oligomycins and Myocardial-Ischemia

The mitochondrial F1F0 ATP synthase is a critical enzyme that works by coupling the proton motive force generated by the electron transport chain via proton transfer through the F0 or proton-pore forming domain of this enzyme to release ATP from the catalytic F1 domain. This enzyme is regulated by calcium, ADP, and inorganic phosphate as well as increased transcription through several pathways. This enzyme is also an ATP hydrolase under ischemic conditions. This "inefficient" hydrolysis of ATP consumes 90% of ATP consumed during ischemia as shown with non-selective ATPase inhibitors oligomycin and Aurovertin B. A benzopyran analog, BMS-199264, selectively inhibits F1F0 ATP hydrolase activity with no effect on ATP synthase activity. BMS-199264 had no effect on ATP before ischemia, but reduced the decline in ATP during ischemia. Selective hydrolase inhibition seen with the small molecule BMS-199264 suggests a conformational change in the F1F0 ATPase enzyme when switching from synthase to hydrolase activity.

Topics: Adenosine Triphosphate; Animals; Aurovertins; Enzyme Inhibitors; Forecasting; Humans; Hydrolysis; Imidazoles; Mitochondria; Mitochondrial Proton-Translocating ATPases; Models, Biological; Myocardial Ischemia; Oligomycins; Signal Transduction

The mitochondrial F1F0 ATP synthase is responsible for the majority of ATP production in mammals and does this through a rotary catalytic mechanism. Studies show that the F1F0 ATP synthase can switch to an ATP hydrolase, and this occurs under conditions seen during myocardial ischemia. This ATP hydrolysis causes wasting of ATP that does not produce work. The degree of ATP inefficiently hydrolyzed during ischemia may be as high as 50-90% of the total. A naturally occurring, reversible inhibitor (IF-1) of the hydrolase activity is in the mitochondria, and it has a pH optimum of 6.8. Based on studies with the nonselective (inhibit both synthase and hydrolase activity) inhibitors aurovertin B and oligomycin B reduce the rate of ATP depletion during ischemia, showing that IF-1 does not completely block hydrolase activity. Oligomycin and aurovertin cannot be used for treating myocardial ischemia as they will reduce ATP production in healthy tissue. We generated a focused structure-activity relationship, and several compounds were identified that selectively inhibited the F1F0 ATP hydrolase activity while having no effect on synthase function. One compound, BMS-199264 had no effect on F1F0 ATP synthase function in submitochondrial particles while inhibiting hydrolase function, unlike oligomycin that inhibits both. BMS-199264 selectively inhibited ATP decline during ischemia while not affecting ATP production in normoxic and reperfused hearts. BMS-191264 also reduced cardiac necrosis and enhanced the recovery of contractile function following reperfusion. These data also suggest that the reversal of the synthase and hydrolase activities is not merely a chemical reaction run in reverse.

Topics: Animals; Aurovertins; Enzyme Inhibitors; Imidazoles; Mitochondria; Mitochondrial Proton-Translocating ATPases; Myocardial Ischemia; Oligomycins; Prokaryotic Initiation Factor-1; Proton-Translocating ATPases; Structure-Activity Relationship

The antiapoptotic protein Bcl-2 is targeted to the mitochondria, but it is uncertain whether Bcl-2 affects only myocyte survival after ischemia, or whether it also affects metabolic functions of mitochondria during ischemia. Hearts from mice overexpressing human Bcl-2 and from their wild-type littermates (WT) were subjected to 24 minutes of global ischemia followed by reperfusion. During ischemia, the decrease in pH(i) and the initial rate of decline in ATP were significantly reduced in Bcl-2 hearts compared with WT hearts (P<0.05). The reduced acidification during ischemia was dependent on the activity of mitochondrial F1F0-ATPase. In the presence of oligomycin (Oligo), an F1F0-ATPase inhibitor, the decrease in pH(i) was attenuated in WT hearts, but in Bcl-2 hearts, Oligo had no additional effect on pH(i) during ischemia. Likewise, addition of Oligo to WT hearts slowed the rate of decline in ATP during ischemia to a level similar to that observed in Bcl-2 hearts, but addition of Oligo had no significant effect on the rate of decline in ATP in Bcl-2 hearts during ischemia. These data are consistent with Bcl-2-mediated inhibition of consumption of glycolytic ATP. Furthermore, mitochondria from Bcl-2 hearts have a reduced rate of consumption of ATP on uncoupler addition. This could be accomplished by limiting ATP entry into the mitochondria through the voltage-dependent anion channel, and/or the adenine nucleotide transporter, or by direct inhibition of the F1F0-ATPase. Immunoprecipitation showed greater interaction between Bcl-2 and voltage-dependent anion channel during ischemia. These data indicate that Bcl-2 modulation of metabolism contributes to cardioprotection.

