The combination of laser poration and microelectrode arrays (MEA) allows action potential-like recordings of cultivated primary and stem cell-derived cardiomyocytes. The waveform shape provides superior insight into test compounds’ mode of action than standard recordings. It links patch-clamp and MEA readout to further optimize cardio safety research in the future.
Life-threatening drug-induced cardiac arrhythmia is often preceded by prolonged cardiac action potentials (AP), commonly accompanied by small proarrhythmic membrane potential fluctuations. The shape and time course of the repolarizing fraction of the AP can be pivotal for the presence or absence of arrhythmia.
Microelectrode arrays (MEA) allow easy access to cardiotoxic compound effects via extracellular field potentials (FP). Although a powerful and well-established tool in research and cardiac safety pharmacology, the FP waveform does not allow to infer the original AP shape due to the extracellular recording principle and the resulting intrinsic alternating current (AC) filtering.
A novel device, described here, can repetitively open the membrane of cardiomyocytes cultivated on top of the MEA electrodes at multiple cultivation time points, using a highly focused nanosecond laser beam. The laser poration results in transforming the electrophysiological signal from FP to intracellular-like APs (laser-induced AP, liAP) and enables the recording of transcellular voltage deflections. This intracellular access allows a better description of the AP shape and a better and more sensitive classification of proarrhythmic potentials than regular MEA recordings. This system is a revolutionary extension to the existing electrophysiological methods, permitting accurate evaluation of cardiotoxic effect with all advantages of MEA-based recordings (easy, acute, and chronic experiments, signal propagation analysis, etc.).
The electrical contribution of a heartbeat results from a complex and precisely timed interplay of many cardiac channels and transporters, as well as the precisely tuned propagation of electrical signals through the myocardium1. Alteration of these closely coordinated mechanisms (e.g., using drugs) can result in severe consequences for the function of the heart (i.e., life-threatening arrhythmia)2,3. Arrhythmias are defined as irregular heartbeats that alter the normal rhythm of the heart, which can have life-threatening consequences. They may be caused either by impaired initiation of a wave of cardiac excitation or by abnormal propagation of cardiac excitation4, which in turn results in a dysfunction of the heart’s pumping mechanism.
Many highly potent drug candidates must be excluded from further investigations during the early drug development phase due to their (pro-) arrhythmic potential2,3. They modulate key cardiac channels (e.g., the human ether-a-go-go-related gene channel [hERG]) that are responsible for normal cardiac action potential formation and termination as well as subsequent signal propagation5.
Pharmaceutical companies routinely use patch-clamp measurements or microelectrode arrays (MEA) to investigate potential cardiotoxic off-target effects induced by drug candidates. Patch-clamp recordings allow to decipher the impact of substances on cardiac ion channels and to analyze the transcellular cardiac action potential with high spatio-temporal resolution6,7. However, disadvantages of this technique include low throughput with manual patch-clamp and limited applicability of automation due to the dependence of this method on cells in suspension. Furthermore, chronic effects cannot be investigated due to the invasiveness of the method. Finally, typically only single cells are studied simultaneously rather than the entire cardiac syncytium, making it impossible to address information about signal propagation.
Voltage-sensitive dyes are valuable for noninvasively investigating cardiac action potentials and drug-induced arrhythmias8. They allow the investigation of both single-cell and syncytium activity. Drawbacks of this method are cytotoxic effects of either the dyes per se or of the reaction product during illumination. They are used for acute experiments and are hardly applicable for long-term studies9,10,11. Voltage-sensitive proteins as alternatives have made significant progress over the last couple of years in terms of usability and sensitivity but require genetic modification of the cells of interest and lack high temporal resolution compared to electrophysiological techniques12.
Information from the most recent CiPA initiative13 states that MEAs are widely used in cardiac safety screenings as an alternative electrophysiological approach as they represent a powerful and well-established tool to investigate cardiac function and safety pharmacology. Cardiomyocytes are cultivated as a syncytium directly on top of the chips, and extracellular field potentials (FPs) are recorded noninvasively via substrate-integrated microelectrodes. This recording principle allows conducting increased throughput screenings over several days, which makes them suitable for pharmaceutical research on chronic effects. The resulting FP waveform is a derivative of the intracellular AP14. Parameters such as beat rate, the amplitude of the initial part of the FP, and FP duration are easily accessible15. Other essential criteria such as the differentiation between prolongation and triangulation of the FP (an important marker of proarrhythmia16,17) are inaccessible due to the AC filtering effect of the technique. Furthermore, detecting other small proarrhythmic events such as early and delayed afterdepolarizations (EAD and DAD, respectively) are often easily overlooked due to their small amplitude.
