Confocal imaging of CICR events from isolated and immobilized SR vesicles

The Ca²⁺ concentration inside the sarcoplasmic reticulum ([Ca²⁺]_SR) is a difficult parameter to measure in ventricular cardiac myocytes. Interference from Ca²⁺ -sensitive dye loading into cellular compartments other than the SR interferes with free Ca²⁺ measurement. In addition, the composition of the cytosol surrounding the SR in intact cells cannot be easily controlled. We have developed a method to measure localized [Ca²⁺]_SR in immobilized membrane vesicles during rapid solution switches. Ca²⁺ uptake and release in rat SR membrane vesicles was monitored using confocal microscopy. Vesicles were immobilized on a coverslip using an agarose matrix. Perfusion with a Ca²⁺-containing solution supplemented with ATP initiated SR Ca²⁺ uptake, causing a rise in intravesicular fluorescence in vesicles containing the low-affinity Ca²⁺ indicator fluo-5N. Perfusion with caffeine caused SR Ca²⁺ release and a decrease in intravesicular flourescence. Although caffeine-dependent release was readily visible with extravesicular Ca²⁺-green, Ca²⁺ which leaked from the SR was detected only indirectly as eventless release. We conclude that SR Ca²⁺ uptake and release can be selectively measured in functional SR vesicles using a confocal microscope. Caffeine-dependent release is directly measurable though SR Ca²⁺ leak can only be inferred as subresolution events, presumably because channels in separate vesicles were not close enough to result in concerted Ca²⁺-induced Ca²⁺ release.