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Am J Physiol Heart Circ Physiol
Am J Physiol Heart Circ Physiol
AJPHEART
American Journal of Physiology - Heart and Circulatory Physiology
0363-6135
1522-1539
American Physiological Society Rockville, MD

38488520
H-00063-2024
H-00063-2024
10.1152/ajpheart.00063.2024
Short Report
Cardiac Excitation and ContractionSingle-cell ionic current phenotyping explains stem cell-derived cardiomyocyte action potential morphology
SINGLE-CELL IONIC CURRENT PHENOTYPING OF iPSC-CMs
https://orcid.org/0000-0002-5200-6573
Clark Alexander P. 1
https://orcid.org/0000-0003-1085-6788
Wei Siyu 2
https://orcid.org/0000-0002-4810-6125
Fullerton Kristin 3
https://orcid.org/0000-0003-2298-6918
Krogh-Madsen Trine 3 4
https://orcid.org/0000-0003-0852-2922
Christini David J. 1 2
1Department of Biomedical Engineering, Cornell University, Ithaca, New York, United States
2Department of Physiology and Pharmacology, SUNY Downstate Health Sciences University, Brooklyn, New York, United States
3Department of Physiology and Biophysics, Weill Cornell Medicine, New York, New York, United States
4Institute for Computational Biomedicine, Weill Cornell Medicine, New York, New York, United States
Correspondence: D. J. Christini (david.christini@downstate.edu).
1 5 2024
15 3 2024
15 3 2024
326 5 H1146H1154
1 2 2024
26 2 2024
1 3 2024
Copyright © 2024 The Authors.
2024
The Authors
https://creativecommons.org/licenses/by/4.0/ Licensed under Creative Commons Attribution CC-BY 4.0. Published by the American Physiological Society.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are a promising tool to study arrhythmia-related factors, but the variability of action potential (AP) recordings from these cells limits their use as an in vitro model. In this study, we use recently published brief (10 s), dynamic voltage-clamp (VC) data to provide mechanistic insights into the ionic currents contributing to AP heterogeneity; we call this approach rapid ionic current phenotyping (RICP). Features of this VC data were correlated to AP recordings from the same cells, and we used computational models to generate mechanistic insights into cellular heterogeneity. This analysis uncovered several interesting links between AP morphology and ionic current density: both L-type calcium and sodium currents contribute to upstroke velocity, rapid delayed rectifier K+ current is the main determinant of the maximal diastolic potential, and an outward current in the activation range of slow delayed rectifier K+ is the main determinant of AP duration. Our analysis also identified an outward current in several cells at 6 mV that is not reproduced by iPSC-CM mathematical models but contributes to determining AP duration. RICP can be used to explain how cell-to-cell variability in ionic currents gives rise to AP heterogeneity. Because of its brief duration (10 s) and ease of data interpretation, we recommend the use of RICP for single-cell patch-clamp experiments that include the acquisition of APs.

NEW & NOTEWORTHY We present rapid ionic current phenotyping (RICP), a current quantification approach based on an optimized voltage-clamp protocol. The method captures a rich snapshot of the ionic current dynamics, providing quantitative information about multiple currents (e.g., ICa,L, IKr) in the same cell. The protocol helped to identify key ionic determinants of cellular action potential heterogeneity in iPSC-CMs. This included unexpected results, such as the critical role of IKr in establishing the maximum diastolic potential.

arrhythmias
computer simulation
induced pluripotent stem cells
iPSC-CMs
patch clamp
HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI) 10.13039/100000050 F31HL154655 Alexander P. ClarkHHS | NIH | National Heart, Lung, and Blood Institute (NHLBI) 10.13039/100000050 U01HL136297 David J. Christini
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pmcINTRODUCTION

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are a promising model to study congenital and acquired cardiac arrhythmias. The depth of insights from electrophysiological studies with these cells is limited by their immature phenotype and cell-to-cell variability (1). There is substantial laboratory-to-laboratory heterogeneity in the action potential (AP) morphology of iPSC-CMs, and even genetically identical iPSC-CMs derived from the same donor display significant variability (2, 3). Such shortcomings make it difficult to glean meaningful physiological information from patient-specific iPSC-CMs (4) and have led to inconsistent results in multisite drug cardiotoxicity screening studies (5). Developing an understanding of iPSC-CM heterogeneity is an essential step to improve the utility of these cells as a tool for use in precision medicine.

