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.

In an earlier study we showed a transient doubling of mitochondrial matrix (m) [Ca 2+ ] in response to added ADP and proposed that greater buffering of Ca 2+ by ATP than by ADP is largely responsible for the increase. Previous studies have described mitochondrial volume contraction in response to ADP. Thus we explored if reduced mitochondrial volume with ADP also contributes to the apparent increase in m[Ca 2+ ]. Guinea pig heart mitochondria were isolated by differential centrifugation. Mitochondria were suspended in respiration media, including 1 mM EGTA, 1 mM MgCl 2 , 140 mM KCl, and 5 mM K 2 HPO 4 . Mitochondria were energized with pyruvic acid followed by 250 μM ADP. Mitochondrial volume was measured by light scattering at a wavelength of 520 nm. Alamethicin (5 μg/ml) was added at the end to attain maximal matrix volume. Changes in matrix volume after addition of ADP were calculated as a percentage of maximal volume change. We showed a matrix contraction in response to ADP of approximately 5% of the maximal change in matrix volume. We conclude that although matrix contraction can contribute to the increase in [Ca 2+ ] after addition of ADP, it explains only a small proportion of the rise in [Ca 2+ ] during phosphorylation of ADP, suggesting differential Ca 2+ buffering of ADP and ATP remains a major factor.

Mitochondrial free Ca2+ ([Ca2+]m) is regulated by Ca2+ influx via Ca2+ uniporter and Ca2+ efflux via Na+/Ca2+ exchanger as well as by Ca2+ buffering via mitochondrial metabolites and proteins. However, [Ca2+]m regulation during transient state‐3 respiration is not well understood. To test competing hypotheses and gain a quantitative understanding of [Ca2+]m regulation, we developed a detailed computational model by integrating our recent biophysical models of cation transporters into our existing model of mitochondrial bioenergetics. The model also accounts for binding and buffering of cations with proteins and metabolites, including ATP, ADP and Pi. Experiments were conducted to measure [Ca2+]m, [Ca2+]e, ΔΨm, [NADH]m, and matrix volume in guinea pig heart mitochondria suspended in Na+, Mg2+, and Ca2+ free buffer (1 mM EGTA) respiring with 0.5 mM pyruvic acid. Dynamics were inferred by adding CaCl2 (0.25, 0.5, 0.65, 0.75, 0.85, 1.0 mM) followed by 250 μM ADP with or without 25 μM RuR, 1 mM MgCl2, and ANT and F1F0‐ATPase blockers. Model analyses of the data suggest (a) [Ca2+]m is regulated by Ca2+ phosphate precipitation, (b) [Ca2+]m is a feedback inhibitor and [Mg2+]e is a competitive inhibitor of Ca2+ influx, (c) matrix [ADP] may activate Ca2+ influx during ADP phosphorylation, and (d) ATP synthesis leads to matrix contraction and contributes to the transient increases in [Ca2+]m during state‐3 respiration.

Mitochondrial electron transport chain complexes can be major sources of ROS. Several mechanisms are responsible for modulating ROS production, possibly including mitochondrial Ca2+ uptake. Here we tested effects of added buffer CaCl2 on ROS generation from complex I in the presence of rotenone, and from complex III in the presence of antimycin A. Guinea pig heart mitochondria (n=6) were isolated by differential centrifugation and suspended in respiration media containing amplex red and horseradish peroxidase to measure the rate of H2O2 generation. Increasing concentrations of buffered CaCl2 were added to the mitochondrial suspension. Complex I substrate pyruvate (10 mM) or complex II substrate succinate (10 mM) was added followed by either rotenone (10 μM) or antimycin A (5 μM) to block complex I or III, respectively. Compared to no added CaCl2 in the respiratory buffer, the slope of the H2O2 signal in the presence of pyruvate + rotenone increased respectively by 1.3±0.1, 2.1±0.2, 3.4±0.4, 4.5±0.3 times with 10, 25, 50, and 100 μM added external CaCl2. In contrast, H2O2 generation from complex III in the presence of antimycin A did not change with increasing CaCl2, whereas H2O2 generation from complex I in the presence of succinate (due to reversed electron flow) decreased with increasing buffer CaCl2. Moreover, H2O2 generation from complex III in the presence of antimycin A and rotenone in mitochondria supported with succinate did not change with increased buffer CaCl2. We conclude that adding CaCl2 to the buffer enhances H2O2 generation from complex I only during blocked downstream electron transport. This emphasizes the impact of matrix Ca2+ loading on electron leak leading to free radical formation only under conditions of inhibited electron flow at complex I.

