In situ monolayer patch clamp of acutely stimulated human iPSC-derived cardiomyocytes promotes consistent electrophysiological responses to SK channel inhibition

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) represent an in vitro model of cardiac function. Isolated iPSC-CMs, however, exhibit electrophysiological heterogeneity which hinders their utility in the study of certain cardiac currents. In the healthy adult heart, the current mediated by small conductance, calcium-activated potassium (SK) channels (ISK) is atrial-selective. Functional expression of ISK within atrial-like iPSC-CMs has not been explored thoroughly. The present study therefore aimed to investigate atrial-like iPSC-CMs as a model system for the study of ISK. iPSCs were differentiated using retinoic acid (RA) to produce iPSC-CMs which exhibited an atrial-like phenotype (RA-iPSC-CMs). Only 18% of isolated RA-iPSC-CMs responded to SK channel inhibition by UCL1684 and isolated iPSC-CMs exhibited substantial cell-to-cell electrophysiological heterogeneity. This variability was significantly reduced by patch clamp of RA-iPSC-CMs in situ as a monolayer (iPSC-ML). A novel method of electrical stimulation was developed to facilitate recording from iPSC-MLs via In situ Monolayer Patch clamp of Acutely Stimulated iPSC-CMs (IMPASC). Using IMPASC, > 95% of iPSC-MLs could be paced at a 1 Hz. In contrast to isolated RA-iPSC-CMs, 100% of RA-iPSC-MLs responded to UCL1684, with APD50 being prolonged by 16.0 ± 2.0 ms (p < 0.0001; n = 12). These data demonstrate that in conjunction with IMPASC, RA-iPSC-MLs represent an improved model for the study of ISK. IMPASC may be of wider value in the study of other ion channels that are inconsistently expressed in isolated iPSC-CMs and in pharmacological studies.

APD30 (A) and APD50 (B) recorded from isolated D35 RA-iPSC-CMs, isolated D65 RA-iPSC-CMs and RA-iPSC-MLs.All action potentials recorded using whole-cell current clamp at 37 °C.Data presented as mean ± SEM.Statistics represent Brown-Forsythe ANOVA test (as appropriate).Data recorded from one batch of RA-iPSC-CMs.These data are calculated from the same recordings as presented in Fig. 3.

Figure S2: Stimulation of iPSC-MLs.
A) Whole-cell current clamp recording of a spontaneous (red) and triggered (black) APs from an RA-iPSC-ML.AP firing was triggered by using a current injection via the patch electrode.B) Capacitive current recording of a spontaneous (red) and triggered (black) AP from an RA-iPSC-ML.AP firing was triggered by field stimulation with a platinum electrode placed at both edges of the bath.For all recordings, stimulation threshold was confirmed by visual check of the monolayer for contraction and a stimulation of threshold +20% was applied.iPSCs were 'preconditioned' ~12 hours before the main differentiation protocol began.In order to push monolayers towards an atrial-like phenotype, retinoic acid (RA) was included in a proportion of monolayers from day 2 to 6. DMSO was used as a control in the absence of RA.Spontaneous beating typically began between day 8 and 10.

Figure S1 :
Figure S1: APD30 and APD50 in isolated RA-iPSC-CMs and RA-iPSC-MLs.APD30 (A) and APD50 (B) recorded from isolated D35 RA-iPSC-CMs, isolated D65 RA-iPSC-CMs and RA-iPSC-MLs.All action potentials recorded using whole-cell current clamp at 37 °C.Data presented as mean ± SEM.Statistics represent Brown-Forsythe ANOVA test (as appropriate).Data recorded from one batch of RA-iPSC-CMs.These data are calculated from the same recordings as presented in Fig. 3.

Figure S3 :
Figure S3: Triggered action potentials in iPSC-MLs.All action potentials from RA-iPSC-MLs (A) and DMSO-iPSC-MLs (B) used to generate the data presented in Table 1 and Fig. S3C.Traces shown in the three panels represent the three individual differentiation batches of iPSC-MLs used for experiments.In A, n = 23, 15 & 15 from left to right.In B, n = 15, 17 & 13 from left to right.C) Comparison of properties between RA-iPSC-MLs (reds; n = 53) and DMSO-iPSC-MLs (blues; n = 45).Diastolic potential (i; membrane potential prior to AP firing), maximum diastolic potential (ii), after-hyperpolarisation (iii; difference between the diastolic and maximum diastolic potential), APD30 (iv), APD50 (v), APD90 (vi), AP amplitude (vii), plateau amplitude (viii; AP amplitude 20 ms after the AP peak) and plateau fraction (ix) are shown.Different shades represent the three differentiation batches.Batches were pooled for comparisons between RA and DMSO.Data presented as mean ± SEM.Statistics represent unpaired Student's t-test, with Welch's correction where appropriate(i, v, viii, ix).APs were recorded using whole-cell current clamp at 37 °C.

Figure S4 :
Figure S4: Spontaneous action potentials in iPSC-MLs.Representative spontaneous action potentials from RA-iPSC-MLs (A) and DMSO-iPSC-MLs (B) used to generate the data presented in Table S1 and Fig. S4C.Traces shown in the three panels represent the three individual differentiation batches of iPSC-MLs used for experiments.C) Comparison of properties between RA-iPSC-MLs (reds; n = 53) and DMSO-iPSC-MLs (blues; n = 58).Maximum diastolic potential (i), cycle length (ii), maximal upstroke velocity (iii), APD30 (iv), APD50 (v), APD90 (vi), AP amplitude (vii), plateau amplitude (viii) and plateau fraction (ix) are shown.Different shades represent the three differentiation batches.Batches were pooled for comparisons between RA and DMSO.Statistics represent unpaired Student's t-test, with Welch's correction where appropriate(ii, iv, vi,  viii & ix).APs were recorded using whole-cell current clamp at 37 °C.

Figure S5 :
Figure S5: EADs triggered by application of 1 µM E-4031.Ai) Number of iPSC-MLs in which EADs occurred following application of E-4031.Aii) E-4031-induced prolongation of APD90 in RA-iPSC-MLs and DMSO-iPSC-MLs, separated by the occurrence of EADs.Incidence of EADs did not appear to be associated with the degree of APD prolongation.Data presented as mean ± SEM.Statistics represent an unpaired Student's t-test comparing change in APD90 between monolayers in which EADs did and did not occur.B) Representative traces from RA-iPSC-MLs (i) and DMSO-iPSC-MLs (ii) exhibiting EADs in the presence of 1 μM E-4031.

Figure S7 :
Figure S7: Culture protocol for the differentiation of iPSCs into cardiomyocytes.iPSCswere 'preconditioned' ~12 hours before the main differentiation protocol began.In order to push monolayers towards an atrial-like phenotype, retinoic acid (RA) was included in a proportion of monolayers from day 2 to 6. DMSO was used as a control in the absence of RA.Spontaneous beating typically began between day 8 and 10.