Ca2+ activation of heart mitochondrial oxidative
phosphorylation: role of the
F0/F1-ATPase
Paul R.
Territo,
Vamsi K.
Mootha,
Stephanie A.
French, and
Robert
S.
Balaban
Laboratory of Cardiac Energetics, National Heart, Lung, and Blood
Institute, National Institutes of Health, Bethesda, Maryland
20892-1061
 |
ABSTRACT |
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 |
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 Pi generation (8). Through
this mechanism, ATP synthesis was envisioned to follow ATP hydrolysis
in the cytosol, paced by ADP concentration ([ADP]) and
[Pi]. 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 Pi 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
[Pi] (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 Pi. 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
(m
O2)]
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 |
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
Pi, 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 m
1.
Respiratory rate (m
O2),
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 Pi, 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
(m
O2) was determined in a
modified closed-system respirometer described previously (51) and was
used to estimate steady-state ATP production rate. Briefly, the
ADP-Pi-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-Pi-driven respiration in substrate-energized
mitochondria, whereas state 4 was respiration in the absence of
ADP-Pi (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
bO 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, Vc 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
m
O2 at state 3
and state 4 in buffer B, which contained 5 mM glutamate,
5 mM malate, 2 mM Pi, 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
(MNADH) and CF (MCF) 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 (MEBT). Model spectra
(MNADH, MCF, and MEBT) 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
FNADH, FCF, and FEBT refer to the
corresponding fits for NADH, CF, and EBT, respectively.
The algorithm determined the coefficients INADH,
ICF, and IEBT 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
INADH/ICF 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+, Vm
is the volume of the mitochondria (in microliters), Vt is the total volume of the system (in microliters), Vc 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), Rc is the partition
coefficient for nonspecific binding, Ev is the
millivolt reading from the electrode,
Ed is the
electrode drift in millivolts (empirically determined before each run),
and Yo, mo, 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 m
O2 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
[Cyta]dil is the cytochrome A
dilution factor for Ca2+-stimulated respiration
[(m
O2)Ca2+]
to achieved identical respiratory rates when compared with
controls[(m
O2)0]. This dilution resulted in identical absolute oxygen consumption rates
in the chamber with both control and Ca2+-stimulated
mitochondria. Provided that
m
O2 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
m
O2 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 |
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 Pi, 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 Km 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
m
O2. In
Ca2+-depleted mitochondria, state 3 m
O2 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 (K0.5) 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 m
O2 could be due to
increased driving force for ATP production, via increases in NADH,
and/or mitochondrial uncoupling by Ca2+ transport.

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Fig. 1.
Ca2+ dose-response curves at state 3 respiration.
A: data are plotted as state 3 mitochondrial
O2
(m O2) 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.
|
|
To further evaluate the possibility of Ca2+-dependent
uncoupling and select a useful [Ca2+] for this
study, m
O2 and NADH
production rates were evaluated at state 4 where the effects should be
amplified. Like state 3, m
O2
showed saturation kinetics with [Ca2+] studied
(Fig. 2A); however, the
K0.5 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 m
O2 observed. Based
on the combined state 3 and 4 data, 535 nM was used as the optimal
[Ca2+], with minimal deleterious effects.

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Fig. 2.
Ca2+ dose-response curves at state 4 respiration.
A: data are plotted as state 4 m O2 vs.
[Ca2+]. B: plot of state 4 NADH vs.
[Ca2+]. Statistical analysis and regression
coefficients for m O2 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.
|
|
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 m
O2 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.

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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
(m O2) 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
(m
O2
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
m
O2 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.

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Fig. 4.
Representative force-flow (F-F) relationships at state 3 respiration
with variable Ca2+ and substrates. A: data are
plotted as m O2 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 m O2 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.
|
|
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
m
O2 compared with CaDH
controls at the same [NADH], indicating that some step
beyond the generation of NADH was inhibiting
m
O2 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
m
O2 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
m
O2 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,
m
O2 was inhibited in
the absence of significant [Ca2+], despite
adequate NADH driving force and identical [G/M].

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Fig. 5.
Representative F-F relationships at state 3 respiration with variable
substrates in presence and absence of Ca2+. A: data
are plotted as m O2 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
m O2 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+].
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 m
O2
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
m
O2. If Ca2+
stimulates m
O2 through ANT
and/or F0/F1-ATPase, then the difference between FCCP-uncoupled and ADP-Pi-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-Pi-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+.

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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
m O2 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-Pi-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
(Pi), 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.

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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
m O2 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-Pi-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.

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Fig. 8.
Typical state 3 activation plot of F0/F1-ATPase
by Ca2+. A: data are plotted as
m O2 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 m O2 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 |
Ca2+ activation of oxidative
phosphorylation.
Collectively, the current study suggests that Ca2+ enhanced
ADP-Pi-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 INADH/ICF), 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
Km. Because the current experiments were conducted
at saturating levels of substrates and ADP-Pi, 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 K0.5 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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