ADP influx and ADP phosphorylation may alter mitochondrial free [Ca2+] ([Ca2+](m)) and consequently mitochondrial bioenergetics by several postulated mechanisms. We tested how [Ca2+](m) is affected by H2PO4(-) (P(i)), Mg2+, calcium uniporter activity, matrix volume changes, and the bioenergetic state. We measured [Ca2+](m), membrane potential, redox state, matrix volume, pH(m), and O2 consumption in guinea pig heart mitochondria with or without ruthenium red, carboxyatractyloside, or oligomycin, and at several levels of Mg2+ and P(i). Energized mitochondria showed a dose-dependent increase in [Ca2+](m) after adding CaCl2 equivalent to 20, 114, and 485 nM extramatrix free [Ca2+] ([Ca2+](e)); this uptake was attenuated at higher buffer Mg2+. Adding ADP transiently increased [Ca2+](m) up to twofold. The ADP effect on increasing [Ca2+](m) could be partially attributed to matrix contraction, but was little affected by ruthenium red or changes in Mg2+ or P(i). Oligomycin largely reduced the increase in [Ca2+](m) by ADP compared to control, and [Ca2+](m) did not return to baseline. Carboxyatractyloside prevented the ADP-induced [Ca2+](m) increase. Adding CaCl2 had no effect on bioenergetics, except for a small increase in state 2 and state 4 respiration at 485 nM [Ca2+](e). These data suggest that matrix ADP influx and subsequent phosphorylation increase [Ca2+](m) largely due to the interaction of matrix Ca2+ with ATP, ADP, P(i), and cation buffering proteins in the matrix.

Mg2+ is known to limit Ca2+ uptake by mitochondria through the Ca2+ uniporter. Changes in matrix Ca2+ concentration are an important signaling pathway in mitochondrial function as well as in apoptosis. In a previous study we showed an increase in matrix free Ca2+ in response to added ADP in MgCl2 free buffer. Because of the presumed role of Mg2+ in mitochondrial regulation of Ca2+ we explored the effects of buffer Mg2+ on matrix Ca2+ uptake and buffering in isolated mitochondria. Guinea pig heart mitochondria were isolated by differential centrifugation, loaded with the fluorescent dye Indo 1 AM and then suspended in respiration media, containing 1 mM of EGTA, with or without added 1 mM MgCl2. To the mitochondrial suspension was added 0.5 mM pyruvic acid, either 0.25, 0.5 or 0.75 mM CaCl2, and 250 μM ADP. Adding 0.25, 0.5 and 0.75 mM Ca2+ caused a dose-dependent increase in matrix Ca2+ of 14, 35 and 45%, respectively, in the group without Mg2+ in the buffer, and 6, 18 and 42%, respectively, in the group with Mg2+ in the buffer. The differences in uptake between Mg2+ and no Mg2+ groups were significant in the 0.25 and 0.5 mM groups, but not in the 0.75 mM group. The additional increase in matrix free Ca2+ in response to ADP without Mg2+ was 9, 11 and 9% for the 0.25, 0.5 and 0.75 mM Ca2+ groups, respectively. These additional increases in matrix free Ca2+ with ADP were not significantly altered by Mg2+. We conclude that external Mg2+ alters the uptake of Ca2+ into the mitochondrial matrix, but does not alter the increase in matrix ionized Ca2+ after addition of ADP.

