Am J Physiol Heart Circ Physiol 287: H1747–H1755, 2004;
10.1152/ajpheart.01019.2003.
Excessive ATP hydrolysis in ischemic myocardium by mitochondrial
F1F0-ATPase: effect of selective pharmacological inhibition
of mitochondrial ATPase hydrolase activity
Gary J. Grover, Karnail S. Atwal, Paul G. Sleph, Feng-Li Wang,
Hossain Monshizadegan, Thomas Monticello, and David W. Green
Metabolic and Cardiovascular Drug Discovery, Bristol-Myers-Squibb
Pharmaceutical Research Institute, Pennington, New Jersey 08534
Submitted 15 December 2003; accepted in final form 26 May 2004
is responsible for the majority of
ATP production in the heart. Synthesis of ATP in the mitochondria requires a proton gradient across the inner membrane,
which is generated by the respiratory proteins as electrons are
transferred to oxygen. ATP synthesis by the catalytic F1 domain is driven by transport of protons into the matrix through
the F0 domain (10, 28). Oxygen deprivation collapses the
mitochondrial electrochemical gradient, which switches F1F0ATPase from ATP synthesis to ATP hydrolysis (11, 25, 32). In
ischemic myocardium, this may cause excessive and wasteful
hydrolysis of ATP, which does not contribute to cardiac work.
Studies using severely ischemic dog hearts showed that the
nonselective (inhibits both ATP synthase and hydrolase activities) F1F0-ATPase inhibitor oligomycin reduced the rate of
ATP depletion during ischemia (11). It is estimated that 50 –
80% of ATP consumed during severe ischemia, depending on
the species, may be attributed to mitochondrial ATP hydrolase
activity (25, 27). Because oligomycin inhibits both F1F0-ATP
hydrolase and synthase activities, such an agent cannot not be
used clinically but nevertheless serves as a useful experimental
tool.
In “slow” heart rate species such as dogs, humans, and
rabbits, the amount of ATP hydrolyzed by mitochondrial
ATPase during ischemia is thought to be significantly reduced by the reversible binding of the IF1 protein (26). This
small 80-amino acid protein specifically inhibits the ATP
hydrolase activity of F1F0-ATPase (5, 20, 26). The binding
of IF1 to F1F0-ATPase is optimal at pH 6.7 and requires the
hydrolysis of ATP in the absence of an electrochemical
gradient, conditions also seen during myocardial ischemia
(13, 19). The inhibition of the ATP hydrolase activity by IF1
is reversible upon restoration of the mitochondrial electrochemical gradient and increased pH as might be seen during
reperfusion (30). IF1 may not play an important role in
inhibiting ATPase activity in “fast” heart rate species such
as the rat, where it is not as highly expressed compared with
the dog and rabbit (23, 24, 26). Nevertheless, the unique
feature of IF1 is that it specifically inhibits ATP hydrolase
activity rather than ATP synthetic activity, showing that it is
possible to selectively inhibit hydrolase activity. In addition,
IF1 does not completely inhibit hydrolase activity so that
further inhibition might be of benefit (11). It is currently
thought that the ATP synthase activity of F1F0-ATPase is a
reversible molecular mechanism, although some evidence
suggests that this ATP synthesis is not a reversal of its
ATPase activity (for a review, see Ref. 32). Therefore, it is
possible that an agent that inhibits hydrolase activity such as
azide may do so specifically without affecting synthase
acivity or require dissociation of the hydrolase inhibitor as
is seen for IF1. Modeling IF1 activity is not a likely scenario
for a small organic molecle that selectively inhibits ATP
hydrolase activity without affecting synthase activity because it is unlikely for such an agent to dissociate from the
complex at the “right” time (i.e., when reoxygenation oc-
Address for reprint requests and present address of G. J. Grover: Product
Safety Laboratories, 2394 Highway 130, Dayton, NJ 08810 (E-mail:
[email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ischemia; heart; reperfusion
MITOCHONDRIAL F1F0-ATPASE
http://www.ajpheart.org
0363-6135/04 $5.00 Copyright © 2004 the American Physiological Society
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Grover, Gary J., Karnail S. Atwal, Paul G. Sleph, Feng-Li
Wang, Hossain Monshizadegan, Thomas Monticello, and David
W. Green. Excessive ATP hydrolysis in ischemic myocardium by
mitochondrial F1F0-ATPase: effect of selective pharmacological inhibition of mitochondrial ATPase hydrolase activity. Am J Physiol
Heart Circ Physiol 287: H1747–H1755, 2004; 10.1152/ajpheart.
01019.2003.—Mitochondrial F1F0-ATPase normally synthesizes
ATP in the heart, but under ischemic conditions this enzyme paradoxically causes ATP hydrolysis. Nonselective inhibitors of this
enzyme (aurovertin, oligomycin) inhibit ATP synthesis in normal
tissue but also inhibit ATP hydrolysis in ischemic myocardium. We
characterized the profile of aurovertin and oligomycin in ischemic and
nonischemic rat myocardium and compared this with the profile of
BMS-199264, which only inhibits F1F0-ATP hydrolase activity. In
isolated rat hearts, aurovertin (1–10 ␮M) and oligomycin (10 ␮M), at
concentrations inhibiting ATPase activity, reduced ATP concentration
and contractile function in the nonischemic heart but significantly
reduced the rate of ATP depletion during ischemia. They also inhibited recovery of reperfusion ATP and contractile function, consistent
with nonselective F1F0-ATPase inhibitory activity, which suggests
that upon reperfusion, the hydrolase activity switches back to ATP
synthesis. BMS-199264 inhibits F1F0 hydrolase activity in submitochondrial particles with no effect on ATP synthase activity. BMS199264 (1–10 ␮M) conserved ATP in rat hearts during ischemia while
having no effect on preischemic contractile function or ATP concentration. Reperfusion ATP levels were replenished faster and necrosis
was reduced by BMS-199264. ATP hydrolase activity ex vivo was
selectively inhibited by BMS-199264. Therefore, excessive ATP
hydrolysis by F1F0-ATPase contributes to the decline in cardiac
energy reserve during ischemia and selective inhibition of ATP
hydrolase activity can protect ischemic myocardium.