Here we describe a method for gaining access to the intracellular membrane potential by opening the membrane of cardiomyocytes. The IntraCell device (hereafter referred to as intracellular recording device) allows repeated membrane openings of cardiomyocytes cultivated on top of the MEA electrodes using a highly focused nanosecond laser beam via a specific physical phenomenon (surface plasmon resonance)18. As a result, the recording transitions from a regular FP into an intracellular-like AP (laser-induced AP, liAP). The protocol shows how this allows gaining access to kinetic aspects of the waveform that cannot easily be captured by analyzing FPs. This method represents a bridge between traditional intracellular patch-clamp and MEA recordings. The technology is therefore a powerful extension of current cardiac safety assessment methods.
1. Induced pluripotent stem cell-derived cardiomyocytes preparation
NOTE: iCell cardiomyocytes2 (referred to as induced pluripotent stem cell [iPSCs]-derived cardiomyocytes) were prepared according to the protocol provided by the supplier. The protocol will be briefly summarized in the following section.
2. MEA recordings
NOTE: The device used to transform the FP signal to liAP consists of an upright microscope and a 1064 nm laser.
3. Laser-induced cell poration
4. Drug handling and application
5. Data export
6. Data handling and statistical analysis
The recording system used to record electrical activity from cultivated cardiomyocytes consisted of a standard MEA system equipped with a heater and a chamber for carbogen attached to a computer. The system was set on top of the intracellular recording device, which in turn was mounted on top of a small anti-vibration unit (Figure 1A–B).
iPSC-derived cardiomyocytes2 started beating spontaneously within 2-3 days after thawing (days in vitro, DIV) and were visible under a microscope. From DIV 4 onward, the beating frequency became regular, and extracellular field potentials (FP) with peak-to-peak amplitudes of the depolarizing component between 1 to 5 mV could be detected on most of the electrodes within the respective wells of the MEA chips. The electrical activity could be detected in more than 95% of the wells under investigation. From DIV 7 onward, the probability of cell detachment increased, making further use of these wells impossible.
The software to control the laser-induced membrane opening allows adjusting both the power and process time of the laser that mediates the opening of the cell membrane only on the electrode under investigation (Figure 1C), whereas other electrodes in the respective well are unaffected. While too conservative settings did not change the waveform of the FP, too high settings resulted in the putative injury of the cardiomyocytes, indicated by vigorous but transient beating or loss of the signal. When adjusted to a setting of 40% power and 25% process time well-tolerated by the cells, triggering of the laser pulse resulted in multiple changes to the recorded waveform (see Figure 1D for an exemplary recording). Under these conditions, no alteration of the electrode material was macroscopically observed. The recorded signal amplitude massively increased by 4.1 ± 0.41 (n = 20, range 1.34-8.83) times, analyzed from a randomly picked subset of recordings, resulting in amplitudes between 7 and 22 mV. Furthermore, the waveform transformed from a standard FP shape with rapid, biphasic, and transient voltage deflection at the beginning, followed by a plateau phase back at baseline and a small deflection indicating the end of the FP to a shape that was closer to an intracellularly recorded AP with a rapid rise, extended depolarized plateau phase and a repolarization phase with an undershoot below the baseline (Figure 1E). We defined these voltage deflections as laser-induced AP (liAP). In most cases, the transition was transient and at least partially inverted within 5 min. Signal propagation within the cardiac syncytium remained unaltered after liAP induction (Figure 2), indicating that the remaining syncytium was not affected by potential damage from the laser pulse.
Similarities to intracellularly recorded APs allowed for extracting parameters of the liAP that are not accessible to FPs (for exemplary parameters see e.g., Figure 3A), most prominently the measurement of the duration of the liAP at specific time points (e.g., at 20%, 50%, and 90%) (Figure 3B), analogous to APD20/50/90 commonly used for the description of APs.
