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

Cardiac mitochondrial matrix (m) free Ca2+ ([Ca2+](m)) increases primarily by Ca2+ uptake through the Ca2+ uniporter (CU). Ca2+ uptake via the CU is attenuated by extra-matrix (e) Mg2+ ([Mg2+](e)). How [Ca2+](m) is dynamically modulated by interacting physiological levels of [Ca2+](e) and [Mg2+](e) and how this interaction alters bioenergetics are not well understood. We postulated that as [Mg2+](e) modulates Ca2+ uptake via the CU, it also alters bioenergetics in a matrix Ca2+-induced and matrix Ca2+-independent manner. To test this, we measured changes in [Ca2+](e), [Ca2+](m), [Mg2+](e) and [Mg2+](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 (Delta I-m), and respiration. We found that > 0.125 mM MgCl2 significantly attenuated CU-mediated Ca2+ uptake and [Ca2+](m). Incremental [Mg2+](e) did not reduce initial Ca(2+)uptake but attenuated the subsequent slower Ca2+ uptake, so that [Ca2+](m) remained unaltered over time. Adding CaCl2 without MgCl2 to attain a [Ca2+](m) from 46 to 221 nM enhanced state 3 NADH oxidation and increased respiration by 15 %; up to 868 nM [Ca2+](m) did not additionally enhance NADH oxidation or respiration. Adding MgCl2 did not increase [Mg2+](m) but it altered bioenergetics by its direct effect to decrease Ca2+ uptake. However, at a given [Ca2+](m), state 3 respiration was incrementally attenuated, and state 4 respiration enhanced, by higher [Mg2+](e). Thus, [Mg2+](e) without a change in [Mg2+](m) can modulate bioenergetics independently of CU-mediated Ca2+ transport.