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MITOCHONDRIAL ATPASE INHIBITION
Fig. 1. The chemical structures for aurovertin B, oligomycin B, and two selective ATP
hydrolase inhibitors, BMS-199264 and
BMS-250685.
AJP-Heart Circ Physiol • VOL
METHODS
Isolated rat heart preparation. Male Sprague-Dawley rats (400 –
500 g) were anesthetized using 100 mg/kg pentobarbital sodium (ip).
The trachea was intubated, and the jugular vein was injected with
heparin (1,000 U/kg). Hearts were perfused in situ via retrograde
cannulation of the aorta and then excised and moved to a Langendorff
apparatus, where they were perfused with oxygenated Krebs-Henseleit solution containing (in mM) 112 NaCl, 25 NaHCO3, 5 KCl, 1.2
MgSO4, 1 KH2PO4, 1.2 CaCl2, 11.5 glucose, and 2 pyruvate at a
constant perfusion pressure (85 mmHg). A water-filled latex balloon
attached to a metal cannula was inserted into the left ventricle for
measurement of left ventricular pressure. End-diastolic pressure
(EDP) was adjusted to 5 mmHg, and this balloon volume was
maintained for the duration of the experiment. Preischemia or predrug
function, heart rate, and coronary flow (extracorporeal electromagnetic flow probe, Carolina Medical Electronics; King, NC) were then
measured. Contractile function was calculated by subtracting EDP
from left ventricular peak systolic pressure, giving left ventricular
developed pressure (LVDP). Cardiac temperature was maintained by
submerging the hearts in 37°C buffer, which accumulated in a stoppered, heated tissue chamber. This technique has been previously
described in detail (6, 8).
All drug treatments were given before ischemia directly via the
perfusate. Ischemia was initiated by completely shutting off the
perfusate flow for 25 min for most studies. This duration of ischemia
was chosen because it is severe enough to cause contracture and a
switch to hydrolase activity, yet established agents that conserve ATP
during ischemia can be clearly shown to be active in a dose-dependent
manner (7, 9, 18). At the end of the reperfusion period, contractile
function, coronary flow, and cumulative lactate dehydrogenase (LDH)
release were measured. Severity of ischemia was determined from
time to the onset of ischemic contracture, recovery of contractile
function at 30 min into reperfusion, and cumulative LDH release into
the reperfusate. Cumulative LDH release has previously been shown
to be well correlated with necrosis (16). Time to the onset of ischemic
contracture was defined as the time (in min) during global ischemia in
which the first 5-mmHg increase in EDP was observed.
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curs). Indeed, development of a small organic molecule that
selectively inhibits hydrolase activity might suggest the
possibility that this is not merely a reversible reaction but
that different conformational states operate in the hydrolase
or synthase modes.
Nonselective inhibitors of F1F0-ATPase such as aurovertin
B and oligomycin B (Fig. 1) would be expected to reduce ATP
production in nonischemic tissue and possibly in reperfused
tissue (not well studied) while conserving ATP in ischemic
tissue. The first goal of this study was to characterize the
effects of aurovertin and oligomycin on nonischemic, ischemic, and reperfused rat myocardial function and energetic
status. Aurovertin and oligomycin are structurally distinct and
inhibit the ATPase at different sites: aurovertin binds to the
catalytic F1 domain, whereas oligomycin binds to the F0
membrane domain.
Structure-activity studies from our laboratories showed several small molecule chemotypes that selectively inhibit F1F0ATP hydrolase activity in vitro in submitochondrial particles
(SMPs) (1) (Fig. 1). Our second goal was to determine the
pharmacological profile of one of these agents (BMS-199264;
Fig. 1) in a model of ischemia and reperfusion in rat hearts and
if this is consistent with its selective inhibitory effects on
F1F0-ATP hydrolase activity.
This study showed that the nonselective F1F0-ATPase
inhibitors aurovertin and oligomycin (inhibit both ATP
synthase and hydrolase activities) reduced ATP depletion
during ischemia but also reduced ATP synthesis in nonischemic and reperfused rat myocardium. The selective F1F0ATP hydrolase inhibitor BMS-199264 reduced ATP decline
during ischemia and exerted significant cardioprotective
effects without affecting ATP synthesis in normal or reperfused tissue. This further suggests that the ATP hydrolysis
by this enzyme does not contribute to useful work or to cell
survival during ischemia.
MITOCHONDRIAL ATPASE INHIBITION
Table 1. Effect of aurovertin or oligomycin on pre- and
postischemic cardiac function (LVDP) and coronary
flow in isolated rat hearts
Predrug
HR, beats/min
Vehicle
1 ␮M Aurovertin
3 ␮M Aurovertin
10 ␮M Aurovertin
10 ␮M Oligomycin
LVDP, mmHg
Vehicle
1 ␮M Aurovertin
3 ␮M Aurovertin
10 ␮M Aurovertin
10 ␮M Oligomycin
Coronary flow, ml䡠min⫺1䡠g⫺1
Vehicle
1 ␮M Aurovertin
3 ␮M Aurovertin
10 ␮M Aurovertin
10 ␮M Oligomycin
Postdrug
Reperfusion
(30 min)
286⫾11
301⫾9
286⫾8
291⫾7
283⫾6
285⫾14
302⫾12
248⫾13
275⫾10
278⫾15
246⫾26
215⫾23*
179⫾2*†
78⫾33*†
36⫾36*†
126⫾6
120⫾5
130⫾5
132⫾5
138⫾7
116⫾15
86⫾4*†
67⫾4*†
56⫾4*†
62⫾5*†
9⫾2*
12⫾4*
13⫾0.7*
9⫾3*
9⫾2*
19.3⫾1.9
19.3⫾1.9
20.8⫾1.1
20.1⫾0.9
20.3⫾1.0
19.2⫾2.9
22.9⫾2.3
36.5⫾1.6*†
31.6⫾1.2*†
28.5⫾1.3*†
13.8⫾1.5*
9.8⫾1.6*
15.4⫾0.6*
14.9⫾0.9*
12.2⫾0.9*
Values are means ⫾ SE. HR, heart rate; LVDP, left ventricular developed
pressure. *Significantly different from the respective predrug value (P ⬍ 0.05);
†signficantly different from the respective vehicle group value (P ⬍ 0.05).
