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.
Division of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Belgium.
Niall
N
Macquaide
Division of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Belgium.
Virginie
V
Bito
Division of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Belgium.
Karin R
KR
Sipido
D002118:Calcium / Q000032:analysis*; D003584:Cytological Techniques / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods*; D032383:Myocytes, Cardiac / Q000737:chemistry* / Q000378:metabolism; D018408:Patch-Clamp Techniques / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*
D005456:Fluorescent Dyes; D002118:Calcium