Selective inhibition of late sodium current suppresses ventricular tachycardia and fibrillation in intact rat hearts

doi:10.1016/j.hrthm.2013.11.026

Heart Rhythm

Volume 11, Issue 3, March 2014, Pages 492-501

https://doi.org/10.1016/j.hrthm.2013.11.026 Get rights and content

Background

Enhanced late inward Na current (I_Na-L) modulates action potential duration (APD) and plays a key role in the genesis of early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs) and triggered activity.

Objective

The purpose of this study was to define the influence of selective block of I_Na-L on EAD- and DAD-mediated triggered ventricular tachycardia (VT) and ventricular fibrillation (VF) in intact hearts using (GS967), a selective and potent (IC₅₀ = 0.13 ± 0.01 μM) blocker of I_Na-L.

Methods

VT/VF were induced either by local aconitine injection (50 μg) in the left ventricular muscle of adult (3–4 months) male rats (N = 21) or by arterial perfusion of 0.1 mM hydrogen peroxide (H₂O₂) in aged male rats (24–26 months, N = 16). The left ventricular epicardial surface of the isolated-perfused hearts was optically mapped using fluorescent voltage-sensitive dye, and microelectrode recordings of action potentials were made adjacent to the aconitine injection site. The suppressive and preventive effects of GS967 (1 μM) against EAD/DAD-mediated VT/VF were then determined.

Results

Aconitine induced VT in all 13 hearts studied. Activation map (N = 6) showed that the VT was initiated by a focal activity arising from the aconitine injection site (cycle length [CL] 84 ± 12) that degenerated to VF (CL 52 ± 8 ms) within a few seconds. VF was maintained by multifocal activity with occasional incomplete reentrant wavefronts. Administration of GS967 suppressed the VT/VF in 10 of 13 hearts (P < .001). Preexposure to GS967 for 15 minutes before aconitine injection prevented initiation of VT/VF in 5 of 8 additional hearts (P < .02). VF reoccurred within 10 minutes on washout of GS967. Microelectrode recordings (N = 7) showed that VT/VF was initiated by EAD- and DAD-mediated triggered activity at CL of 86 ± 14 ms (NS from VT CL) that preceded the VF. GS967 shortened APD, flattened the slope of the dynamic APD restitution curve, and reduced APD dispersion from 42 ± 12 ms to 8 ± 3 ms (P < .01). H₂O₂ perfusion in eight fibrotic aged hearts promoted EAD-mediated focal VT/VF, which was suppressed by GS967 in five hearts (P < .02).

Conclusion

The selective I_Na-L blocker GS967 effectively suppresses and prevents aconitine and oxidative stress-induced EADs, DADs, and focal VT/VF. Suppression of EADs, DADs, and reduction of APD dispersion make GS967 a potentially useful antiarrhythmic drug in conditions of enhanced I_Na-L.

Introduction

An emerging new strategy to suppress the initiation of triggered ventricular tachycardia (VT) and ventricular fibrillation (VF) is to prevent pathologic increases in the late inward Na current (I_Na-L).1, 2, 3, 4 Enhanced I_Na-L seen in human heart failure,5, 6 patients with long QT syndrome type 3,⁷ and diverse animal models including aconitine8, 9 and oxidative stress10, 11 plays an important role in promoting early depolarizations (EADs) and delayed afterdepolarizations (DADs).¹² These afterpotentials initiate triggered activity1, 2, 12, 13, 14 that may cause focal VT15, 16 and VF¹⁷ maintained by multifocal mechanisms.2, 18 Previous studies have shown that inhibition of I_Na-L with sodium channel–blocking drugs such as ranolazine, flecainide, and mexiletine suppresses EAD- and DAD-mediated triggered arrhythmias.1, 2, 19 However, none of these drugs, including ranolazine, are sufficiently selective inhibitors of the I_Na-L as they also affect the conductances of other ion channels. As a result, the exclusive role played by enhanced I_Na-L in the genesis of focal VT and multifocal VF in intact hearts remains unclear. The recent synthesis of GS-458967 (GS967), a highly selective and potent (IC₅₀ = 0.13 μM) blocker of I_Na-L, which completely eliminates I_Na-L while having minimal or no effect on the conductances of other ion channels,1, 20 made the study of the exclusive role of enhanced I_Na-L in the genesis of focal VT/VF possible. Table 1 (modified from Belardinelli et al¹) describes the comparative potencies of GS967 against I_Na-L relative to ranolazine and flecainide. In this study, we hypothesize that focal EAD/DAD-mediated VT/VF associated with aconitine and oxidative stress is suppressed by selective inhibition of I_Na-L with GS967. Preliminary data have been reported in abstract form.²¹