Here, we present a new method called rapid ionic current phenotyping (RICP) that we use to study variations in the ionic currents contributing to iPSC-CM AP heterogeneity. In this study, we use recently published iPSC-CM patch-clamp data from our laboratory (6), including short (10 s) voltage-clamp (VC) recordings that provide insight into the presence and relative size of several key cardiac ionic currents. The novelty of this study is in the use of such a brief VC protocol to draw the following set of conclusions about the iPSC-CMs used here: •  L-type calcium current (ICa,L) drives upstroke in iPSC-CMs with a depolarized AP morphology.

•  Rapid delayed rectifier K+ current (IKr) plays a role in establishing the maximal diastolic potential.

•  Seal-leak current contaminates VC recordings and contributes to a depolarized maximal diastolic potential.

•  Large positive currents present at potentials >40 mV correlate with a shortened AP duration.

•  Several cells contain a strong outward current at 6 mV that is not present in computational AP models and correlates with AP duration.

METHODS

iPSC-CM Cell Culture and Electrophysiological Setup

The in vitro data were previously published (6).

Frozen iPSC-CMs were thawed from five different vials that were purchased from the Stanford Cardiovascular Institute Biobank. Before patch-clamp recording, each cell was plated on a coverslip and had been independently cultured for 3–13 days in a 24-well plate. These cells were derived from an African-American female donor in a process approved by Stanford University Human Subjects Research Institutional Review Board.

Cells were prepared for electrophysiological experiments following the steps described in Ref. 6. Briefly, cells were thawed and cultured as a monolayer in one well of a six-well plate precoated with 1% Matrigel. Cells were cultured with RPMI media (Fisher/Corning 10–040-CM) containing 5% FBS and 2% B27 and kept in an incubator at 37°C, 5% CO2, and 85% humidity. After 48 h, cells were lifted with 1-mL Accutase, diluted to 100,000 cells/mL, and replated on 124-sterile, 8-mm coverslips precoated with 1% Matrigel. Cells were cultured with RPMI media that was swapped every 48 h. Cells were patched between days 5 and 15 after thaw.

Voltage-clamp and current-clamp recordings were acquired from 40 cells using the perforated patch technique and an amplifier equipped with a voltage follower circuit (Model 2400; A-M Systems, Sequim, WA). We excluded one cell from the analyses in this study because it had spontaneous alternans with inconsistent AP features. All cells had a prerupture seal of >300 MΩ.

Voltage-Clamp Protocol

We have previously developed a voltage-clamp protocol consisting of multiple short segments, each designed to isolate one key ionic current (6). The protocol was designed using optimization techniques and a mathematical model of iPSC-CMs (7) to maximize, one at a time, the contribution to total current by each of seven key currents: IKr, ICa,L, sodium current (INa), transient outward K+ current (Ito), inward rectifier K+ current (IK1), funny current (If), and slow delayed rectifier K+ current (IKs).

During our analysis, we found that the current measured 100 ms after a depolarizing step to 6 mV (I6mV) was substantially different in many cells from that predicted by the mathematical model and we therefore included I6mV as an 8th current measure. For each current-isolating segment, we quantified the recorded total current Iout using either the minimum or the average over a 2 ms span centered at the following values: I6mV (600 ms, average), IKr (1,262 ms, average), ICa,L (1,986 ms, minimum), INa (2,760 ms, minimum), Ito (3,641 ms, average), IK1 (4,300 ms, average), If (5,840 ms, average), and IKs (9,040 ms, average).

AP Feature Calculations

The transmembrane potential of each cell was recorded for 10 s. Of the 39 cells, 12 were not spontaneously beating. We computed a minimal potential (MP) for these nonspontaneous cells.

