Measuring Ca²⁺ sparks in cardiac myocytes

This protocol describes the measurement of CA(2+) sparks in intact myocytes by using a CA(2+)-sensitive dye and imaging using laser scanning confocal microscopy. It takes advantage of spontaneous CA(2+)-release events-sparks-using them as a measure of the activity of ryanodine receptors (RyRs). Two methodologies are described: One requires that cardiomyocytes be stimulated, preferably under voltage clamp by depolarizing pulses, until steady-state is reached, and then stimulation is stopped and CA(2+) sparks are recorded. The second requires that cells be permeabilized and bathed in a solution to load the cell with CA(2+) sufficient to elicit CA(2+) sparks, but not CA(2+) waves. These are then analyzed offline to quantify spark frequency and morphology. The advantages and disadvantages of each approach are discussed.