Ischemia-reperfusion injury (IRI) is associated with mitochondrial permeability transition pore (mPTP) opening and impaired mitochondrial respiration. Hypothermia attenuates IRI. We examined mitochondrial function in mitochondria obtained from isolated hearts subjected to warm or cold ischemia. Guinea pig isolated hearts were perfused at constant pressure with Krebs-Ringer's solution at 37°C and subjected to 30 min global ischemia at 37°C or 17°C. After 5 min of reperfusion mitochondria were isolated. Mitochondrial [Ca2+]m, membrane potential (ΔΨm), and NADH were measured by spectrophotometry at appropriate wavelengths with indo-1, BCECF, rhodamine 123 fluorescent dyes, and autofluorescence, respectively. After energizing with pyruvic acid, 0-100 μM CaCl2 (0.03-60 μM free [Ca2+]e) was added followed by 250 μM ADP. Ca2+ -induced mPTP opening was assessed by collapse of ΔΨm. 10 μM [Ca2+]e resulted in mPTP opening after 37°C IRI, but only at 35 μM [Ca2+]e after 17°C IRI. ADP decreased ΔΨm and NADH and increased [Ca2+]m in all mitochondria, but the fall in ΔΨm was greater and the responses to ADP with Ca2+ overloading were worse after 37°C IRI vs. 17°C IRI. The incidence of no state 4 respiration was 25% with no added CaCl2 after 37°C IRI and 0% after 17°C IRI. This study shows that hypothermia prevents IRI damage through pathways restricting mitochondrial Ca2+ loading and preserves mitochondrial redox state and respiration. Moreover, mitochondria protected during ischemia with hypothermia were more resistant to Ca2+-induced mPTP opening and oxidative phosphorylation was better preserved. Hypothermia might prevent conformational changes in the F1F0-ATPsynthase and the ADP/ATP carrier, leading to better mitochondrial function and a resistance to mPTP opening as the ADP/ATP carrier is associated with mPTP opening.

Hypothermia decreases ischemia‐reperfusion (IR) injury. IR injury has been associated with mitochondrial permeability transition pore (PTP) opening, and decreased activity of the electron transport chain. We studied mitochondria from isolated hearts subjected to normothermic and hypothermic ischemia. Guinea pig isolated hearts were perfused at constant pressure with Krebs‐Ringer's solution at 37 ºC and subjected to 30 min of ischemia at 37ºC or 17ºC. After 5 min of reperfusion mitochondria were isolated. Mitochondrial [Ca2+]m, pH, membrane potential (ΔΨm) and NADH were measured spectrophotometrically with indo 1, BCECF, rhodamine 123 and autofluorescence. After energizing with pyruvic acid, [CaCl2] (0‐100 μM) and ADP were added. Adding CaCl2 caused a dose dependent [Ca2+]m increase. Ca2+ induced PTP opening, assessed by collapse of ΔΨm, and prevented by cyclosporine A, occurred after adding 50 μM CaCl2 after 37ºC IR, and adding 100 μM CaCl2 after 17ºC IR. ADP decreased ΔΨm, pH and NADH and increased [Ca2+]m in all mitochondria. These bioenergetic markers recovered after 17ºC IR, but not after 37ºC IR. Our study shows that hypothermia protects mitochondria against IR injury as shown by resistance to Ca2+ induced PTP opening and appropriate responses to ADP. This study emphasizes the importance of mitochondrial function in protecting against IR injury, and gives insight into how hypothermia reduces IR injury.