AJP-Heart Circ Physiol • VOL
nitrogen and were assayed for ATP concentration (dry weight) as
previously described (6, 8).
The effect of oligomycin on ATP concentrations was also determined in a separate series of hearts. For this study, a more detailed
time course during ischemia was determined with a single concentration. Rat hearts were given either vehicle or 10 ␮M oligomycin for 2
min in the perfusate before ischemia. The hearts were collected and
frozen in liquid nitrogen at the following times (n ⫽ 5 for all groups):
2 min after drug or vehicle infusion, 10 min into global ischemia, at
15 min of ischemia, at 25 min of ischemia, and 10 and 30 min into
reperfusion after 25 min of global ischemia for both vehicle- and
oligomycin-treated hearts.
We determined the histological or morphological effects of oligomcyin on globally ischemic-reperfused isolated rat hearts. This was
done because the time to contracture was increased and LDH release
was reduced by this agent despite poor postischemic recovery of
function and ATP to further substantiate reduced necrosis. Hearts
were prepared as described above, treated with either vehicle or
oligomycin for 2 min (10 ␮M, n ⫽ 4), and subjected to global
ischemia for 25 min and reperfusion for 30 min. At this time, the
hearts were perfusion fixed for 60 s with McDowell-Trumps solution
followed by immersion-fixation. Hearts were cut in cross section
midway between the base and apex, and the left ventricular free wall
was isolated. The epicardium from each sample was removed, and six
blocks of left ventricular free wall, 1–2 mm in diameter, were
prepared from each heart. Tissues were processed into epoxy resin,
sectioned to 1-␮m thickness, and stained with toluidine blue. All
slides were blinded and randomized before light microscopic evaluation. With the use of a ⫻40 objective lens and a reticle, each field of
myocardial injury was graded as follows: grade 0, no significant
microscopic lesion; grade 1, myofiber degeneration (granular or dense
myoplasm, swollen myofibers, vacuoles, loss of detail, etc.); grade 2,
myofibrillary lysis; and grade 3, myofibrillary lysis with hypercontraction band formation. Each field of the myocardium was scored
with an injury grade and the percent area involvement, and the overall
severity score was the product of myocardial injury and percent tissue
involvement. For each heart section, a mean severity score was
determined. An average of 40 fields/heart sample were graded with a
total of 240 fields evaluated and graded per heart. Slides were
decoded, and a mean score from the six samples per heart was
determined. This technique has been published previously from our
laboratories (16).
Effect of oligomycin on F1F0-ATPase activity in SMPs from ischemic rat hearts. Mitochondrial F1F0-ATPase activity was measured in
isolated rat hearts ex vivo treated with 10 ␮M oligomycin or vehicle
(n ⫽ 4 hearts/group) at 10 min into ischemia. The hearts were
pretreated for 2 min preischemia as performed above. Previous work
has shown inhibition of mitochondrial F1F0-ATPase when SMPs were
treated in vitro, but we wished to show whether this enzyme could be
inhibited when given into the coronary arteries and therefore whether
it can effectively penetrate cells and mitochondria. Mitochondria were
prepared according to Gasiner et al. (4) and SMPs as described by
Matsuno-Yagi and Hatefi (14). The hearts from either group were
immediately put on ice and individually homogenized in 50 mM
Tris 䡠 HCl (pH 7.4), 250 mM sucrose, and 1 mM EDTA (2 ml/g tissue)
using a Waring blender. The insoluble material was pelleted at 960 g
for 15 min at 4°C. The pellet was redissolved in 10 mM Tris 䡠 HCl (pH
7.4), 250 mM sucrose, and 1 mM EDTA and repelleted twice. The
final pellet was resuspended in 1 ml buffer and layered onto a mixture
of 2.2 ml of 2.5 M sucrose, 12.25 ml of 10 mM Tris 䡠 HCl (pH 7.4), 1
mM EDTA, and 6.55 ml Percoll. The mixture was centrifuged at
60,000 g for 45 min at 4°C in a Beckman R50:2 Tri rotor. The second
band from the top of the tube was removed with a Pasteur pipet,
diluted to 30 ml with 10 mM Tris 䡠 HCl (pH 7.4), 250 mM sucrose, and
10 mM EDTA, and pelleted at 10,000 g to remove the Percoll. The
mitochondrial pellet was resuspended in 10 mM Tris-acetate (pH 7.5),
250 mM sucrose, and 10 mM MgCl2 and stored at ⫺70°C. The frozen
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Aurovertin and oligomycin studies. Little is known about the
pharmacological effects of nonselective F1F0-ATPase inhibitors on
preischemic and ischemic myocardium, especially during reperfusion.
Oligomycin is the most studied inhibitor; therefore, we evaluated
aurovertin in more detail and used oligomycin as a reference agent.
For the purpose of comparison, the effects of oligomycin were also
studied because it is the standard reference agent in the few studies
that have been done in the heart.
For the aurovertin studies, isolated rat hearts were subjected to one
of several treatments: 0.04% DMSO (the vehicle used for all studies,
n ⫽ 8 hearts) or 1, 3, or 10 ␮M aurovertin B (n ⫽ 5 hearts/group;
Sigma Chemical, St. Louis, MO) for 2 min via the perfusate. The
hearts were rendered totally and globally ischemic for 25 min followed by 30 min of reperfusion without aurovertin. Cardiac function
and coronary flow were determined throughout the procedure. For
comparison, the standard reference agent oligomycin was tested in
another series of rat hearts. The hearts were prepared as described
above and were subjected to 2 min of vehicle (n ⫽ 8), 10 ␮M
oligomycin B (n ⫽ 5, Sigma Chemical), or 10 ␮M aurovertin (n ⫽ 5)
before the onset of ischemia. The hearts were then made globally
ischemic for 25 min followed by 30 min of reperfusion (without drug).
