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Am J Physiol Heart Circ Physiol 287: H1747-H1755, 2004; doi:10.1152/ajpheart.01019.2003
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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


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 µM) and oligomycin (10 µM), 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. BMS-199264 (1–10 µM) 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.

ischemia; heart; reperfusion

MITOCHONDRIAL F1F0-ATPASE 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 F1F0-ATPase 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 occurs). 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.



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  		Fig. 1. The chemical structures for aurovertin B, oligomycin B, and two selective ATP hydrolase inhibitors, BMS-199264 and BMS-250685.

 
Structure-activity studies from our laboratories showed several small molecule chemotypes that selectively inhibit F1F0-ATP 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 F1F0-ATP 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.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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.

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 µM 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 µM oligomycin B (n = 5, Sigma Chemical), or 10 µM 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 µM aurovertin values were pooled and the oligomycin data are shown together with the aurovertin data in Table 1.


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  		Table 1. Effect of aurovertin or oligomycin on pre- and postischemic cardiac function (LVDP) and coronary flow in isolated rat hearts

 
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 µM 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 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 µM 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 µM, 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-µm thickness, and stained with toluidine blue. All slides were blinded and randomized before light microscopic evaluation. With the use of a x40 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 µM 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·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·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·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·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 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·mol–1·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 µmol ADP·min–1·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 µM) 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 µM BMS-199264 (n = 5 hearts/group), or 3 µM BMS-199264 + 0.3 µM 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 µM 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 BMS-199264) (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 µM BMS-199264 (n = 5), 2 min before ischemia with 10 µM 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.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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 µM 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 ischemic-reperfusion damage. At the onset of reperfusion in vehicle-treated 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 aurovertin-treated hearts. At 10 µM, oligomycin displayed a profile that was comparable to 10 µM 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 µM 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 µM. 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 µM), 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.



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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 µM), whereas LDH release was significantly reduced. Data are shown as means ± SE.

 
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 µM 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 µM aurovertin) or higher (10 µM 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 tissue and with inhibition of the hydrolase activity in ischemic tissue.



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  		Fig. 3. Effect of 3 or 10 µM 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 concentration-dependent 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 µM aurovertin are significantly different from vehicle, P < 0.05. Data are shown as means ± SE. ', Minutes.

 
We also measured myocardial ATP concentrations in the presence or absence of 10 µM 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 time-matched vehicle-treated hearts. Upon reperfusion, ATP partially recovered in vehicle-treated hearts, whereas oligomycin inhibited the recovery of ATP during reperfusion.



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  		Fig. 4. Effect of 10 µM 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.

 
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 vehicle-treated hearts. It was interesting that cardioprotection was observed for oligomycin even when reperfusion ATP was not restored. The profile of morphological damage in vehicle-treated animals is virtually identical to that previously published for this model, and the profile of protection was also similar to that seen with other cardioprotective agents (see micrographs in Ref. 16).


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  		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

 
Mitochondrial ATP hydrolase activity ex vivo was also determined in rat hearts treated with vehicle or 10 µM 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 µmol ADP·min–1·mg–1) at 10 min into ischemia compared with vehicle (0.36 ± 0.02 µmol ADP·min–1·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 µM BMS-199264, but the cardioprotective concentration of 3 µM was not cardiodepressant. The degree of cardiac depression seen with 10 µM 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 vehicle-treated 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·min–1·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.


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  		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

 
BMS-199264 significantly increased the time to onset of ischemic contracture starting at the 3 µM 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 µM cromakalim (tested in parallel with these studies) protected in this model to a similar degree as to that seen with 10 µM BMS-199264, although the cardioprotection was completely blocked by glyburide as expected (9).



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  		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.

 
The effect of 3 µM 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 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.



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  		Fig. 6. Effect of 3 µM 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.

 
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 µM BMS-199264 given 10 min before ischemia or 10 µM oligomycin given 2 min before ischemia, ATP hydrolase activity in SMPs was significantly 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.


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  		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

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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 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.

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, F1F0-ATPase 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 F1F0-ATP 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 µM) 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.

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.


   FOOTNOTES
 

Address for reprint requests and present address of G. J. Grover: Product Safety Laboratories, 2394 Highway 130, Dayton, NJ 08810 (E-mail: GaryGrover{at}productsafetylabs.com)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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