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

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.


Introduction
The late cardiac Na + current can be recorded 10-100 milliseconds after membrane depolarization as a sustained inward current component (I sus ) in cardiomyocytes 1 . This late current represents a fraction of voltage-dependent Na + channels that fail to inactivate after the initial opening. Instead, these channels change to a conformation with frequent late re-openings in a so-called burst mode 2 . Among other mechanisms, calmodulin kinase II overexpression in chronic heart failure increases the rate of transition to the late bursting mode conformation 2 . The phenomenon is also observed in Na v 1.5 inactivation-deficient mutants, such as ΔKPQ 3 or 1795InsD 4,5 , leading to a specific form of congenital long QT syndrome, LQT-3 6-8 .
Given its role in cardiac arrhythmogenesis 9,10 , pharmacological inhibition of the late Na + current component (I Na late ) is seen as an anti-arrhythmic strategy. Novel late sodium channel blockers, such as the partial fatty acid beta-oxidation inhibitor, ranolazine 11-15 , are used for both myocardial ischemia and to alleviate neuropathic pain and show a preferential affinity for the burst mode conformation of Na + channels [16][17][18][19][20][21] . Other compounds, such as veratridine, a steroidderived alkaloid extracted from rhizomes of Veratrum album or seeds of Schoenocaulon officinale 22,23 , preferentially bind to activated Na + channels, impeding inactivation and leading to increased nerve excitability.
The objectives of this study were to (1) record the veratridinedependent increase in the late Na + current using a HEK293 cell line stably transfected with human Na v 1.5 24 , (2) measure the ranolazineinduced inhibition of basal and veratridine-activated I Na late , and (3) asses the effects of veratridine at the same concentrations on action potentials (APs) in hiPSC-cardiomyocyte preparations externally paced in current-clamp mode. In parallel we also tested the inhibitory effects of veratridine on hERG1 current in stably transfected HEK293 cells. All these experiments were performed using CytoPatch™2 automated patch-clamp equipment.

Materials and methods
All experiments were performed using the CytoPatch™2, using standard dual-channel Cytocentrics chips with embedded quartz pipette tips 2 μM in diameter. For whole-cell late Na + current recordings, the voltage-clamp protocol consisted of repeated depolarizing pulses of -10 mV amplitude and 300 ms duration, from a holding potential of -100 mV, to allow a substantial removal from inactivation of Na v 1.5 channels. The peak and late Na + current were plotted and monitored over the entire duration of experiment, and extracted from recorded data for further analysis. Pharmacological compounds were applied in a predefined sequence using the dispensing needle of automated equipment. All experiments were performed at room temperature (21-22°C).
For whole-cell hERG current recordings, cells were held at -70 mV. After a brief 100-ms prepulse to -50 mV to determine the current leak, a 2-s depolarizing voltage step to +40 mV was followed by a 2-s step to -50 mV to elicit hERG tail currents every 10 s. Peak tail current amplitude was corrected by the leak current and the corrected peak tail current was averaged over the last three pulses of the control phase (I ctrl ) and the application phase (I cpd ), respectively.
From these averaged values the hERG tail current inhibition was calculated as follows: iPSC-CM recordings were performed in current-clamp mode, using repeated sweeps consisting of 3 injected current pulses of 2000 pA amplitude, 0.5 ms duration, at 3-s intervals. During solution uptake the system was switched to voltage-clamp mode.
Cell cultures and preparations HEK293 cells stably expressing either the human Na v 1.5 channel or the hERG K + channel were used as ready-to-use frozen Instant cells (product of Cytocentrics). Cells, kept in liquid nitrogen, were quickly thawed, centrifuged, resuspended in extracellular solution at a density of ~10 6 cells/ml, and used for experiments within 3 hours.
hiPSC-derived iCell ® Cardiomyocytes were kindly provided by Cellular Dynamics International (Madison, WI) as frozen samples, and cultured in monolayers in 12-well plates coated with 0.1% gelatin, for up to 41 days. The thawing/plating and maintenance media were provided by the cell supplier. For detachment, the monolayers were rinsed twice with calcium and magnesium-free PBS, then incubated for 2 min with trypsin 0.1% at 37°C. 1.5 ml of medium was added per well, the cells were suspended, centrifuged at 180 × g for 5 min, and resuspended in a 1:1 mixture of culture medium and hERG extracellular solution.

Solutions and chemicals
For recordings in Na v 1.

