Download Regulation of Mitochondrial ATP Synthase Activity

Clinical Science (1998) 94,499-50) (Printed in Great Britain)
499
Regulation of mitochondrial ATP synthase activity in
human myocardium
Anibh M. DAS
Deportment of Paediatrics, University Hospital Hamburg, Martinistrasse 52, D-20246 Hamburg, Germany
1. In previous studies regulation of the FIFO-ATPase
of
mitochondrial complex V (ATP synthase) has been
demonstrated in rat cardiomyocytes, canine mycocardium and skeletal muscle from children. The aim of
the present study was to examine regulation of ATP
synthase in human myocardium in response to different
metabolic states.
2. Biopsy material was obtained from 10 children
undergoing cardiac surgery. Mitochondria in the postnuclear supernatant were incubated under different
metabolic conditions for 15min and then broken by
sonication. ATP synthase was measured spectrophotometrically using a coupled enzyme assay.
3. ATP synthase can be rapidly measured in sonicated
preparations of heart mitochondria from children. We
show that direct regulation at the level of ATP synthase
occurs in these mitochondria. ATP synthase capacity is
decreased in response to blocking of the respiratory
chain by cyanide (mimicking anoxia) or uncoupling of
mitochondria, falling to 76 % and 66% of control values
respectively. Upregulation of ATP synthase can be
demonstrated in heart mitochondria when the calcium
concentration in the incubation medium is increased to
5 pM (130% of control).
4. ATP synthase is actively regulated in heart mitochondria from children. The enzyme is upregulated in
response to increased calcium. This transition may
reflect the increased energy demand when cardiac
workload is increased.
INTRODUCTION
Mitochondrial ATP synthase (FIFO-ATPase)(for a
review see [l]) is responsible for the bulk of ATP
synthesis in myocardium. It is now well established
that myocardial ATP production closely matches
myofibrillar ATP hydrolysis [2]. As cellular energy
demand changes, flux through mitochondrial ATP
synthase must also change to keep cellular ATP
constant. In the past, mitochondrial respiration was
believed to be ‘controlled’ by substrate levels, higher
ADP levels leading to greater substrate saturation of
the ATP synthase. Recently, however, regulation of
ATP synthase has been demonstrated in cultured
cardiomyocytes from rat [3,4]. These results were
subsequently confirmed in intact canine myocardium
[5] and human skeletal muscle [6]. The capacity of the
mitochondrial ATP synthase was increased in rat
heart cells subjected to high-energy demand (beating,
positive inotropic substances) and decreased in anoxic
cells.
The aim of the present study was to demonstrate
regulation at the level of ATP synthase in myocardium
from healthy children (i.e. children without metabolic
disease). Biopsy specimens were obtained from children undergoing routine cardiac surgery for correction
of structural heart defects. It cannot be completely
ruled out that the heart defects led to secondary
changes in respiratory chain enzyme activity and ATP
synthase regulation. However it is difficult to obtain
heart tissue from children with normal hearts.
In anoxia or when mitochondria are functionally
uncoupled ATP synthase works in reverse, hydrolysing
ATP. Uncontrolled hydrolysis of ATP would be
deleterious to the cell. Switching off ATP synthase
under these conditions helps to conserve cellular ATP.
Calcium ions are the only secondary messenger
known to enter mitochondria. Mitochondrial enzymes
like pyruvate dehydrogenase or the Krebs cycle dehydrogenases [7,8] have been shown to be activated by
raised intramitochondrial calcium. Activation of these
enzymes leads to an increased electron flux. Upregulation of ATP synthase by calcium would lead to
co-ordinate activation of mitochondrial enzymes linking increased electron flux with increased ATP synthesis. Raised intramitochondrial calcium occurs in
increased contraction and in the presence of positive
inotropic substances which may lead to upregulation
of ATP synthase.
If the control of ATP synthase demonstrated previously also exists in the intact human heart this would
represent a potentially important step in the regulation
of myocardial energy production.
Defects in the regulation of ATP synthase might be
responsible for myocardial dysfunction in some
patients with mitochondria1 disorders. In recent
studies we have shown regulation of ATP synthase in
cultured skin fibroblasts [9] and skeletal muscle [6]
from healthy children, which can be used as a
diagnostic method in mitochondrial disorders. Mito-
~
Keywords: ATP synthase, calcium ions, heart muscle. mitochondrion. regulation.