For cells that were spontaneously beating, we computed their MP (in this case, the minimum voltage during the AP), action potential duration at 90% repolarization (APD90), cycle length (CL), and maximal upstroke velocity (dV/dtmax). The average of each feature was calculated for all cells that produced more than one AP during the 10-s recording.

iPSC-CM Mathematical Models

For comparison and to guide the analysis of our experimental data, we used two different mathematical models of iPSC-CM electrophysiology: the Paci et al. model (8) and the Kernik et al. model (7). We set the cell capacitance (Cm) of these models to 45 pF, which is centrally located in the range (18–98 pF) of the capacitances for cells used in this study. To avoid long transients and to better simulate our perforated patch experimental setup, we fixed intracellular sodium and potassium concentrations ([Na+]i and [K+]i) to their baseline steady-state values (taken after 1,000 s of spontaneous or paced current-clamp simulation). Because the leak through the imperfect pipette-membrane seal during single-cell patch-clamp experiments can substantially impact the electrophysiological recordings in these cells, we included a linear leak current (Ileak) in the mathematical models (3). We used a baseline value of 2 GΩ for the seal resistance.

In addition to the seal-leak current, for voltage-clamp simulations, we included explicit modeling of the following experimental artifacts: liquid junction potential offset (−2.8 mV), access resistance (20 MΩ), and series resistance (Rs) compensation (70%), including supercharging (9, 10).

Population of Models and Sensitivity Analysis

A population of 500 individuals with unique parameters sets was generated using both the Paci and Kernik models with experimental artifact equations by randomly sampling conductances of INa, ICa,L, IKr, IKs, Ito, IK1, If, Ileak, sodium-calcium exchange current (INa,Ca), sodium potassium pump current (INa,K), sodium background current (Ib,Na), calcium background current (Ib,Ca), as well as Cm and Rs from a log uniform distribution between 0.25 and four times their baseline values. Of the 500 Paci models, 18 were excluded because of numerical integration issues. The Kernik and Paci populations produced 150 and 194 spontaneously beating individuals, respectively. All individuals (whether spontaneously beating or not) were used to calculate the MP. Only spontaneously beating individuals were used to calculate dV/dtmax, APD90, and CL. We used a Spearman correlation to determine the sensitivity of the current-isolating time points to these parameters.

Linear Regression

A linear least-squares regression was used to compare ionic currents and AP features for both the in vitro and the in silico data. A Spearman correlation coefficient and P value were calculated for these data.

Software and Simulations

Simulations were performed in Myokit v1.33.7 (11). Additional analysis was done in Python using NumPy v1.21.6 and SciPy v1.7.3 (12). All data, code, and models can be accessed from GitHub (https://github.com/Christini-Lab/ap-vc-correlations.git).

RESULTS

iPSC-CMs Are Heterogeneous

We used data from 39 perforated patch-clamped cells (6): 27 were spontaneously beating, and 12 were quiescent and depolarized (Fig. 1, B and C). There is substantial cell-to-cell heterogeneity. Reporting the variation as the standard deviation (SD), we calculate a minimum potential (MP) of −52 ± 10 mV, action potential duration at 90% repolarization (APD90) of 127 ± 70 ms, and maximum upstroke velocity (dV/dtmax) of 10.5 ± 6.6 V/s.

Figure 1. Voltage-clamp (VC) variations and membrane potential in 39 cells. A: optimized voltage-clamp protocol (top) with lines overlaid that indicate time points designed to isolate each current. Responses to the voltage-clamp protocol (bottom) for spontaneous (black), quiescent (blue), and the outlier (pink). B: action potentials (APs) from spontaneously beating cells (n = 27). Pink AP is an outlier with an AP duration at 90% repolarization (APD90) >2× longer than the rest of the population. C: voltage recordings from quiescent and depolarized cells (n = 12).

Within the subset of spontaneously beating cells, there is additional heterogeneity in that some have a predictable and consistent cycle length (CL), while the CL of others is highly variable (Supplemental Fig. S1; all Supplemental Figures are available at https://doi.org/10.6084/m9.figshare.25289914.v3). This level of heterogeneity is consistent with variations observed in other iPSC-CM electrophysiology studies (18, 22).

Rapid Ionic Current Phenotyping Provides Insight into AP Outliers

As detailed in the methods, we recently published a voltage-clamp protocol (6) that was designed to maximize the isolation for each of the following seven key ionic currents: IKr, ICa,L, INa, Ito, IK1, If, and IKs. The protocol works by stepping to voltages designed to maximize the contribution of each current at different time points. The target current (e.g., IKr) during an isolating segment, however, can be contaminated with other ionic (e.g., INa,K) or artifact (Ileak) currents. Yet, we show here and in our previous study (6) that we can draw conclusions about target currents based on the information gleaned from these isolating segments. Because of its short duration (10 s) and design to target multiple currents, we are calling this approach rapid ionic current phenotyping (RICP).

