Atria are More Sensitive Than Ventricles to GS-458967-Induced Inhibition of Late Sodium Current

Arterially Perfused Canine Right Atrium

The entire right atrium (RA) with a thin rim of right ventricular (RV) tissue was dissected from the isolated dog heart and then the preparation was unfolded. The ostium of the right coronary artery was cannulated with polyethylene tubing (inner diameter, 1.75 mm; outer diameter, 2.1 mm), and the preparation was perfused with cold Tyrode solution (12°C-15°C) containing 8 mmol/L [K⁺]_o. Ventricular right coronary branches and the cut atrial branches were ligated using silk thread. The preparation was placed in a temperature-controlled bath (8 × 6 × 3 cm³) and perfused with Tyrode solution (37.0°C ± 0.5°C).

Arterially Perfused Canine Left Ventricular and RV Wedge

Transmural wedges with dimensions of approximately 3 × 1.5 × 1.5 cm³ were dissected from the anterior-apical aspects of the left ventricle (LV) or anterior-basal part of RV. The tissues were cannulated and perfused with cardioplegic solution. The preparations were then placed in a temperature-controlled tissue bath and perfused with Tyrode solution (37.0°C ± 0.5°C).

For both atrial and ventricular preparations, the composition of the Tyrode solution was the following (in mmol/L): NaCl 129, KCl 4, NaH₂PO₄ 0.9, NaHCO₃ 20, CaCl₂ 1.8, MgSO₄ 0.5, and d-glucose 5.5, buffered with 95% O₂ and 5% CO₂. The perfusate was delivered to the artery by a roller pump (Cole Parmer Instrument Co, Niles, Illinois).

Transmembrane action potential (AP) recordings were obtained using the floating microelectrode technique (10-25 MΩ direct current [DC] microelectrode resistance) connected to a high input impedance intracellular amplifier (World Precision Instruments, Sarasota, Florida). The signals were further amplified and digitized (sampling rate = 40 kHz) using Spike2 acquisition system (Cambridge Electronic Design, Cambridge, England).

An electrocardiogram (pseudo-ECG) was recorded using 2 electrodes consisting of AgCl half cells placed in the bath solution 1.0 to 1.5 cm from the opposite ends of the atrial and ventricular preparations.

Effective refractory period (ERP) was measured by delivering premature stimuli after every 10th regular beat at a pacing cycle length (CL) of 500 (with 5-10 ms resolution; stimulation with twice diastolic threshold of excitation (DTE) amplitude).

Postrepolarization refractoriness (PRR) was defined as the difference between ERP and AP duration at 75% (APD₇₅) in atria and APD₉₀ in ventricles (ERP corresponds to APD_70-75 in atria and APD₉₀ in ventricles). ⁷

Maximum Rate of Rise in the AP Upstroke

Stable AP recordings and maximum rate of rise in the AP upstroke (V_max) measurements are difficult to obtain in vigorously contracting perfused preparations. A large variability in V_max measurements is normally encountered at any given condition, primarily due to variability in the amplitude of phase 0 of the AP. The effects of GS-458967 on V_max were determined by comparing the changes in V_max values upon acceleration from a CL of 500 to 300 ms. Due to a substantial interpreparation variability, V_max values were normalized for each experiment and then averaged.

Experimental Protocols

Coronary-perfused RA and LV/RV preparations were equilibrated in the tissue bath until electrically stable (30-120 min). The RA beat spontaneously and LV/RV preparations were paced using a pair of thin silver electrodes insulated except at their tips (bipolar rectangular pulses of 2-millisecond duration and twice DTE intensity). Electrophysiological parameters were recorded under baseline conditions and after the addition of progressively higher concentrations of GS-458967 to the coronary perfusate (10, 30, 100, 300, and 1000 nmol/L). A period of at least 20 to 30 minutes was allowed for each concentration of GS-458967 to act before the start of data collection. In a subset of the experiments with LV and RV wedge preparations, E-4031 (1 µmol/L; a specific rapidly activating delayed rectifier potassium channel [I_Kr] blocker) was added to the coronary perfusate first, followed by addition of progressively higher concentrations of GS-458967 (300 and 1 μmol/L) to the perfusate containing E-4031.

