Regulation of cardiac mitochondrial calcium by average extramitochondrial calcium

A system to perifuse isolated rat heart mitochondria was designed to study the relationship between mitochondrial matrix free Ca²⁺ and extramitochondrial free Ca²⁺ under conditions in which the latter concentration could oscillate over a range typical of that expected in vivo. We tested the hypothesis that the level of intramitochondrial Ca²⁺ responds to the average extramitochondrial Ca²⁺ in the heart. Mitochondria were immobilized within an optical chamber for measurement of endogenous NAD(P)H and fura 2 fluorescence. NAD(P)H increased significantly on provision of substrates and decreased reversibly in the presence of ADP, indicating maintenance intact coupled respiration by this preparation. Matrix free Ca²⁺ was measured using fura 2-loaded mitochondria and, in parallel experiments, media free Ca²⁺ was measured with fura 2 in the absence of mitochondria. Oscillation of extramitochondrial Ca²⁺ from <0.1 μM to ~2 μM at frequencies of 0.5, 1.0, and 1.25 cycles/s produced steady-state levels of matrix Ca²⁺ that were independent of frequency but proportional to the average media free Ca²⁺ concentration. Matrix Ca²⁺ increased to a steady state on an increase in the extramitochondrial average Ca²⁺ concentration with a half-time (t( 1/2 )) of ~2 min at 22°C. Oscillation of mitochondrial Ca²⁺ was not observed under any conditions tested. The data are taken to indicate that in vivo, the concentration of mitochondrial matrix free Ca²⁺ is a steady state that is proportional to the average extramitochondrial Ca²⁺ concentration and that changes in the latter represent a mechanism of signal transduction from the cytosol to the mitochondrial matrix.