Video-Analysis of Monolayer and Spheroid Beating Activity

A modified GoPro HeroBlack 6 camera (Back-Bone Gear Inc., Kanata, Canada) was used to record short video sequences at high frame rate, 240 frames per second, of spheroids in a heating chamber with warmed lid and temperature controller (Ibidi, GmbH, Martinsried, Germany) on the stage of an inverted microscope (Nikon Eclipse TE2000-U) with a Nikon Plan Fluo 10×/0.3 phase contrast lens. Cultures were allowed to warm up to 37°C for 10 min and DMEM containing 25 mM HEPES buffer was used for recordings. For some experiments, a self-made pacing chamber with platinum wires inserted in a glass-bottom dish was used with a MyoPacer (Ionoptix, USA), set to different field-pacing frequencies at 10 V, bipolar pulses of 4 ms duration. The open-source macro “Musclemotion” for ImageJ by Sala et al. (2017) was then used on an Apple MacPro equipped with 64 GB RAM to extract motion data from videos after format conversion and data reduction steps. This software has been validated with a number of cardiac models where beating activity is observed and we have additionally verified the software in our lab by using freshly isolated adult rat ventricular cardiomyocytes that precisely follow an electrically induced contraction frequency (Supplementary Movie 1). For 3D cultures, videos were used for the analysis that show the entire circumference of the spheroids. For 2D cultures, regions of interest were defined that comprised single cells or small groups of cells.