Intramitochondrial free calcium in cardiac myocytes in relation to dehydrogenase activation

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 K_0.5 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 β adrenergic activation (>500 nmol·litre⁻¹) would be associated with significant dehydrogenase activation. The effect of β 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. Cardiovascular Research 1993;27:1840-1844