Assessment of the Effects of Online Linear Leak Current Compensation atDifferent Pacing Frequencies in a Dynamic Action Potential Clamp System
Alan Fabbri1 , Adrianus Prins1 ,Teun P. de Boer1
1

Department of Medical Physiology, Division of Heart & Lungs, University Medical Center Utrecht,Utrecht, The Netherlands
Abstract

Dynamic action potential (AP) clamp (dAPC) is an electrophysiology technique that allows one to study in realtime the effects of a biological current included in a computational AP model. During an experiment, the seal resistance between the cell membrane and the pipette is finite
and a leak current (Ileak ) occurs. Its reduction is crucial
to properly assess the effect of a drug.
Our work aims to quantify the impact of Ileak on a ventricular AP model and to evaluate the benefits of an online
compensation.
We carried out the experiments using a prototype Nanion Patchliner Dynamite8 in dAPC mode, running the ten
Tusscher human ventricular AP model. We use a passivemodel cell (Cm = 19.8pF , Rseal = 500M Ohm) and online compensate the leak current adopting a linear model.
Vmax , RM P and AP D20,50,90 are measured at several
degrees of compensation (within 0 and 100%), at different
pacing frequencies (0.5, 1, 2 Hz), and compared with theAP model in the open loop condition (i.e. with no connection between the model cell and the AP model).
Ileak decreases Vmax , depolarizes RMP (up to +6.1mV
at 1 Hz ) and prolongs AP D both during the plateau andthe late repolarization; a full compensation of Ileak brings
the AP biomarkers close to the open loop condition.
With this test, we show that online compensation of Ileakis beneficial for proper assessment of AP biomarker. Thecorrection of RMP is key, as it affects the following phasesof the AP.

1.

Introduction

Dynamic Clamp (DC) is a hybrid electrophysiology
technique that allows one to link a real cell with a computational model, in real time [1]. In the dynamic Action
Potential clamp (dAPC) configuration, a cell overexpressing a specific ion channel (e.g Human Embryonic Kidney(HEK, [2]) or Chinese Hamster Ovary (CHO, [3]) cells),
is coupled with a whole cell action potential (AP) compu-tational model. In this setting, the membrane potential of
the computational model stimulates the ion channel overexpressed in the real cell, as would happen in a real cardiomyocyte. In turn, the ion current generated in the realcell is scaled appropriately and fed back into the computational model, thereby contributing to action potential formation, and therefore to the final AP waveform, forming aclosed-loop system. As result, dAPC improves on classical electrophysiology techniques as it enables observationof the effects of a drug on the current of interest, and on
the AP at the same time.
During a dAPC experiment, the resistance between thepipette and the cell membrane (RSeal ) is finite and a leakcurrent (Ileak ) occurs. In voltage clamp experiments, theIleak artifact can be corrected for during post-processing.
Since dAPC is a real time technique, the Ileak must be
handled during the experiments. An online compensation
avoids misleading effects on the AP waveform.
This study aims to quantify the effects of Ileak and thebenefits of online compensation when using a ventricular
computational AP model in a dAPC experiment.
To do that, we compared five AP biomarkers (RestingMembrane Potential, RMP, Maximal upstroke Velocity,Vmax , and AP Duration at 20,50 and 90% of repolarization, AP D20,50,90 ) for several degrees of Ileak online
compensation (0% (No compensation), 25%, 50%, 75%
and 100%) at three pacing frequencies (0.5 Hz, 1 Hz and 2Hz), adopting the open loop configuration (no Ileak injection) as a reference.

2.

Methods

2.1.

The dynamic AP clamp system

In this study we used a prototype developed in our lab ofthe Nanion Patchliner Dynamite8 [4], adapted to the dAPC
configuration and running the endocardial version of theten Tusscher and Panfilov 2006 ventricular model (TP06,endo) [5]. The adoption of the Rush-Larsen method [6]allows one to provide a real time stable solution of TP06,
splitting the set of ordinary differential equations in two

groups: the time course of the gating variables is described
via their analytic, exponential solutions; all the other statevariables are integrated through the Forward Euler methodwith a step time of 50 µs.

2.2.