Data storage and analysis
The software files generated during the recordings were stored on computer hard disks. Patch clamp data were analyzed using the CytoPatch TM software and exported to Microsoft Excel or pClamp10 (Axon Instruments, part of Molecular Devices, Sunnyvale, CA) for further analysis, using a proprietary conversion tool. APD90 analysis for current-clamp recordings in iPSC-derived cardiomyocytes was performed with self-written software routines. APD90 was computed as the duration between the point of maximal AP upstroke speed during phase 0 to recovery of 90% of the difference between peak upstroke potential and resting potential. In the case of veratridine application, if recovery was incomplete during the 3-s interstimulus interval, APD90 was computed over several pacing cycles. For statistical analysis, one-way ANOVA for independent samples with Dunnett's post-hoc comparison, as well as Student's t tests where appropriate, were applied, at a level of significance of p ≤ 0.05, using the GraphPad Prism software (La Jolla, CA).

Late Nav1.5 sodium current component
Using automated voltage-clamp protocols applied to human Nav1.5expressing HEK293 cells in the above mentioned conditions, we routinely recorded, in high-quality seal conditions (both seal and membrane resistance > 1 GΩ), and with stability for at least 20 min, whole-cell Na + currents. These currents were elicited by membrane depolarization to -10 mV from a holding potential of -100 mV, required for the proper removal from inactivation of cardiac Na + channels. The late Na + current component (I Na late ) was automatically computed and plotted as time average over the last 100 ms of the 300-ms depolarizing pulse of each sweep. Figure 1A shows the time course of 5 averaged experiments including repeated applications of veratridine 1 μM with different durations (2 min, 1 min, 5 min), as well as a 1-min application of TTX 30 μM, resulting in the rapid complete block of late Na + current. The average current level during TTX application in each experiment was subsequently subtracted from all other averaged steady-state levels to obtain unbiased estimations of I Na late in different experimental conditions. In general, reversibility was good and rapid upon TTX wash-out.
2. Late sodium current activation by veratridine and block by ranolazine in Nav1.5 cells Under control conditions, a 5-min application of veratridine 1 μM induced an increase of the late Na + current by 269.1 ± 16.1% (mean ± SEM, n = 28) of initial values (Table 1 and Figure 2). Figure 1B shows representative current traces of selected sweeps, as well as   magnified inserts of the peak and late phase, within an individual experiment. The application of veratridine 1 μM increased the late current amplitude by more than two-fold relative to the TTX 30 μM reference trace, without any noticeable effect on peak current, while the co-application of ranolazine 10 μM reduced the peak current with apparently no effect on the late component. Systematic experiments with ranolazine applied alone demonstrated I Na late reduction to 75.0 ± 2.7% (mean ± SEM, n = 17) of initial values at 10 μM, and 58.4 ± 3.5% (mean ± SEM, n = 18) at 30 μM (Table 1 and Figure 2). σ 2 = npi 2np 2 i 2 = np(1p)i 2 and I = npi We used a unitary Na + current amplitude i at -10 mV of 1.43 pA, based on the unitary current amplitude of 2 pA at -40 mV observed in single-channel recordings of Nav1.5 late currents in HEK293 cells 26 and a reversal potential of +65 mV. In doing so, we could evaluate the mean open probability p and further the average number of Na + channels n open in the late mode in selected traces, over the last 100 ms of activity during the 300-ms depolarizing pulses. It was observed that 30 μM ranolazine application resulted in a decrease of mean open probability from 0.6 to 0.38, without affecting the average number of channels contributing the late Na + current (n = 4), in agreement with estimates obtained from averaged macroscopic currents. On the other hand, veratridine 1 μM increased the number of channels in late gating mode to n = 10, without influencing the mean open probability.

di-APD prolongation in iPSC-CM by application of veratridine
To get a better understanding of the role that the late Na + current may play in arrhythmogenesis and in the complex mechanisms of di-LQT syndrome, we performed a series of automated patch-clamp  experiments on hiPSC-derived cardiomyocytes with current-clamp recordings of stimulus-triggered APs. Each recorded sweep contained responses elicited by three injected current stimuli (0.5 ms duration, 2 nA amplitude). As shown in Figure 4, veratridine 1 μM induced a marked prolongation of AP duration, reaching saturation in less than 3 min. Further application of 5 μM veratridine resulted in further APD90 prolongation, exceeding the interstimulus interval. There was a slow trend to reversibility during the wash-out phase. Quantitative results for veratridine 1 μM application are summarized in Table 2. Thus, in n = 6 experiments at room temperature the relative APD90 prolongation induced by veratridine was 2.12 ± 0.41 fold (mean ± SEM), reversible to 1.21 ± 0.50 fold at wash-out (mean ± SEM, n = 4).

