Extra-matrix Mg2+ limits Ca2+ uptake and modulates Ca2+ uptake-independent respiration and redox state in cardiac isolated mitochondria.

Cardiac mitochondrial matrix (m) free Ca(2+) ([Ca(2+)]m) increases primarily by Ca(2+) uptake through the Ca(2+) uniporter (CU). Ca(2+) uptake via the CU is attenuated by extra-matrix (e) Mg(2+) ([Mg(2+)]e). How [Ca(2+)]m is dynamically modulated by interacting physiological levels of [Ca(2+)]e and [Mg(2+)]e and how this interaction alters bioenergetics are not well understood. We postulated that as [Mg(2+)]e modulates Ca(2+) uptake via the CU, it also alters bioenergetics in a matrix Ca(2+)-induced and matrix Ca(2+)-independent manner. To test this, we measured changes in [Ca(2+)]e, [Ca(2+)]m, [Mg(2+)]e and [Mg(2+)]m spectrofluorometrically in guinea pig cardiac mitochondria in response to added CaCl2 (0-0.6 mM; 1 mM EGTA buffer) with/without added MgCl2 (0-2 mM). In parallel, we assessed effects of added CaCl2 and MgCl2 on NADH, membrane potential (ΔΨm), and respiration. We found that ≥0.125 mM MgCl2 significantly attenuated CU-mediated Ca(2+) uptake and [Ca(2+)]m. Incremental [Mg(2+)]e did not reduce initial Ca(2+)uptake but attenuated the subsequent slower Ca(2+) uptake, so that [Ca(2+)]m remained unaltered over time. Adding CaCl2 without MgCl2 to attain a [Ca(2+)]m from 46 to 221 nM enhanced state 3 NADH oxidation and increased respiration by 15 %; up to 868 nM [Ca(2+)]m did not additionally enhance NADH oxidation or respiration. Adding MgCl2 did not increase [Mg(2+)]m but it altered bioenergetics by its direct effect to decrease Ca(2+) uptake. However, at a given [Ca(2+)]m, state 3 respiration was incrementally attenuated, and state 4 respiration enhanced, by higher [Mg(2+)]e. Thus, [Mg(2+)]e without a change in [Mg(2+)]m can modulate bioenergetics independently of CU-mediated Ca(2+) transport.