Abbreviations: CaBI. calcium binding inhibitor; FCCP, carbonyl cyanlde gtrifluoromethoxyphenylhydmne.
Correspondence: D r A. M. Das.
A. M. Das
500
chondrial diseases have heterogeneous clinical phenotypes, the heart being one of the most commonly
involved organs [lo]. Enzyme deficiencies expressed in
heart are often not present in skeletal muscle or
cultured fibroblasts [l 11. Therefore, it seems more
promising to look for enzyme deficiencies in myocardium from patients suspected of having metabolic
heart disease rather than in fibroblasts or skeletal
muscle. We studied ATP synthase control in children
as most patients with severe deficiency of respiratory
chain enzymes do not reach adulthood.
METHODS
Patients
Subepicardial biopsy samples were obtained from
10 children with heart defects undergoing corrective
cardiac surgery. Informed consent was obtained from
the parents and the investigation conformed with the
principles outlined in the Declaration of Helsinki. Two
children had ventricular septal defect, six had atrial
septal defect and two had tetralogy of Fallot. In total,
10 samples from the right atrium and two from the
right ventricle were obtained. From the two patients
with tetralogy of Fallot biopsy material from both
atrium and ventricle could be obtained. The wet weight
of the samples ranged from 7 to 26 mg. Biopsy samples
were taken just before cardioplegia was instituted.
Children were aged 2 weeks to 12 years and had no
clinical or biochemical signs of mitochondrial disorders.
General anaesthesia has been reported to lead to
inhibition of respiratory chain complexes [ 121. Therefore, activities of all respiratory chain complexes were
determined and found to be normal [ 131 in all biopsies
(Table 1).
Tissue preparation
A slight modification of the method described by
Rustin et al. [14] was used. Biopsy specimens were
collected into 10 ml of ice-cold centrifugation buffer
containing 250 mM sucrose, 40 mM KCI, 3 mM
EDTA, 20 mM Tris/HCl and 1 mg/ml essential-fattyacid-free BSA adjusted to pH 7.4 (KOH). The biopsy
material was trimmed of connective tissue and
weighed. The tissue was then cut into small slices using
a scalpel and homogenized by 20 strokes in a Potter
Table I Activities of respiratory chain complexes and
citrate synthase in myocardium (postnuclear supernatant)
Values are meansf5.E.M.. n = 10.
_____
Activity
(nmol' min .mg-' protein)
Enzymes
Complex 1-111
Complex II 111
Complex IV (COX)
Citrate synthase
+
236 f 28
159f31
835 f I24
325 f 62
homogenizer. A postnuclear supernatant was obtained
by centrifugation at 600 g.All steps were carried out at
4 "C. The pellet was resuspended in 2 ml of centrifugation buffer and stored in aliquots of 150 pl on ice.
Because the amount of biopsy material from heart is
very limited experiments were performed in postnuclear supernatant rather than in isolated mitochondria.
The isolation procedures used for obtaining the
postnuclear supernatant may lead to an alteration of
ATP synthase capacity by interfering with the binding
of regulatory proteins to the enzyme. Therefore, we
looked at ATP synthase capacity in intact myocardium
in comparison to enzyme capacity in postnuclear
supernatant. We used a method for 'freezing' the
binding state of the naturally occurring inhibitor
protein IF, by using rapid sonication in a neutral
buffer of low ionic strength (20 mM HEPES, 1 mM
MgCl,, 2 mM EGTA, pH 7.0) as in previous studies
[3-61.
Enzyme assays
Aliquots of 150 pl postnuclear supernatant were
incubated under different metabolic conditions for
15 min on ice. Incubation ofcontrol mitochondria was
carried out in the abovementioned centrifugation
buffer plus 5 mM glutamate as substrate without
further additions. Mitochondria were incubated with
1 mM KCN to inhibit cellular respiration (mimicking
anoxia), 2 pM FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) to uncouple mitochondria
or various concentrations of calcium (10 nm-1 mM).
Mitochondria were broken by sonication with a
Bandelin probe sonicator (set at 20 W of power) for a
total of 2 x 10 s with single pulses of 0.3 s duration.
The time between pulses was 0.7 s. Sonication was
carried out on ice.
