Ca²⁺ activation of heart mitochondrial oxidative phosphorylation: role of the F₀/F₁-ATPase

Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1061

	ABSTRACT

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Ca²⁺ has been postulated as a cytosolic second messenger in the regulation of cardiac oxidative phosphorylation. This hypothesis draws support from the well-known effects of Ca²⁺ on muscle activity, which is stimulated in parallel with the Ca²⁺-sensitive dehydrogenases (CaDH). The effects of Ca²⁺ on oxidative phosphorylation were further investigated in isolated porcine heart mitochondria at the level of metabolic driving force (NADH or ) and ATP production rates (flow). The resulting force-flow (F-F) relationships permitted the analysis of Ca²⁺ effects on several putative control points within oxidative phosphorylation, simultaneously. The F-F relationships resulting from additions of carbon substrates alone provided a model of pure CaDH activation. Comparing this curve with variable Ca²⁺ concentration ([Ca²⁺]) effects revealed an approximate twofold higher ATP production rate than could be explained by a simple increase in NADH or via CaDH activation. The half-maximal effect of Ca²⁺ at state 3 was 157 nM and was completely inhibited by ruthenium red (1 µM), indicating matrix dependence of the Ca²⁺ effect. Arsenate was used as a probe to differentiate between F₀/F₁-ATPase and adenylate translocase activity by a futile recycling of ADP-arsenate within the matrix, catalyzed by the F₀/F₁-ATPase. Ca²⁺ increased the ADP arsenylation rate more than twofold, suggesting a direct effect on the F₀/F₁-ATPase. These results suggest that Ca²⁺ activates cardiac aerobic respiration at the level of both the CaDH and F₀/F₁-ATPase. This type of parallel control of both intermediary metabolism and ATP synthesis may provide a mechanism of altering ATP production rates with minimal changes in the high-energy intermediates as observed in vivo.

metabolism; ATP synthesis; dehydrogenase; force-flow analysis

	INTRODUCTION

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MOST BIOLOGICAL SYSTEMS are capable of maintaining a steady-state metabolism by balancing work with biochemical energy conversion (21, 35, 40). How this metabolic steady state is achieved and controlled in the intact cell is still actively debated. The current study focuses on the regulation and control of mitochondrial energy conversion, a key element in energy metabolism of many tissues.

The mitochondrion, a putative symbiont (10), has been domesticated to perform a primary role in cellular energy metabolism. This organelle produces the major high-energy intermediate in the cytosol, ATP, by oxidative phosphorylation of ADP (4). Indeed, most of the external work performed by the mitochondrion is the production and delivery of ATP to the cytosol. The regulation of mitochondrial ATP production has been suggested to occur by a cytosolic feedback mechanism, relying on ATP hydrolysis with work for ADP and P_i generation (8). Through this mechanism, ATP synthesis was envisioned to follow ATP hydrolysis in the cytosol, paced by ADP concentration ([ADP]) and [P_i]. Studies in the heart (1, 40), brain (20), liver (41), kidney (73), and smooth muscle (72) have demonstrated that the dynamic changes in ADP and P_i with work do not adequately support a simple kinetic feedback model. Most notably are cardiac ATP hydrolysis/synthesis rates, which can change severalfold without a change in cytosolic [ADP] or [P_i] (40). These results suggest that the cytosolic network controlling work and ATP hydrolysis contains elements that modulate mitochondrial ATP production in parallel. This would permit changes in workload with minimal alterations in metabolic intermediates, such as ATP, ADP, or P_i. For this scheme to work in the heart, a cytosolic signaling system must exist that can activate both the contractile activity and oxidative phosphorylation in parallel.

An example of a single cytosolic transducer that could activate both work and biochemical energy conversion in the heart is Ca²⁺ (for reviews see Refs. 29, 33, and 34). Cytosolic [Ca²⁺] ([Ca²⁺]_c) is important in the activation of cardiac muscle contraction (56, 70). Elevation of [Ca²⁺]_c results in an electrophoretic uniport (13, 61) and RaM-dependent (28) matrix accumulation of Ca²⁺ (reviewed in Ref. 27). This accumulation enhances substrate conversion via the Ca²⁺-sensitive dehydrogenases (CaDH), i.e., pyruvate, isocitrate, and 2-oxoglutarate dehydrogenases (32, 49). CaDH activation increases the maximum rate of oxidative phosphorylation by augmenting the delivery of NADH to the respiratory chain, thereby increasing the thermodynamic driving force for ATP synthesis. Augmenting NADH delivery increases the maximum respiratory rate of heart mitochondria in a near linear fashion with concentration (51, 54, 60, 65). In addition, Ca²⁺ has been suggested to modify the ATP synthetic enzyme complex of the mitochondrion, the F₀/F₁-ATPase (62, 77), and the adenine nucleotide translocase in the liver (52, 53). These latter effects suggest that Ca²⁺ may control mitochondrial work (i.e., ATP production) in a pattern similar to cytosolic work (i.e., muscle contraction in the heart) by stimulating both the supporting intermediary metabolism and work in parallel. This parallel scheme of activation, within the cytosol and mitochondrion, could result in an increased ATP production rate for work with minimal changes in high-energy intermediates, as previously described in intact heart (5, 35, 40, 67).

The purpose of this study was to test the hypothesis that physiological [Ca²⁺] activates the F₀/F₁-ATPase, in addition to CaDH, resulting in a parallel stimulation of mitochondrial ATP production at both the carbon substrate oxidation and ADP phosphorylation steps. To perform this task, the effects of [Ca²⁺] on isolated porcine heart mitochondria were studied while simultaneously monitoring oxygen consumption [mitochondrial O₂ (mO₂)] and the metabolic driving forces at NADH and mitochondrial membrane potential (). These studies permitted the separation of net effects of CaDH activation and F₀/F₁-ATPase activity in the intact mitochondrion.