The model cell and the leak current
compensation

In our dAPC experiment, the HEKA MC10 model cell
(HEKA Elektronik, Germany) provides the external current that affects the final shape of the AP. The MC10model cell consists in a passive linear parallel RC circuit (RM C = 500M Ω and C = 19.8pF ). We assumed
RM C = Rleak , therefore, the model cell contributes to theAP shape providing Ileak . The linear components of themodel cells allowed us to perform the online Ileak compensation through a linear model [7]:Icomp = αcomp gleak (Vm − Eleak )(1)

If inal = Ileak − Icomp(2)−84.2mV , ∆RM P = +6.1mV , ∆RM P % =+7.2%). Vmax monotonically decreases with faster pacing(∆Vmax = −18.3, −26.9 and −30.7% at 0.5, 1 and 2 Hzrespectively.). For a complete overview see Table 1). The
APD undergoes to prolongation both in early and in late repolarization (see Table 2). Surprisingly AP D20 lengthens
between +24 and +26.2% (at 2 and 0.5 Hz respectively). At
first sight, indeed, one could expect a faster repolarization
due to the outward Ileak , instead of a slower one. The underlying mechanisms will be explained in the Discussionsection. Finally, AP D50 and AP D90 show smaller increases (∆AP D50 = 4.4% at 0.5 Hz, ∆AP D90 = 5.5%
at 2 Hz.)During the diastolic interval, the current density provided by the model cell was ≈ 8pA/pF , a value thatwouldn’t allow the TP06 to repolarize. For this reason theinjected current was scaled down of a factor 1/10.

2.3.

Hardware and software

We carried out the dAPC experiments on a EPC10-USB
amplifier controlled by Patchmaster v2x90.2 (HEKA Electronik, Germany). Data analysis and plots (extraction ofthe AP biomarkers AP D20,50,90 , RM P and Vmax ) are
performed with custom code using Matlab 2017b (TheMathworks, Natick MA, USA).
To provide a more in depth analysis of the mechanisms
responsible for the changes of the AP biomarkers, we ran
simulations of the TP06 model using OpenCOR [8], reproducing the effects of the leak current with the same properties of the HEKA MC10 model cell.

3.

Figure 1. A-B) AP and injected current. C) RMP, D)
Upstroke and peak, E) late depolarization of TP06 at 0.5
HzResults

The effects of Ileak on the AP morphology at 0.5, 1 and
2 Hz are shown in Figure 1,2 and 3. If not compensated,
Ileak depolarizes RMP (see panels C, Figure 1,2 and 3),
dampens the peak (panels D) and prolongs the late repolarization phase (panels E). The online compensation ofIleak progressively brings the AP shape back to the open
loop condition, with a good superimposition when a fullcompensation is performed.
The biomarker extraction reveals a RMP depolarization
similar for the three frequencies, with the highest variation at 1 Hz (RM P0% = −78.1mV , RM POpenLoop =Figure 2. A-B) AP and injected current. C) RMP, D)
Upstroke and peak, E) late depolarization of TP06 at 1 Hz

Table 2. AP D20,50,90 and relative changes with respect tothe open loop condition at 0.5, 1 and 2 Hz

0.5 Hz1 HzFigure 3. A-B) AP and injected current. C) RMP, D)
Upstroke and peak, E) late depolarization of TP06 at 2 Hz

2 Hz

0%
25%
50%
75%
100%Open Loop
0%
25%
50%
75%
100%
Open Loop0%
25%
50%
75%
100%
Open Loop

AP D20 ∆AP D20AP D50 ∆AP D50AP D90 ∆AP D90
ms%
ms%
ms%
179.2
26.2
276.2
4.4
309.4
5.4
165.2
16.4
268.3
1.4
300.6
2.4
157.7
11.1
272.7
3.1
303.6
3.5
148.3
4.5
275.3
4.1
305.8
4.2
143.9
1.4
265.2
0.2
293.7
0.1
142.0
264.6
293.4
179.8
23.6
275.0
2.3
309.9
7.2
180.6
24.1
278.0
3.4
310.9
6.8
160.7
10.4
269.1
0.1
301.8
2.8
153.7
5.7
268.0
-0.3
299.3
1.1
148.4
2.0
268.6
-0.1
298.7
-0.3
145.5
268.8
299.1
169.0
24.0
256.3
4.2
292.8
5.5
161.7
18.6
251.3
2.2
286.0
3.1
156.9
15.2
249.5
1.5
282.4
1.8
145.5
6.8
247.9
0.8
280.3
1.0
140.8
3.4
244.6
-0.5
275.9
-0.6
136.3
245.9
277.5

Table 1. Vmax , RMP and relative changes with respect tothe open loop condition at 0.5, 1 and 2 Hz

0.5 Hz1 Hz2 Hz

4.

