Measurement of mitochondrial free Ca²⁺ concentration in living single rat cardiac myocytes

A technique that allows the continuous measurement of mitochondrial free Ca²⁺ ([Ca²⁺](m)) in a single living cardiac myocyte is described. It involves the introduction of the fluorescent chelating agent indo-1 into the cell by exposure to the acetoxymethyl ester, followed by selective quenching of the fluorescence of indo-1 in the cytosol by Mn²⁺. The identity of the remaining fluorescence due to intramitochondrial indo-1 is established by its resistance to treatment of the cell with digitonin at concentrations that release cytosolic but not mitochondrial enzymes and by the finding that ruthenium red and carbonyl cyanide p-trifluoromethoxyphenylhydrazone prevent its response to elevated cytosolic free Ca²⁺ ([Ca²⁺](c)). [Ca²⁺](m) is found to be low (< 100 nM) in unstimulated cells and to rise in procedures that chronically elevate [Ca²⁺](c), such as Na⁺ replacement. The gradient [Ca²⁺](m)/[Ca²⁺](c) is less than unity at values of [Ca²⁺](c) of < 500 nM but rapidly increases at higher values of [Ca²⁺](c). Although there is no detectable increase in [Ca²⁺](m) during a single electrical stimulation, [Ca²⁺](m) increases up to 600 nM as the pacing frequency is raised to 4 Hz in the presence of norepinephrine; this increase occurs over the course of many contractions. It is concluded that these findings are consistent with an increase in [Ca²⁺](m) acting as a signal to increase dehydrogenase activity, and hence flux through oxidative phosphorylation, in response to increased work loads.