	MATERIALS AND METHODS

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Heart isolation and perfusion. Porcine hearts were harvested from heparinized (250 IU/kg iv) stage III (plane 4) -chloralose anesthetized animals (100 mg/kg iv) through a midline thoracotomy. All procedures performed were in accordance with the guidelines listed in the Animal Care and Welfare Act (7 U.S.C. 2142 § 13). The isolated heart was quickly perfused retrogradely via the aorta with 500 ml (4°C) buffer A (280 mM sucrose, 10 mM HEPES, and 0.2 mM EDTA at pH 7.2) to facilitate removal of blood and extracellular Ca²⁺. The perfused heart was cleared of all epicardial fat, blood vessels, atria, and right ventricular myocardium. The left ventricular myocardium was weighed and quartered for use in mitochondrial isolation.

Mitochondrial isolation and fluorescence standard loading. Mitochondria were isolated according to methods described in Ref. 25. Briefly, four 25-g sections of left ventricle were finely minced using scissors in 50 ml of buffer A. The minced ventricle was then treated for 15 min with 0.5 mg bovine pancreas trypsin per gram of tissue (Sigma, St. Louis, MO). The supernatant was then poured off and replaced with buffer A containing 1 mg/ml BSA and trypsin inhibitor (2.6 mg/g tissue), and maintained at 4°C for 5 min. On completion, the supernatant was replaced with buffer A containing 1 mg/ml BSA, and the suspension was quickly homogenized with two grades of Teflon homogenizers (Thomas Scientific, Swedesboro, NJ). This homogenate was centrifuged in 60-ml aliquots for 10 min (600 g) and the pellets discarded. The supernatant was recentrifuged and washed with buffer A (containing 1 mg/ml BSA) at 8,000 g for 15 min. The pelleted mitochondria were resuspended in 4 ml of buffer B (137 mM KCl, 10 mM HEPES, 2 mM P_i, 2.5 mM MgCl₂, 0.5 mM EDTA, pH 7.2) and stored on ice. Of the 4 ml of isolated mitochondria, 3.5 ml were loaded at 1 nmol/nmol cytochrome A (for assay see below) with 5-(6)-carboxy-2'-7'-dichlorofluoresceindiacetate succinimidyl ester (CF; Molecular Probes, Eugene, OR) at 4°C for 10 min for light-scattering corrections (14). Loaded and unloaded mitochondria were washed and repelleted (8,000 g for 10 min) three times (twice in buffer A containing 1 mg/ml BSA and once in plain buffer A) to remove any unloaded CF. The third mitochondrial pellet was resuspended in plain buffer B at ~15 nmol cytochrome A/ml and kept on ice until use.

Mitochondrial cytochrome A assay. Mitochondrial cytochrome A content was determined spectrophotometrically as previously described (2). Briefly, mitochondria were solubilized with a 2% solution of Triton X-100 in 100 mM Na₃PO₄ buffer (pH 7), and difference spectra were obtained with a spectrophotometer (Lambda 3B; Perkin-Elmer) between oxidized and hydrosulfide reduced mitochondrial solutions. The cytochrome content was determined as described previously (2) using the 605-nm, 630-nm wavelength pair, and a molar extinction coefficient of 12 m¹.

Respiratory rate (mO₂), membrane potential (), and NADH fluorescence. To determine the effects of Ca²⁺ on metabolism, it was necessary to deplete heart mitochondria of endogenous Ca²⁺. This was achieved with 6 min of incubation in buffer C (125 mM KCl, 20 mM HEPES, 15 mM NaCl, 5 mM MgCl₂, 1 mM K₂EDTA, 1 mM EGTA, 2 mM P_i, 0.1 mM malate, and 4 µM TPP⁺, with 3.4 mM Na₂ATP added fresh daily, pH 7) in the absence of extraneous carbon substrates. Postincubation, substrates and Ca²⁺ were added to the mitochondrial suspension to establish new steady states. Free [Ca²⁺] in buffer C was determined from binding affinity constants previously reported (23). In all cases except where noted, [Ca²⁺] are presented as calculated free concentrations.

The rate of mitochondrial O₂ consumption (mO₂) was determined in a modified closed-system respirometer described previously (51) and was used to estimate steady-state ATP production rate. Briefly, the ADP-P_i-driven rate of O₂ consumption (state 3) and subsequent state 4 rate were monitored in a custom-thermostatted chamber (37°C) with a polarographic oxygen electrode calibrated to room air. State 3 was defined as steady-state maximal ADP-P_i-driven respiration in substrate-energized mitochondria, whereas state 4 was respiration in the absence of ADP-P_i (8). Experiments were performed at mitochondrial concentrations of 1 nmol cytochrome A/ml. A magnetic stirring bar provided mixing of the mitochondrial suspension.

Oxygen consumption has the units of nanomoles of oxygen per nanomole of cytochrome A per minute and was calculated as follows

(1)

where b_O is the calculated slope from digitized oxygen recordings in change in percentage of oxygen per second,  is the solubility of oxygen in buffer for a given salt content in nanomoles per milliliter, V_c is the volume of the chamber in milliliters, and cytochrome A is the cytochrome A content in nanomoles. The oxygen solubility used was 199 nmol/ml at 37°C (9). The ATP production rate was estimated to be 2.8 moles ATP per mole of oxygen (O) consumed (see ADP/O calculations below). Estimates of mitochondrial integrity were determined from the respiratory control ratios (RCR) of mO₂ at state 3 and state 4 in buffer B, which contained 5 mM glutamate, 5 mM malate, 2 mM P_i, and 4 µM TPP⁺, and were stimulated with a single addition of 1.3 mM ADP (final). Where applicable, glutamate (G) and malate (M) were added in equimolar proportions, where 5 mM G/M represents the concentration of each substrate in the mixture.

