Sodium-Induced Calcium Flux via Mitochondrial NCE is Dissimilar when Derived from Matrix and Extra-Matrix Calcium Ion Concentrations

Abstract

Free Ca2+ plays a pivotal role in regulating cellular and mitochondrial processes in cardiomyocytes. Effective regulation of mitochondrial Ca2+ (mCa2+) is vital for mitochondrial bioenergetics. Altered mCa2+ homeostasis resulting from cytosolic Ca2+ overload can lead to mitochondrial and cellular injury. mCa2+ dynamics are regulated primarily by influx via the mitochondrial Ca2+ uniporter (mCU) and efflux via the mitochondrial Na+/Ca2+ exchanger (mNCE). Ca2+ flux across the inner mitochondrial membrane is modulated by the mΔΨ and mΔpH and by the Ca2+ and Na+ ion gradients. How the mNCE dynamically regulates mCa2+ remains to be elucidated. Previous attempts to characterize mNCE kinetics were limited in scope, because they focused on monitoring changes only on extra-matrix [Ca2+] or matrix [Ca2+] statically or under limited Ca2+ loading conditions. Here we explored in detail the kinetics of mNCE in energized mitochondria isolated from guinea pig hearts. We first added CaCl2 (no buffer Na+) to increase extra-matrix [Ca2+], and observed dynamic time-dependent changes in extra-matrix [Ca2+] and matrix [Ca2+], measured by indo-1 PP and indo-1 AM fluorescence, respectively. Then the mCU was blocked by ruthenium red and mCa2+ efflux was induced by adding increasing extra-matrix amounts of NaCl. We observed that the kinetics of mNCE depend both on the preceding uptake of Ca2+ and on the subsequent uptake of extra-matrix Na+. Moreover, we found that Ca2+ efflux rates were dissimilar when derived from measurements of matrix [Ca2+] vs. extra-matrix [Ca2+], suggesting differential Ca2+ buffering. Thus, the amount and buffering of mCa2+ are crucial factors in the dynamics of Ca2+ efflux by mNCE. Our approach should yield novel findings in understanding the dynamics of Ca2+ efflux by mNCE.