Cardiac function and coronary flow were determined throughout the
procedure.
Because the respective functional values were similar between the
first aurovertin study (dose-response study) and this second set of
studies, vehicle group values and 10 ␮M aurovertin values were
pooled and the oligomycin data are shown together with the aurovertin data in Table 1.
To correlate functional changes due to aurovertin, we performed a
separate series of studies in rat hearts to determine changes in cardiac
ATP levels. Because we determined ATP biochemically, only one
measurement could be made per heart and separate hearts were used
for the different time periods of the protocol. In this study, vehicle or
3 or 10 ␮M aurovertin B (n ⫽ 5 hearts/group) was given 2 min via the
perfusate before ischemia. For these three treatments, separate hearts
were used to measure ATP concentrations at the end of the drug or
vehicle perfusion (preischemia), 10 min into ischemia, 25 min into
ischemia, or 30 min into reperfusion after 25 min of global ischemia.
For each respective time, the hearts were rapidly frozen in liquid
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MITOCHONDRIAL ATPASE INHIBITION
AJP-Heart Circ Physiol • VOL
RESULTS
Effect of aurovertin or oligomycin on isolated rat hearts.
The effects of aurovertin and oligomyin on pre- and postischemic cardiac function and coronary flow in rat hearts are shown
in Table 1. Although separate vehicle groups were run for these
two compounds, we pooled the values (both groups were
similar, total n ⫽ 16) and included the oligomycin data in
Table 1 for comparison. We also pooled the two separate tests
on 10 ␮M aurovertin because the data were similar (total n ⫽
10). Before ischemia, aurovertin reduced cardiac function in a
concentration-dependent manner with no effect on heart rate.
Aurovertin significantly increased preischemic coronary flow.
At 30 min into reperfusion, cardiac function recovered poorly
in vehicle-treated hearts, indicating severe ischemicreperfusion damage. At the onset of reperfusion in vehicletreated animals, modest recovery of contractile function was
seen in the first minute after reperfusion but afterward rapidly
declined and was stable at a low level well before 30 min of
reperfusion. This has been a uniform finding in this model over
many years, and, after 30 min of reperfusion, no further
improvement in function was seen (6 –9). Aurovertin did not
significantly improve reperfusion contractile function, and the
immediate postreperfusion recovery of function seen in vehicle
groups was not seen for aurovertin (LVDP was stable throughout the reperfusion period). Reperfusion coronary flow did not
recover to preischemic values in either vehicle- or aurovertintreated hearts. At 10 ␮M, oligomycin displayed a profile that
was comparable to 10 ␮M aurovertin. Briefly, oligomycin
reduced preischemic function without affecting heart rate, but
a significant increase in coronary flow was observed. Oligomycin did not improve recovery of contractile function or
coronary flow and was comparable to 10 ␮M aurovertin.
The effects of aurovertin on LDH release and time to the
onset of contracture during ischemia are shown in Fig. 2. The
time to the onset of contracture during global ischemia was
increased by aurovertin in a concentration-dependent manner,
suggesting conservation of ATP during ischemia (inhibition of
rigor). Interestingly, reperfusion LDH release was markedly
and significantly reduced by aurovertin, despite a poor recovery of contractile function. The peak inhibitory effect of
aurovertin on LDH release was observed starting at 3 ␮M.
Despite the poor contractile functional recovery, these data
paradoxically suggest reduced myocardial necrosis. Reperfusion EDP for vehicle-treated hearts was markedly elevated
(77 ⫾ 5 mmHg), and, at 30 min of reperfusion, a similar
number was seen for aurovertin (69 ⫾ 6 mmHg for 10 ␮M),
suggesting a poor recovery of ATP for both treatments. The
time to the onset of contracture during global ischemia was
significantly increased by oligomycin from 17.2 ⫾ 0.9 min for
vehicle to 23.8 ⫾ 0.7 min, and these data are comparable to
aurovertin. Reperfusion LDH release was significantly reduced
by oligomycin from 23 ⫾ 4 U/g in vehicle to 7 ⫾ 1 U/g.
Therefore, oligomycin and aurovertin possess similar pharmacological profiles in ischemic, nonischemic, or reperfused rat
hearts.
The pharmacological profile of aurovertin or oligomycin in
ischemic versus nonischemic hearts may be explained by the
inhibition of both the synthase and hydrolase reactions under
normoxic and ischemic conditions. We measured myocardial
ATP concentrations in the presence or absence of 3 and 10 ␮M
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mitochondria were thawed and homogenized in storage buffer solution containing 1 mM potassium succinate and 10 mM MnCl2. The
suspension was sonicated using a Branson sonifier at maximum output
and 50% pulse mode for 1 min at 0°C. The pH of the suspension was
adjusted to 7.4, and it was centrifuged at 15,000 g for 6 min before the
supernatent was decanted and the SMPs were pelleted at 100,000 g for
40 min at 4°C. The SMPs were washed in 0.25 M sucrose and 10 mM
Tris-acetate (pH 7.5) and repelleted before being stored at ⫺70°C or
assayed directly. ATP hydrolase activity was measured using a coupled assay to follow the oxidation of NADH to NAD⫹ at 340 nm as
described by Pullman et al. (20). The assay was done in 50 mM
Tris-acetate (pH 7.5) containing 2 mM MgCl2, 0.5 mM ATP, 2 mM
phosphoenolpyruvate, 0.2 mM NADH, and pyruvate kinase and LDH.