The redox state of pyridine dinucleotides was monitored concurrently using a commercial spectroflurometer (LS50B; Perkin-Elmer) connected via an external fiber-optic bundle that was coupled to an embedded sapphire window in the experimental chamber. The entire respirometry system was housed in a light-tight box to minimize extraneous light. Mitochondrial fluorescence spectra were collected using an excitation of 340 nm (10-nm slit, 350-nm cutoff filter) and emission of 360-660 nm at 1,500 nm/s with a 15-nm slit. Control spectra were obtained in fully oxidized (0.067 mM ADP) and reduced (5 mM G/M) mitochondria, both with and without CF. From these difference spectra, model spectra of NADH (M_NADH) and CF (M_CF) were constructed. Additionally, spectra were obtained from G-10 sephadex beads (size 40-120 µm) at 1 mg/ml suspended in buffer B to model excitation light bleedthrough (M_EBT). Model spectra (M_NADH, M_CF, and M_EBT) were then fitted with a multiple linear regression and compared against experimental spectra to eliminate both primary and secondary inner filter effects as previously described (25). The linear fit was described by the following relationships

(2)

(2.1)

(2.2)

(2.3)

where F is the combined fit for all model (M) components and F_NADH, F_CF, and F_EBT refer to the corresponding fits for NADH, CF, and EBT, respectively. The algorithm determined the coefficients I_NADH, I_CF, and I_EBT as estimates of each peak's contribution to the total fluorescence spectrum. All regressions were performed iteratively until the sum of squares convergence was achieved using the Marquardt-Levenberg algorithm written in Interactive Data Language (version 5.1, Research Systems). The algorithm produces 1) the coefficients of the model spectra, 2) SD for the coefficients, 3) an F test for fit between model and experimental data, and 4) multiple linear correlation coefficients for the fitted spectra. In all cases, model and experimental spectra had a high degree of concordance (0.99 ± 0.0001, n = 1,340). Data for each experimental spectrum was presented as I_NADH/I_CF ratios to correct for inner filter effects, and normalized within each preparation to mitochondria with 5 mM G/M + 535 nM free [Ca²⁺] at state 3.

Mitochondrial membrane potential () was determined from the Nernst equilibrium of 4 µM TPP⁺ across the mitochondrial membrane. This lipophilic cation was detected using a TPP⁺ ion-selective electrode (model MEH2SW20; World Precision Instruments) and an Ag-AgCl reference electrode (model MI 402; Microelectrodes). Both electrodes were connected to a high-impedance pH meter (model 901; Orion Research) and the output amplified. The resulting signal was digitally sampled at 2 Hz via an analog-to-digital (A/D) converter and recorded using a custom program written in Workbench-Mac (Strawberry Tree). The correlation between absolute [TPP⁺] and electrode voltages was determined daily using an automated micropipetter (Microlab 500; Hamilton) to establish standard curves. It was determined that the electrode drifted over time; however, this drift could be accounted for by changes in the intercept of the standard curve over time and did not significantly alter the slope. Nonspecific binding of TPP⁺ to mitochondrial membranes was corrected for according to the methods previously described (59). Drift- and binding-corrected membrane potential were calculated according to the following equation

(3)

where RT/F is the Nernst factor (R is gas constant, T is temperature, and F is the Faraday constant), Z is the valence of TPP⁺, V_m is the volume of the mitochondria (in microliters), V_t is the total volume of the system (in microliters), V_c is the volume of the experimental chamber (in microliters), [TPP⁺]_T is the total concentration of TPP⁺ in the experimental buffer (in micromolar), K is the nonspecific binding constant for TPP⁺ (6 µl/nmol cytochrome A), R_c is the partition coefficient for nonspecific binding, E_v is the millivolt reading from the electrode, E_d is the electrode drift in millivolts (empirically determined before each run), and Y_o, m_o, and m₁ are the coefficients describing the standard curve using a three-parameter exponential growth regression (Sigma Plot version 4.0; SPSS). Although mitochondrial volume has been shown to change with ATP production rate and [Ca²⁺] (25), volume estimates in these preparations varied by only 5.9 ± 0.7% (n = 12), which equated to <1% error in estimating over the entire range of substrate and Ca²⁺ concentrations used. The sensitivity of the TPP⁺ electrode to [Ca²⁺] was evaluated over the entire range of [Ca²⁺] used in these studies. With the extramitochondrial [TPP⁺] of 4 µM, no dependence of [Ca²⁺] was found using this ion-selective electrode system (data not shown).

ADP-to-oxygen ratio. ADP-to-oxygen ratios (ADP/O) were calculated according to methods previously described (8, 36). Briefly, the oxygen consumed from the single addition of ADP (500 µM final concentration) was determined from the amount of oxygen consumed per mole of ADP added to mitochondria in buffer C. Analysis was performed using a custom program written in IDL.

Luminometric ATP determination. In an effort to validate the ADP/O ratio as a reliable estimate of ATP production rate and to evaluate the possibility that [Ca²⁺] used in this study significantly uncoupled oxidative phosphorylation, luciferin/luciferase luminescent assays of ATP production were performed in the presence and absence of Ca²⁺ at state 3. Mitochondria were incubated in buffer C (minus ATP) containing 40 µg/ml luciferase and 715 µM D-luciferin for 6 min. Postincubation, mitochondria were reduced with 5 mM G/M, and maximal state 3 respiration was initiated with a 1.3 mM bolus of ADP (final concentration). At the end of each experiment, a known standard of ATP (160 µM) was added to the reaction mixture serving as an internal control for interexperimental variation. Total photons were collected with a custom-built photomultiplier tube assembly optically coupled to the thermostatted 37°C reaction chamber via a liquid light guide. Simultaneous measures of oxygen consumption were collected using a polarographic oxygen electrode (see above). Voltages were digitally sampled at 10 Hz with a 12-bit A/D converter and recorded using a tailor-made program written in Workbench-Mac.