Figure 1A shows the voltage-clamp protocol (top) and heterogeneity in ionic current responses (bottom) from the 39 cells included in this study. On the voltage-clamp protocol, we have overlaid dashed lines to highlight current-isolating time points that we use to study ionic current dynamics. In addition to the seven designed current-isolating segments, we included another time point measure (I6mV, the current recorded 100 ms after a depolarizing step to 6 mV), as we found that it provides insight into dynamics that are not present during other portions of the voltage-clamp protocol.

Spontaneously beating cells are plotted in black and quiescent cells are in blue (Fig. 1, A–C). The pink spontaneous cell is an outlier, with an APD90 value greater than two times longer than the rest of the population. When compared with the rest of the population, the outlier cell appears to have the smallest total current during the regions of the protocol designed to isolate IK1, If, and IKs (Supplemental Fig. S2).

RICP Identifies Ica,L as Driver of Upstroke in Depolarized Cells

Figure 2A displays the relationship between the total current during each current-isolating segment and the dV/dtmax for all cells. None of the eight current-isolating segments correlate with dV/dtmax. Figure 2A shows the likely presence of INa in many of these cells: we draw this conclusion because INa is the only current expected to generate a total recorded current (Iout) of < −40 A/F within 2 ms after the INa voltage step. However, the upstroke velocity is small in most cells and does not correlate with the current measured during the INa-isolating segment. The small upstroke velocities and the lack of correlation with INa was an unexpected finding, given that INa is present in many of these cells.

Figure 2. ICa,L helps drive upstroke in depolarized human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). A: there is no correlation between Iout from the eight current-isolating segments and maximal upstroke velocity (dV/dtmax). B: dV/dtmax decreases as minimal potential (MP) depolarizes. Red boxes denote the two cells with the most hyperpolarized MP and largest dV/dtmax. C: two highlighted cells from B share few action potential (AP) commonalities with one another other than their relatively hyperpolarized MP and large dV/dtmax. D: a trend emerges between Iout during the ICa,L-isolating segment and dV/dtmax when the two hyperpolarized cells are removed from the analysis. ICa,L, L-type calcium current.

Although there is no significant relationship with the VC segments, dV/dtmax does correlate with MP (Fig. 2B); dV/dtmax increases as MP becomes more hyperpolarized. The two cells with MP below −70 mV (denoted with red squares) stand out as having much larger upstroke velocities than the rest of the population. These two cells likely repolarize enough to make some sodium channels available for an INa-driven upstroke. Other than having relatively hyperpolarized MP values, these two cells produce APs with few morphological similarities (Fig. 2C).

Interestingly, when we exclude these cells from the regression analysis, and only consider iPSC-CMs with MP > −70 mV, a significant relationship emerges between the ICa,L-isolating segment and dV/dtmax (Fig. 2D). This indicates that ICa,L may be, at least partly, responsible for the upstroke in cells with MP > −70 mV.

RICP Identifies IKr-Isolating Segment as Predictor of MP

Four current-isolating segments (IKr, Ito, IK1, and If) of the VC protocol correlate with MP (Fig. 3A), with the IKr-isolating segment being the strongest correlate (R = 0.72). We selected cells at the two MP extremes to illustrate the relationship between the IKr-isolating segment and MP (Fig. 3B): cell 1 (blue) is the most hyperpolarized in the population (MP = −73 mV and APD90 = 81 ms), and cell 2 is the most depolarized (quiescent with MP of −27 mV).

Figure 3. Correlations between ionic current segments and minimal potential (MP). A: Iout from the IKr, Ito, IK1, If, and IKs segments correlate significantly (P < 0.05) with MP. B: current-clamp recordings from all cells. The human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with the most hyperpolarized (blue, cell 1) and depolarized (green, cell 2) MP values are compared. C: traces from the IKr-isolating segment of the voltage-clamp (VC) protocol for all cells. VC protocol (top) includes a 750-ms prestep at 6 mV, followed by a 7-ms step at −41 mV, and then a step to 9 mV. VC protocol was designed to isolate IKr at 1,262 ms (red dashed line). Cell 1 (blue) and cell 2 (green) are highlighted. If, funny current; IKr, rapid delayed rectifier K+ current; IK1, inward rectifier K+ current; IKs, slow delayed rectifier K+ current; Iout, total recorded current; Ito, transient outward K+ current.

