ca by GS967, in addition to its decrease of I_NaL, is not expected to impair A-V conduction. However, this effect can contribute to 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^[38–40].

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^{[16–19, 49–54]} and in vivo^{[51, 55]} 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. Although GS967 had high brain penetration and caused a profound use-dependent block on all sodium channel isoforms studied, making the compound prone for possible central nervous system side effects^[56], 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, suppressed Ca²⁺ 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 (or probably I/B + IV) 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.

Acknowledgements

This work was funded by the National Research Development and Innovation Office (NKFIH-PD120794 and NKFIH-FK128116 to B.H., NKFIH-K119992 to A.V. and NKFIH K 128851 to I.B.) and by the Hungarian Academy of Sciences (János Bolyai Research Scholarship to B.H.). Further support was obtained from the Thematic Excellence Programme of the Ministry for Innovation and Technology in Hungary (ED_18-1-2019-0028), within the framework of the Space Sciences thematic programme of the University of Debrecen, and from the GINOP-2.3.2.-15-2016-00040 and GINOP-2.3.2.-15-2016-00048, projects, co-financed by the European Union and the European Regional Development Fund. The work was also supported by the Ministry of Human Capacities Hungary (EFOP-3.6.2-16-2017-00006, EFOP 3.6.2 16 2017-00006, EFOP 3.6.3-VEKOP-16-2017-00009, 20391-3/2018/FEKUSTRAT, ÚNKP-20-2 and ÚNKP-20-3 projects).

Author contribution statement

Preparation of the manuscript: PPN, AV, BH.

Design of experimental protocols: TB, JM, IB.

Action potential voltage clamp experiments: TH, CsD, DK.

Conventional voltage clamp experiments: MNK, JP, NSz..

Action potential recording from multicellular preparations: TáL, RV, EF, LT.

Action potential recording from isolated canine myocytes preparations: KK, TM.

Statistical analysis: NJ, JA, LV.

Conflict of interest: none declared.

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You are reading this latest preprint version

Is Selective Late Na+ Current Inhibition Different From Class I/B Antiarrhythmic Action? Comparison of The Effects of GS967 to Mexiletine in Canine Ventricular Myocardium

Status:

Version 1

Abstract

Figures

1. Introduction

2. Methods

2.1. Animals

2.2. Isolation of cardiomyocytes

2.3. Electrophysiology

2.3.1. Action potential voltage clamp

2.3.2. Conventional voltage clamp

2.3.3. Recording of APs from multicellular preparations

2.3.4. Determination of beat-to-beat variability of APD in isolated myocytes

2.4. Statistics

3. Results

3.1. Effects of GS967, mexiletine and TTX under action potential voltage clamp conditions

3.2. Effects of GS967 and mexiletine on I_NaL under conventional voltage clamp conditions

3.3. Effects of GS967 and mexiletine on I_Ca under conventional voltage clamp conditions

3.4. Effects of GS967 and mexiletine on action potential upstroke

3.5. Effects of GS967 and mexiletine on APD

3.6. Effects of GS967 and mexiletine on beat-to-beat variability of APD

4. Discussion

Declarations

References

Status:

Version 1

Is Selective Late Na+ Current Inhibition Different From Class I/B Antiarrhythmic Action? Comparison of The Effects of GS967 to Mexiletine in Canine Ventricular Myocardium

Status:

Version 1

Abstract

Figures

1. Introduction

2. Methods

2.1. Animals

2.2. Isolation of cardiomyocytes

2.3. Electrophysiology

2.3.1. Action potential voltage clamp

2.3.2. Conventional voltage clamp

2.3.3. Recording of APs from multicellular preparations

2.3.4. Determination of beat-to-beat variability of APD in isolated myocytes

2.4. Statistics

3. Results

3.1. Effects of GS967, mexiletine and TTX under action potential voltage clamp conditions

3.2. Effects of GS967 and mexiletine on INaL under conventional voltage clamp conditions

3.3. Effects of GS967 and mexiletine on ICa under conventional voltage clamp conditions

3.4. Effects of GS967 and mexiletine on action potential upstroke

3.5. Effects of GS967 and mexiletine on APD

3.6. Effects of GS967 and mexiletine on beat-to-beat variability of APD

4. Discussion

Declarations

References

Status:

Version 1

3.2. Effects of GS967 and mexiletine on I_NaL under conventional voltage clamp conditions

3.3. Effects of GS967 and mexiletine on I_Ca under conventional voltage clamp conditions