Determination of absolute [ATP] above 200 µM is difficult with this approach due to the nonlinear photon emission rates caused by product inhibition (i.e., oxyluciferin) and loss of quantum yield (18, 19, 44, 45). To maintain constant state 3 respiratory rates where substrates are not limiting, the [ADP] and, subsequently, synthesized [ATP] were used at concentrations >200 µM (51). It was reasoned that if the ATP production rate was held constant by appropriate dilution of the mitochondrial concentration in the presence of Ca²⁺, the time intensity curves of the control and Ca²⁺-stimulated conditions could be directly compared, thus minimizing the kinetic complications of the luciferin/luciferase assay. The rate of ATP production was assumed to be proportional to the mO₂ under both conditions, with no coupling loss caused by Ca²⁺ at 535 nM. Ca²⁺-stimulated mitochondria were diluted relative to controls (Ca²⁺-depleted) by the following equation

(4)

where [Cyt_a]_dil is the cytochrome A dilution factor for Ca²⁺-stimulated respiration [(mO₂)_Ca²⁺] to achieved identical respiratory rates when compared with controls[(mO₂)₀]. This dilution resulted in identical absolute oxygen consumption rates in the chamber with both control and Ca²⁺-stimulated mitochondria. Provided that mO₂ was associated with the same ATP/O ratio with and without Ca²⁺ stimulation, the luciferin/luciferase photon emission time courses should be identical for both conditions.

Statistical analysis. Slope determinations between treatment groups were calculated by a first-order least-squares linear regression (Statistica version 5.0; Statsoft). This analysis determines 1) the regression coefficient, 2) the equation of the line describing the relationship, and 3) the probability that the slope of the line is significantly different from zero. Individual slopes (between mO₂ and NADH, or ), ADP/O ratios, ATP production, and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) dose-response curves at each level were then compared using a single-factor-dependent variable t-test (Statistica version 5.0). Where appropriate, values are means ± 1 SE. In all cases, the fiduciary level of significance was taken at P  0.05.

	RESULTS

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Mitochondrial characterization. Isolated mitochondria in this preparation produced a high yield of tightly coupled mitochondria with an average yield of 0.9 ± 0.06 nmol cytochrome A/g wet wt myocardium (n = 29), corresponding to 2.5% of the total cytochrome A in pig heart (51). Estimates of mitochondrial integrity were determined by the RCR and were performed in buffer B with 5 mM G/M, 2 mM P_i, 4 µM TPP⁺, and a single addition of 1.3 mM ADP (final concentration) (37°C), which allowed for comparison with previous work. The average RCR was 10.5 ± 0.3 (n = 29) and ranged between 8 and 14. Mitochondria with RCR of <8 were not used in these studies.

Effects of Ca²⁺ depletion. To determine the physiological affects of Ca²⁺ on mitochondrial energetics, it was necessary to deplete the organelle of endogenous Ca²⁺ and substrates to minimize interexperimental variability. In all cases, mitochondria were Ca²⁺ and substrate depleted with a 6-min incubation in buffer C (state 1) (8), which resulted in a significant reduction in from 143 ± 3 to 121 ± 3 mV (P  0.05, n = 29). This condition is defined as Ca²⁺ depleted with nominally zero [Ca²⁺], because some residual Ca²⁺ is likely to present in the system. Addition of substrates (G/M or succinate) and exogenous Ca²⁺ resulted in a repolarization of the mitochondrial membrane to preincubation levels. In studies where minimal CaDH effects were desired, succinate was used as the oxidizable carbon source. Dosing studies with succinate without Ca²⁺ revealed Michaelis-Menten kinetics for state 3 respiratory rates in buffer C, with an apparent K_m value 2.46 mM. Maximum ADP-stimulated respiratory rates were attained with 15 mM succinate.

Ca²⁺ optimization. Steady-state kinetics at state 3 and state 4 for Ca²⁺ were determined for NADH driving force and mO₂. In Ca²⁺-depleted mitochondria, state 3 mO₂ and NADH increased with [Ca²⁺] over the range of 1.54 to 1,810 nM (Fig. 1). The kinetics showed saturation with a half-maximal activation (K_0.5) at 157 nM free [Ca²⁺] while oxidizing G/M as the substrate. The equations and regression statistics describing these trends are presented in Table 1. The increase in NADH is consistent with the known increase in CaDH activity with Ca²⁺. The increase in mO₂ could be due to increased driving force for ATP production, via increases in NADH, and/or mitochondrial uncoupling by Ca²⁺ transport.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Ca2+ dose-response curves at state 3 respiration. A: data are plotted as state 3 mitochondrial O2 (mO2) vs. [Ca2+]. B: plot of state 3 NADH vs. [Ca2+]. Statistical analysis and regression coefficients are presented in Table 1. In all cases, mitochondria were Ca2+ depleted as described in MATERIALS AND METHODS, and experiments were performed with 5 mM G/M (glutamate/malate) as the oxidizable carbon source. Cyta, cytochrome A; INADH, NADH coefficient; ICF, 5-(6)-carboxy-2'-7'-dichlorofluoresceindiacetate succinimidyl ester coefficient.

View this table: [in this window] [in a new window]   Table 1.   Regression analysis of state 3 condition with [Ca2+]

To further evaluate the possibility of Ca²⁺-dependent uncoupling and select a useful [Ca²⁺] for this study, mO₂ and NADH production rates were evaluated at state 4 where the effects should be amplified. Like state 3, mO₂ showed saturation kinetics with [Ca²⁺] studied (Fig. 2A); however, the K_0.5 was 376.1 nM, which is more than double that seen at state 3 (Table 2). NADH in mitochondria oxidizing G/M at state 4 increased with [Ca²⁺] <600 nM, whereas at higher concentrations NADH decreased. The calculated optima for NADH levels occurred at 535 nM [Ca²⁺] (Fig. 2B). The decrease in NADH above 600 nM [Ca²⁺] suggests that significant mitochondrial uncoupling might be occurring above this concentration, dissipating the driving force for ATP synthesis. However, it is unclear to what extent this contributes to the total mO₂ observed. Based on the combined state 3 and 4 data, 535 nM was used as the optimal [Ca²⁺], with minimal deleterious effects.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Ca2+ dose-response curves at state 4 respiration. A: data are plotted as state 4 mO2 vs. [Ca2+]. B: plot of state 4 NADH vs. [Ca2+]. Statistical analysis and regression coefficients for mO2 are presented in Table 2. Due to nonsaturating kinetics, statistical analyses for NADH were not performed. In all cases, mitochondria were Ca2+ depleted as described in MATERIALS AND METHODS, and experiments were performed with G/M as the oxidizable carbon source.