The SMPs were added and preincubated before NADH oxidation was
measured on a Molecular Devices UVMax plate reader with a 340-nm
filter interfaced to a computer. The change in absorbance at 340 nm
was converted into micromoles of ADP per mininute using the
extinction coefficient of NADH (3,220 䡠 mol⫺1 䡠 cm⫺1). ATP synthase
activity was determined by measuring the increase in absorbance at
340 nm using a hexokinase-glucose-6-phosphate dehydrogenase coupled assay (1). Protein concentrations were determined using the
Bradford assay and used to calculate specific activity (in ␮mol
ADP 䡠 min⫺1 䡠 mg⫺1).
Selective ATP hydrolase inhibitor BMS-199264 in ischemic rat
hearts. BMS-199264 (Fig. 1) was identified in our laboratories from
a focused structure-activity study as a selective F1F0-ATPase hydrolase inhibitor (IC50 ⫽ 0.5 ␮M) with minimal effects on mitochondrial
ATP synthase activity (1). This compound is structurally related to
ATP-sensitive K⫹ (KATP) channel openers, although we found it to
have no KATP activity (1). In our first series of studies, we determined
the pharmacological and cardioprotective profile of this agent in
ischemic-reperfused rat hearts. Isolated rat hearts were given vehicle
(0.04% DMSO, n ⫽ 5 hearts) or 1–10 ␮M BMS-199264 (n ⫽ 5
hearts/group), or 3 ␮M BMS-199264 ⫹ 0.3 ␮M glyburide (n ⫽ 5
hearts) over 10 min before the initiation of global ischemia. Global
ischemia was maintained for 25 min and reperfusion was instituted for
30 min without drug. Preischemic and reperfusion cardiac function
and coronary flow were measured. Glyburide was used to ensure that
BMS-199264 was not having effects secondary to KATP activation (9).
The effect of BMS-199264 on cardiac ATP levels was also determined in a separate group of isolated rat hearts. The hearts were
pretreated for 10 min with either vehicle or 3 ␮M BMS-199264 and
were subdivided according to the experimental design described next.
Hearts were collected for measurement of ATP concentration after 10
min of drug or vehicle treatment (n ⫽ 5 hearts/group, nonischemic
tissue), after 10 min of drug or vehicle administration ⫹ 15 min of
global ischemia (n ⫽ 5 hearts/group), or after 30 min of reperfusion
after 25 min of global ischemia in hearts pretreated for 10 min with
BMS-199264 or vehicle (n ⫽ 5 hearts/group). After the appropriate
treatment, the hearts were frozen in liquid nitrogen, and ATP levels
were determined as described above.
We previously showed the intrinsic effect of BMS-199264 in vitro
on the hydrolase and synthase activities of F1F0-ATPase in SMPs
from normal rat hearts (SMP preparation was superfused with BMS199264) (1). We now determined the effect of BMS-199264 on
ATPase activity in normal or ischemic rat hearts ex vivo. This was
necessary to show that this compound could penetrate whole heart
cardiomyocytes and reach the mitochondria. Isolated rat hearts were
prepared as described above and were treated 10 min before ischemia
with 3 ␮M BMS-199264 (n ⫽ 5), 2 min before ischemia with 10 ␮M
oligomycin (n ⫽ 5), or 10 min before ischemia with vehicle (n ⫽ 5,
0.04% DMSO). The hearts were then rendered globally ischemic for
15 min, at which time the hearts were analyzed for mitochondrial ATP
hydrolase or synthase activity as described above.
MITOCHONDRIAL ATPASE INHIBITION
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Fig. 2. Effect of increasing concentrations of aurovertin on the time to onset
of contracture during global ischemia (Isch) in isolated rat hearts (A). Also
shown is the cumulative lactate dehydrogenase (LDH) release during 30-min
reperfusion (Reper; after 25-min global ischemia) (B), which is an index of
necrosis. Time to contracture was significantly increased (*P ⬍ 0.05) from
vehicle (0 ␮M), whereas LDH release was significantly reduced. Data are
shown as means ⫾ SE.
aurovertin before, during, or after global ischemia (Fig. 3).
Before ischemia, ATP concentrations were reduced by aurovertin by ⬃30%, which explains the negative inotropic effects
of this agent as well as its coronary dilator effect. During
ischemia, ATP was rapidly reduced in vehicle-treated hearts.
Aurovertin slowed the rate of decline of ATP during ischemia
such that ATP levels were similar (3 ␮M aurovertin) or higher
(10 ␮M aurovertin) in drug-treated hearts compared with
vehicle-treated hearts at 10 min into ischemia. At 25 min into
ischemia, aurovertin significantly conserved ATP in the myocardium compared with vehicle-treated hearts in a dose-dependent manner. During reperfusion, ATP recovered partially in
vehicle-treated hearts, whereas aurovertin further depressed
ATP levels. These results are consistent with inhibition of
F1F0-ATP synthase activity in both normal and reperfused
AJP-Heart Circ Physiol • VOL
Fig. 3. Effect of 3 or 10 ␮M aurovertin on myocardial ATP levels in the left
ventricular free wall before, during, or after global ischemia. Aurovertin
reduced ATP before ischemia in a concentration-dependent manner, which is
expected for ATP synthase inhibition. Despite the initial reduction in preischemic ATP, aurovertin conserved ATP in ischemic tissue in a concentrationdependent manner. Unfortunately, aurovertin also inhibited recovery of ATP
during reperfusion, suggesting inhibition of mitochondrial ATP synthase
activity. *Significantly different from vehicle, P ⬍ 0.05. **Both 3 and 10 ␮M
aurovertin are significantly different from vehicle, P ⬍ 0.05. Data are shown
as means ⫾ SE. ⬘, Minutes.
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tissue and with inhibition of the hydrolase activity in ischemic
tissue.
We also measured myocardial ATP concentrations in the
presence or absence of 10 ␮M oligomycin before, during, or
after global ischemia (Fig. 4), although a more detailed time
course study was done during ischemia compared with that
done for aurovertin. Before ischemia, ATP concentrations were
reduced 30% by oligomycin, which is consistent with the
negative inotropic effects of this agent. During ischemia, ATP
was rapidly reduced in vehicle-treated hearts and oligomycin
significantly increased ATP compared with those in timematched vehicle-treated hearts. Upon reperfusion, ATP partially recovered in vehicle-treated hearts, whereas oligomycin
inhibited the recovery of ATP during reperfusion.
