Mg\u3csup\u3e2+\u3c/sup\u3e Differentially Regulates Two Modes of Mitochondrial Ca\u3csup\u3e2+\u3c/sup\u3e Uptake in Isolated Cardiac Mitochondria: Implications for Mitochondrial Ca\u3csup\u3e2+\u3c/sup\u3e Sequestration

The manner in which mitochondria take up and store Ca2+ remains highly debated. Recent experimental and computational evidence has suggested the presence of at least two modes of Ca2+ uptake and a complex Ca2+ sequestration mechanism in mitochondria. But how Mg2+ regulates these different modes of Ca2+ uptake as well as mitochondrial Ca2+ sequestration is not known. In this study, we investigated two different ways by which mitochondria take up and sequester Ca2+ by using two different protocols. Isolated guinea pig cardiac mitochondria were exposed to varying concentrations of CaCl2 in the presence or absence of MgCl2. In the first protocol, A, CaCl2 was added to the respiration buffer containing isolated mitochondria, whereas in the second protocol, B, mitochondria were added to the respiration buffer with CaCl2 already present. Protocol A resulted first in a fast transitory uptake followed by a slow gradual uptake. In contrast, protocol B only revealed a slow and gradual Ca2+ uptake, which was approximately 40 % of the slow uptake rate observed in protocol A. These two types of Ca2+ uptake modes were differentially modulated by extra-matrix Mg2+. That is, Mg2+ markedly inhibited the slow mode of Ca2+ uptake in both protocols in a concentration-dependent manner, but not the fast mode of uptake exhibited in protocol A. Mg2+ also inhibited Na+-dependent Ca2+ extrusion. The general Ca2+ binding properties of the mitochondrial Ca2+ sequestration system were reaffirmed and shown to be independent of the mode of Ca2+ uptake, i.e. through the fast or slow mode of uptake. In addition, extra-matrix Mg2+ hindered Ca2+ sequestration. Our results indicate that mitochondria exhibit different modes of Ca2+ uptake depending on the nature of exposure to extra-matrix Ca2+, which are differentially sensitive to Mg2+. The implications of these findings in cardiomyocytes are discussed

Extra-matrix Mg\u3csup\u3e2+\u3c/sup\u3e Limits Ca\u3csup\u3e2+\u3c/sup\u3e Uptake and Modulates Ca\u3csup\u3e2+\u3c/sup\u3e 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 (ΔΨm), and respiration. We found that \u3e0.125 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

Slow Ca2+ Efflux by Ca2+/H+ Exchange in Cardiac Mitochondria Is Modulated by Ca2+ Re-uptake via MCU, Extra-Mitochondrial pH, and H+ Pumping by FOF1-ATPase

Mitochondrial (m) Ca2+ influx is largely dependent on membrane potential (ΔΨm), whereas mCa2+ efflux occurs primarily via Ca2+ ion exchangers. We probed the kinetics of Ca2+/H+ exchange (CHEm) in guinea pig cardiac muscle mitochondria. We tested if net mCa2+ flux is altered during a matrix inward H+ leak that is dependent on matrix H+ pumping by ATPm hydrolysis at complex V (FOF1-ATPase). We measured [Ca2+]m, extra-mitochondrial (e) [Ca2+]e, ΔΨm, pHm, pHe, NADH, respiration, ADP/ATP ratios, and total [ATP]m in the presence or absence of protonophore dinitrophenol (DNP), mitochondrial uniporter (MCU) blocker Ru360, and complex V blocker oligomycin (OMN). We proposed that net slow influx/efflux of Ca2+ after adding DNP and CaCl2 is dependent on whether the ΔpHm gradient is/is not maintained by reciprocal outward H+ pumping by complex V. We found that adding CaCl2 enhanced DNP-induced increases in respiration and decreases in ΔΨm while [ATP]m decreased, ΔpHm gradient was maintained, and [Ca2+]m continued to increase slowly, indicating net mCa2+ influx via MCU. In contrast, with complex V blocked by OMN, adding DNP and CaCl2 caused larger declines in ΔΨm as well as a slow fall in pHm to near pHe while [Ca2+]m continued to decrease slowly, indicating net mCa2+ efflux in exchange for H+ influx (CHEm) until the ΔpHm gradient was abolished. The kinetics of slow mCa2+ efflux with slow H+ influx via CHEm was also observed at pHe 6.9 vs. 7.6 by the slow fall in pHm until ΔpHm was abolished; if Ca2+ reuptake via the MCU was also blocked, mCa2+ efflux via CHEm became more evident. Of the two components of the proton electrochemical gradient, our results indicate that CHEm activity is driven largely by the ΔpHm chemical gradient with H+ leak, while mCa2+ entry via MCU depends largely on the charge gradient ΔΨm. A fall in ΔΨm with excess mCa2+ loading can occur during cardiac cell stress. Cardiac cell injury due to mCa2+ overload may be reduced by temporarily inhibiting FOF1-ATPase from pumping H+ due to ΔΨm depolarization. This action would prevent additional slow mCa2+ loading via MCU and permit activation of CHEm to mediate efflux of mCa2+.HIGHLIGHTS-We examined how slow mitochondrial (m) Ca2+ efflux via Ca2+/H+ exchange (CHEm) is triggered by matrix acidity after a rapid increase in [Ca2+]m by adding CaCl2 in the presence of dinitrophenol (DNP) to permit H+ influx, and oligomycin (OMN) to block H+ pumping via FOF1-ATP synthase/ase (complex V).-Declines in ΔΨm and pHm after DNP and added CaCl2 were larger when complex V was blocked.-[Ca2+]m slowly increased despite a fall in ΔΨm but maintained pHm when H+ pumping by complex V was permitted.-[Ca2+]m slowly decreased and external [Ca2+]e increased with declines in both ΔΨm and pHm when complex V was blocked.-ATPm hydrolysis supports a falling pHm and redox state and promotes a slow increase in [Ca2+]m.-After rapid Ca2+ influx due to a bolus of CaCl2, slow mCa2+ efflux by CHEm occurs directly if pHe is low

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+) transpor