We next tested the response of the laser-opened cardiomyocytes to commonly used cardioactive pharmacological tool compounds. An exemplary protocol design can be found in Figure 3C. Since the transformation of the liAP did not always persist throughout the entire experiment, the compound application was performed as a single-concentration-per-well rather than in a cumulative manner to reduce the total recording time. Nevertheless, it was necessary to either reopen the cells or open another electrode area prior to applying the test compound.
The addition of the specific L-type Ca2+ channel blocker, Nifedipine23,24, reduced the plateau phase of the liAP in a concentration-dependent manner and thereby shortened the entire liAP (Figure 4A,B). This shortening was comparable to the analysis obtained from FPs of cardiomyocytes from unmanipulated electrodes (Figure 4C), indicating that this recording method did not have adverse effects compared to classical FP recordings.
E4031 inhibits the repolarization of relevant Kv1.11 (hERG) potassium channel25 and leads to arrhythmic behavior of cardiomyocytes at increased concentrations. Similar to the analysis obtained from FP recordings, E4031 increased the liAP duration in a concentration-dependent manner (Figure 5). Additionally, at concentrations of 0.01 µM and higher, small positive voltage deflections at the end of the liAP were visible. These deflections became more prominent with higher concentrations, indicating a transient new depolarization, unlike in the FPs, where these deflections were virtually invisible (see Figure 5B–C, upper (FP) vs lower (liAP) traces). This behavior is known as early afterdepolarization (EAD). At the highest concentration of 0.1 µM, these EADs escalated over time into ectopic beats, which are premature action potentials (Figure 5C). Both EAD and ectopic beats are key indicators of proarrhythmic activity. At the end of the example shown in Figure 5D, the electrical activity resulted in arrhythmic beating. Also, concentration-response-relationships displayed between FP and liAP recordings were matching (Figure 5E). However, there is more considerable variability in the FP data resulting from the weak repolarization component of the FPs at higher concentrations of the test compound. It seems to be the characteristic nature of iPSC-derived cardiomyocytes2 to tend to generate unphysiologically long APs under control conditions also (AP durations > 700 ms). The MEA system applied an intrinsic 0.1 Hz AC filtering, which in turn resulted in a partially filtered shape of the liAP, although without occluding the qualitative information about the onset and termination of the underlying AP.
It turned out that the initial occurrence of proarrhythmic voltage deflections could be detected at lower concentrations in liAPs in comparison to FP recordings. Shown in Figure 6 is the recording of electrical activity during the application of Dofetilide at a concentration of 3 µM. The recording was obtained in the same well. Although both FP and liAP displayed durations of approximately 2 s, the FP waveform was unobtrusive, presenting regular repolarizing deflections. At the same time, at the end of liAPs, EADs at different magnitudes became visible. This increase in relevant safety-pharmacological sensitivity further supports the finding that liAPs induced by surface plasmon resonance allow improved qualification of the repolarizing phase and thereby help to learn more about the mode of action of the test compounds under investigation.
Figure 1: The intracellular recording setup and exemplary recordings. (A) Setup overview. (B) Top view of the recording system with open MEA recording amplifier. (C) Initialization software with the virtual MEA map on the right-hand side. 1: anti-vibration table, 2: intracellular recording system, 3: laser protection lid, 4: humidified carbogen chamber, 5: MEA heating system, 6: MEA interface board, 7: 1-well MEA chip inside the recording amplifier. (D) Recording examples from one electrode before and after induction of liAPs. Top: recording of approximately 6 min. Dotted lines mark the expanded areas shown at the bottom. (E) Magnified FP (top) and liAP (bottom). Please click here to view a larger version of this figure.
Figure 2: Signal propagation pattern remains conserved after liAP induction. False-color coding of the signal propagation of the excitation wave within the syncytium. Blue indicates early (starting at -4 ms); red indicates late time points (+3 ms) from the signal obtained at reference electrode E54 as indicated by the color bar. The signal travels from top right to bottom left of the electrode array. (A) Before liAP induction, (B) 1 min after liAP induction. (C) 4 min after liAP induction. The Flash symbol indicates the laser induction point at electrode 64. Note that no difference in the overall propagation direction is visible. Black rectangles indicate invalid data. Please click here to view a larger version of this figure.