View this table: [in this window] [in a new window]   Table 2.   Regression analysis of state 4 oxygen consumption with [Ca2+]

ADP/O values were determined as a function of [Ca²⁺] to further evaluate the possibility of Ca²⁺ uncoupling (for data, see Table 4). The ADP/O ratio was constant up to 535 nM [Ca²⁺], suggesting that any uncoupling caused by Ca²⁺-dependent transport was not significant at state 3 ATP production rates.

To confirm this indirect measurement, the production of ATP was monitored directly using the luciferin/luciferase assay in intact mitochondria. For this assay, the concentration of mitochondria stimulated by [Ca²⁺] was reduced relative to control, using Eq. 4. Under these conditions, if the ATP/O ratio was constant under Ca²⁺-depleted and Ca²⁺-stimulated conditions, the time course of the photon emission should be identical. The magnitude and time courses of the photon emission are presented in Fig. 3A. ATP production rate with 535 nM Ca²⁺ was identical for the two conditions (P > 0.05, n = 4) despite a 1.8-fold lower mitochondrial content. The observation that state 3 mO₂ accurately predicted the proper dilution of the mitochondria to match the ATP production rates also suggests that the ATP/O ratio was identical in the presence and absence of Ca²⁺. These data combined, the ADP/O and ATP production kinetics in the presence and absence of Ca²⁺ demonstrate that Ca²⁺ was not significantly uncoupling respiration at 535 nM.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Mitochondrial ATP production and uncoupling optimization. A: luminometric analysis of photon production (ATP) in Ca2+-depleted mitochondria oxidizing 5 mM G/M. State 3 respiration was initiated with 1.3 mM bolus of ADP in presence (535 nM) and absence of Ca2+. Metabolic rates from paired experiments were matched in all cases using Eq. 4. Data are means ± SE, where SE data are presented at every 50th data point for clarity. B: plot of uncoupled respiration (mO2) vs. titration of metabolic uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; in nM). In all cases, mitochondria were incubated at 37°C for 6 min without substrates, as described in MATERIALS AND METHODS. Data are means ± SE; * significant differences from proceeding condition (P  0.05). PMT, photomultiplier tube.

Dose response to uncoupler. In an effort to evaluate maximally stimulated rates, independent of F₀/F₁-ATPase and adenylate transport (ANT), state 4 Ca²⁺-depleted mitochondria oxidizing succinate (30 mM) + 535 nM Ca²⁺ were titrated with FCCP in buffer C. The results of these studies are shown in Fig. 3B. The optimum concentration of FCCP was 33 nM FCCP. Some variation in the optimal concentration of FCCP was found in the absence of Ca²⁺, with the apparent optimal concentration of FCCP increasing to 50 nM with nominally zero [Ca²⁺] present.

Ca²⁺ activation of oxidative phosphorylation. The initial study attempted to establish the effect of CaDH activation alone on mitochondrial ATP production. Because Ca²⁺ stimulates CaDH activity, which augments ATP production rate via increasing [NADH] (32, 49), these effects could be simulated by titrating carbon substrates oxidized by CaDH. Using this approach, the relationship between the driving force ([NADH] and/or ) and flow (mO₂  ATP synthesis rate as estimated by ADP/O and confirmed by luminometry) would provide a standard curve for [NADH] and -dependent CaDH effects. These force-flow (F-F) relationships are presented in Fig. 4 as the NADH levels or vs. maximal state 3 rate, with G/M as the CaDH oxidizable substrates and succinate as the substrate with minimal CaDH contributions in its oxidation pathway. As illustrated by Fig. 4, linear F-F relationships were obtained for NADH and with mO₂ and were in agreement with previous studies (43, 51). Regression statistics and F-F slopes for mitochondria oxidizing G/M and succinate are presented in Table 3. Because the above F-F functions establish the benchmark for the CaDH/NADH response, any deviation from these F-F curves for NADH or with [Ca²⁺] would suggest a mechanism other than simple CaDH activation.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Representative force-flow (F-F) relationships at state 3 respiration with variable Ca2+ and substrates. A: data are plotted as mO2 vs. NADH for G/M (squares) and succinate (SUC; circles). Filled symbols represent fixed [Ca2+] with variable [substrate] (control), whereas open symbols are variable [Ca2+] with fixed [substrate]. B: plot of mO2 vs. with either G/M or SUC. Symbols and conditions are as stated in A. Solid (G/M) and dashed (SUC) lines are linear regressions, with statistical summary presented in Table 3. Substrate concentrations ranged from 5 to 0.5 mM with G/M and 15 to 1.67 mM with SUC, whereas Ca2+ used in these studies was fixed at 535 nM free for control conditions. Under experimental conditions [Ca2+] ranged from 535 to 0 nM free, whereas substrate concentrations were fixed at 5 and 15 mM for G/M and SUC, respectively. In all cases, mitochondria were incubated at 37°C for 6 min, as described in MATERIALS AND METHODS, for Ca2+ depletion before commencement of experiments.

View this table: [in this window] [in a new window]   Table 3.   Mean force-flow slopes and regression statistics with substrate and Ca2+ titration

The effects of increasing [Ca²⁺] on state 3 respiration with a fixed level of carbon substrate are also shown in Fig. 4 for comparison. Both G/M and succinate with nominal [Ca²⁺] failed to support higher mO₂ compared with CaDH controls at the same [NADH], indicating that some step beyond the generation of NADH was inhibiting mO₂ and/or ATP production at low [Ca²⁺]. NADH levels increased proportionately with [Ca²⁺], consistent with the activation of CaDH with G/M, and to a lesser extent with succinate. However, the NADH F-F slopes more than doubled with Ca²⁺ for G/M and succinate (Fig. 4A and Table 3), indicating a disproportionate increase in mO₂ for a given [NADH]. Ruthenium red (1 µM), a Ca²⁺ uniport inhibitor, completely blocked the Ca²⁺ effects, suggesting that matrix [Ca²⁺] is necessary for activation (data not shown). The significant increases in the NADH F-F slopes with Ca²⁺ indicated that mO₂ increased more than could be predicted by a simple increase in NADH through CaDH activation. Moreover, these data also show that, at the same NADH driving force, respiration is augmented severalfold with the addition of Ca²⁺.

