Cytoplasmic Na+-dependent modulation of mitochondrial Ca2+ via electrogenic mitochondrial Na+-Ca2+ exchange

To clarify the role of mitochondrial Na+-Ca2+ exchange (NCXmito) in regulating mitochondrial Ca2+ (Ca-mito(2+)) concentration at intact and depolarized mitochondrial membrane potential (Delta Psi(mito)), we measured Ca-mito(2+) and Delta Psi(mito) using fluorescence probes Rhod-2 and TMRE, respectively, in the permeabilized rat ventricular cells. Applying 300 nM cytoplasmic Ca2+ (Ca-c(2+)) increased Ca-mito(2+) and this increase was attenuated by cytoplasmic Na+ (Na-c(+)) with an IC50 of 2.4 mM. To the contrary, when Delta Psi(mito) was depolarized by FCCP, a mitochondrial uncoupler, Na-c(+) enhanced the Ca-c(2+)-induced increase in Ca-mito(2+) with an EC50 of about 4 mM. This increase was not significantly affected by ruthenium red or cyclosporin A. The inhibition of NCXmito by CGP-37157 further increased Ca-mito(2+) when Delta Psi(mito) was intact, while it suppressed the Ca-mito(2+) increase when Delta Psi(mito) was depolarized, suggesting that Delta Psi(mito) depolarization changed the exchange mode from forward to reverse. Furthermore, Delta Psi(mito) depolarization significantly reduced the Ca-mito(2+) decrease via forward mode, and augmented the Ca-mito(2+) increase via reverse mode. When the respiratory chain was attenuated, the induction of the reverse mode of NCXmito hyperpolarized Delta Psi(mito), while Delta Psi(mito) depolarized upon inducing the forward mode of NCXmito. Both changes in Delta Psi(mito) were remarkably inhibited by CGP-37157. The above experimental data indicated that NCXmito is voltage dependent and electrogenic. This notion was supported theoretically by computer simulation studies with an NCXmito model constructed based on present and previous studies, presuming a consecutive and electrogenic Na+-Ca2+ exchange and a depolarization-induced increase in Na+ flux. It is concluded that Ca-mito(2+) concentration is dynamically modulated by Na-c(+) and Delta Psi(mito) via electrogenic NCXmito.