Figure 3: FP/liAP parameter definition and recording protocol. (A) Parameters that can be extracted from the classical FP. (B) Additional parameters that can be obtained from liAPs. (C) Timeline of drug measurement. From left to right: control recording for 60 s, induction of liAP, recording of liAP for 60 s, drug application, wash-in time 300 s, re-induction of liAP, recording of liAP for 60 s. Please click here to view a larger version of this figure.
Figure 4: Nifedipine shortens the cardiac liAP in a concentration-dependent manner. (A) Top: FP traces at control (blue) and in the presence of 0.3 µM Nifedipine (red). Traces are displayed with a y-axis offset for better visualization. Bottom: liAP traces from the same MEA recording, resulting in a shortening of the liAP combined with an increase in the beat rate. (B) Superposition of single liAPs during control (blue) and application of different concentrations of Nifedipine (red). a: 0.01 µM, b: 0.1 µM, and c: 0.3 µM. Note the shortening of the liAP duration with increasing concentrations. (C) The concentration-response relationship of signal width obtained from FP (black) and liAP (red) recordings. Data is from n = 3 experiments and normalized to control. Error bars indicate the standard error of the mean. Please click here to view a larger version of this figure.
Figure 5: E4031 induces (pro-) arrhythmic behavior. (A) FP (top) and liAP (bottom) at control conditions. (B–C) FPs and liAPs were recorded at different time points after applying E4031 (0.1 µM). After 80 s, the first EADs are visible at the end of the liAP (B; marked by a red arrow). EADs convert into ectopic beats after 320 s in the presence of the test compound (C). (D) After 530 s, the cardiac syncytium enters a tachycardic state. Trace depicted from liAP recording. (E) Concentration-response relationship of FP (black) and liAP (red) width. Data from n = 4 experiments, normalized to control. Error bars indicate the standard error of the mean. Please click here to view a larger version of this figure.
Figure 6: Detection of proarrhythmic events is more sensitive in liAPs than FPs. (A) FP recording and (B) liAP recording within the same well during application of 3 µM Dofetilide. While at the end of some liAPs, EADs are detectable, they remain undiscoverable in FP recordings. Please click here to view a larger version of this figure.
This innovative method demonstrates a new way to investigate in vitro the pharmacological modulation of the cardiac action potential during the application of cardioactive pharmacological tool compounds.
Classical MEA recordings allow FP recordings, which are the derivative of the cardiac AP14. This indirect recording convolves the time course of the de- and repolarization and thereby eliminates essential characteristics of the AP. Furthermore, although the transcellular voltage change of an AP typically reaches values of approximately 100 mV, the overall FP amplitude remains comparably low, with peak amplitudes between several 100 µV and low single-digit mV values. Due to the recording principle, the repolarizing phase is small; in many cases, it is merely detectable and often of unclear shape, making it difficult to define the end of the FP. The opening of the cell membrane allows us to gain access to the intracellular voltage, thereby uncovering the time course of the cardiac AP. There are multiple advantages of this recording method in comparison to FP recordings. Firstly, the signal amplitude is more prominent, providing a superior signal-to-noise ratio. Secondly, the waveform results in better detection of the repolarization. Thirdly, the shape of the repolarization phase contributes insights into the mode of action of the test compound, provided by the steepness of the signal relaxation. And lastly, this method offers an improved sensitivity to detect critical adverse drug effects, demonstrated by the recording example displayed in Figure 6 for the occurrence of EADs in the liAP but not in the FP.
So far, there are two ways of gaining access to the intracellular AP. The first one is achieved by electroporation26,27. Here, short and strong voltage pulses applied via the recording electrodes can open the cell membrane28. The second possibility is the membrane opening via a laser pulse, making use of a physical phenomenon named surface plasmon resonance, as demonstrated here. One of the advantages compared to electroporation is the increased likelihood of consecutive openings. Due to the highly focused laser spot (1-3 µm) this effect is very locally limited to the electrode of interest. Interestingly, the initiation of the liAP did not alter the signal propagation of the cultivated syncytium. This indicates that, although the cell integrity is damaged, the cardiomyocytes do not seem to depolarize via the hole in the membrane.
