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

H. Miyata, H. S. Silverman, S. J. Sollott, E. G. Lakatta, M. D. Stern, R. G. Hansford

Research output: Contribution to journalArticlepeer-review

316 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)H1123-H1134
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Issue number4 30-4
StatePublished - 1991
Externally publishedYes


  • cytosolic calcium concentration
  • indo-1
  • manganese

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)


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