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Imaging of Mitochondrial Ca^ Concentration in Saponin Permeabilized Rat Ventricular Myocytes

We investigated the changes in mitochondrial Ca 2 + concentration ([Ca 2 + ] m i t o ) by using laser scanning confocal microscopy in rat ventricular myocytes. Cells were loaded with the acetoxymethyl ester form of fluo-3, a fluorescent Ca 2 + indicator, and perfused with saponin (0.05 mg/ml) in 0 Ca 2 + plus EGTA solution to permeabilize the sarcolemmal membrane. The application of internal solution (196 nM Ca 2 + ) caused a significant increase in the fluorescent signal. When cells were pretreated with ruthenium red (RuR, 1 μM, an inhibiter of the mitochondrial Ca 2 + uniporter), the increase in fluorescent signal intensity by Ca 2 + containing internal solution was abolished. These results indicated that permieabilization by saponin in combination with fluo-3/AM provided a useful method of monitoring [Ca 2 + ] m i t o in isolated myocytes.