Ventricular-Isolated Myocytes

Single myocytes were obtained by enzymatic dissociation from coronary-perfused ventricular wedge preparations of LV free wall as described previously. ¹⁴ Cells from the epicardial, M cell, and endocardial regions of the LV were used in the study. The composition of external solution was (in mml/L) as follows: 140 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 10 glucose, and 10 HEPES. Transmembrane AP recordings were obtained using high-resistance microelectrode technique (40-60 MΩ DC microelectrode resistance).

Chemicals

GS-458967, synthesized at Gilead Sciences, Inc (Foster City, California), is a triazolopyridine derivative, 6-(4-(trifluoromethoxy)phenyl)-3-(trifluoromethyl)-[1,2,4]triazolo[4,3-a] pyridine, with a molecular weight of 347.

Drugs

GS-458967 (Gilead Sciences, Inc, dissolved in 100% DMSO) and E-4031 (Enzo Life Sciences, Inc, Farmingdale, New York; dissolved in distilled water) were prepared fresh before each experiment (stock solution = 0.3 or 1 mmol/L for GS-458967 and 5 mmol/L for E-4031).

Statistics

Statistical analysis was performed using a Student t test or 1-way analysis of variance for paired or unpaired data, as appropriate. All data are expressed as mean ± standard deviation.

Electrophysiological Effects of GS-458967 in Atria and Ventricles

In the initial experimental series, we compared the effect of GS-458967 (10-300 nmol/L) in arterially perfused atrial and ventricular preparations paced at a CL of 500 ms (approximating the resting heart rate in canine in vivo). At a pacing CL of 500 ms, GS-458967 (100-300 nmol/L) significantly abbreviated APD_50-90 in atria but did not significantly alter APD in the ventricles (Figure 1). Despite its effect to abbreviate APD in atria, GS-458967 (100-300 nmol/L) prolonged atrial ERP secondary to the development of PRR (Figure 2). GS-458967 did not affect ERP or induce PRR in the coronary-perfused ventricular wedge (Figure 2). In the presence of GS-458967 (100 and 300 nmol/L), acceleration of pacing rate from a CL of 500 to 300 ms caused a significant use-dependent reduction in V_max in the atrium but not in the ventricle (Figure 3). The shortest S₁-S₁ permitting 1:1 activation was significantly increased in atria but not in the ventricles (Figure 4).

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Figure 1.

GS-458967 abbreviates action potential duration (APD) in the atria but causes little change in APD in the ventricles at a pacing cycle length (CL) of 500 ms. Upper panels: shown are superimposed action potentials recorded in atrial and ventricular preparations under baseline condition (control) and following the addition of 100 and 300 nmol/L of GS-458967. Bottom panels: summary data of the effect of GS-458967 on APD at 50% repolarization (APD₅₀) and APD at 90% repolarization (APD₉₀) in atria and ventricles. Pacing CL = 500 ms. *P < .05 versus respective control (n = 8-10). CT indicates crista terminalis; PM, pectinate muscle; Epi, epicardium.

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Figure 2.

Atrial-selective induction of postrepolarization refractoriness (PRR) by GS-458967. PRR, estimated by the difference between ERP and action potential duration at 75% repolarization (ADP₇₅) in atria and APD at 90% repolarization (ADP₉₀) in ventricles, increases with increasing concentrations of GS-458967 in atria but not in the ventricles. *P < .05 versus respective control (n = 7-8). Atrial data are from the pectinate muscle region of the right atrium. Ventricular ERP was measured in the endocardial surface and APD₉₀ is from the subendocardial region (ie, M cell region).

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Figure 3.