Section snippets

Surgical preparation and electrophysiologic recordings

We studied 21 adult (~3 months) and 16 aged (23–25 months) male Fisher344 rats. This investigation conforms to the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No.85-23, revised 1996) and was approved by the Animal Research Committee of UCLA. The hearts of the anesthetized rats were removed and cannulated and perfused through the aorta at 5 mL/min in a Langendorff setting. Simultaneous ECG, ventricular and atrial electrograms, and

Activation maps during aconitine-induced VT

Figure 1 shows the electrical propagation pattern in an adult isolated heart during sinus rhythm. No conduction slowing and/or block developed. After local injection of aconitine, monomorphic VT at a mean cycle length (CL) of 84 ± 12 ms (N = 13) arose (Figure 2). We optically mapped six hearts to study the mechanism of VT onset. The VT in all these studies originated from focal activation from the near aconitine injection site and propagated over the entire mapped LV surface. This pattern of

Discussion

The results of this study show that selective inhibition of I_Na-L with GS967 suppresses and prevents VT/VF associated with the two stressors aconitine¹⁶ and H₂O₂.²² Both stressors initiate rapid focal VT that degenerates to VF within seconds2, 16, 18 that is maintained by multifocal activation (Figure 3) with occasional incomplete reentrant wavefronts wandering between the LV epicardial foci.2, 18 Aconitine directly interferes with Na channel inactivation,8, 9 whereas H₂O₂10, 11 activates

Acknowledgment

We thank Hong Cao, BS, for preparing the manuscript.

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Aconitine is the main active component of Aconitum plants. Although aconitine has effects that include strengthening the heart, analgesia, anti-tumor, and immune-regulating effects, aconitine has both efficacy and toxicity, especially cardiotoxicity. Severe effects can include arrhythmia and cardiac arrest, which limits the clinical application of aconitine-containing traditional Chinese medicine. Ginsenoside Rb1(Rb1) is mainly found in plants, such as ginseng and Panax notoginseng, and has cardiovascular-protective and anti-arrhythmia effects.
This study aimed to investigate the detoxifying effects of Rb1 on aconitine cardiotoxicity and the electrophysiological effect of Rb1 on aconitine-induced arrhythmia in rats.
Pathological analysis, myocardial enzymatic indexes, and Western blotting were used to investigate the ameliorating effect of Rb1 on aconitine cardiotoxicity. Optical mapping was used to evaluate the effect of Rb1 on action potential and calcium signaling after aconitine-induced arrhythmia.
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Cardiovascular diseases are the top killer of human beings. The ventricular arrhythmia, as a type of malignant cardiac arrhythmias, typically leads to death if not treated within minutes. The multi-scale virtual heart provides an idealized tool for exploring the underlying mechanisms, by means of incorporating abundant experimental data at the level of ion channels and analyzing the subsequent pathological changes at organ levels. However, there are few studies on building a virtual heart model for rats—a species most widely used in experiments.
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Selective INaL inhibition is considered to be a new strategy in the treatment of atrial fibrillation (Daniels and Hill, 2015) and ventricular arrhythmias (Belardinelli et al., 2013). GS967 as a selective INaL inhibitor can effectively suppress EADs and delayed afterdepolarizations DADs, and minimize spatial-temporal dispersions of membrane repolarization or APD in the ventricle (Pezhouman et al., 2014). A recent study indicated that Eleclazine (GS6615), a novel selective INaL inhibitor, exerts anti-arrhythmic effects (Zablocki et al., 2016).
Studies demonstrated that the incidence of atrial fibrillation is significantly increased in patients with diabetes mellitus. Increase of late sodium current (I_NaL) has been associated with atrial arrhythmias. However, the role of I_NaL in the setting of atrial fibrillation in diabetes mellitus remained unknown. In this study, we investigated the alteration of I_NaL in the atria of diabetic mice and the therapeutic effect of its inhibitor (GS967) on the susceptibility of atrial fibrillation. The whole-cell patch-clamp technique was used to detect single cell electrical activities. The results showed that the density of I_NaL in diabetic cardiomyocytes was larger than that of the control cells at the holding potential of −100 mV. The action potential duration at both 50% and 90% repolarization, APD₅₀ and APD₉₀, respectively, was markedly increased in diabetic mice than in controls. GS967 application inhibited I_NaL and shortened APD of diabetic mice. High-frequency electrical stimuli were used to induce atrial arrhythmias. We found that the occurrence rate of atrial fibrillation was significantly increased in diabetic mice, which was alleviated by the administration of GS967. In GS967-treated diabetic mice, the I_NaL current density was reduced and APD was shortened. In conclusion, the susceptibility to atrial fibrillation was increased in diabetic mice, which is associated with the increased late sodium current and the consequent prolongation of action potential. Inhibition of I_NaL by GS967 is beneficial against the occurrence of atrial fibrillation in diabetic mice.
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Thus, the blunting of epinephrine-induced increases in TWA levels along with a reduction in ventricular arrhythmias is consistent with a protective action of the drug in reducing arrhythmogenic nonuniformities in repolarization. Pezhouman et al5 demonstrated in intact rat hearts that another selective cardiac late INa inhibitor, GS-967, is effective in decreasing spatial dispersion of repolarization and flattening the action potential restitution curve, consequently resulting in decreased propensity for wavebreak and reentrant activation. The potential role of cardiac late INa–mediated amelioration of intracellular calcium excess during epinephrine infusion by GS-967 is accordingly a plausible though unproven basis for the reduction in TWA level in the present study, as calcium overload is a well-established factor responsible for this phenomenon.10,22
The capacity of catecholamines to induce ventricular tachycardia (VT) is well documented.
The effectiveness of the novel cardiac late sodium inhibitor eleclazine in suppressing catecholamine-induced VT in a large animal model was compared with that of flecainide.
In 13 closed-chest anesthetized Yorkshire pigs, spontaneous VT and surges in T-wave alternans (TWA) level measured using the Modified Moving Average method were induced by epinephrine (2.0 µg/kg, i.v., bolus over 1 minute). Effects of eleclazine (0.3 mg/kg, i.v., infused over 15 minutes; n = 6) or flecainide (1 mg/kg, i.v., bolus over 2 minutes followed by 1 mg/kg/hr, i.v., for 1 hour; n = 7) on VT incidence and TWA level were measured from right intraventricular electrogram recordings.
Epinephrine reproducibly elicited hemodynamically significant spontaneous VT in all 13 pigs and increased TWA level by 33-fold compared to baseline (P < .001). Eleclazine reduced the incidence of epinephrine-induced ventricular premature beats and couplets by 51% (from 31.3 ± 1.91 to 15.2 ± 5.08 episodes; P = .038) and the incidence of 3- to 7-beat VT by 56% (from 10.8 ± 3.45 to 4.7 ± 3.12 episodes; P = .004). Concurrently, the drug reduced the peak epinephrine-induced TWA level by 64% (from 217 ± 22.2 to 78 ± 15.3 µV; P < .001). Flecainide also reduced the incidence of epinephrine-induced ventricular premature beats and couplets by 53% (from 40.4 ± 6.37 to 19.0 ± 2.73 episodes; P = .024) but did not affect the incidence of VT (from 15.0 ± 3.08 to 11.6 ± 2.93 episodes; P = .29) or the peak TWA level (from 207 ± 30.6 to 172 ± 26.2 µV; P = .34).
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: This study was supported by NIH Grant P01 HL079831, the Laubisch Endowments, and a research grant from Gilead Sciences Inc., Foster City California. Dr. Belardinelli is an employee of Gilead Sciences Inc. Dr. Karagueuzian is a recipient of an unrestricted research award from Gilead.