Histopatholgical evaluation of ischemic-reperfusion damage
was done in a separate group of rat hearts pretreated with
oligomcyin (25-min ischemia ⫹ 30-min reperfusion). Individual severity scores for each heart and group mean scores are
shown in Table 2. Myocardial lesions were consistently less
severe in oligomycin-treated heart sections compared with
vehicle-treated hearts. The degree of myofibrillary lysis with or
without formation of hypercontraction bands was significantly
less in the oligomycin-treated hearts. Myofiber degenerative
changes, which included swollen myofibers, granular or dense
myoplasm, and vacuolation, were observed in both groups. The
group mean severity score in oligomycin-treated hearts was
significantly different (P ⬍ 0.05) compared with vehicletreated hearts. It was interesting that cardioprotection was
observed for oligomycin even when reperfusion ATP was not
restored. The profile of morphological damage in vehicletreated animals is virtually identical to that previously pub-
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MITOCHONDRIAL ATPASE INHIBITION
Table 3. Effect of BMS-199264 on pre- and postischemic
cardiac function (LVDP) in isolated rat hearts subjected
to 25-min global ischemia followed by 30-min reperfusion
Predrug
Reperfusion
(30 min)
281⫾11
279⫾8
275⫾10
280⫾10
277⫾14
276⫾11
270⫾6
277⫾10
221⫾25*
249⫾22
277⫾12†
275⫾11*†
282⫾12
271⫾12
272⫾10*†
124⫾6
127⫾5
126⫾8
125⫾6
117⫾4
116⫾4
109⫾6
98⫾2*†
11⫾1*
18⫾4*
38⫾4*†
46⫾3*†
118⫾6
108⫾13
39⫾6*†
Values are means ⫾ SE. *Significantly different from the respective predrug
value (P ⬍ 0.05); †signficantly different from the respective vehicle group
value (P ⬍ 0.05).
Fig. 4. Effect of 10 ␮M oligomycin given 2 min before the onset of ischemia
on ATP concentrations in rat hearts before, during, and after global ischemia.
Oligomycin significantly reduced ATP (*P ⬍ 0.05) in normal myocardium but
significantly reduced ATP hydrolysis during ischemia. Upon reperfusion, ATP
began to recover in vehicle-treated hearts, and this effect was abolished by
oligomycin. These data are consistent with the inhibition of both mitochondrial
ATP synthase and hydrolase activities in these hearts. Data are shown as
means ⫾ SE.
lished for this model, and the profile of protection was also
similar to that seen with other cardioprotective agents (see
micrographs in Ref. 16).
Mitochondrial ATP hydrolase activity ex vivo was also
determined in rat hearts treated with vehicle or 10 ␮M oligomycin. Mitochondrial ATP hydrolase activity was determined
to show that ATP preservation during ischemia and reduced
necrosis induced by oligomycin are accompanied by reduced
hydrolase activity during ischemia. Mitochondrial ATP hydrolase activity was significantly attenuated by oligomycin
(0.19 ⫾ 0.0.01 ␮mol ADP䡠min⫺1 䡠mg⫺1) at 10 min into
Table 2. Quantitative myocardial histopatholical severity
scores for 10 oligomycin-treated hearts compared with
vehicle-treated hearts after 25-min global ischemia
and 25-min reperfusion
Group
Severity Score
Vehicle
Vehicle
Vehicle
Vehicle
Mean ⫾ SE
4.92
6.34
5.46
5.36
5.52⫾0.3
Oligomycin
Oligomycin
Oligomycin
Oligomycin
Mean ⫾ SE
1.44
1.01
1.46
1.55
1.37⫾0.12*
*Significantly different from the vehicle-treated group (P ⬍ 0.05).
AJP-Heart Circ Physiol • VOL
ischemia compared with vehicle (0.36 ⫾ 0.02 ␮mol
ADP䡠min⫺1 䡠mg⫺1).
BMS-199264 in isolated rat hearts. The effect of increasing
concentrations of BMS-199264 on preischemic cardiac function are shown in Table 3. LVDP was slightly reduced by 10
␮M BMS-199264, but the cardioprotective concentration of 3
␮M was not cardiodepressant. The degree of cardiac depression seen with 10 ␮M BMS-199264 was so minor as to be
physiologically irrelevant. BMS-199264 had no effect on preischemic coronary flow in this model (data not shown). During
reperfusion, little functional recovery was observed in vehicletreated hearts. BMS-199264 significantly improved the recovery of contractile function in a concentration-dependent manner. This was accompanied by a proportional increase in
reperfusion coronary flow (10.9 ⫾ 1.9 vs. 16.2 ⫾ 1.1
ml䡠min⫺1 䡠g⫺1 for vehicle- and drug-treated groups, respectively), and in this model such enhanced recovery of coronary
flow is typically associated with improved recovery of contractile function and does not imply a direct coronary dilator effect
(7, 8). Therefore, the pharmacological profile for BMS-199264
was distinct from that seen for the nonselective agents aurovertin and oligomycin.
BMS-199264 significantly increased the time to onset of
ischemic contracture starting at the 3 ␮M concentration (Fig.
5). This effect was not abolished by glyburide; therefore, the
protective effect was not due to KATP activation. BMS-199264
reduced reperfusion LDH release in a concentration-dependent
manner (Fig. 5), and this effect was also not abolished by
glyburide. Although the data are not shown, 10 ␮M cromakalim (tested in parallel with these studies) protected in this
model to a similar degree as to that seen with 10 ␮M BMS199264, although the cardioprotection was completely blocked
by glyburide as expected (9).
