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

Abstract

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 (less than 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 less than 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.

Mitochondrial free calcium transients during excitation‐contraction coupling in rabbit cardiac myocytes

“…Species differences may account for the differences between our findings in rabbit myocytes showing rapid changes of intramitochondrial Ca 2+ during the excitation-contraction cycle and the results described by Miyata et al [16] in rat myocytes, which did not reveal rapid mitochondrial transients. Alternatively, the differences may result from the use by Miyata and co-workers of Mn 2+ to quench cytosolic fluorescence of the Ca 2+ indicating fluorophore.…”

“…Using Fura-2 ratio imaging and Mn 2+ to quench cytosolic fluorescence, Miyata and co-workers [16] reported that mitochondiral free Ca 2+ did not increase and decrease in parallel with transients of cytosolic free Ca 2+ accompanying the excitation-contraction cycle. Rather, mitochondrial Ca 2+ increased gradually in response to an increase of contractile frequency.…”

Excitation-contraction coupling and mitochondrial energetics

“…In all studies using the Mn 2+ -quench method [59,60,74,136,137], no beat-to-beat oscillations of [Ca 2+ ] m in response to cytosolic Ca 2+ -transients could be detected ( Fig. 5A and B).…”

“…Depending on the loading protocol of indo-1/AM, MnCl 2 reduced its overall fluorescence by ∼30-55%, indicating that ∼45-70% of fluorescence stemmed from noncytosolic compartments, i. e., primarily from mitochondria [137,222].…”

“…Instead, a slow, cumulative increase of [Ca 2+ ] m from resting levels (∼90 nmol/L) to ∼400-600 nmol/ L was observed in response to β-adrenergic stimulation and an increase of stimulation frequency to 4 Hz ( Fig. 5C; [60,137] (Fig. 6A-D).…”