In the experiments with intact heart tissue the biopsy
specimens were first disrupted in the low-ionic strength
buffer (see above) using an Ultraturrax disintegrator
set at 3000 rpm for 2 x 10 s. Mitochondria were then
broken by sonication with a Bandelin probe sonicator
set at 100 W of power for three intervals of 45 s
duration.
ATP synthase activity, measured in the direction of
ATP hydrolysis (ATPase activity), was assayed by the
continuous spectrophotometric assay of Rosing et al.
[15] except that 2 mM EGTA replaced EDTA in the
reaction medium. Aliquots of sonicate ( 2 W O p g in
20p1 sample volume) were added to the reaction
medium containing 60 mM sucrose, 50 mM triethanolamine-HCl,50 mM KCl, 4 mM MgCl,, 2 mM
ATP, 2 mM EGTA, 1 mM KCN, pH 8.0 (KOH) with
l00pM NADH, 5 units/ml pyruvate kinase and 5
units/ml lactate dehydrogenase. The total volume in
the cuvette was 1 ml. The blank cuvette contained air.
The linear reaction was followed for 2 min at 340 nm
and 37 "C. Oligomycin (8 pglml) was added to inhibit
oligomycin-sensitive ATPase (of mitochondrial origin).
Regulation of ATP synthase in myocardium
Rotenone-sensitive NADH :0, oxidoreductase was
determined according to the method of Fischer et al.
[ 161. Antimycin-sensitive succinate :cytochrome-c oxidoreductase activity was measured under the different
metabolic conditions as a mitochondrial marker using
a slight modification of the method described by
Stumpf and Parks [ 171. Cytochrome-c oxidase activity
in sonicates of postnuclear supernatant was determined according to the method of Wharton and
Tzagoloff [ 181. Citrate synthase was determined according to the method of Srere [19]. Protein was
measured as described by Bensadoun and Weinstein
[201.
Materials
ATP, lactate dehydrogenase, pyruvate kinase, phosphoenolpyruvate and NADH for enzyme assays were
purchased from Boehringer (Mannheim, Germany).
Oligomycin, FCCP, HEPES, fatty-acid-free BSA fraction V and all other chemicals, of the highest purity
available, were from Sigma (Deisenhofen, Germany).
Statistical analysis
All values for ATP synthase are given as means?
S.E.M. Data were compared using Student’s t-test.
RESULTS
Activity of respiratory chain complexes and citrate
synthase
The activities of the respiratory chain complexes
and of the mitochondrial marker enzyme citrate
synthase are shown in Table 1.
Measurement of ATP synthase capacity in heart
mitochondria
The activity of the mitochondrial ATP synthase was
measured in the direction of ATP hydrolysis (ATPase
activity). In uncoupled and broken mitochondria ATP
synthase will hydrolyse (rather than synthesize) ATP.
The ATP synthase activity in the mitochondria can
thus be monitored by breaking open mitochondria
and measuring mitochondrial ATPase activity (hydrolysis) at saturating ATP levels (ATP synthase
‘capacity ’).
Several procedures to break open heart mitochondria have been tested. Detergents (deoxycholate)
would not completely break open the mitochondria
except at concentrations so high as to yield an unstable
ATPase preparation. Freeze-thawing also led to incomplete breakage of mitochondria. Ultrasonic treatment with pulses for 2 x 10 s led to complete exposure
of the mitochondrial ATPase. Breaking of mitochondria was judged by addition of FCCP to the sonicate
and measurement of ATPase activity before and after
addition of the uncoupler. In previous studies sonic-
50 I
ation was found to be the method of choice for
breaking mitochondria in cardiomyocytes from rats,
canine myocardium and human fibroblasts and skeletal muscle [3-6,9].
The assay buffer contained 2 mM EGTA to minimize any Ca2+-dependent ATPase activity and less
than 5 mM sodium to minimize Na+/K+-dependent
ATPase activity. Under these conditions about 75 YO
of total ATPase activity was sensitive to oligomycin
and hence of mitochondrial origin.
Variation between duplicates from the same sonicate was 5 %. ATP synthase capacity in control cells
incubated in the abovementioned centrifugation buffer
without any further additions was 14 m-units/mg of
protein (mean, n = 12). This value is of the same order
of magnitude as observed in previous studies [21,22].
No significant difference in the ATP synthase capacity
could be observed in the two hearts where both atrium
and ventricle were examined. Data of atria and
ventricles were pooled.
