Measuring calcium transients in isolated cardiomyocytes

To characterize calcium transients, we isolated ventricular cardiomyocytes from the hearts of male Sprague Dawley rats with a weight of 250–300 g (Charles River Laboratories, Wilmington, MA). We anesthetized the rats with inhaled isoflurane and quickly removed the hearts from the chest after euthanasia. We retrograde perfused the hearts with Ca²⁺-free Tyrode buffer (140 mM NaCl, 5.4 mM KCl, 0.33 mM NaH₂PO₄, 0.5 mM MgCl₂, 11 mM glucose, and 5 mM HEPES (pH7.4)) at 1.0 mL/min for 3 min, followed by an enzyme solution containing collagenase (1.0 mg/mL collagenase type II; Worthington Biochemical, Lakewood, NJ), protease (0.05 mg/mL, type XIV; Sigma-Aldrich, St. Louis, MO), and 0.1 mM Ca²⁺ for 7 min. To harvest the cardiomyocytes, we cut the ventricular tissue into small pieces and filtered it with a 250 μm nylon mesh. We gradually increased the calcium concentration of the Tyrode solution to 1.0 mM for the physiologic analysis and incubated the cardiomyocytes for 15 min with 1 μM Fura-2-AM (Invitrogen, Carlsbad, CA) in Tyrode (1.0 mM, Ca²⁺). We mounted the cardiomyocytes into a recording chamber on the stage of an Olympus IX-71 inverted microscope (Olympus, Center Valley, PA), where we stimulated them electrically at a frequency of 0.5 Hz. Using a galvanometer-driven mirror (HyperSwitch; IonOptix, Westwood, MA), we excited Fura-2 at a wavelength of 340 or 380 nm and recorded the emission at 510 nm using a photomultiplier (IonOptix ). After 5 min of incubation with the drug dofetilide at concentrations of 4, 8, 16, 38, and 130 nM, we recorded cardiomyocyte calcium fluorescence at 250 Hz for 8 min for n = 6 cells each and analyzed the recordings in real time.