Weak hERG inhibition by veratridine
Last, we performed experiments using HEK293 cells stably expressing hERG1 to assess the effects of veratridine at different concentrations on this current component, in order to better understand the complex effects of this compound on the action potential of iPSC-derived cardiomyocytes. As shown in Figure 5, veratridine concentrations up to 5 μM did not produce levels of hERG inhibition significantly different than the diluting vehicle alone (ethanol 0.1%), while at 50 μM there was a 28.9 ± 6.0% (mean ± SD, n = 10) inhibition of peak hERG current, a statistically significant effect (p < 0.0001, one-way ANOVA for independent samples with Dunnett's multiple comparison test vs. control).

Discussion
The main findings of the present study using the CytoPatch TM 2 instrument are the following: (1) inhibition of cardiac late Na + current to 75.0% and 58.4% of initial values by ranolazine 10 and 30 μM, respectively; (2) activation of the same current by veratridine 1 μM to 269.1% of initial values; and (3) prolongation of APD90 by veratridine 1 μM to 212% of initial values in human iPSC-derived cardiomyocytes. We have succeeded in recording and analysing the small (a few picoA) late Na + current component in a human cell line stably transfected with wild-type human SCN5A, using automated patch-clamp technology, with a voltage protocol different from that used in the first electrophysiology characterization of ΔKPQ LQT-3 mutant Na + channels 3 . We observed that instrumental noise does not significantly affect the average current value over the last 100 ms of a 300-ms depolarizing pulse from -100 to -10 mV, therefore this value can be computed and plotted in real-time, representing a sensitive indicator for evidencing pharmacological effects in automated patch-clamp assays. Another critical parameter is the frequency of repetition of depolarizing stimuli, because it may reveal use dependency for compounds exerting state-dependent binding effects 27 . Although in the present set of experiments we kept a fixed value of 0.33 Hz, inclusion of different pacing rates in future variants of the assay may offer supplementary information of pharmacological relevance. An important tool in the precise assessment of I Na late levels is the application of TTX 30 μM, which results in an almost complete late Na + current inhibition, and offers a current reference used for the correction by subtraction of all other values in different experimental conditions. An excellent seal stability and access resistance, such as those offered by the Cytocentrics microfluidic chips 28 , is another prerequisite for successful late Na + pharmacology experiments.
Due to the limited amplitude of late Na + current under basal conditions, we increased it using low concentrations of veratridine, a steroid-derived natural alkaloid known to bind to an intramembrane site of Na + channel pore subunits and, in doing so, stabilize it in the open conformation 29,30 , contributing to the I Na late . Although veratridine at 1 μM resulted in a consistent 2.5 -3-fold increase in late Na + current, compared to initial levels ( Table 1 and Figure 1- Figure 3), we would not recommend this procedure for pharmacology assays because of possible interactions between veratridine and test compounds. Although we have not systematically explored this phenomenon, preliminary observations, as shown in Figure 1B, suggest a reduced effectiveness of ranolazine 10 μM when co-applied with veratridine, compared to application of ranolazine alone at the same concentration. The value of relative inhibition of the late Na + current by ranolazine 10 μM in the single compound application obtained in our study (75.0% of control levels) is larger than those obtained with the same concentration of ranolazine on late Na + current activated by veratridine 40 μM (89% and 81% for holding potentials of -110 mV and -90 mV, respectively) 31 . An elegant method that may offer pharmacologically relevant information concerning the site and mechanism of action of a certain compound is fluctuation analysis. As shown in Figure 3

Competing interests
None of the authors has competing interests concerning the present study, except for the participation of TK, OS and BA in the Cytocentrics company.