The effect of 3 ␮M BMS-199264 on myocardial ATP
concentration before, during, and after global ischemia is
shown in Fig. 6. Before ischemia, BMS-199264 had no effect
on ATP concentration, unlike the effect of oligomycin. At 15
min into global ischemia, ATP was significantly reduced in
vehicle-treated hearts, whereas ATP levels were significantly
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HR, beats/min
0 ␮M BMS-199264
1 ␮M BMS-199264
3 ␮M BMS-199264
10 ␮M BMS-199264
3 ␮M BMS-199264 ⫹
0.3 ␮M glyburide
LVDP, mmHg
0 ␮M BMS-199264
1 ␮M BMS-199264
3 ␮M BMS-199264
10 ␮M BMS-199264
3 ␮M BMS-199264 ⫹
0.3 ␮M glyburide
Postdrug
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MITOCHONDRIAL ATPASE INHIBITION
icantly inhibited at 15 min into global ischemia. ATP synthase
activity was significantly reduced by oligomycin while being
unaffected by BMS-199264, confirming its selective inhibition
of hydrolase activity.
DISCUSSION
Fig. 5. Effect of increasing concentrations of the mitochondrial ATP hydrolase
inhibitor BMS-199264 on time to the onset of ischemic contracture (A) and
reperfusion cumulative LDH release (B) in isolated rat hearts after 25-min
global ischemia followed by 30-min reperfusion. BMS-199264 increased time
to contracture and reduced LDH release in a concentration-dependent manner,
which was not blocked by glyburide (Gly). *P ⬍ 0.05. Data are shown as
means ⫾ SE.
increased by BMS-199264. Reperfusion ATP was significantly
higher in BMS-199264- versus vehicle-treated hearts, unlike
oligomycin. The enhanced recovery of ATP is consistent with
the improved recovery of contractile function seen for this
compound.
In another series of rat hearts, the effect of BMS-199264 and
oligomycin on F1F0-ATP hydrolase or synthase activity was
tested ex vivo in ischemic rat hearts (Table 4). The intact hearts
were pretreated with oligomycin or BMS-199264, and SMPs
were subsequently extracted and analyzed as discussed above
but without superfusion of drug. At 3 ␮M BMS-199264 given
10 min before ischemia or 10 ␮M oligomycin given 2 min
before ischemia, ATP hydrolase activity in SMPs was signifAJP-Heart Circ Physiol • VOL
Mitochondrial F1F0-ATPase is a critical enzyme for production of ATP (28). ATP generated from oxidative phosphorylation, and consequently mitochondrial F1F0-ATPase supplies
virtually all of the energy needed for normal cardiac contractile
function as well as ionic homeostasis. Reduction of the oxygen
supply-to-demand ratio will cause myocardial dysfunction and
ATP levels will rapidly drop. Consumption of ATP will continue (albeit at a lower rate) to maintain cellular homeostasis,
although anaerobic glycolysis can only maintain this for short
periods of time (15). Myocardial utilization of ATP during
ischemia is not efficient and ATP “wastage” has been hypothesized (8, 18, 24, 25, 33). F1F0-ATP synthase activity is not
only inhibited during ischemia but its ATP hydrolase activity
will predominate because this enzyme can run in the reverse
mode.
This ATP hydrolysis does not contribute to useful work or to
the maintenance of ionic homeostasis in the heart (11, 33).
Previous studies using the nonselective F1F0-ATPase inhibitor
oligomycin show that inhibition of this hydrolase activity
Table 4. Effect of BMS-199264 or oligomycin on ATP
synthase or hydrolase activity in submitochondrial particles
from rat hearts subjected to 15-min global ischemia
Treatment
Synthase Activity, ␮mol
ATP䡠min⫺1䡠mg⫺1
Hydrolase Activity, ␮mol
ADP䡠min⫺1䡠mg⫺1
Vehicle
3 ␮M BMS-199264
10 ␮M Oligomycin
0.22⫾0.02
0.23⫾0.03
0.07⫾0.01*
0.31⫾0.03
0.18⫾0.01*
0.19⫾0.01*
*Significantly different from vehicle (P ⬍ 0.05).
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Fig. 6. Effect of 3 ␮M BMS-199264 on myocardial ATP concentrations
before and during ischemia and 30 min into reperfusion in isolated rat hearts.
In normal myocardium, BMS-199264 had no effect on ATP while significantly
conserving ATP (*P ⬍ 0.05) at 15 min into global ischemia. BMS-199264
significantly enhanced the recovery of ATP during reperfusion, and this is
consistent with its enhanced recovery of contractile function. Data are shown
as means ⫾ SE.
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MITOCHONDRIAL ATPASE INHIBITION
AJP-Heart Circ Physiol • VOL
The increased time to onset of contracture caused by oligmycin or aurovertin is consistent with ATP conservation as
contracture is caused by rigor. During reperfusion, F1F0ATPase activity might be expected to revert to synthase activity and therefore potentially further enhance the decrement of
ATP by oligomycin and aurovertin. The data showed this to be
the case, suggesting that recovery of ATP was hindered.
Similarly, reperfusion did not cause a recovery of contractile
function in the drug-treated hearts, which is consistent with
energy limitation. Because of the reduced rate of reduction
ATP during ischemia by aurovertin and oligomycin, IF1 does
not completely protect these hearts nor completely inhibits the
hydrolase activity.
Despite a poor recovery of ATP during reperfusion, oligomcyin reduced reperfusion LDH release, which suggests
reduced necrosis (16), which was confirmed histologically.
Oligomycin-treated hearts showed significantly reduced ischemic lesions and necrosis. These data suggest the possibility that
selective inhibition of mitochondrial ATP hydrolase activity
can confer cardioprotective effects during ischemia per se.
Nonselective inhibition of both synthase and hydrolase activities is contraindicated, and agents such as oligomcyin are not
clinically useful. These data also suggest that, although recovery of ATP is necessary for recovery of contractile function
during reperfusion, it does not appear to be necessary for
short-term cell survival during reperfusion. Reduction of the
severity of ischemia preceding the reperfusion most likely
reduced the immediate deleterious effects of reperfusion injury
(necrosis and/or apoptosis), and other factors such as explosive
reintroduction of calcium or oxygen are important and their
effects may be mitigated by a reduced severity of damage
during ischemia.