Introduction: Mitochondrial (m) Ca2+ uptake occurs mainly via the Ca2+ uniporter (CU) and is dependent on the electrical and chemical gradient. We measured Ca2+ uptake at deceasing membrane potentials (ΔΨm) in isolated mitochondria. Methods: m[Ca2+], ΔΨm, pH, and NADH fluorescence were measured using indo-1, rhodamine 123, BCECF and autofluorescence, respectively, in isolated guinea pig heart mitochondria. After energizing with pyruvic acid, 0, 10, 20, 30 or 100 μM of the protonophore dinitrophenol (DNP) was added to reduce ΔΨm to 0, 3, 5, 9 and 80% of the maximal depolarization elicited by the protonophore CCCP, after which 10 and 25 mM [CaCl2] ([Ca2+] = 80 and 130 nM) were added. Results: Partial depolarization resulted in decreased Ca2+ uptake. Adding 25 μM Ca2+ without DNP gave a Ca2+ uptake of 44 nM/s. Partial depolarization decreased Ca2+ uptake in a dose dependent fashion (30, 28, 18, 10 nM/s with 10, 20, 30 or 100 nM DNP). Adding 10 μM CaCl2 gave an uptake of 4.1, 3.6, 2.5, 2.8, 0.9 nM/s with 0, 10, 20, 30, 100 μM DNP, respectively. After 10, 20 and 30 nM DNP, m[Ca2+] did not attain a steady state after the initial Ca2+ uptake. DNP alone decreased matrix pH, and addition of CaCl2 caused additional decreases in pH. Conclusion: We demonstrate the importance of the electrical and chemical gradients for Ca2+ uptake. We show that mild depolarization reduced the rate of Ca2+ influx, but it did not decrease total steady-state m[Ca2+] after 10 min. Only full depolarization of ΔΨm, as observed with 100 μM DNP, resulted in a lower total m[Ca2+]. These results provide additional insight in understanding the dynamic vs steady-state transport of Ca2+ via the CU and its mutual dependence on ΔΨm and extra-matrix [Ca2+].

Introduction: Several processes influence mitochondrial matrix pH such as the state of respiration (states 2,3,4), uncouplers, proton leak, flux of other ions, and substrate utilized. We compared changes in matrix pH during phosphorylation of ADP to ATP (state 3) in the presence of NADH-linked substrate pyruvate (10 mM) or FADH2-linked substrate succinate (10 mM+rotenone). Methods: Guinea pig heart mitochondria were isolated through differential centrifugation and loaded with BCECF-AM to measure matrix pH by fluorescence spectrophotometry. Respiration, NADH, and Δψm were also measured. Results: Addition of either substrate caused matrix alkalinization. Addition of ADP (250 μM) to initiate state 3 respiration caused a marked decrease in matrix pH, which was larger (% max ΔpH with CCCP) and longer in succinate/rotenone (46±1%, 55±4 s) vs. pyruvate (20±3%, 28±2 s). Decreases in NADH and Δψm during state 3 were also larger and longer with succinate/rotenone than pyruvate. On conversion of all ADP to ATP (state 4), all variables returned to state 2 levels. Corresponding values for O2 consumption (states 2,3,4 in μmol/hr/mg) for succinate/rotenone and pyruvate, respectively, were: 3.4±0.1, 12.7±0.4, 4.4±0.2, and 0.9±0.04, 12.8±0.6, 1.2±0.06. Conclusion: The degree and extent of matrix acidity is dependent on ADP phosphorylation rate, TCA turnover rate, and the number of reducing equivalents produced (proton pumping). Per electron pair, there are 10 H+ pumped per NADH and 6 H+ pumped per FADH2. The substrate -induced differences in pH during state 3 may be due to the differences in number of protons pumped by pyruvate vs. succinate (+rotenone). A mechanistic model of mitochondrial bioenergetics and pH handling may help to characterize these differences.

Mitochondrial free Ca2+ ([Ca2+]m) is regulated by cation fluxes via Ca2+ uniporter (CU), Na+/Ca2+ exchanger (NCE), Na+/H+ exchanger (NHE), and Ca2+/H+ exchanger (CHE) as well as via Ca2+ buffering by mitochondrial proteins. However, the regulation of [Ca2+]m via metabolite dependent dynamic Ca2+ buffering in the matrix during transient state‐3 respiration is not well known. To gain a quantitative understanding of this Ca2+ buffering, we developed a computational model by integrating our recent biophysical models of CU, NCE, NHE and CHE into our existing model of mitochondrial bioenergetics. The model also accounts for binding and buffering of cations with metabolites, including ATP, ADP and Pi. Experiments were performed to spectrofluorometrically measure [Ca2+]m, pHm, ΔΨm and NADH redox state in guinea pig heart mitochondria suspended in Na+ and Ca2+ free buffer (ensured by ~50 μM EGTA) with 0.5 mM pyruvic acid (HPyr). Dynamics were inferred with various addition of CaCl2 (0, 10, 25 μM CaCl2; 16, 88, 130 nM [Ca2+]m) followed by 250 μM ADP with or without ANT and F1F0‐ATPase blockers. Model analysis of the data on (i) initial decrease of [Ca2+]m with addition of HPyr, and (ii) transient increases of [Ca2+]m with addition of ADP suggests metabolite dependent dynamic [Ca2+] buffering in the matrix. This model will be helpful to understand mechanisms by which [Ca2+]m regulates mitochondrial energy metabolism.

