Extra-matrix Mg^sup 2+^ limits Ca^sup 2+^ uptake and modulates Ca^sup 2+^ uptake-independent respiration and redox state in cardiac isolated mitochondria

Cardiac mitochondrial matrix (m) free Ca^sup 2+^ ([Ca^sup 2+^]^sub m^) increases primarily by Ca^sup 2+^ uptake through the Ca^sup 2+^ uniporter (CU). Ca^sup 2+^ uptake via the CU is attenuated by extra-matrix (e) Mg^sup 2+^ ([Mg^sup 2+^]^sub e^). How [Ca^sup 2+^]^sub m^ is dynamically modulated by interacting physiological levels of [Ca^sup 2+^]^sub e^ and [Mg^sup 2+^]^sub e^ and how this interaction alters bioenergetics are not well understood. We postulated that as [Mg^sup 2+^]^sub e^ modulates Ca^sup 2+^ uptake via the CU, it also alters bioenergetics in a matrix Ca^sup 2+^-induced and matrix Ca^sup 2+^-independent manner. To test this, we measured changes in [Ca^sup 2+^]^sub e^, [Ca^sup 2+^]^sub m^, [Mg^sup 2+^]^sub e^ and [Mg^sup 2+^]^sub m^ spectrofluorometrically in guinea pig cardiac mitochondria in response to added CaCl^sub 2^ (0-0.6 mM; 1 mM EGTA buffer) with/without added MgCl^sub 2^ (0-2 mM). In parallel, we assessed effects of added CaCl^sub 2^ and MgCl^sub 2^ on NADH, membrane potential (ΔΨ^sub m^), and respiration. We found that >0.125 mM MgCl^sub 2^ significantly attenuated CU-mediated Ca^sup 2+^ uptake and [Ca^sup 2+^]^sub m^. Incremental [Mg^sup 2+^]^sub e^ did not reduce initial Ca^sup 2+^uptake but attenuated the subsequent slower Ca^sup 2+^ uptake, so that [Ca^sup 2+^]^sub m^ remained unaltered over time. Adding CaCl^sub 2^ without MgCl^sub 2^ to attain a [Ca^sup 2+^]^sub m^ from 46 to 221 nM enhanced state 3 NADH oxidation and increased respiration by 15 %; up to 868 nM [Ca^sup 2+^]^sub m^ did not additionally enhance NADH oxidation or respiration. Adding MgCl^sub 2^ did not increase [Mg^sup 2+^]^sub m^ but it altered bioenergetics by its direct effect to decrease Ca^sup 2+^ uptake. However, at a given [Ca^sup 2+^]^sub m^, state 3 respiration was incrementally attenuated, and state 4 respiration enhanced, by higher [Mg^sup 2+^]^sub e^. Thus, [Mg^sup 2+^]^sub e^ without a change in [Mg^sup 2+^]^sub m^ can modulate bioenergetics independently of CU-mediated Ca^sup 2+^ transport.[PUBLICATION ABSTRACT]