Atrial-selective use-dependent depression of the maximum rate of rise of the action potential upstroke (V_max) in the presence of GS-458967. Left panel: representative atrial and ventricular action potentials and accompanying V_max tracings recorded following acceleration of pacing rate from a CL (cycle length) of 500 to 300 ms. Right panel: summary data expressed as percentage of V value at 500 ms following acceleration of pacing rate from a CL of 500 to 300 ms. *P < .05 versus V_max change in control (n = 8-10).

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Figure 4.

GS-458967 significantly increases the shortest S₁-S₁ permitting 1:1 activation in atria but not in ventricles. The shortest S₁-S₁ was measured using 2 × diastolic threshold intensity (as determined at a cycle length [ CL] of 500 ms). *P < .05 versus control (n = 4-9).

Because the effects of late I_Na inhibition on APD are most evident when APD is prolonged, ^{3 –5} additional experiments were performed in ventricular wedge preparations paced at slow rates. A higher concentration of GS-458967 (1 μmol/L) was tested as well. At a CL of 500 to 2000 ms, GS-458967 (300-1000 nmol/L) produced no significant effect on APD of subendocardial M cells, the cells traditionally displaying the longest APD ¹⁵ (Figure 5). When ventricular APD was further prolonged using E-4031 (mimicking long QT type 2 syndrome), addition of GS-458967 (1 μmol/L) significantly abbreviated APD in the M cell region of the ventricular wedge preparation at a CL of 2000 ms (Figure 5). At a CL of 500 milliseocnds, GS-458967 (300-1000 nmol/L) did not significantly abbreviate ventricular APD (207 ± 13 ms in the presence of E-4031 and 204 ± 17 ms following 1 μmol/L GS-458967).

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Figure 5.

GS-458967 does not significantly affect repolarization in control ventricles but abbreviates it in a long QT2 model (in the presence of E-4031). Upper panel: superimposed action potentials recorded from M cell regions of left ventricle wedge preparations before and after progressively higher concentrations of GS-458967 (300 and 1000 nmol/L) at a pacing cycle length (CL) of 2000 ms. Data from 2 different preparations are shown. Bottom panel: summary effect of GS-458967 on action potential duration at 90% APD₉₀ in subendocardial M cell regions (combined data from left and right ventricular wedge preparations) at a pacing CL of 2000 ms. *P < .05 versus control (n = 4-5).

In contrast to the lack of effect of the drug in untreated ventricular multicellular preparations (Figure 5, upper left panel), GS-458967 (300 nmol/L) consistently and significantly abbreviated APD when studied in myocytes isolated from the epicardial, M cell, and endocardial regions of the LV, with greater APD shortening of the cells isolated from the M region (Figure 6).

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Figure 6.

GS-458967 consistently shortens action potential duration (APD) in isolated single ventricular myocytes. Upper panel: superimposed action potentials recorded from isolated left ventricular midmyocardium cell (M cell) myocytes before (control) and after addition of 300 nmol/L of GS-458967 at a pacing cycle length (CL) of 2000 ms. Bottom panels: summary data of the effect of GS-458967 on APD at 90% (APD₉₀) in isolated ventricular myocytes at pacing CLs of 500 and 2000 ms. *P < .05 versus control (n = 4-8). Epi indicates epicardium; Endo, endocardium.

Is GS-458967an Atrial Preferential Peak and Late I_Na Inhibitor?