The preparation procedures for obtaining the
postnuclear supernatant could lead to an alteration of
FIFO-ATPase activity. Therefore, ATP synthase activity was measured in intact myocardium as well. The
F,P-ATPase activity was normalized to citrate synthase activity. In anoxic mitochondria the F,-ATPase/
citrate synthase ratio was 1.97f0.12 (mean f S.E.M.,
n = 3), and in the tissue homogenate it was 1.80 0.1 1
(n = 3). These values are similar to those found in
canine myocardium [5].
Effects of cyanide and uncoupler on the
mitochondrial ATP synthase capacity of heart
mitochondria
Figure 1 shows that cyanide, which was added in
order to mimic anoxia by inhibiting complex IV
activity as in previous studies [3,6,9], in the incubation
medium led to a decrease in ATP synthase capacity to
76% of control values (14 m-units/mg of protein).
Addition of the mitochondrial uncoupler FCCP to the
incubation medium decreased ATP synthase capacity
to 66 YOof control values.
These transients might reflect changes in mitochondrial recovery under the different metabolic conditions. However, succinate: cytochrome c-reductase
as a mitochondrial marker enzyme remained constant
under the different incubation conditions (results not
shown). When cyanide or FCCP were added after
sonication ATP synthase activity remained unchanged. It is concluded that mitochondrial ATP
synthase capacity did indeed vary with the metabolic
conditions imposed on the mitochondria.
Effects of calcium concentration on the
mitochondrial ATP synthase capacity of heart
mitochondria
Under physiological conditions the heart meets its
energy demand mainly by oxidative phosphorylation.
A. M. Das
502
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CONTROL
CN (1mM)
-7.5
-7
-6.5
FCCP (2pM)
-6
-5.5
-5
-4.5
-4
-3.5
-3
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-2.5
lg [Calcium ions] [MI
Figure I Effect of cyanide (mimicking anoxia) and the
mitochondrial uncoupler FCCP on the mitochondria1ATP
synthase. measured as oligomycin-sensitive ATPace
Figure 2 Modulation of ATP synthase by calcium concentration in the incubation medium, plotted on a semilogarithmic scale
Heart mitochondria in the postnuclear supernatant were incubated for 15 min on ice without further additions (control) or in
the presence of either I mM K C N or 2pM FCCP. Mitochondria
were broken by sonication and ATP synthase assayed spectrophotometrically (n = 12). 'Significant difference (P < 0.02) versus
control mitochondria.
Heart mitochondria were incubated in the presence of different
calcium concentrations for 15 min on ice, broken by sonication
and ATP synthase assayed spectrophotometrically (n = 12).
Contraction of the heart and action of positive
inotropic substances is mediated by an increase in
cytosolic calcium concentration. The increased cytosolic calcium leads to a rise in intramitochondrial
calcium concentration, which is a potential signal for
switching on mitochondrial energy production [23].
Figure 2 shows the effect of increased calcium
concentration in the incubation medium on the mitochondrial ATP synthase capacity. ATP synthase capacity rose to a maximum of 130 YOof control values at
5pM calcium. This rise in ATP synthase capacity
could be prevented by incubating the mitochondria
with Ruthenium Red (4 pg/ml) before incubation with
calcium (Figure 3). Ruthenium Red inhibits transport
of calcium into mitochondria.
Supraphysiological concentrations of calcium in the
incubation medium led to a decrease in ATP synthase
capacity. Again mitochondrial recovery was constant
under the various incubation conditions as judged by
succinate: cytochrome c-reductase activity (results not
shown).
The fact that the calcium transients may simply
reflect the calcium optimum of ATP synthase was
excluded by running the enzyme assay at different
calcium concentrations in the cuvette. No changes in
ATP synthase capacity could be observed in the
presence of the different calcium concentrations.
DISCUSSION
As has already been shown for cultured human
fibroblasts, skeletal muscle from children and rat
cardiomyocytes [3-6,9], we demonstrate in this work
that the mitochondrial ATP synthase capacity can also
be rapidly measured in heart muscle mitochondria
from children. A substantial amount of total ATPase
activity is of mitochondrial origin as measured by
oligomycin sensitivity. Tissue processing for isolation
of mitochondria in the postnuclear supernatant does
not significantly alter ATP synthase activity as judged
Regulation of ATP synthase in myocardium
T .