These findings were further confirmed by similar experiments in mitochondria titrated with G/M in the presence (535 nM) and absence (nominally zero) of [Ca²⁺] (Fig. 5). In both cases, mitochondria exhibit a linear dependence with [G/M]; however, the NADH F-F slope more than doubled with the addition of optimal Ca²⁺ (Table 3). As with previous experiments, mO₂ was inhibited in the absence of significant [Ca²⁺], despite adequate NADH driving force and identical [G/M].

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Representative F-F relationships at state 3 respiration with variable substrates in presence and absence of Ca2+. A: data are plotted as mO2 vs. NADH, with filled symbols representing fixed [Ca2+] (535 nM) and variable substrate concentrations, whereas open symbols are variable substrate concentrations minus additional Ca2+. B: plot of mO2 vs. in mitochondria oxidizing G/M (0.5-5 mM). In all cases, mitochondria were incubated at 37°C for 6 min, as described in MATERIALS AND METHODS, to Ca2+ deplete before commencement of experiments. Statistical analyses and degree of fit are presented in Table 3.

Both of these studies with constant or variable [G/M] demonstrated an increase in NADH with Ca²⁺ consistent with CaDH activation described previously (42, 51). However, the degree of NADH increase with Ca²⁺ was not adequate to explain the increases in state 3 ATP synthesis rate observed, suggesting a mechanism in addition to CaDH.

More revealing was the data, where, instead of increasing with [Ca²⁺] as occurred with CaDH controls, decreased, resulting in a change in sign and reversal of the F-F slope (Fig. 4B; Table 3). Similar results were obtained with succinate in the presence of 5.8 µM rotenone (Table 3), an inhibitor of site 1, thus eliminating NADH contributions completely. The fact that the F-F slopes were identical, within statistical limits, in the presence and absence of rotenone indicates that the activation by Ca²⁺ is downstream of site 1. Clearly, the major driving force for ANT and ATP synthesis decreased with Ca²⁺, the opposite of what would be predicted from a simple increase in CaDH activity. Provided these interpretations are correct, titrating substrates (G/M) with and without Ca²⁺ should result in a similar F-F slope direction; however, the absolute magnitude should be considerably lower without Ca²⁺. The results of these experiments are presented in Fig. 5B and Table 3, confirming these predictions. The drop in (less negative) observed with increasing ATP production suggests that ATP synthesis (F₀/F₁-ATPase) and ANT have increased in the presence of Ca²⁺, despite significantly lower driving forces. These increases occurred without a decrease in the ADP/O ratio with increasing [Ca²⁺], thus providing no evidence for significant uncoupling via inner membrane recycling of Ca²⁺ (Table 4). Luminometric estimates of ATP synthesis rates further support this contention and directly illustrates that ATP synthesis per mole of cytochrome A is augmented by Ca²⁺ (Fig. 3). When combined, these data demonstrate that the activity of ANT and/or F₀/F₁-ATPase increased with [Ca²⁺].

View this table: [in this window] [in a new window]   Table 4.   ADP/O ratios with substrate and Ca2+ titration

To further evaluate the rate limitations associated with ANT, F₀/F₁-ATPase, and cytochrome flux, the effects of the uncoupler FCCP were evaluated. FCCP, a proton ionophore, collapses the electrochemical gradient across the inner mitochondrial membrane, effectively short-circuiting ANT and F₀/F₁-ATPase (3, 47). Under these conditions, it has been shown that mO₂ is dominated by the rate of NADH or FADH₂ formation and subsequent cytochrome oxidation. Thus FCCP effectively removes any rate limitation of ANT and F₀/F₁-ATPase on mO₂. If Ca²⁺ stimulates mO₂ through ANT and/or F₀/F₁-ATPase, then the difference between FCCP-uncoupled and ADP-P_i-driven respiration should decrease as the inhibition of these enzymes is relieved with increasing [Ca²⁺]. With the use of succinate to minimize the influence of CaDH, the percent difference between FCCP and maximal ADP-P_i-stimulated respiration was compared as a function of [Ca²⁺] (Fig. 6). The percent difference between Ca²⁺-stimulated and FCCP-uncoupled respiration decreased with increasing [Ca²⁺], consistent with the notion that ANT and/or F₀/F₁-ATPase are activated by Ca²⁺.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Comparison of uncoupled and Ca2+-stimulated coupled respiration. Plot of percentage difference between state 3 and FCCP-uncoupled respiration with increasing [Ca2+]. State 3 respiration, oxidizing SUC (15 mM), was initiated with ADP (500 µM) after Ca2+ depletion. Maximally uncoupled respiration was achieved by titration of [FCCP] (33.3 nM final). Percent increase in mO2 with uncoupler is plotted vs. free [Ca2+]. Buffers and Ca2+ depletion were as indicated in Fig. 1. Data are means ± SE; * significant differences from state 3 (P  0.05).

The effects of Ca²⁺ on the uncoupled rate of respiration yielded some insight into the Ca²⁺ effects and cytochrome flux. We estimated, using succinate, that only 10% of the Ca²⁺ stimulation of respiration was due to residual CaDH activation and was based on the increases in NADH observed with varying [Ca²⁺] compared with the NADH standard curve (Fig. 4). Thus any adverse effects of Ca²⁺ on the uncoupled rate with succinate should be due to cytochrome flux effects, such as direct activation of cytochrome oxidase (39). Uncoupled respiration increased by 10% with [Ca²⁺]; however, these changes could be fully attributed to the small CaDH activation observed with succinate oxidation. Thus no evidence for Ca²⁺ stimulation of cytochrome activity was observed.

Interestingly, at the highest [Ca²⁺] tested, the difference between uncoupled and ADP-P_i-driven respiration was not significantly different (P  0.05, n = 7). For this to occur, all rate limitations upstream (i.e., dehydrogenases and cytochromes) would have to be negligible. These latter data suggest that a minimal limitation to ATP production rate exists at the level of ANT and/or F₀/F₁-ATPase when optimally activated by Ca²⁺.

