Optical action potential measurements

For optical action potential imaging a genetically encoded Förster resonance energy transfer (FRET)-mediated membrane potential sensor (voltage-sensitive fluorescent protein, VSFP) was used as previously described (Chen et al., 2017). Briefly, day 58 porcine CMs were reseeded on a 3.5 cm glass-bottom cell culture dishes (MatTek Life Science) using papain-based dissociation and 2 days later transduced with a lentiviral vector encoding the VSFP sensor under the control of the ubiquitous phosphoglycerate kinase 1 (PGK) promoter. Five days after infection, CMs were incubated with Tyrode’s solution and subjected to imaging at 100 frames per second on an inverted epifluorescence microscope (DMI6000B, Leica Microsystems) equipped with a Zyla V sCMOS camera (Andor Technology). Electrical stimulation was performed at 0.5 Hz using field stimulation electrodes as described above. The VSFP was excited at 480 nm, and the emitted GFP and RFP fluorescence signals were separated using an image splitter (OptoSplit II, Caim Research) equipped with CAIRN HQ535.50 566DCXR E570LP filters (Chen et al., 2017; Goedel et al., 2018). The fluorescence over cells and over background regions was quantified in GFP and RFP channels using ImageJ (National Institutes of Health). Custom-written scripts were applied for further analysis in RStudio Team (2020). After background correction, the RFP/GFP ratio corresponding to APs was derived. Cardiomyocytes based on their action potentials were classified into 2 groups: Ventricular-like cardiomyocytes (V-CMs), and immature ventricular-like cardiomyocytes (iV-CMs) based on APD₉₀/₅₀ ratio. iV-CMs = APD₉₀/APD₅₀ ratio between 1.4 and 1.8. V-CMs = APD₉₀/APD₅₀ ratio between 1.0 and 1.4.