A plot of the IKr-isolating section, which was previously designed and subsequently validated with drug studies (see Fig. 5 from Ref. 6 for details), shows the voltage command (top) and current responses (bottom) for all cells in the population (Fig. 3C). During this section of the protocol, cells were clamped to 6 mV for 750 ms, then stepped to −41 mV for 7 ms, and then back up to 9 mV. At the time point designed to isolate IKr (red dashed line), cell 1 conducts a small positive total current (0.3 A/F) that increases in size, which is consistent with IKr recovering from rectification. In contrast, cell 2 conducts a net negative current (−2.5 A/F) and does not show IKr recovery characteristics, suggesting that this cell has much less IKr. These cells are examples of the population-level correlation seen between the IKr-isolating segment and MP. They indicate that IKr, and other currents that may be present during the step to −40 mV, likely play a critical role in establishing MP in these cells.

We used an iPSC-CM mathematical model with a linear seal-leak current (see methods) to further study the potential role of IKr in establishing an MP. Supplemental Fig. S3 shows the effect of scaling IKr conductance (gKr) on the MP for a model with a 2 GΩ seal resistance. While holding all other parameters constant, reducing gKr by >70% of baseline results in substantial depolarization. The 10–90% range of our experimental data corresponds to a roughly 70–85% reduction in gKr (Supplemental Fig. S3B). These model findings are consistent with the correlations seen between the experimental IKr-isolating Iout measurements and MP.

Seal-Leak Current Likely Contributes to the Depolarized MP

In addition to the IKr-isolating segment, the Ito, IK1, If, and IKs-isolating segments also weakly correlate with MP (Fig. 3A). Some of these correlations are unexpected and counterintuitive as, e.g., Ito is typically inactivated during phases 3 and 4 of the AP. We hypothesized that these correlations may be influenced by a leak artifact current caused by an imperfect seal between the pipette tip and cell membrane; we recently demonstrated that this leak current can substantially depolarize the MP of iPSC-CMs (3).

To investigate the role of Ileak on these segments, we developed a population of computational models that include patch-clamp experimental artifact equations (Supplemental Fig. S4). In this in silico population, the IKr segment is also the main correlate with MP, and Ito, IKs, and If are more weakly correlated (Supplemental Fig. S6).

A sensitivity analysis of the population of models shows that the segments weakly correlating with MP in the experiments (i.e., the Ito, IK1, If, and IKs segments) are all sensitive to the conductance of Ileak (i.e., gleak, Supplemental Fig. S4C). Ileak is modeled as a linear current with a reversal potential of zero, and so it will increase when cells are clamped to voltages that are far from 0 mV. Seeing as IKs and Ito are elicited by stepping to large positive voltages and IK1 and If to large negative voltages, this is consistent with these segments being contaminated by Ileak. Increased gleak increases the total (net outward) currents computed at positive voltages (e.g., IKs and Ito segments) and depolarizes the MP, consistent with the positive correlation seen between MP and the IKs and Ito segments in our experiments. Similarly, the negative correlations between MP and IK1 and If in our cells is consistent with the in silico results where increased gleak during these hyperpolarized segments in the models contribute to the large (net inward) current and depolarized MP values.

RICP Identifies Strong Outward Currents as Drivers of APD90

Four segments (I6mV, IK1, If, and IKs) of the VC protocol correlate with APD90 (Fig. 4A). Three of the segments (IK1, If, and IKs) were designed to isolate potassium-conducting currents, each of which is clamped to voltages far from 0 mV. The I6mV segment was added to quantify observed current dynamics different from those produced by the mathematical models and which we hypothesized could be explanatory of AP morphological differences.