GS-458967 is reported to be a highly selective late I_Na inhibitor when studied in isolated ventricular myocytes. ^13,16 The half maximal inhibitory concentration (IC₅₀) for GS-458967 block of endogenous late I_Na is 0.46 µmol/L in guinea pig ventricular myocytes ¹⁶ and of ATX-II-augmented late I_Na is 0.13 ± 0.01 µmol/L in rabbit ventricular myocytes. ¹³ Only 7.5% of peak I_Na and 16% of I_Kr are inhibited by 10 µmol/L of GS-458967 in rabbit ventricular myocytes. Moreover, GS-458967 does not significantly inhibit I_Ca-L, I_Ca-T, I_K-ATP, and I_Na-Ca (at 1-3 µmol/L). ¹³ Of note, the potency of GS-458967 to block peak I_Na was measured at a CL of 0.1 Hz and holding potential of −120 mV, which may significantly underestimate its effect under physiological conditions. Under these voltage clamp conditions, 285 to 1329 µmol/L of ranolazine has been reported to cause a 50% reduction (IC₅₀) in peak I_Na, ^6,13 whereas at faster rates and “physiological” holding potentials, IC₅₀ is less than 10% of the lower value. ⁸ The QRS interval, an peak I_Na-dependent parameter, was not affected by 3 µmol/L of GS-486967 at pacing rates of 1 to 4 Hz in isolated rabbit heart, indicating lack of effect of this compound in rabbit ventricles at physiologically relevant heart rates. ¹³

Atrial-selective reduction in V_max, induction of PRR, and an increase in the shortest S₁-S₁ interval by GS-458967 in our study are consistent with atrial-selective block of peak I_Na by this agent. Atrial selectivity of peak I_Na block has been described for sodium channel blockers that dissociate rapidly from the sodium channel including ranolazine, amiodarone, dronedarone, vernakalant, AZD1305, AVE0118, and Wenxin Keli. ^{7,9 –12,17 –21} The factors underlying the atrial-selective effects of I_Na blockers include a more negative steady state inactivation relationship, a less negative resting membrane potential, and a more gradual phase 3 of the AP in atrial versus ventricular cells as well as a relatively rapid dissociation of the drug from the sodium channel. ^7,8,22 GS-458967 dissociates from sodium channels (HEK 293 and atrial myocytes) rapidly with a time constant of 300 to 500 ms (holding potential of −120 mV at 23°C), a value approaching that of lidocaine (L. Belardinelli et al, PhD unpublished observation, 2014). Because GS-458967 has rapid unbinding kinetics and considering the fact that all I_Na blockers inhibit both peak and late I_Na, commonly with a higher potency for late versus peak I_Na inhibition, atrial-selective block of late I_Na by GS-458967 is not unexpected.

GS-458967-induced APD abbreviation in atria but not in ventricles could be due to atrial-selective inhibition of late I_Na. In support of this hypothesis, ranolazine inhibits sea anemone toxin-enhanced late I_Na more effectively in atrial versus ventricular myocytes. ²³ The atrial-selective abbreviation of APD could also be due to atrioventricular differences in other factors, including AP morphology, late I_Na density, input resistance, and cellular coupling. Maximum late I_Na density recorded under voltage clamp conditions has been reported to be greater in atrial versus ventricular cells in one study, ²³ but greater in ventricular versus atrial in another (G.X. Yan MD, PhD, and L. Belardinelli PhD, unpublished observation, 2013), and of similar density in a third study. ²⁴ A greater density of late I_Na in atria is expected based on the finding that peak I_Na density is much greater in canine atrial versus ventricular cells under identical recording conditions. ^7,25 The voltage dependence of late I_Na peaks at about −20 mV¹⁴ approximating the voltage of the AP plateau in atrial but not ventricular cells (Figure 1). Thus, the contribution of late I_Na during the AP plateau might be greater in atrial versus ventricular cells. A greater input resistance in atria likely contributes to the greater effect of GS-458967 on the APD of atrial cells. ^{26 –28} Moreover, membrane resistance of single isolated myocytes is much higher than that of cells that are well coupled to each other in a multicellular preparation. These findings can explain the effect of GS-458967 to abbreviate APD in isolated ventricular myocytes but not in multicellular preparations (Figures 1 and 6). Note that isolated single myocytes and tissue slices are generally more responsive than isolated coronary-perfused cardiac preparations to agents that either prolong or abbreviate APD. ²⁹

Atria have an intrinsically greater level of fibrosis than ventricles, ³⁰ contributing to reduced cell to cell coupling. Cellular uncoupling is known to amplify the effect of drugs that prolong or abbreviate APD. ^29,31 It is expected that GS-458967 would shorten APD in structurally compromised ventricles.

