Calcium Transient Analysis in SARS-CoV-2 S-Expressing hiPSC-CMs

Untransfected and SARS-CoV-2 S transfected hiPSC-CMs cultured on fibronectin-coated 35mm glass-bottom dishes (MatTek Corporation, Ashland, MA) at 37°C, 5% CO₂ were loaded with 5μM of Fluo-4 AM (Thermo Fisher Scientific, Waltham, MA) with 0.02% F-127 (Thermo Fisher Scientific, Waltham, MA) in Tyrode’s Solution (Alfa Aesar, Tewksbury, MA) for 30 minutes. Following wash-out, Tyrode’s solution was added, and cells were imaged. During imaging, cells were kept in a heated 37°C stage-top environment chamber supplied with 5% CO₂. Imaging of Ca²⁺ transients was taken under a 40X objective using a Nikon Eclipse Ti (Melville, NY) light microscope. Human iPSC-CMs were paced at 1 Hz using an IonOptix MyoPacer Field Stimulator (Westwood, MA). Time-lapse videos were taken at a speed of 20ms per frame for 20s. Each video recording was analyzed for the percent area exhibiting pacing, calcium sparks, and “calcium tsunamis”. The raw data was exported to Excel software (Microsoft, Redmond, WA) and analyzed with a custom Excel-based program in order to normalize for photo bleaching and movement. All values are reported as mean ± SEM. Statistical analysis was performed using GraphPad Prism 8 software (San Diego, CA). An unpaired two-tailed Student’s t-test was used to determine statistical significance between two groups, and a one-way ANOVA followed by Tukey’s multiple comparisons test was used to determine statistical significance between 3 groups. A P <0.05 was considered to be significant.