Figure 4. Correlations between ionic currents and action potential (AP) duration at 90% repolarization (APD90). A: Iout from the I6mV, IK1, If, and IKs segments correlate significantly (P < 0.05) with APD90. B: cells that generated the shortest (short) and second longest (long) APs were investigated. Current responses from cells during I6mV (C) and IKs (D) step. If, funny current; IK1, inward rectifier K+ current; IKs, slow delayed rectifier K+ current; Iout, total recorded current.

To illustrate the relationship between currents and APD90, we investigated the current responses from two different cells (Fig. 4B): one with the shortest APD90 (cell 3) and one with the second longest APD90 (cell 4).

The strong correlation between the IKs-isolating segment and APD90 indicates that the net outward current at large positive voltages affects APD90. Cell 3 has a slightly larger outward current during this IKs-isolating segment when compared with cell 4 (Fig. 4D).

If and IK1-isolating segments correlate less strongly with APD90 and in the opposite direction than what would be expected. Although both currents are expected to influence AP duration, their current-isolating segments are likely contaminated with Ileak (Supplemental Fig. S4) that is contributing in the opposite direction (3).

The outward current present during the I6mV segment is much larger in cell 1 than any other in the population and is very likely driving the rapid repolarization shown in this cell. In contrast, cell 2 has a nearly balanced net current during the I6mV segment. Comparing our population of models to experimental current responses shows that the experimental responses for many of the cells (11/39) have a strong repolarizing ionic current at 6 mV that is not represented in the iPSC-CM electrophysiological model (Supplemental Fig. S5).

DISCUSSION

Here, we propose RICP as a novel approach to study the ionic current underpinnings of AP heterogeneity. By analyzing data from a brief, optimized 10-s VC protocol, we generate the following insights about the iPSC CMs used in this study: •  ICa,L is partly responsible for driving upstroke when MP is >−70 mV.

•  IKr contributes to establishing the MP.

•  Ileak contaminates VC responses at very positive and negative voltages and contributes to MP.

•  Strong outward current elicited in the range of IKs activation correlates with AP duration.

•  A subset of these cells has an unidentified outward current present at 6 mV that is not recapitulated by iPSC-CM mathematical models.

Overall, this study demonstrates both the large cell-to-cell heterogeneity in iPSC-CMs and provides a tractable method to study the ionic current determinants of such variability during patch-clamp experiments.

RICP Is a Tool to Understand Ionic Current Mechanisms of iPSC-CM Heterogeneity

We used our recently published VC protocol to capture a rich snapshot of current dynamics that is explanatory of iPSC-CM AP morphology. This 10-s protocol provided information about several currents (e.g., ICa,L, IKr) and their effects on AP morphology (dV/dtmax, MP). Other studies have collected VC data for one or two currents and shown correlations with certain AP morphological features from the same cells (13). However, we believe this is the first study that attempts to collect information about several currents with a <10-s protocol and use these data to explain AP features.

The VC protocol was designed to identify the presence and relative size of seven individual currents, but each of the segments is contaminated, at least to some extent, by off-target currents. For example, Ileak contaminates every segment of the protocol, as it contributes current at all voltages, with increased Ileak at voltages far from 0 mV. The sensitivity analysis shows how the IK1, Ito, If, and IKs segments, all of which are clamped far from 0 mV, are sensitive to changes in the seal-leak resistance (Fig. 4C). Some currents (e.g., INa) have distinct dynamics that provide for relatively easy isolation, but others (e.g., IKr) always open when several currents conduct ions.

The isolation of currents could potentially be improved through the development of new VC protocols with different optimization methods or by using new computational models that better capture the dynamics of iPSC-CMs used in a specific experimental context. However, we believe new protocols will only result in marginal improvements. Instead, we think there is far more to gain by developing methods to tease apart contributions from the different currents. To further validate the RICP approach, and depending on the objective of the experiments, one could sequentially dissect current contributions (14) or fit computational models to individual traces (15), either of which could provide estimates of cell-specific ionic current densities. These approaches could be used in conjunction with a leak compensation current [see Clark et al. (3) for details] during current-clamp recordings to probe the independent contributions of seal-leak and ionic-current densities to AP morphology.