There are limitations to this method. Like with electroporation, the membrane opening does, in most cases, not last over the entire experimental course. The minimal power and duration settings of the laser pulse required for stable opening of the specific cell type of interest need to be defined independently prior to the experiments. We found (not shown) that the parameters drastically vary between different cell types (in our case, several hiPS-derived and primary cardiomyocytes). This avoids unnecessary stress on the cells during the compound test experiment and results in more reliable and reproducible data. It is of critical importance to adjust the z-axis to provide a clear focus on the cells and the electrodes. An unfocused camera picture yields in a laser spot located at a suboptimal level, potentially resulting in the inability to open the cell membrane. Even with best-adjusted parameters, the liAP effect is transient, and the amplitude reduces over time. Furthermore, the access to the intracellular space of the cells varies between liAP inductions, both within consecutive openings at the same electrode and between electrodes. This results in high variability of the liAP amplitude. The reason is not yet fully understood. Possible explanations include mechanical issues such as a drift of the laser focus or different subcellular localization of the membrane opening. This makes the analysis of amplitude effects of test compounds complicated at this point in time. Also, recording electrical activity by a MEA system requires high pass filtering to compensate for unavoidable baseline drift. Although in the system used here, this filtering was set to 0.1 Hz (the lowest filter setting available for this system), filtering effects during the plateau phase were still visible, resulting in a slow trend of the voltage deflection toward the baseline during the plateau phase of the cardiac AP. This is especially problematic with extensively long underlying APs like the iPSC-derived iCell cardiomyocytes2 used here, which already generate AP >700 ms under control conditions. The use of systems with lower filtering may better conserve the shape of the AP and allow even better access to the time course of the repolarization phase.
The authors have nothing to disclose.
The authors would like to thank Foresee Biosystems for lending the IntraCell system during the studies. They would also like to thank Hae In Chang for technical assistance. This work has received funding from the European Union's Horizon 2020's research and innovation program under the grant agreement no. 964518 (ToxFree), from the EU Horizon Europe European Innovation Council Programme, project SiMulTox (grant agreement No 101057769), and from the State Ministry of Baden-Wuerttemberg for Economic Affairs, Labour and Tourism.
1 well MEA chip | Multi Channel Systems MCS GmbH | 890301 | |
6 well MEA chip | Multi Channel Systems MCS GmbH | 7600069 | |
DMSO | Merck KGaA | 20-139 Sigma-Aldrich |
solvent for drugs |
Dofetilide | ALOMONE LABS ISRAEL HEADQUARTERS |
D-100 | Drug-Measurement |
dPBS | Fisher Scientific GmbH | 12037539 | Coating |
E4031 | ALOMONE LABS ISRAEL HEADQUARTERS |
E-500 | Drug-Measurement |
Falcon | Fisher Scientific GmbH | 10788561 | |
FB Alps version 0.5.005 | Foresee Biosystems | ||
Fibronectin | Merck KGaA | 11051407001 | Coating |
iCell cardiomyocytes | FUJIFILM Cellular Dynamics, Inc. (FCDI) |
C1016 | |
IntraCell | Foresee Biosystems | ||
IntraCell | Foresee Biosystems | ||
Isopropanol | Carl Roth GmbH + Co. KG | CN09.1 | For cleaning of MEA contact pads |
Maintenance Medium | FUJIFILM Cellular Dynamics, Inc. (FCDI) |
#M1003 | For cell-culture |
MC_Data Tool | Multi Channel Systems MCS GmbH | Data export | |
MC_Rack | Multi Channel Systems MCS GmbH | MEA recording | |
MEA 2100 – 2×60 – system | Multi Channel Systems MCS GmbH | 890485 | For MEA-recordings |
Nifedipine | Merck KGaA | N7634 Sigma-Aldrich |
Drug-Measurement |
Plating Medium | FUJIFILM Cellular Dynamics, Inc. (FCDI) |
M1001 | For cell-culture |
Tergazyme | VWR International, LLC | 1304-1 | cleaning of MEAs |