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Selective inhibition of late sodium current suppresses ventricular tachycardia and fibrillation in intact rat hearts

Background

Objective

Methods

Results

Conclusion

Introduction

Section snippets

Surgical preparation and electrophysiologic recordings

Activation maps during aconitine-induced VT

Discussion

Acknowledgment

J Am Coll Cardiol

Heart Rhythm

Prog Biophys Mol Biol

Heart Rhythm

A novel, potent, and selective inhibitor of cardiac late sodium current suppresses experimental arrhythmias

J Pharmacol Exp Ther

Ranolazine, an antianginal agent, markedly reduces ventricular arrhythmias induced by ischemia and ischemia-reperfusion

Am J Physiol Heart Circ Physiol

Late sodium current inhibition reverses electromechanical dysfunction in human hypertrophic cardiomyopathy

Circulation

Molecular mechanism for an inherited cardiac arrhythmia

Nature

The effect of aconitine on the membrane current in cardiac muscle

Pflugers Arch Gesamte Physiol Menschen Tiere

Properties of aconitine-modified sodium channels in single cells of mouse ventricular myocardium

Gen Physiol Biophys

Blocking late sodium current reduces hydrogen peroxide-induced arrhythmogenic activity and contractile dysfunction

J Pharmacol Exp Ther

Ionic mechanism of the effects of hydrogen peroxide in rat ventricular myocytes

J Physiol

The arrhythmogenic consequences of increasing late INa in the cardiomyocyte

Cardiovasc Res

Aconitine-induced delayed afterdepolarization in frog atrium and guinea pig papillary muscles in the presence of low concentrations of Ca2+

Jpn J Physiol

Effects of sodium-calcium exchange inhibitors, KB-R7943 and SEA0400, on aconitine-induced arrhythmias in guinea pigs in vivo, in vitro, and in computer simulation studies

J Pharmacol Exp Ther

Studies on auricular tachycardia caused by aconitine administration

Proc Exp Biol Med

Action potential duration restitution and ventricular fibrillation due to rapid focal excitation

Am J Physiol Heart Circ Physiol

The arrhythmogenic consequences of increasing late I_Na in the cardiomyocyte

Aconitine-induced delayed afterdepolarization in frog atrium and guinea pig papillary muscles in the presence of low concentrations of Ca²⁺