*3.3. EHT with Notch*/*Oscillations in the AP Showed Large BKCa Expression*

The BKCa channel is formed as a tetramer of the channel forming alpha-subunit KCa1.1 encoded by the *KCNMA1* gene. We performed retrospectively expression analysis in order to investigate whether there was an association between appearance of oscillations and the expression level of *KCNMA1*. The mRNA level for the channel forming alpha-subunit KCa1.1 showed very low expression in human LV tissue. The same holds true for the in-house control cell line ERC018 and the commercially available cell lines iCell and iCell2, where notch or oscillations in the AP have never been detected [5]. In contrast, all EHTs from C25 from which we could record notch/oscillations showed a substantial increase in *KCNMA1* mRNA, which is encoding for KCa1.1 (Figure 4). In addition, RNA sequencing showed a 12-fold higher expression level for *KCNMA1* in C25 than in the control cell line ERC001 (Supplementary Figure S4). Other genes related to *KCNMA1* did not show major alterations in expression. Immunofluorescence analysis of hiPSC-CMs revealed the expression of the alpha-1 subunit of the BKCa channel in the C25 cell line with enhanced signal intensity at cell-cell contacts, whereas no signal was detected in the control cell line [30] (Supplementary Figure S5).

**Figure 4.** Expression analysis for the alpha subunit (*KCNMA1)* of big conductance calcium activated potassium channel. Individual expression levels of *KCNMA1* in left ventricular tissue (LV) and engineered heart tissue (EHT) from cell line C25 (without and with notch/oscillations), ERC018, iCell and iCell2. Mean values <sup>±</sup> SEM, \*\*\* *<sup>p</sup>* <sup>&</sup>lt; 0.001, 1-way ANOVA with multiple comparison, *<sup>n</sup>* <sup>=</sup> number of patients for LV and *n* = number of EHT/number of individual differentiation batches.