A greater effect of GS-458967 on M cell versus epicardial and endocardial myocytes (Figure 6) is likely due to a larger late I_Na density in the M cells. ¹⁴ The effect of GS-458967 to abbreviate APD is reported to be prominent in ventricular myocytes isolated from guinea pig ¹⁶ but not from rabbit. ¹³ GS-458967 (1 µmol/L) causes only a small statistically nonsignificant APD₅₀ abbreviation in rabbit isolated ventricular myocytes (−5.9%, at a pacing CL of 1000 ms). ¹³ The effect of GS-458967 (30-300 nmol/L) to abbreviate APD in Purkinje fibers at all pacing rates ³² may be due to its intrinsically longer APD, relatively high level of late I_Na and higher input resistance. ³³ GS-458967 induced abbreviation of APD in Purkinje fibers but not in ventricular muscle, thus reducing dispersion of repolarization at the Purkinje–muscle junction, which could diminish arrhythmic potential when this parameter is augmented.

The atrioventricular differences in the effect of GS-458967 on APD are not observed with ranolazine. ⁷ This is likely due to the fact that ranolazine blocks both the depolarizing late I_Na and repolarizing I_Kr. ² Inhibition of I_Kr causes a greater APD prolongation in atria versus ventricles at normal pacing rates. ⁹ However, like GS-458967, ranolazine produces atrial-selective depression of V_max- and I_Na-dependent parameters. ^7,34

It remains to be determined whether other ion channel-specific sodium channel blockers can produce atrial-selective APD abbreviation. The specific I_Na blocker lidocaine, at concentrations that significantly reduce peak I_Na in atria and ventricles, abbreviates APD in both ventricular and atrial multicellular preparations. ^7,35 Unlike ranolazine, lidocaine is a sodium channel blocker with relatively poor atrial selectivity. ⁷

Pathology-Specific Effect of GS-458967 in Ventricular Muscle

Our results suggest that the effect of late I_Na block in the ventricle is enhanced under conditions in which APD is significantly prolonged, suggesting a pathology-specific role for late I_Na inhibition in cardiac diseases in which APD is prolonged, including heart failure, long QT syndromes, and hypertrophic cardiomyopathy. ⁵ Heart rate can significantly affect the contribution of late I_Na in cardiac electrophysiology. The magnitude of late I_Na is reverse-rate dependent, that is, the faster the rate the smaller is the magnitude of this current. ^2,14,36 Late I_Na blockers commonly produce significant APD abbreviation in ventricular multicellular preparations only when APD is significantly prolonged (eg, at slow rates and/or in the presence of I_Kr blockers or late I_Na augmenters), ^2,4,13,37 consistent with the effect of GS-458967 in the present study.

Limitations of the Study

Our experiments were performed in isolated coronary-perfused atrial and ventricular preparations perfused with Tyrode solution or in isolated single-ventricular myocytes. These preparations lack autonomic influences and other factors present in vivo, which could influence our results. The atrial selectivity of GS-458967 was determined acutely in “healthy” cardiac preparations. Clinical cardiac arrhythmias are normally associated with pathology-mediated electrical and structural abnormalities, which may significantly modulate pharmacological response and antiarrhythmic efficacy. Thus, the pathology specificity of the drug’s effect remains to be more fully explored. We cannot totally dismiss the possibility that GS-458967 affects other currents in ventricular myocardium that may prevent the expected shortening of APD in response to its inhibition of late I_Na. Lidocaine and mexiletine, specific I_Na blockers, are reported to cause small but significant abbreviation of APD in canine coronary-perfused and superfused tissue slice ventricular preparations in control (nonlong QT) setting, ^7,35 and this effect is most likely due to inhibition of late I_Na.