F Di Lisa1, G Gambassi, H Spurgeon, R G Hansford. 1. Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224.
Abstract
OBJECTIVE: The aim was to quantitate intramitochondrial free Ca2+ ([Ca2+]m) in cardiac myocytes under conditions of stimulation previously shown to cause activation of pyruvate dehydrogenase. METHODS: [Ca2+]m was monitored in single, isolated rat cardiac myocytes using fluorescence microscopy following the loading of the cells with the fluorescent chelating agent indo-1, in its permeant acetoxymethylester form, and the selective quenching of cytosolic fluorescence with MnCl2. The extent of contraction upon electrical stimulation was also measured. RESULTS: Electrical stimulation at 2 Hz and higher frequency raised [Ca2+]m significantly, and this was potentiated by exposure to isoprenaline. However, isoprenaline had no effect in quiescent cells, in which [Ca2+]m was raised above resting values by partial replacement of Na+ in the medium. The mitochondrial uncoupling agent carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) raised [Ca2+]m in unstimulated cells, but lowered it in cells subjected to electrical stimulation at 2 Hz or more, to partial Na+ replacement, or to the alkaloid veratridine. CONCLUSIONS: The values of [Ca2+]m in quiescent myocytes (approximately 100 nmol.litre-1) would be associated with very little activation by Ca2+ of pyruvate dehydrogenase phosphatase, based on determination of K0.5 value of 650 nmol.litre-1 in work with mitochondrial suspensions. By contrast, the values of [Ca2+]m associated with electrical stimulation at 2 Hz or greater in the presence of beta adrenergic activation (> 500 nmol.litre-1) would be associated with significant dehydrogenase activation. The effect of beta adrenergic activation is only seen in the presence of electrical stimulation and probably involves enhancement of systolic transients in cytosol [Ca2+]. Effects of uncoupling agents validate the conclusions on the direction and magnitude of the mitochondrial Ca2+ gradient in situ in living myocytes.
OBJECTIVE: The aim was to quantitate intramitochondrial free Ca2+ ([Ca2+]m) in cardiac myocytes under conditions of stimulation previously shown to cause activation of pyruvate dehydrogenase. METHODS: [Ca2+]m was monitored in single, isolated rat cardiac myocytes using fluorescence microscopy following the loading of the cells with the fluorescent chelating agent indo-1, in its permeant acetoxymethylester form, and the selective quenching of cytosolic fluorescence with MnCl2. The extent of contraction upon electrical stimulation was also measured. RESULTS: Electrical stimulation at 2 Hz and higher frequency raised [Ca2+]m significantly, and this was potentiated by exposure to isoprenaline. However, isoprenaline had no effect in quiescent cells, in which [Ca2+]m was raised above resting values by partial replacement of Na+ in the medium. The mitochondrial uncoupling agent carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) raised [Ca2+]m in unstimulated cells, but lowered it in cells subjected to electrical stimulation at 2 Hz or more, to partial Na+ replacement, or to the alkaloid veratridine. CONCLUSIONS: The values of [Ca2+]m in quiescent myocytes (approximately 100 nmol.litre-1) would be associated with very little activation by Ca2+ of pyruvate dehydrogenase phosphatase, based on determination of K0.5 value of 650 nmol.litre-1 in work with mitochondrial suspensions. By contrast, the values of [Ca2+]m associated with electrical stimulation at 2 Hz or greater in the presence of beta adrenergic activation (> 500 nmol.litre-1) would be associated with significant dehydrogenase activation. The effect of beta adrenergic activation is only seen in the presence of electrical stimulation and probably involves enhancement of systolic transients in cytosol [Ca2+]. Effects of uncoupling agents validate the conclusions on the direction and magnitude of the mitochondrial Ca2+ gradient in situ in living myocytes.