Calcium Measurements on Isolated Cardiomyocytes

Intracellular calcium measurements were performed as previously described by Oakley et al.13 Briefly, fresh isolated cardiomyocytes were incubated with a calcium sensitive indicator, fluo‐4 (Invitrogen). Following isolation, cardiomyocytes were allowed to attach to Laminin‐coated glass bottom wells in complete AW medium (Cellutron, Baltimore, MD) for at least 1 hour. Cells were incubated in serum‐free AW medium containing 1 μmol/L fluo‐4 for 15 minutes at room temperature in the dark. Cells were washed 3 times then incubated for 15 minutes at room temperature in a HEPES‐buffered Tyrode salt solution (135 mmol/L NaCl; 4 mmol/L KCl; 1.0 mmol/L MgCl₂; 20 mmol/L HEPES; 1.0 mmol/L CaCl₂ and 10 mmol/L glucose, with pH 7.4 adjusted by NaOH). Calcium measurements were performed on a Nikon Eclipse Ti‐E inverted confocal microscope equipped with 20× 0.75 NA objective. Fluo‐4 fluorescence was monitored by exciting the indicator at 488 nm, and collecting the emission wavelength at 500 to 630 nm. Data were collected in a Galvano frame mode at 2 frames per second. The change in fluo‐4 fluorescence emission intensity represents the change in intracellular calcium. Regions of interest were selected with cardiomyocytes displaying spontaneous intracellular calcium changes in the form of local calcium transients. Intracellular calcium changes were monitored at the single cell level. For each experiment 4 regions of interest were monitored with a total of at least 20 cells. A caffeine solution was diluted over the samples for stimulation of intracellular calcium signals. Cells were monitored at baseline and final concentrations of caffeine bathing the cells at 0.25 and 10 mmol/L.