Cardiomyocyte calcium imaging and contractility.

Adult murine ventricular cardiomyocytes from wild-type and transgenic mice were isolated as previously described (16) and loaded with 0.25 μg fura 2-AM for subsequent calcium transient analysis using the MMSYS IonOptix imaging system. Isolated cardiomyocytes were stimulated with 15 V at a frequency of 5 Hz to induce uniform cell contraction at 37°C. Contractility measurements were calculated using sarcomere shortening distances from real-time phase-contrast images. Intracellular calcium concentrations were calculated from the ratio of bound to unbound fura 2-AM These measurements were used to determine contractile and calcium transients depicting the cell’s overall contractility or the flux of calcium across the cell membrane. Both contractility measurements and calcium transients were recorded for 5–10 min, at which point each curve was averaged to generate a representative contractile or calcium transient plot for each cell recorded. These plots were then analyzed using a monotransient data analysis algorithm to generate surrogate parameters for systolic and diastolic function for that cell.