Dynamic Calcium Movement in Cardiac Sarcoplasmic Reticulum during Release

METHODS Cell isolation Single ventricular myocytes were isolated from New Zealand White rabbits using standard enzymatic techniques. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and approved by the Institutional Animal Care and Use Committee. Animals were anaesthetized with sodium pentobarbital (50 mg/kg i.v.). Hearts were excised, rinsed in cold Ca-free solution, and mounted on a Langendorff perfusion system. Hearts were perfused at 37°C with Ca free solution (DMEM, Dulbecco’s minimal essential medium, Gibco/Invitrogen, USA, gassed with 95% O25% CO2) for 5 min. Solution was then switched to a collagenase (0.3-0.7 mg/ml; Boehringer Mannheim, Germany) and Ca (10-20 μM) containing solution. After 15–25 min, perfusion was stopped and ventricles were minced into small pieces. Optionally the pieces were incubated for 5–20 min in fresh enzyme. Finally, enzyme activity was stopped with DMEM solution containing bovine serum albumin (BSA; 0.5-1%), and tissue was agitated or triturated to liberate single myocytes. Cells were washed and stored in DMEM solution adjusted to [Ca] = 150 μM. All experiments were performed at room temperature (21-24C).