The effect of Ca²⁺ on oxidative phosphorylation observed could be due to direct activation of either ANT or F₀/F₁-ATPase as discussed above; however, the difficulty in isolating the effects of Ca²⁺ on intact mitochondrial ANT or F₀/F₁-ATPase is due to the close coupling of these enzyme complexes in the synthesis of ATP. To resolve this difficulty, arsenate (AsO₄) was used as a substrate for F₀/F₁-ATPase to remove the influence of ANT. In the absence of exogenous phosphate (P_i), AsO₄ uncouples ATP production from adenylate translocation, after initial loading with ADP, by synthesizing a metastable complex of ADP and AsO₄ within the matrix via the F₀/F₁-ATPase. The resulting ADP-AsO₄ complex undergoes rapid hydrolysis to re-form ADP and AsO₄, where the cycle is repeated (12, 26, 52, 68) (Fig. 7A). Oligomycin inhibits uncoupling by AsO₄; however, oligomycin does not prevent uncoupling by dinitrophenol (DNP) (22), therefore illustrating F₀/F₁-ATPase dependence of this agent. In all cases, mitochondria were preloaded with ADP (200 µM), where atractyloside (8.34 µM) was added to eliminate ANT contributions to the reaction. CaDH activation was minimized using succinate as the carbon substrate. Ca²⁺ stimulation of F₀/F₁-ATPase could be assessed directly using these conditions.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   A: schematic diagram of AsO4-stimulated respiration. AsO4-stimulated respiration is as described in text and was performed in presence of sufficient atractyloside to inhibit adenylate transport function (not shown) postloading with ADP. All other experimental protocols were performed as described in Fig. 4 legend with SUC as oxidizable carbon source. B: plot of mO2 with serial additions of substrates and metabolic inhibitors. Respiration was stimulated with 2 mM AsO4 addition, after serially preloading with 200 µM ADP and 8.34 µM atractyloside, and was inhibited with excess oligomycin B (oligo B; 310 µM), illustrating F0/F1-ATPase dependence. Data are means ± SE; * significant differences from state 2 (P  0.05).

Addition of AsO₄ (2 mM) stimulated respiration by 2.4 ± 0.19-fold over the state 4 rate (P  0.05, n = 7) and was totally inhibited with the addition of excess oligomycin B (310 µM), a specific inhibitor of the F₀/F₁-ATPase (Fig. 7B). These results are consistent with AsO₄ stimulating the F₀/F₁-ATPase directly as outlined above. The effects of [Ca²⁺] on AsO₄-stimulated respiration are shown in Fig. 8. Ca²⁺ increased AsO₄-stimulated respiration above the CaDH effects on NADH or driving force and resulted in a greater than twofold higher NADH F-F slope in mitochondria oxidizing succinate as the carbon source (Fig. 8A; Table 3). Consistent with the effects of Ca²⁺ on ADP-P_i-driven respiration, the F-F function in AsO₄-stimulated mitochondria resulted in a decrease in and a reversal of F-F slope, with increasing [Ca²⁺] (Fig. 8B; Table 3), and is consistent with direct activation of the F₀/F₁-ATPase. These results provide evidence that Ca²⁺ directly increases F₀/F₁-ATPase activity independent of CaDH- and ANT-mediated mechanisms.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Typical state 3 activation plot of F0/F1-ATPase by Ca2+. A: data are plotted as mO2 vs. NADH. In all cases, mitochondria were Ca2+ depleted, where 200 µM ADP and 8.34 µM atractyloside were added serially. Respiration was driven with a single addition of 2 mM AsO4. Filled symbols represent fixed [Ca2+] with variable [SUC] (control), whereas open symbols are mitochondria with variable [Ca2+] with fixed [SUC]. B: plot of state 3 mO2 and . All other conditions are as stated in Fig. 4A with SUC as oxidizable carbon source. Statistical analysis and results are presented in Table 3.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ca²⁺ activation of oxidative phosphorylation. Collectively, the current study suggests that Ca²⁺ enhanced ADP-P_i-driven respiration in heart mitochondria, and is not limited to CaDH activation alone. These data also suggest that a significant fraction of the matrix Ca²⁺-stimulated ATP production is caused by direct activation of the F₀/F₁-ATPase with minimal metabolic uncoupling. With the use of the standard and experimental data with zero and optimal free [Ca²⁺] (535 nM) at the same NADH driving force (i.e., 0.4 I_NADH/I_CF), it was calculated that the fraction of Ca²⁺-stimulated ATP production by non-CaDH mechanisms was >60% (Fig. 4A).

The effects of Ca²⁺ on the CaDH have been well characterized in the literature (32, 48); however, evidence demonstrating direct Ca²⁺ stimulation of the F₀/F₁-ATPase and its mechanism to date is less clear (reviewed by Ref. 34). Several indirect studies from in situ cardiac biopsies (63), sonicated cardiomyocytes (14-17), and submitochondrial particles (62, 77) have implicated Ca²⁺ in the activation of F₀/F₁-ATPase; however, in most cases, ATP synthesis rates were estimated from ATP hydrolysis rates in the absence of , a known regulator of oxidative phosphorylation (see Ref. 34). The meaning of these studies is, therefore, difficult to evaluate because the sites for hydrolysis and synthesis and their mechanisms are known to be distinct (see Ref. 4). Ca²⁺ is also known to bind to a number of matrix proteins, which include the Ca²⁺-binding protein (CaBI) (75-77), cyclophilin D (11), and calmodulin (55), which in turn are known to alter membrane transport (11, 74) and/or ATP hydrolysis (77). Although these protein complexes interact with matrix Ca²⁺, it is unclear how these translate to metabolic changes in heart mitochondria. Interestingly, matrix accumulation of Ca²⁺ has been shown to change the volume of heart mitochondria (25), which in turn is speculated to modify matrix enzyme activity (31) and, potentially, the F₀/F₁-ATPase. Any of the protein-binding modifications or volume changes could be responsible for the Ca²⁺ effects observed in this study.

