*3.1. Outward Potassium Currents in hiPSC-CMs, Appearance of IBK,Ca*

Large, transient outward currents were elicited in hiPSC-CMs (Figure 1A) by depolarizing test pulses. We found in several C25-hiPSC-CMs a large, inactivating outward current followed by a late sustained current with an irregular shape during the entire depolarizing test pulse. The irregular-shaped "noisy" currents were similar to BKCa currents, which were reported previously in mesenchymal stem cells [26]. Similar to that report we used rather high test pulse potentials (+70 mV) from physiological resting membrane potential of <sup>−</sup>80 mV and increased the free Ca2<sup>+</sup> concentration of the pipette solution from 2 to 4.4 mM to facilitate the detection of BKCa [26]. The selective IBK,Ca blocker IBTX (100 nM) was used to identify the IBK,Ca (Figure 1A). Of the hiPSC-CMs 76% (19 out of 25) showed IBTX-sensitive outward currents suggesting the presence of BKCa; the area under the curve was reduced by IBTX from 30.6 ± 5.1 pAs/pF to 20.2 ± 4.1 pAs/pF (*n* = 19, *p* < 0.0001, paired *t* test, Figure 1B). IBTX inhibited both the peak and late current density (peak from 82.9 ± 11.5 pA/pF to 44.8 ± 7.6 pA/pF, *p* < 0.0001, Figure 1C and late: from 29.2 ± 5.4 pA/pF to 17.8 ± 3.4 pA/pF; *n* = 19, *p* = 0.004, Figure 1D). In IBTX-insensitive hiPSC-CMs, baseline values of outward peak and late currents were smaller compared to IBTX-sensitive hiPSC-CMs. Furthermore, in hiPSC-CMs without the irregular-shaped outward current IBTX did not change peak or late currents (peak: 45.4 ± 6.4 pA/pF baseline vs. 43.4 ± 3.9 pA/pF IBTX, *p* = 0.594, *n* = 6 and late: 8.3 ± 2.6 pA/pF baseline vs. 7.5 ± 1.8 pA/pF IBTX, *n* = 6, *p* = 0.363; paired *t* test). HiPS-CMs from the commercially available iCell cell line did not show irregular-shaped "noisy" currents or IBTX-sensitivity (Supplementary Materials Figure S1).

**Figure 1.** Outward currents in C25 human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and the effect of iberiotoxin (IBTX). (**A**) Original outward traces before (black) and after (green) exposure of 100 nM IBTX in insensitive (upper, black directly underlying green curve) and sensitive (lower panel) hiPSC-CMs. (**B**–**D**). Summary of IBTX (100 nM) effects in insensitive (left panel) and sensitive (right panel) hiPSC-CMs quantified by area under the curve (**B**), peak current (**C**) and current at the end of the test pulse (late current, **D**). Mean values ± SEM. \* *p* < 0.05, unpaired Student's *t* test for basal values in insensitive vs. sensitive hiPSC-CMs; ## *p* < 0.01, ### *p* < 0.001; paired Student's *t* test for basal vs. IBTX; *n* = number of isolated cells/number of individual differentiation batches.

#### *3.2. Action Potentials with Strong Initial Repolarization and Oscillations Are Sensitive to Iberiotoxin*

APs recorded in C25-EHTs, exhibiting the IBTX-sensitive irregular-shaped outward current, showed a pronounced initial repolarization ("notch") below the later plateau level of the AP and the initial notch was followed by a (very) early afterdepolarization (Figure 2). In some APs the notch was followed by a peculiar oscillation during plateau phase of the AP. This peculiarity of notch/oscillation was only observed in some of independent differentiation batches of the C25 cell line, but never in any other of three in-house cell lines or five commercial cell lines investigated with sharp microelectrodes as previously described [5,9,27–30]. When the notch/oscillation was detected in one EHT, all impalements showed this peculiarity including all EHTs from this differentiation batch. From all C25 batches investigated with sharp microelectrode, we detected seven with notch/oscillations and 11 without. The passage number of individual differentiation batches was not significantly different with or without notch/oscillations (77.1 ± 5.9, *n* = 7 vs. 67.9 ± 7.9, *n* = 9; *p* = 0.391) as well as the hiPSC-CM differentiation efficiency (81% ± 5% vs. 85% ± 3% TnT + cells, *p* = 0.51). In addition, spontaneous beating frequency was not different between EHTs with or without notch/oscillations (1.09 ± 0.19 Hz, *n* = 9 vs. 0.92 ± 0.07 Hz; *n* = 14; *p* = 0.34). EHTs were treated with 300 nM ivabradine to allow pacing at 1 Hz as previously described [5]. Under these conditions, APD90 was slightly longer in EHTs with notch/oscillations than without (279 ± 12 ms, *n* = 18/9/6 vs. 226 ± 6 ms, *n* = 30/17/9; *p* = 0.0044). Take-off potential, upstroke velocity and AP amplitude were not significantly different. The number of oscillations within one AP varied between 1 and 6. In case of several oscillations, there was a rather constant cycle length of oscillations in different EHTs (22.8 ± 0.9 ms, *n* = 11). In case of multiple oscillations, the amplitude decreased with each subsequent oscillation (ranging from 50 to

10 mV, Figure 2B). We used the specific inhibitor IBTX (100 nM) in order to evaluate whether oscillations originate from IBK,Ca. To quantify effects of IBTX on early repolarization we analyzed the membrane potential when the initial repolarization reached its lowest point (on average 9.2 ± 0.7 ms after AP upstroke).

**Figure 2.** Pronounced notch with oscillation in the plateau phase of action potential (AP) recorded from engineered heart tissue (EHT) derived by C25 human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). (**A**) Original AP recordings without (**A**) and with (**B**) notch followed by oscillating afterdepolarizations in EHT from cell line C25. (**C**) Summary of the results for take-off potential (TOP), AP amplitude (APA), maximum upstroke velocity (Vmax), AP duration at 90% repolarization (APD90), \*\* *p* < 0.01, unpaired Student's *t* test; *n* = number of impalements/number of EHTs/number of individual differentiation batches.

In case of notch/oscillations, IBTX (100 nM) lifted the membrane potential at this time point from −43.5 ± 5.6 to −13.2 ± 8.3 mV (*n* = 7, *p* = 0.001, paired *t*-test). APD10, APD20, APD50 and APD70 but not APD90 was significantly prolonged by IBTX (Figure 3). IBTX did not affect AP duration in EHTs without notch/oscillations. In addition, IBTX did not show any effect on AP in human LV tissue (Figure 3).

**Figure 3.** Effects of iberiotoxin (IBTX) on action potentials (APs) in C25 engineered heart tissue (EHT) and in human left ventricular tissue (LV). Original AP recordings before and after exposure to IBTX (100 nM, green) in EHTs with (**A**) and without (**B**) notch/oscillations from cell line C25 and in LV (**C**). Mean values of AP duration at different levels of repolarization (APD10-90) before and after exposure to IBTX (100 nM) in EHTs with (**D**) and without (**E**) notch in cell line C25 and in LV (**F**). Mean values ± SEM, \* *p* < 0.05, \*\* *p* < 0.01, paired Student's *t* test.