Introduction: Matrix free [Ca2+] (m[Ca2+]) is believed to be a key regulator of mitochondrial function. The effect of differential buffering of calcium by ADP, ATP and Pi on m[Ca2+] levels has not been examined. We tested how m[Ca2+] is increased by ADP/ATP transport and phosphorylation, and if increased m[Ca2+] alters the bioenergetic state. Materials and Methods: Guinea pig heart mitochondria were isolated by differential centrifugation. Respiration and m[Ca2+], using indo-1 fluorescence, and corrected for NADH autofluorescence, were measured. After energizing mitochondria with 0.5 mM pyruvic acid, 0, 10, 25 μM CaCl2 (16, 88, 130 nM [Ca2+]) was added to the suspension before adding 250 μM ADP, in the presence or absence of ADP/ATP carrier blocker carboxyatractyloside (CATR) or F1F0ATPase blocker oligomycin (OMN). Results: m[Ca2+] increased proportionately with addition of CaCl2. ADP caused an additional increase to 100±6% in m[Ca2+] after 25 μM CaCl2. This was due to lesser binding of ADP vs. ATP to Ca2+. The rise in m[Ca2+] after ADP was reversed after all ADP was converted to ATP. With OMN the increase after ADP was lower (18±6%), but remained elevated as ADP was not phosphorylated to ATP. CATR completely blocked the ADP -induced increases in m[Ca2+] because matrix ADP transport was blocked. State 2 and 4 respiration, but not state 3, increased 14% and 18% with 25 μM CaCl2. NADH decreased with ADP alone, but NADH was not altered by adding CaCl2. Discussion: These results show that ADP transport into mitochondria and ADP conversion to ATP have significant effects on m[Ca2+]. Acutely changing buffer [CaCl2] has limited effects on redox state, although m[Ca2+] is believed to stimulate several dehydrogenases. However the k0.5 (1 μM) for this effect is only reached by adding ADP after 25 μM CaCl2.

Application of mild hypothermia during the initial critical period of reperfusion after cardiac ischemia may slow mitochondrial and cell metabolism sufficiently to attenuate cardiac damage and better preserve mechanical function. To test this we isolated guinea pig hearts for perfusion at constant pressure with Krebs‐Ringer's solution at pH 7.4. Left ventricular pressure (LVP), coronary flow (CF), and O2 consumption were measured and continuous NADH/FAD and mitochondrial (m) [Ca2+] were assessed in the LV wall by fluorescence spectrophotometry in the absence or presence of indo 1 + MnCl2 to quench cell Ca2+. Hearts were subjected to 30 min ischemia at 37ºC, and then reperfused at either 32ºC or 37ºC for 60 min followed by additional 60 min reperfusion at 37ºC. Values up to reperfusion were similar in both groups. Developed LVP and m[Ca2+] were both lower at 32ºC vs. 37ºC. At the end of reperfusion the 32ºC group showed improved developed LVP (62 % of baseline) when compared to the non‐treatment group (50 %). There were no noticeable improvements in CF, NADH, FAD, and mCa2+ loading during later perfusion. Lower contractility and m[Ca2+] by hypothermia during the critical early reperfusion period may protect the heart during later warm reperfusion.

