Calcium handling

Tissues within culture platforms were loaded with Fluo-4 NW (50% v/v, Life Technologies) in RPMI+B27 media containing 5 μM blebbistatin (Sigma) as necessary to reduce movement artifacts for 30 minutes at 37°C. Videos were acquired at a rate of 150 frames per second using a Pike F-032 camera (Allied Vision Technologies) as described above for contractility analysis. Videos were analyzed in MATLAB using a custom script that calculated the temporal changes in calcium fluorescence intensity. Specifically, each frame was normalized to a baseline background region chosen by the user to give baseline-corrected changes in minimum and maximum fluorescence values for each frame. The temporal change in fluorescence intensity was presented as a calcium transient trace from which the measurements were obtained. Briefly, the calcium transient “timing” was determined as the peak-to-peak values of two successive beats as defined by the peak maxima. Calcium transient “amplitude” was determined by numerically integrating the area below the peak maxima relative to the baseline. Calcium transient traces were analyzed during 5 mM caffeine stimulation of tissues previously treated with either 1 mM verapamil (Sigma-Aldrich) or 1 μM thapsigargin (Sigma-Aldrich). Caffeine responses were quantified by comparing this calcium transient amplitude before and after the addition of 5 mM caffeine (Sigma-Aldrich).