The selective inhibitory activity of IF1 protein shows that the
mitochondrial synthase and hydrolase activities can be differentially modulated, suggesting the possibility of developing
small molecules that selectively inhibit F1F0-ATP hydrolase
activity without interfering with normal ATP synthetic activity.
We established a screen determining both mitochondrial F1F0ATP synthase and hydrolase activities using a coupled assay
using SMPs in vitro (1). Interestingly, several hits came out of
our KATP opener series, which are known to effectively penetrate into mitochondria. While clear structure activity relationships could be shown (1), the mechanism for selective hydrolase activity is presently unknown. Interestingly, the substituted benzopyrans, which selectively inhibited the hydrolase
activity, resulted from an inversion of the stereochemistry of
the benzopyrans, which open KATP channels (1).
BMS-199264 is one of the substituted benzopyrans that
selectively inhibit ATP hydrolase activity (IC50 ⫽ 0.5 ␮M)
with no effect on ATP synthesis or KATP (potential benzopyran
effect) activity (1). BMS-199264 had no effect on preischemic
ATP concentrations and cardiac function, suggesting a selective effect on mitochondrial hydrolase activity. Interestingly,
BMS-199264 increased the time to ischemic contracture, and
this was associated with conservation of ATP during ischemia.
During reperfusion, contractile function was significantly improved by BMS-199264, and this was accompanied by improved ATP recovery. Therefore, this compound can be clearly
distinguished from the nonselective ATPase inhibitors such as
oligomycin.
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during ischemia can reduce the rate of ATP depletion in
globally ischemic dog hearts ex vivo (11, 33). Because oligomycin will inhibit both synthase and hydrolase activities, this
beneficial effect will only be seen during ischemia and oligomycin will inhibit ATP synthesis under normal or even reperfused conditions when oxygenation is returning to normal. Few
studies have determined whether the conservation of ATP
during ischemia by agents such as oligomycin is protective,
although this is difficult to show as this agent is a toxin in
normal tissue. Vuorinen et al. (33) showed that inhibition of
F1F0-ATP hydrolase activity during ischemia could account for
some of the protective effects of preconditioning (17), and this
may be related to endogenous inhibition of ATP hydrolase
activity. Even though these results are of great interest, subsequent results have been mixed (2, 6, 29).
Pullman and Monroy (20) discovered an endogenous, soluble, heat-stable protein that selectively inhibits the ATP hydrolase activity of F1F0-ATPase. Characterization of this IF1
protein showed it to be active under conditions when hydrolysis of ATP predominates over synthesis (3). Binding of IF1 to
F1F0-ATPase requires hydrolysis of ATP in the absence of an
electrochemical gradient (pH optimum of 6.7) as seen under
ischemic conditions. IF1 is a reversible inhibitor that binds to
the F1 domain in a 1:1 stoichiometry and is thought to work by
trapping adenine nucleotides within the catalytic sites in the F1
domain (5). Determination of mitochondrial concentrations of
IF1 show some species differences. Rouslin et al. (24, 26)
grouped different species into several categories according to
heart rate, IF1 content, or IF1 affinity. Larger animals (slow
heart rate species) such as dogs express relatively higher
amounts of IF1, although the study by Jennings et al. (11)
showed that during ischemia, inhibition of the ATP hydrolase
activity was incomplete. Rats and mice, which have fast heart
rates, have relatively lower expression levels of IF1. Guinea
pigs have a full complement of IF1, but the nature of the
interaction of IF1 with the hydrolase is not clear. Nevertheless,
IF1 is expressed in all of these species and would be expected
to inhibit ATP hydrolysis and potentially protect ischemic
tissue. The data from this study and others show that IF1 does
not completely inhibit hydrolase activity and that further inhibition will confer added benefit.
The first goal of our studies was to characterize the nonselective F1F0-ATPase inhibitors aurovertin and oligomycin.
Aurovertin is a fungal metabolite containing substituted pyrone
and aglycone rings connected by a six-carbon spacer with
conjugated double bonds. Aurovertin binds between the subunits of the catalytic F1 domain of F1F0-ATPase, where it is
postulated to prevent conformational changes required for the
catalytic cycle of the enzyme (31). Oligomycin is a macrocyclic compound produced by actinomycetes that binds to the F0
domain and blocks proton flow through the ATPase (12). We
chose concentrations of aurovertin and oligomycin that inhibited ATPase activity but were not lethal in normal tissue, at
least under our experimental conditions. As expected, both
aurovertin and oligomycin reduced cardiac function in normoxic tissue, and this was accompanied by modest ATP
depletion, suggesting ATPase synthase inhibition. During ischemia, the rapid rate of ATP depletion seen during global
ischemia was attenuated by both oligomycin and aurovertin as
seen previously in rats and dogs (11, 33). This activity was
associated with F1F0-ATPase hydrolase inhibition ex vivo.
MITOCHONDRIAL ATPASE INHIBITION
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These studies clearly show the potential for selective inhibition of mitochondrial ATP hydrolase activity for treating
ischemia or as an experimental tool for determining the importance of F1F0-ATP hydrolase in the pathogenesis of ischemia. Inhibition of ATP hydrolysis during ischemia beyond that
already conferred by IF1 appears useful in a variety of species,
although further work is warranted. Inhibition of this excessive
and wasteful use of ATP would not have a significant cost in
terms of side effects so long as no inhibition of ATP synthase
activity is evident. Another potential advantage of selective
inhibition of this hydrolase activity is the utility for treating
ischemic tissue other than myocardium because F1F0-ATPase
is ubiquitously expressed. The data with BMS-199264 also
bring up the possibility that the ATPase and synthase reactions
do not merely run in reverse but may require different conformational states and that such a transformation would allow a
small organic molecule to selectively inhibit one activity over
the other (32). BMS-199264 may also be a useful tool for
learning more about the question of whether F1F0-ATPase is a
“reversible molecular machine” (32) or how these two activities may be independently controlled either physiologically or
through pharmacological agents.
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