Author:

Keywords:

Calcium, Cytological Techniques, Fluorescent Dyes, Microscopy, Confocal, Myocytes, Cardiac, Patch-Clamp Techniques, Staining and Labeling, 0306 Physical Chemistry (incl. Structural), 0601 Biochemistry and Cell Biology, 1103 Clinical Sciences, 3101 Biochemistry and cell biology

Abstract:

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.