Mitochondrial (m) Ca2+ uptake occurs mainly via the Ca2+ uniporter and is highly dependent on the membrane potential (Δ Ψ m). We measured Ca2+ uptake at decreasing ΔΨ m in isolated mitochondria in the presence and absence of oligomycin (OMN) to test the effect of complex V reversal. [Ca2+]m, ΔΨm, pHm, and NADHm were measured spectrophotometrically using indo‐1, rhodamine 123, BCECF fluorescence and autofluorescence, respectively, in guinea pig heart isolated mitochondria. After energizing with pyruvic acid, 0, 10, 20, 30 or 100 µM dinitrophenol (DNP) was added to stepwise reduce Δ?m, after which 80 and 130 nM [Ca2+]e was added. OMN was added in some experiments to block complex V. The effect of DNP to decrease Δ?m, was exaggerated in the presence of OMN. Partial depolarization decreased the rate of matrix Ca2+ uptake, while uptake of Ca2+ resulted in further depolarization. With partial depolarization [Ca2+]m did not reach steady state but increased despite further depolarization. OMN reversed this increase and actually caused a decrease in [Ca2+]m with greater depolarization. pHm and NADHm were also lower with OMN. Our study demonstrates the importance of 1) electrical and chemical gradients for Ca2+ uptake, and 2) a likely effect of ATP hydrolysis at complex V to support ΔΨ m even during mild, graded uncoupling. Proton leaks may lead to reversal of ATP synthesis to maintain membrane potential. NIH, AHA, VA Merit grants.

Introduction: Mitochondrial free [Ca2+] ([Ca2+]m) is regulated by cation fluxes through the Ca2+ uniporter (CU), Na+/Ca2+ exchanger (NCE), Na+/H+ exchanger (NHE), and Ca2+/H+ exchanger (CHE) as well as via Ca2+ buffering by the mitochondrial proteins. However, the regulation of [Ca2+]m via ATP/ADP-dependent dynamic Ca2+ buffering mechanism inside the mitochondrial matrix during transient state-3 respiration is not well known. Methods: To gain a quantitative understanding of this Ca2+ buffering phenomenon, we developed a computational model of mitochondrial bioenergetics and Ca2+ handling by integrating our recent biophysical models of the CU, NCE, NHE, and CHE into our well-validated model of mitochondrial oxidative phosphorylation, TCA cycle, and electrophysiology. The model also accounts for binding and buffering of cations with metabolites, including ATP, ADP and Pi. Experiments were performed to spectrofluorometrically measure [Ca2+]m, pHm, membrane potential (ΔΨm), and NADH redox state in guinea pig heart mitochondria suspended in Na+ and Ca2+ free buffer medium (ensured with ∼50 μM of EGTA) with 0.5 mM pyruvic acid (HPyr). Dynamics were inferred with various addition of CaCl2 (0, 10, 25 μM of CaCl2; 16, 88, 130 nM of free [Ca2+] followed by 250 μM of ADP in the presence or absence of carboxyatractyloside (ANT blocker) and oligomycin (F1F0-ATPase blocker). Results and Discussion: Model analysis of the data on (i) initial decrease of [Ca2+]m with addition of Na+-independent substrate HPyr, and (ii) transient increases of [Ca2+]m with addition of ADP suggests ATP/ADP-dependent dynamic Ca2+ buffering inside the cardiac mitochondrial matrix. This model will be helpful to understand mechanisms by which [Ca2+]m both regulates, and is modulated by, mitochondrial energy metabolism.

