ExperimentalUse-dependent block of cardiac late Na+ current by ranolazine
Introduction
Local anesthetic and class I antiarrhythmic drugs inhibit peak Na+ current (INa) by binding to voltage-gated Na+ channels. Inhibition of peak INa produced by these drugs is often enhanced by rapid, repetitive stimulation. The resultant block is called use-dependent block (UDB) or frequency-dependent block. Recently, we and others1, 2 have shown that the antianginal drug ranolazine causes UDB of skeletal (Nav1.4), cardiac (Nav1.5) and peripheral (Nav1.7 and Nav1.8) peak INa, but UDB of late INa has not been characterized.
Many excitable tissues have been shown to have a component of INa that is resistant to inactivation. The existence of inactivation-resistant (persistent or late) INa was first identified in cardiac Purkinje fibers of dogs and rabbits.3 Recently, Maltsev et al4 showed the presence of late INa in human mid-myocardial myocytes isolated from normal and failing hearts. Similar to peak INa, late INa has been shown to be blocked by local anesthetics5, 6, 7 and by ranolazine.8 The block of late INa by ranolazine has been shown to occur at significantly lower concentrations than the block of peak INa. The values of IC50 for ranolazine to block late and peak INa in canine ventricular myocytes were reported as 5.98 and 294 μM,9 respectively.
Because late INa may play an important mechanistic role in inducing tachyarrhythmias,10, 11 it is important to understand whether the effect of drugs to inhibit late INa is increased or decreased at rapid heart rates. Therefore, in this study, our goal was to determine whether the effect of ranolazine to inhibit late INa is use-dependent and if the selectivity of ranolazine to inhibit late INa relative to peak INa is maintained at high stimulating frequencies. Because the amplitude of endogenous late INa is normally very small,4, 12 we utilized cells expressing Na+ channels with a long QT syndrome type 3 (LQT3) mutation, R1623Q, a missense mutation13 in the voltage-sensing region of the Na+ channel that leads to an increase of late INa. The endogenous late INa in R1623Q was sufficiently large to allow characterization of the UDB by ranolazine of peak and late INa using the whole-cell patch clamp technique.
Section snippets
Heterologous expression of SCN5A wild-type and R1623Q
Human embryonic kidney (HEK293) cells stably expressing the human heart Na+ channel (hH1a; Nav1.5) clone of SCN5A gene (α-subunit alone) were purchased from Cytomyx, Cambridge, UK. The LQT3 mutation R1623Q was generated by site-directed mutagenesis of WT SCN5A cDNA using overlap extension polymerase chain reaction strategy.13, 14 HEK293 cells were transiently transfected using PolyFect (Qiagen, Valencia, California). After 48 hours following transfection, green fluorescence protein-positive
Use-dependent block of WT and R1623Q Na+ channels by ranolazine
The late component of INa in HEK293 cells transiently expressing R1623Q mutation was greater than the late component of INa in HEK293 cells stably expressing WT channels (Figure 1), as previously shown.13 To study the UDB by ranolazine of peak (WT and R1623Q) and late (R1623Q) INa, a series of 40 pulses (50 msec in duration) to −20 mV from a holding potential of −140 mV were applied at rates of 1, 2, and 5 Hz. Late INa for R1623Q was measured as the mean value of INa between 46 and 48 msec
Discussion
The major new finding of this study was that ranolazine caused a UDB of late INa, in addition to UDB of peak INa. The potencies for ranolazine to cause tonic (0.1 Hz) and UDB (at 5 Hz) of R1623Q peak INa were 95.3 and 24.6 μM, respectively; for R1623Q late INa, the ranolazine potency values for tonic and UDB were 7.45 and 1.94 μM, respectively. Thus, the potencies of ranolazine to cause block of peak and late INa were similarly increased approximately 3-fold with increased stimulating
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2020, Heart Rhythm O2Citation Excerpt :The antianginal agent ranolazine (Ranexa) is a prototypic example of a drug with atrial-selective action against voltage-gated Na+ channels.7 Ranolazine shows use- and voltage-dependent block of voltage-gated Na+ channels through binding to the local anesthetic binding site within the Na+ channel vestibule and is relatively selective for the late Na+ current (INaL).13–16 The atrial-selective action of ranolazine against Na+ channels arises through a preferential block of the activated state and trapping of the drug in the inactivated state in combination with atrial-ventricular differences in (1) the voltage dependence of INa activation and inactivation and (2) resting membrane potential and diastolic interval (DI).7,12,16
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2019, Journal of Pharmacological and Toxicological MethodsCitation Excerpt :This observation is supported by the fact that the size of late INa at 40 ms of the depolarizing pulse was significant larger than the size of late INa during the ramp in the presence of ATX-II (Fig. 2B). In the present study, we found that the well-known late INa blocker, ranolazine, concentration-dependently blocked late INa with an IC50 of 16.7 μM when measured during the ramp (Fig. 5B), which is close to the IC50 values (5.8 to 7.9 μM) of ranolazine on late INa in native myocytes or recombinant cell lines (Antzelevitch et al., 2004; Crumb Jr. et al., 2016; Rajamani et al., 2009). However, the IC50 for ranolazine blocking late INa was slightly right shifted to 34 μM when measured at the 40 ms step pulse during the same recording.
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All experiments were funded by and performed at Gilead Sciences, Inc (formerly CV Therapeutics, Inc.), Palo Alto, California. Drs. Rajamani, El-Bizri, Shryock, and Belardinelli are employees of Gilead Sciences, Inc. (owner of ranolazine).