Grant information
This study was in part funded by a BMBF grant 03WKCC4D, TransCure NCCR Network to HA, and Austrian Science Fund FWF (grant W1232-B11).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
In agreement with the significant activatory effects of veratridine 1 μM on cardiac Nav1.5 channels in late gating mode in stably transfected cell lines, we found an important prolongation of action potential duration when the drug was applied to human iPSCderived cardiomyocytes in current-clamp configuration, using CytoPatch™2 automated patch-clamp equipment. The APD90 was increased over twofold by veratridine 1 μM, with a good reversibility upon wash-out (Table 2), and an even higher prolongation with 5 μM (Figure 4), although in this case we were unable to accurately compute APD90 values for externally paced APs, due to their overlap with the next stimulus. This effect is consistent with the findings of previous studies, showing APD prolongation in bronchial smooth muscle cells upon veratridine application 32 , and the enhancement of AP burst generation by veratridine in rat trigeminal sensory neurons 33 . Furthermore, we have demonstrated that hERG inhibition does not play any role in the observed APD prolongation by veratridine at 1 or 5 μM, since significant hERG inhibition occurred with higher veratridine concentrations ( Figure 5). A recent combined in vivo and in vitro study proved that the augmentation of the myocardial late Na + current during ventricular remodeling induced by pulmonary arterial hypertension in rats was accompanied by significant APD90 increases, and a beneficial effect of repeated ranolazine administration via late Na + current block was demonstrated with a subsequent alleviation of induced intracellular Ca 2+ overload 34 .
In conclusion, the present study demonstrates that automated patchclamp, as implemented by the CytoPatch™2 equipment using our proprietary CYTOCENTERING technology and quartz pipette tips embedded in silicon microfluidic chips 28,35 , which allow highquality stable seals, lead to reliable late Na + current pharmacology recordings, with results at least comparable to those obtained in manual patch-clamp experiments. In addition, automation may prove advantageous given the small amplitude of late Na + current, which therefore requires a large number of experiments for the accurate assessment of pharmacological effects. . They showed that the cardiac I can be inhibited by ranolazine and activated by veratridine. Using fluctuation analysis, they hypothesized that ranolazine would act as an open pore blocker, reducing the open probability without affecting the number of active channels, while veratridine would increase the number of active channels without influencing their open probability. In addition, they also provided additional functional data by performing a series of automated patch-clamp experiments showing that application of veratridine concentrations that increased I but had no effects on HERG current prolonged the action potential duration of human iPSC-derived cardiomyocytes. This is an interesting and well-designed study that reports high quality data. Considering the role of late sodium current in the action potential duration, mainly under pathological conditions, and the difficulty to accurately record this small ionic current, the subject of this study is highly relevant. The demonstration of the suitability of the automated patch clamp technology to accurately measure the late sodium current and to assess the effects of pharmacological agents on this current is helpful. In addition, the use of the hiPSC -derived cardiomyoyctes with this approach is also interesting. I do not have any major issues to address. However, the paper could be improved by attention to the following points.

Data availability
All experiments were performed at room temperature. Considering the limited amplitude of the late Na current under basal conditions, it could have been useful to perform these experiments at a more physiological temperature to help increase the amplitude of the current.
In Figure 1B, it would have been interesting to also present the recordings with ranolaxine alone.
The was elicited by the of 2 nA of . This is a rather large current action potential injection current and is probably not just-suprathreshold depolarizing . Do the hiPSC-derived current cardiomyocytes usually require such a high current injection to elicit action potential?
For the HERG recordings, is there any specific reason why the external concentration of KCl was only 2.5 mM?
Data reported in Table 1  7.
Data reported in Table 1 could have been normalized to the cell capacitance to report the current density instead of the current amplitude.
For some statistical analysis, an ANOVA would have been more appropriate that a Student's tests t as several groups were examined (eg., Tables 1 and 2).
There are few typographic errors in the Abstract: the units for the drugs ranolaxine, veratridine and TTX are all in millimolar concentrations but should be in the micromolar concentrations. Overall, the study is interesting and describes a methodological approach that could help study the late sodium current. The experiments were well designed and presented and the subject of this manuscript is of importance to the field. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
No competing interests were disclosed. Competing Interests: 28  The paper by Chevalier . analyzed whether late sodium current (I ) can be assessed using an et al automated patch-clamp device. To this end, the I effects of ranolazine (a well known I inhibitor) and veratridine (an I activator) were described. The authors tested the CytoPatch automated patch-clamp equipment and performed whole-cell recordings in HEK293 cells stably transfected with human Nav1.5. Furthermore, they also tested the electrophysiological properties of human induced pluripotent stem cell-derived cardiomyocytes (hiPS) provided by Cellular Dynamics International. The title and abstract are appropriate for the content of the text. Furthermore, the article is well constructed, the experiments were well conducted, and analysis was well performed.
I is a small current component generated by a fraction of Nav1.5 channels that instead to entering in the inactivated state, rapidly reopened in a burst mode. I critically determines action potential duration (APD), in such a way that both acquired (myocardial ischemia and heart failure among others) or inherited (long QT type 3) diseases that augmented the I magnitude also increase the susceptibility to cardiac arrhythmias. Therefore, I has been recognized as an important target for the development of drugs with either antiischemic or antiarrhythmic effects. Unfortunately, accurate measurement of I is a time consuming and technical challenge because of its extra-small density. The automated patch clamp device tested by Chevalier resolves this problem and allows to fast and reliable I measurements. et al.
The results here presented merit some comments and arise some unresolved questions. First, in some experiments (such is the case in experiments B and D in Figure 2) current recordings obtained before the ranolazine perfusion seem to be quite unstable. Indeed, the amplitude progressively increased to a maximum value that was considered as the control value (highlighted with arrows). Can this problem be overcome? Is this a consequence of a slow intracellular dialysis? Is it a consequence of a time-dependent shift of the voltage dependence of activation/inactivation? Second, as shown in Figure 2, intensity of drug effects seems to be quite variable. In fact, experiments A, B, C, and D in Figure 2