0%
25%
50%
75%
100%
Open Loop
0%
25%
50%
75%
100%
Open Loop
0%
25%
50%
75%
100%
Open LoopVmax
V/ms248268
291
308
304
304
224
233.
284
291
303
306
187
214
231
260
265
269

∆Vmax%
-18.3
-11.8
-4.3
1.1
0.1
-26.9
-23.9
-7.1
-5.0
-1.1
-30.7
-20.4
-14.1
-3.6
-1.8

RM P
mV
-79.0
-80.6
-81.7
-81.8
-84.9
-84.8
-78.1
-78.5
-81.9
-83.3
-84.5
-84.2
-78.2
-79.7
-80.8
-82.7
-83.8
-84.0

∆RM P%
6.9
4.9
3.6
3.6
-0.1
7.2
6.8
2.8
1.1
-0.3
6.9
5.1
3.8
1.5
0.3

Discussion and Conclusion

With this study, we aimed to quantify the impact of linear Ileak on the AP morphology of an in real time running ventricular computational model (TP06,endo [5]), integrated in a dAPC setup. To do that, we performed theonline Ileak compensation at increasing degrees (from 0 to100%) at 0.5, 1 and 2 Hz, and we compared several APbiomarkers.
The results showed that Ileak depolarizes RMP, decreases
Vmax and prolongs APD both during early and late repolarization in all the explored pacing frequencies. The fullonline compensation brings the biomarkers close to theopen loop condition, where no current is injected.
To investigate the underlying mechanisms, we performed
simulations mimicking the experimental conditions. Us-ing OpenCOR [8], we observed the uncompensated Ileak
scenario (see Figure 4, red lines) with the original model(black lines), and we compared the time course of IN a andall the other membrane currents. The depolarized RMP inactivates IN a ion channels, leading to a lower IN a during
the usptroke (see Figure 4, panel B). The reduced peak ofIN a is therefore responsible for the decreased Vmax .
During the plateau phase, the time course of the net current in the two scenarios is similar (see Figure 4 panel C)
and the repolarization rate is almost the same. This result
is achieved by an interplay between the inward net current
and Ileak : the model reacts to Ileak providing a slightly
stronger inward current during the early stage of the repolarization (see Figure 4, panel D). AP D20 shows an unexpected prolongation due to a smaller AP amplitude (APpeak - RMP), rather than a more intense net inward current.
Ileak slows down the late repolarization due to its inward
contribution, which is responsible for a delayed and lessintense repolarizing current.
The discussed results were obtained using a specific computational model (TP06). As a further step it would be
fruitful to quantify the impact of Ileak on different computational models (e.g. on the O’Hara-Rudy 2011 model [9],one of the latest and most used models). The AP waveform
results from a delicate balance between a specific mix ofcurrents, and the adoption of a different model could leadto different behaviors.
From the experimental perspective, our approach with themodel cell allowed us to assess the effect of Ileak with aRSeal value that is known and remains stable over time.
This is an ideal condition that does not usually occur during an experiment with HEK or CHO cells. Our dAPC
system allows the user to automatically update the current
estimated value of RSeal , which typically decreases overtime. In conclusion, online compensation of Ileak is important in dAPC experiments to avoid inaccurate resultsthat are due to artifacts present in the experimental setup.

Figure 4. Underlying mechanisms responsible for changes
in AP morphology. Time course of A) AP, B) IN a , C)
total net current without IN a , D) total net current (solidlines) and leak current (dashed lines) displayed separately.
Pacing frequency = 1 Hz but the same mechanisms holdtrue at 0.5 and 2 Hz.

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Address for correspondence:
Alan Fabbri
Department of Medical Physiology
University Medical Center Utrecht
Yalelaan 50, 3584CM Utrecht, The Netherlands
afabbri@umcutrecht.nl