Introduction: K+ influx into the respiring mitochondrial matrix is balanced by K+ efflux via K+/H+ exchange (KHE). Quinine (QN) is a reversible inhibitor of KHE. We have shown that QN blocks matrix K+ efflux when the K+ ionophore valinomycin is given to increase matrix [K+]. However QN may have other effects on mitochondria. Here we tested the effects of QN on mitochondrial respiration. Methods: Guinea pig heart mitochondria were isolated by differential centrifugation and then suspended in either KCl or choline Cl media inside a respirometer. Either the complex 1 substrate pyruvate (10 mM) or the complex 2 substrate succinate (10 mM) with rotenone (10 μM) was added to initiate state 2 respiration. QN (500 μM) was added to inhibit KHE. State 3 was initiated by adding ADP (250 μM) and state 4 occurred when ADP was converted to ATP. Results: In KCl buffer with pyruvate, QN increased states 2 and 4 respiration by 56±8% and by 48±10%, respectively, and decreased state 3 by 15±2%. With succinate and rotenone, QN increased states 2 and 4 respiration by 37±3% and by 15±2%, respectively, and decreased state 3 by 26±1%. QN had similar effects on respiration in choline Cl buffer. Conclusion: The similar effects of QN on respiration in both media suggest a K+-independent mechanism of QN, which also may be acting as an uncoupler to bring H+ inside the matrix. Additional experiments show that QN lowers matrix pH without changing membrane potential. More studies with QN and other putative blockers are required to reveal the mechanism by which QN affects mitochondrial transport and bioenergetics.

Ranolazine (RAN) is believed to block a late Na+ (NaL) current that may be activated by reactive oxygen species (ROS) via peroxidation in cardiac cells during ischemia. Na+ loading contributes to cell Ca2+ loading via Na+/Ca2+ exchange (NCE) and cell damage on reperfusion. We tested if RAN treatment during ischemia improves cardiac function on reperfusion and reduces superoxide (O2 ?−) emission. Left ventricular pressure (LVP) and coronary flow (CF) were measured in guinea pig isolated hearts perfused with Krebs‐Ringer's solution; O2 ?− emission was assessed in the LV wall, on‐line, by fluorescence spectrophotometry using DHE. RAN (5 µM), infused for 1 min before 30 min global ischemia, itself decreased heart rate by 20% and LVP by 30% and increased CF by 20%. During late ischemia O2▸− emission increased less with RAN (20% vs. 40% control). At 15 min reperfusion O2▸− emission was reduced 20% by RAN vs. control. At 120 min reperfusion the RAN group (vs. control) exhibited higher LVP (68% vs. 55%) and CF (72% vs. 58%), and reduced incidence of V fibrillation (16% vs. 100%). Ischemia and consequent ROS release are believed to induce a persistent NaL current so blocking this current may reduce Ca2+ loading via NCE. These studies suggest a link between ROS‐induced Na+ loading via NaL and LV function during reperfusion. The decrease in O2▸− emission with RAN during ischemia is likely secondary to improved mitochondrial bioenergetics.

We reported that alkaline buffer activated Na+/H+ exchanger (NHE) in isolated hearts and increased mitochondrial Ca2+ (mCa2+) and ROS production, which were abated by inhibiting NHE. We tested if blocking mCa2+ uniporter with ruthenium red (RuR, 2.5 μM) before and after ischemia, would reduce mCa2+ overload and ROS, and restore NADH and cardiac function during activated NHE. Guinea pig isolated hearts were perfused (HEPES buffer) with or without RuR at pH 7.4 (control), pH 8 to activate, or pH 6.5 to inhibit, NHE for 10 min before, after, and during 35 min ischemia. NADH, mCa2+, and ROS were assessed at the LV wall by fluorescence. We found that mCa2+ increased less during ischemia in all groups in the presence of RuR. NADH was higher with pH 8+RuR vs pH 8 alone, but RuR had no added effect in other pH groups. ROS was higher after pH 8 alone than in other groups, but adding RuR did not decrease ROS. pH 8+RuR did not protect LVP function or restore coronary flow better than pH 8, but pH 7.4+RuR was better than pH 7.4, pH 7.4 was better than pH 8±RuR, and pH 6.5 was most protective. Results indicate that RuR reduced mCa2+ loading (all pHs) and improved redox state (pH 8), did not reduce ROS (all pHs), and improved function (pH 7.4). These data show that blocking the mCa2+ uniporter reduces mCa2+ loading at all NHE activities but suggest that reducing mCa2+ may be less important than reducing ROS production for improving cardiac function after ischemia.