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

Abstract

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 is 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 (ΔΨm), 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 Ca2+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.

Identity and function of a cardiac mitochondrial small conductance Ca 2+ -activated K + channel splice variant

“…After adding 0.2–3 mM CaCl 2 (in buffer with 2.5 mM EGTA), buffer and matrix [Ca 2+ ] can increase only within the nM range. This is apparent from a previous report [33] in which we measured external and matrix [Ca 2+ ] using a similar respiration buffer but containing 0.6 mM CaCl 2 and 1 mM EGTA; this resulted in an external [Ca 2+ ] of 300 nM and a matrix [Ca 2+ ] of 1000 nM. In buffer containing approximately 40 μM EGTA carried over with the mitochondria from the isolation buffer, with no added CaCl 2 , we observed a basal external [Ca 2+ ] of 80–100 nM and a matrix [Ca 2+ ] of 210–250 nM [34,35].…”

Mg2+ differentially regulates two modes of mitochondrial Ca2+ uptake in isolated cardiac mitochondria: implications for mitochondrial Ca2+ sequestration

“…Mitochondria from guinea pig hearts were isolated as previously described (Blomeyer et al 2013; Boelens et al 2013; Aldakkak et al 2013; Haumann et al 2010). Briefly, guinea pigs (250–350 g) were anesthetized with intraperitoneal injection of 30 mg ketamine, and 700 units of heparin for anticoagulation.…”

“…All experiments were conducted at room temperature (25 °C) using the cuvette-based fluorescence spectrophotometer described in our previous studies (Blomeyer et al 2013; Boelens et al 2013; Haumann et al 2010; Agarwal et al 2012, 2014; Aldakkak et al 2011). Pyruvic acid (PA, 0.5 mM), cyclosporine A (CsA, 0.5 μM) and MgCl 2 (0, 0.5, 1.0 mM) were always present in the buffer.…”

“…ΔΨ m was measured using the lipophilic dye TMRM (1 μM, Invitrogen™, Eugene, OR) in a ratiometric excitation approach (Scaduto and Grotyohann 1999). NADH was measured by its autofluorescence (Blomeyer et al 2013; Boelens et al 2013; Haumann et al 2010). To ensure identical conditions as for the [Ca 2+ ] m measurement, mitochondria were incubated with the appropriate concentration of DMSO for 30 min at 25 °C before measuring [Ca 2+ ] e , ΔΨ m , and NADH.…”

“…Interestingly, in a recent study, we reported that mitochondrial Ca 2+ uptake displays two different profiles of matrix Ca 2+ transient when CaCl 2 was added to mitochondrial suspension: a fast uptake of Ca 2+ followed by a slower and more gradual uptake as matrix Ca 2+ plateaus to a steady state (Boelens et al 2013). Addition of Mg 2+ to the respiration buffer appeared to have a differential effect on the two phases of the Ca 2+ transient, with the slow phase attenuated more than the fast uptake phase.…”