T
0 mM Ca2+
5 LM Ca2+
RR + 5
Ca2+
Figure 3 Effect of Ruthenium Red on the activation of
ATP synthase by calcium
When mitochondriawere preincubated for 5 min with Ruthenium
Red (4pg/ml), which blocks mitochondrial calcium uptake, ATP
synthase could not be activated by an increased calcium concentration in the incubation medium (n = 8). *Significant difference ( P < 0.02) versus control mitochondria.
by the ATP synthase/citrate synthase ratio in myocardium and postnuclear supernatant.
We show that ATP synthase capacity is downregulated after treatment with cyanide (mimicking
anoxia). Under anoxic conditions the mitochondrial
ATP synthase works in reverse as an ATPase hydrolysing cellular ATP. Downregulation of ATP synthase
in the anoxic myocardium would therefore help to
conserve cellular ATP. In the uncoupled mitochondrion ATP synthase is switched off as well. Similar
observations have been made in anoxic and uncoupled
mitochondria from rat cardiomyocytes [3], skeletal
muscle [6] and in hypoxic canine myocardium [5].
A likely candidate for downregulation of ATP
synthase is the naturally occurring inhibitor protein of
ATP synthase, IF, [24]. This regulatory protein has
been found in human hearts [22]. In isolated mitochondria IF, has been shown to respond to the
potential across the mitochondrial inner membrane
[25,26],binding to ATP synthase when the potential is
low as in uncoupled or anoxic mitochondria.
Myocardial energy demand under physiological
503
conditions is mainly met by oxidative phosphorylation; in vivo, 5-10-fold changes in cardiac energy
demand are not uncommon. ATP levels in the myocardium hardly change in vivo [27]. Thus, ATP
production must change as well to keep cellular ATP
constant. In beating cultured cardiomyocytes from rat
an increase in ATP synthase capacity has been demonstrated in response to increased mitochondrial calcium
levels [4]. In the present study activation of ATP
synthase in human heart mitochondria could be
observed when calcium concentration in the incubation buffer was increased mimicking stronger contraction of the heart muscle and action of positive
inotropic substances. Other calcium-sensitive intramitochondrial enzymes can be activated by micromolar concentrations of calcium also leading to an
increased NADH level [23]. Activation of the mitochondrial ATP synthase at the same time leads to an
increased capacity for ATP production. By preincubation with Ruthenium Red, which inhibits mitochondrial calcium uptake, it was shown that this
upregulation is mediated by an increase in calcium
concentration inside mitochondria. A calcium binding
inhibitor (CaBI) has been described [28] which dissociates from ATP synthase molecules at high intramitochondrial calcium concentrations. Substantial
amounts of CaBI have been found in heart muscle [29].
Upregulation of ATP synthase by calcium in heart
mitochondria may reflect the action of CaBI. The
mechanism for the interaction of CaBI and ATP
synthase is still unclear.
The upregulation of ATP synthase by increased
intramitochondrial calcium may serve to meet the
increased energy demand of the beating heart muscle
in response to positive inotropic substances. Further
experiments are required to prove this hypothesis.
In summary, we show in this study that ATP
synthase in human heart mitochondria is actively
regulated in response to metabolic conditions of the
heart.
If upregulation of ATP synthase is abnormal under
pathological conditions this could lead to ATP depletion and myocardial dysfunction. Ischaemiareperfusion injury is accompanied by supraphysiological intramitochondrial calcium concentrations. In
this study we show that supraphysiological calcium
concentrations lead to an inhibition of ATP synthase.
This might contribute to myocardial dysfunction in
the reperfused ischaemic heart.
The structural defects of the hearts examined in this
study with alterations of heart pressure and volume
may lead to secondary effects on the respiratory chain
enzymes and ATP synthase regulation. However, it is
difficult to obtain heart tissue from subjects with
normal hearts.
The methods described here may be used diagnostically to identify mitochondrial disorders in which
primary dysregulation of ATP synthase occurs. They
may also prove to be useful as a screening method in
respiratory chain disorders with a decreased potential
across the inner mitochondrial membrane. Another
504
A. M. Das
possible application is monitoring of cardiac metabolism during open heart surgery (e.g. when testing
different methods of cardioplegia).
ACKNOWLEDGMENTS
I am indebted to Professor G . Ziemer, Hannover
Medical School (now at University of Tubingen), for
providing biopsy material from human heart.
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