Ica,L Likely Drives Upstroke in Many iPSC-CM Studies

Using RICP, we identified ICa,L as a contributor to upstroke velocity in the depolarized iPSC-CMs used in this study. Despite the presence of INa in most of our cells, ICa,L appears to be the predominant driver of upstroke. This is likely because, at depolarized MP values (e.g., >−70 mV), INa is inactive and cannot generate an inward current to drive upstroke.

This implies a mechanism like AP generation in SA nodal cells, despite the presence of INa (16), the upstroke is primarily driven by calcium currents (17). This finding also hints at ICa,L as the likely driver of upstroke in many other studies where MP < −65 mV or dV/dtmax is <20 V/s (18, 19). As we and others have shown, it is possible to recover a polarized MP by dynamically clamping a synthetic IK1 (6, 20). Such an approach makes it possible for INa, among other currents, to recover from inactivation and results in APs with a faster upstroke velocity and more mature appearance.

IKr Is Likely an Important Current in Establishing MP in Depolarized iPSC-CMs

IK1 conductance is often thought to be reduced in iPSC-CMs relative to adult cardiomyocytes, resulting in depolarized MP. However, iPSC-CMs can have large amounts of IK1, and yet, still be depolarized compared with adult cells (18). One hypothesis for this discrepancy in MP between adult and iPSC cardiomyocytes is the increased role of Ileak in iPSC-CMs (3, 18) caused by their smaller size relative to adult cardiomyocytes. In this study, we identify IKr as likely playing a role in establishing the MP of iPSC-CMs. The correlation between IKr and MP is unlikely to be substantially influenced by leak as the VC step to isolate IKr is close to the expected seal leak reversal potential.

In both our experimental data and in populations of models based on either the Paci or Kernik models, the IKr-isolating segment has the strongest correlation to MP (−0.66, −0.54, and −0.58 for experimental data, Paci, and Kernik, respectively, Fig. 3 and Supplemental Figs. S6 and S7).

This finding agrees with previous work showing that iPSC-CM MP values are sensitive to E-4031 (21). Our modeling work indicates that with a small Ileak (2-GΩ seal) and substantial reduction in baseline IKr, the model will depolarize to MP values like those shown in our study.

Limitations and Future Directions

The RICP method shows how a brief VC protocol can be used to provide mechanistic insights into AP morphology and heterogeneity. This protocol, however, does not perfectly isolate each of the seven currents, and so the causal relationships of currents with AP morphological features are likely weaker than if we conducted traditional drug block experiments. This is the tradeoff we make in attempting to collect data for seven different ion channels all in the same cell. In the future, we believe methods that tease apart the current contributions at each time point have the potential to improve insights drawn from this VC protocol, such as ion channel coexpression patterns. Furthermore, we were unable to deduce the identity of the outward current at 6 mV, given the voltage and nonlinearity in the current’s response, we hypothesize that this is a potassium-conducting species with a time or state (perhaps concentration) dependence, but further drug studies would be required to definitively identify this current.

In this study, we focus on a set of cells derived from a single individual, using a single differentiation method, within the perforated patch-clamp experimental context used in our laboratory. Although we expect the RICP approach to generalize to other cell lines and contexts, the iPSC-CM characterization here (e.g., ICa,L driving upstroke) may not be descriptive of similar cells in other studies. In the future, it would be interesting to conduct this RICP approach on cells from multiple donors, across multiple differentiation batches, and in multiple laboratory settings.

DATA AVAILABILITY

All data can be accessed or generated from the GH page: https://github.com/Christini-Lab/ap-vc-correlations.

SUPPLEMENTAL DATA

10.6084/m9.figshare.25289914.v3 Supplemental Figs. S1–S7: https://doi.org/10.6084/m9.figshare.25289914.v3.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants U01HL136297 (to D.J.C.) and F31HL154655 (to A.P.C.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.P.C., S.W., K.F., T.K-M., and D.J.C. conceived and designed research; A.P.C. and S.W. performed experiments; A.P.C. and K.F. analyzed data; A.P.C., S.W., K.F., T.K-M., and D.J.C. interpreted results of experiments; A.P.C. and K.F. prepared figures; A.P.C., T.K-M., and D.J.C. drafted manuscript; A.P.C., S.W., K.F., T.K-M., and D.J.C. edited and revised manuscript; A.P.C., S.W., K.F., T.K-M., and D.J.C. approved final version of manuscript.
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