
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.
Cytological Techniques/methods, Microscopy, Myocytes, Microscopy, Confocal, Patch-Clamp Techniques, Calcium/analysis, Staining and Labeling, Patch-Clamp Techniques/methods, Cytological Techniques, Cardiac/chemistry, Genetics and Molecular Biology, Confocal/methods, Fluorescent Dyes/metabolism, General Biochemistry, Calcium, Myocytes, Cardiac, Staining and Labeling/methods, Fluorescent Dyes
Cytological Techniques/methods, Microscopy, Myocytes, Microscopy, Confocal, Patch-Clamp Techniques, Calcium/analysis, Staining and Labeling, Patch-Clamp Techniques/methods, Cytological Techniques, Cardiac/chemistry, Genetics and Molecular Biology, Confocal/methods, Fluorescent Dyes/metabolism, General Biochemistry, Calcium, Myocytes, Cardiac, Staining and Labeling/methods, Fluorescent Dyes
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