Late cardiac sodium current can be assessed using automated patch-clamp

Abstract

The cardiac late Na + current is generated by a small fraction of voltage-dependent Na + channels that undergo a conformational change to a burst-gating mode, with repeated openings and closures during the action potential (AP) plateau. Its magnitude can be augmented by inactivation-defective mutations, myocardial ischemia, or prolonged exposure to chemical compounds leading to drug-induced (di)-long QT syndrome, and results in an increased susceptibility to cardiac arrhythmias. Using CytoPatch™ 2 automated patch-clamp equipment, we performed whole-cell recordings in HEK293 cells stably expressing human Nav1.5, and measured the late Na + component as average current over the last 100 ms of 300 ms depolarizing pulses to -10 mV from a holding potential of -100 mV, with a repetition frequency of 0.33 Hz. Averaged values in different steady-state experimental conditions were further corrected by the subtraction of current average during the application of tetrodotoxin (TTX) 30 μM. We show that ranolazine at 10 and 30 μM in 3 min applications reduced the late Na + current to 75.0 ± 2.7% (mean ± SEM, n = 17) and 58.4 ± 3.5% ( n = 18) of initial levels, respectively, while a 5 min application of veratridine 1 μM resulted in a reversible current increase to 269.1 ± 16.1% ( n = 28) of initial values. Using fluctuation analysis, we observed that ranolazine 30 μM decreased mean open probability p from 0.6 to 0.38 without modifying the number of active channels n, while veratridine 1 μM increased n 2.5-fold without changing p. In human iPSC-derived cardiomyocytes, veratridine 1 μM reversibly increased APD90 2.12 ± 0.41-fold (mean ± SEM, n = 6). This effect is attributable to inactivation removal in Nav1.5 channels, since significant inhibitory effects on hERG current were detected at higher concentrations in hERG-expressing HEK293 cells, with a 28.9 ± 6.0% inhibition (mean ± SD, n = 10) with 50 μM veratridine.

An evaluation of 30 clinical drugs against the comprehensive in vitro proarrhythmia assay (CiPA) proposed ion channel panel

“…In order to observe Nav1.5-late, the normal process of channel inactivation must be altered. This is typically done with a toxin such as ATX-II or veratridine (Chevalier et al, 2014;Belardinelli et al, 2013). In the present experiments, Nav1.5-late was elicited by the application of 50M veratridine to the bath solution.…”

Screening system for drug-induced arrhythmogenic risk combining a patch clamp and heart simulator

“…The inhibition ratio of peak inward current at the 30th pulse was calculated as I Na inhibition. For ranolazine, the inhibitory effect on the late component of I Na was also included using the previously reported parameters ( 30 , 31 ). I Ca was induced by the application of depolarizing step pulses to 0 mV with 150-ms duration from a holding potential of −40 mV every 5 s. The inhibition ratio of peak inward current was calculated as I Ca inhibition, except for ranolazine in which the late component was evaluated ( 30 ).…”

Comprehensive in vitro Proarrhythmia Assay (C i PA): Pending issues for successful validation and implementation

“…I NaLate is a small fraction of the classic "peak" I Na current carried by Na V 1.5 which is generated by voltage-dependent Na + channels entering in burst-gating mode (repeated openings and closures) during myocyte depolarization. Genetic mutations causing defective I Na channel inactivation, myocardial ischemia, and drug treatments can upregulate I NaLate and expose patients to arrhythmic risk (Chevalier, Amuzescu, Gawali, Todt, Knott, Scheel, et al, 2014). Drugs like ranolazine, which is a blocker of this current, can reduce or prevent the proarrhythmic propensity inherent to hERG channel blockade (Gupta, Khera, Kolte, Aronow, & Iwai, 2015).…”

“…A recent report describes the successful use of the CytoPatch™ automated patch-clamp platform and a HEK293 cell line stably expressing human Na V 1.5 to determine I NaLate (Chevalier, et al, 2014). The…”