Another mechanism for F₀/F₁-ATPase activation by Ca²⁺ would involve changes in the apparent K_m. Because the current experiments were conducted at saturating levels of substrates and ADP-P_i, it is therefore probable that Ca²⁺ activation involves an increase in the maximum velocity of F₀/F₁-ATPase or number of active enzyme complexes. As for direct effects on the F₀/F₁-ATPase, current theory holds that the major rate-limiting step in ATP formation is the release of ATP from the -subunit (4). Therefore, if Ca²⁺ were acting directly on F₀/F₁-ATPase, it would likely lower the free energy for ATP release. To date, no evidence exists for direct interaction of Ca²⁺ with the F₀/F₁-ATPase; thus further studies are required to establish the exact mechanism of matrix Ca²⁺ actions.

In the current study, the net effect of Ca²⁺ was an increase in NADH levels and a decrease in at state 3. These data suggest that NAD⁺/NADH and are not in equilibrium under these conditions because the net free energy for these intermediates moved in opposite directions with Ca²⁺ additions (Fig. 4). This suggests that a restriction in energy transfer exists between NADH and at site 1. The site of this restriction is unknown; however, similar results were found in working rat hearts (67), where increases in cardiac work and predictably higher mean cytosolic [Ca²⁺] (6, 24) caused a fall in with increasing NADH. Therefore, the concordance of the data from intact mitochondria, cells, and hearts suggests that these isolated mitochondrial results may be applicable to the heart in vivo.

Despite the similarities with whole heart data, the question remains, are changes in the [Ca²⁺]_c with workload adequate to modify mitochondrial metabolism? Classical models suggest that mitochondrial Ca²⁺ uptake is too slow to match cytosolic Ca²⁺ transients during muscle contraction (69) and would instead track time-averaged [Ca²⁺]_c. Indo 1 fluorescence studies in cardiac myocytes with Mn²⁺ quenching of Ca²⁺ signals seem to support this contention (50); however, these findings are complicated by accumulation and quenching by matrix Mn²⁺ (28). On the contrary, others have reported rapid mitochondrial Ca²⁺ uptake mechanisms (28, 64) and Ca²⁺ transients that track single myocyte contraction (7, 38, 66). Further support comes from work in isolated hepatocytes, where inositol trisphosphate-induced cytosolic Ca²⁺ waves are known to propagate into individual mitochondria (30), thus inducing a self-propagated intramitochondrial wave along the reticular network (37). Irrespective of the mechanism and time course, Ca²⁺ is sequestered by mitochondria (29) when exposed to free [Ca²⁺]_c ranging from 100 to 1,200 nM (69, 71). Integrating these literature data over the cardiac cycle revealed a cytosolic time-averaged and end-diastolic concentrations of 345 and 115 nM, respectively. These findings are consistent with direct measures of mean free [Ca²⁺]_c in adult hamster myocytes at rest (200-300 nM) and with positive inotropic work (~400 nM) using indo 1/AM (6). Based on the integration and direct measurement data described above, it would appear that the K_0.5 for Ca²⁺ activation in this preparation (Table 1) is well within mean physiological levels and is even suited to achieve a reasonable dynamic range if beat-to-beat variations in matrix [Ca²⁺] occur. Of some importance may be the recent work on HeLa cells that demonstrated a close coupling between mitochondria and endo(sarco)plasmic reticulum (SR) [Ca²⁺] (58), which could result in microdomains of high [Ca²⁺]_c (57) in close proximity to the L-type Ca²⁺ channels. These latter results suggest a direct coupling of the SR and mitochondria bypassing the cytosolic pool altogether. This mechanism might permit a rapid transfer of Ca²⁺ to the mitochondrial matrix during contraction not available from classical isolated mitochondrial preparations.

There are several limitations to this study on Ca²⁺ effects on isolated mitochondria. First, any study on isolated mitochondria could suffer from isolation damage due to tissue preparation. Based on our RCR data, isolation damage was minimized because the coupling characteristics of this preparation were excellent (RCR  10), and high ATP production rates per cytochrome A in this preparation were achieved. However, these measures provide only limited information in a very complex process. Second, the depletion of Ca²⁺ is problematic because it is unclear what the resting matrix [Ca²⁺] is in vivo. Therefore, it is difficult to know whether our protocol mimics the intact heart appropriately. Finally, Ca²⁺ transport can uncouple and overload mitochondria, resulting in significant membrane permeability changes or damage (46). As such, care was taken to select a [Ca²⁺] within the physiological range that would not have significant pathophysiological or uncoupling effects. This was evaluated using two separate estimates of the ADP/O ratio, and, in both cases, no evidence for significant uncoupling was observed. Therefore, at 535 nM [Ca²⁺], coupling and reducing equivalent flow from reducing equivalents to ATP production in this preparation were maintained.

In summary, these observations suggest that increased systolic Ca²⁺ and subsequent stimulation of myocardial work at the actin-myosin ATPase could be paralleled by an activation of mitochondrial ATP production at several levels. It is interesting to note that, as in the cytosol where Ca²⁺ increases work and biochemical energy conversion in concert, the present data suggest a similar mode of operation within the mitochondria itself; i.e., Ca²⁺ increases the driving force through CaDH as well as activates the ATP production steps (i.e., work) directly. This form of parallel stimulation in a physiological control network provides a mechanism of increasing flux with minimal perturbations on the potentially important intermediates of the reactions. Finding this type of regulation on two levels of bioenergetics in the heart, the cytosol, and within the mitochondria by Ca²⁺ may suggest that this is an important general mechanism in cellular metabolic regulation.

	ACKNOWLEDGEMENTS

Present address of V. K. Mootha: Dept. of Medicine, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115.

	FOOTNOTES

Address for reprint requests and other correspondence: P. R. Territo, Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bldg. 10, Rm. B1D-416, Bethesda, MD 20892-1061 (E-mail: territop{at}zeus.nhlbi.nih.gov).

Received 20 January 1999; accepted in final form 14 September 1999.

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