Dynamic buffering of mitochondrial Ca^sup 2+^ during Ca^sup 2+^ uptake and Na^sup +^-induced Ca^sup 2+^ release

In cardiac mitochondria, matrix free Ca^sup 2+^ ([Ca^sup 2+^]^sub m^) is primarily regulated by Ca^sup 2+^ uptake and release via the Ca^sup 2+^ uniporter (CU) and Na^sup +^/Ca^sup 2+^ exchanger (NCE) as well as by Ca^sup 2+^ buffering. Although experimental and computational studies on the CU and NCE dynamics exist, it is not well understood how matrix Ca^sup 2+^ buffering affects these dynamics under various Ca^sup 2+^ uptake and release conditions, and whether this influences the stoichiometry of the NCE. To elucidate the role of matrix Ca^sup 2+^ buffering on the uptake and release of Ca^sup 2+^, we monitored Ca^sup 2+^ dynamics in isolated mitochondria by measuring both the extra-matrix free [Ca^sup 2+^] ([Ca^sup 2+^]^sub e^) and [Ca^sup 2+^]^sub m^. A detailed protocol was developed and freshly isolated mitochondria from guinea pig hearts were exposed to five different [CaCl^sub 2^] followed by ruthenium red and six different [NaCl]. By using the fluorescent probe indo-1, [Ca^sup 2+^]^sub e^ and [Ca^sup 2+^]^sub m^ were spectrofluorometrically quantified, and the stoichiometry of the NCE was determined. In addition, we measured NADH, membrane potential, matrix volume and matrix pH to monitor Ca^sup 2+^-induced changes in mitochondrial bioenergetics. Our [Ca^sup 2+^]^sub e^ and [Ca^sup 2+^]^sub m^ measurements demonstrate that Ca^sup 2+^ uptake and release do not show reciprocal Ca^sup 2+^ dynamics in the extra-matrix and matrix compartments. This salient finding is likely caused by a dynamic Ca^sup 2+^ buffering system in the matrix compartment. The Na^sup +^- induced Ca^sup 2+^ release demonstrates an electrogenic exchange via the NCE by excluding an electroneutral exchange. Mitochondrial bioenergetics were only transiently affected by Ca^sup 2+^ uptake in the presence of large amounts of CaCl^sub 2^, but not by Na^sup +^- induced Ca^sup 2+^ release.[PUBLICATION ABSTRACT]