A strategy of cold loading of the Ca2+-indicating fluorophore Rhod 2-AM followed by warm incubation was developed to selectively label mitochondria of adult rabbit cardiac myocytes. After electrical stimulation, mitochondrial Rhod 2 fluorescence observed by confocal microscopy increased and then rapidly decayed to baseline. In regions between mitochondria, the fluorescent transients were small or absent. Subsequent addition of calcium ionophore increased mitochondrial but not cytosolic fluorescence, confirming the mitochondrial localization of Rhod 2. These experiments directly demonstrate rapid mitochondrial free Ca2+ transients during the contractile cycle in rabbit cardiac myocytes.
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