Introduction: Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) represent a useful in vitro model of cardiac function. Isolated iPSC-CMs, however, exhibit significant electrophysiological heterogeneity which hinders their utility as a model system for the study of certain individual cardiac currents [1]. Differentiation of iPSC-CMs using conventional methodologies produces cells which exhibit a ventricular-like phenotype, but the inclusion of retinoic acid (RA) during differentiation produces iPSC-CMs with an atrial-like phenotype [2, 3]. In the adult heart, the current mediated by small conductance, calcium-activated potassium channels (I_SK) is an atrial-selective current [4, 5]. Functional expression of I_SK within atrial-like iPSC-CMs has not been well investigated.

Aims: The present study therefore aimed to investigate atrial-like iPSC-CMs as a model system for the study of I_SK.

Methods: iPSC-CMs were differentiated in the presence of RA or a DMSO control in order to generate cells with a more atrial- or ventricular-like phenotype respectively. Following differentiation, iPSC-CMs were dissociated and plated sparsely as isolated cells (RA- or DMSO-iPSC-CMs) or plated densely to promote reformation of a confluent monolayer (RA- or DMSO-iPSC-MLs). All data are presented as mean ± S.E.M and statistical comparisons represent Student’s t-tests (with Welch’s correction where appropriate), unless otherwise stated.

Results: Although isolated RA-iPSC-CMs exhibited an atrial-like phenotype, they responded poorly to SK channel inhibition by UCL1684, with only 17.6% of cells exhibiting I_SK (n = 17). Isolated RA-iPSC-CMs exhibited substantial heterogeneity of spontaneous action potential (AP) duration (APD). APD heterogeneity was significantly smaller (p < 0.001; F-test) when spontaneous APs were recorded from in situ RA-iPSC-MLs, demonstrating that maintenance of the monolayer reduces electrophysiological variability.

A method for simultaneous electrical stimulation of iPSC-MLs and whole-cell recording has not previously been published to the best of our knowledge. Accordingly, we have developed a novel method for localized stimulation of iPSC-MLs which allows concurrent whole-cell patch clamp recordings to be made at a user-defined stimulation rate. Using this method >95% of RA-iPSC-MLs and DMSO-iPSC-MLs could be paced at 1 Hz. RA-iPSC-MLs (n = 53) paced at 1 Hz exhibited a more atrial-like phenotype than DMSO-iPSC-MLs (n = 45) as characterised by abbreviated repolarisation at APD₃₀ (40.5 ± 4.0 [RA] vs. 128.8 ± 4.6 ms [DMSO]; p < 0.0001) and APD₉₀ (220.8 ± 13.3 [RA] vs. 283.4 ± 10.2 ms [DMSO]; p < 0.001); and a lower AP and plateau amplitude (101.6 ± 1.6 mV and 82.2 ± 2.8 mV respectively) than DMSO-iPSC-MLs (113.0 ± 2.1 mV and 110.0 ± 2.2 mV; p < 0.0001 for both). Prolongation of APD₅₀ by application of UCL1684 was significantly larger in RA-iPSC-MLs (18.7 ± 3.0%; n = 12) than in DMSO-iPSC-MLs (4.2 ± 2.6%; p < 0.01; n = 12). In contrast to data from isolated RA-iPSC-CMs, 100% of RA-iPSC-MLs responded to SK channel inhibition.

Conclusions: These data demonstrate that RA-iPSC-MLs represent a useful model for the study of I_SK. Moreover, this novel method of iPSC-ML stimulation may be of wider value in the study of other ion channels that are inconsistently expressed in isolated iPSC-CMs.