Elsevier

Heart Rhythm

Volume 6, Issue 11, November 2009, Pages 1625-1631
Heart Rhythm

Experimental
Use-dependent block of cardiac late Na+ current by ranolazine

https://doi.org/10.1016/j.hrthm.2009.07.042Get rights and content

Background

Ranolazine is an antianginal drug that inhibits the cardiac late Na+ current (INa). The selectivity of ranolazine to block late INa relative to peak INa at rapid heart rates has not been determined, but is potentially important to drug efficacy and safety.

Objective

This study sought to quantify use-dependent block (UDB) of cardiac peak and late INa by ranolazine.

Methods

Wild-type (WT) and long QT3 mutation R1623Q channels were expressed in HEK293 cells and studied using whole-cell patch-clamp technique.

Results

Ranolazine (1 to 300 μM) caused tonic (0.1 Hz) and UDB (1, 2, and 5 Hz) of WT and R1623Q peak INa. The IC50 values for block of WT and R1623Q peak INa at 0.1, 1, 2, and 5 Hz were 430, 260, 157, and 154 μM, and 95, 77, 37, and 25 μM, respectively. The IC50 values for block of R1623Q late INa at 0.1, 1, 2, and 5 Hz were 7.5, 7.3, 2.2, and 1.9 μM, respectively. Ranolazine (10 μM) caused a hyperpolarizing shift of WT and R1623Q peak INa steady-state inactivation without affecting steady-state activation, suggesting that ranolazine interacts with inactivated states of the channels. Ranolazine (30 μM) significantly slowed the recovery from inactivation of peak INa of both WT and R1623Q and late INa of R1623Q.

Conclusion

Ranolazine slowed recovery of late INa from inactivation and thus caused UDB of late INa. These data suggest that the effect of ranolazine to block late INa may be increased and the selectivity to block late INa relative to peak INa may be retained during tachycardia.

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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    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).

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