Topics: Adenosine Triphosphate; Anaerobiosis; Animals; Apoptosis; Blotting, Western; Cytosol; Energy Metabolism; Female; Gene Expression Regulation; Genes, bcl-2; Glycolysis; Humans; Hydrogen-Ion Concentration; Male; Mice; Mice, Transgenic; Mitochondria, Heart; Myocardial Contraction; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Nuclear Magnetic Resonance, Biomolecular; Oligomycins; Phosphocreatine; Porins; Proto-Oncogene Proteins c-bcl-2; Proton-Translocating ATPases; Recombinant Fusion Proteins; Voltage-Dependent Anion Channels

Mitochondrial F(1)F(0)-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 F(1)F(0)-ATP hydrolase activity. In isolated rat hearts, aurovertin (1-10 microM) and oligomycin (10 microM), 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 F(1)F(0)-ATPase inhibitory activity, which suggests that upon reperfusion, the hydrolase activity switches back to ATP synthesis. BMS-199264 inhibits F(1)F(0) hydrolase activity in submitochondrial particles with no effect on ATP synthase activity. BMS-199264 (1-10 microM) 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 F(1)F(0)-ATPase contributes to the decline in cardiac energy reserve during ischemia and selective inhibition of ATP hydrolase activity can protect ischemic myocardium.

Topics: Adenosine Triphosphate; Animals; Aurovertins; Cell Survival; Enzyme Inhibitors; Hydrolysis; Imidazoles; Male; Mitochondria; Myocardial Ischemia; Myocardium; Oligomycins; Proton-Translocating ATPases; Rats; Rats, Sprague-Dawley; Uncoupling Agents

The physiological role of F(1)F(0)-ATPase inhibition in ischemia may be to retard ATP depletion although views of the significance of IF(1) are at variance. We corroborate here a method for measuring the ex vivo activity of F(1)F(0)-ATPase in perfused rat heart and show that observation of ischemic F(1)F(0)-ATPase inhibition in rat heart is critically dependent on the sample preparation and assay conditions, and that the methods can be applied to assay the ischemic and reperfused human heart during coronary by-pass surgery. A 5-min period of ischemia inhibited F(1)F(0)-ATPase by 20% in both rat and human myocardium. After a 15-min reperfusion a subsequent 5-min period of ischemia doubled the inhibition in the rat heart but this potentiation was lost after 120 min of reperfusion. Experiments with isolated rat heart mitochondria showed that ATP hydrolysis is required for effective inhibition by uncoupling. The concentration of oligomycin for 50% inhibition (I(50)) for oxygen consumption was five times higher than its I(50) for F(1)F(0)-ATPase. Because of the different control strengths of F(1)F(0)-ATPase in oxidative phosphorylation and ATP hydrolysis an inhibition of the F(1)F(0)-ATPase activity in ischemia with the resultant ATP-sparing has an advantage even in an ischemia/reperfusion situation.

Topics: Adenosine Triphosphate; Animals; Biopsy; Cardiac Surgical Procedures; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Male; Mitochondria, Heart; Myocardial Ischemia; Myocardium; Oligomycins; Oxidative Phosphorylation; Perfusion; Proton-Translocating ATPases; Rats; Rats, Sprague-Dawley

Mitochondrial F1F0 adenosinetriphosphatase (ATPase) is responsible for the majority of ATP synthesis during normoxic conditions, but under ischemic conditions it accounts for significant ATP hydrolysis. A previous study showed that preconditioning in isolated rat hearts is mediated by inhibition of this ATPase during ischemia. We tested this hypothesis in our isolated rat heart model of preconditioning. Preconditioning was accomplished by three 5-min periods of global ischemia separated by 5 min of reperfusion. This was followed by 20 min of global ischemia and 30 min of reperfusion. Preconditioning significantly enhanced reperfusion contractile function and reduced lactate dehydrogenase release but paradoxically reduced the time to onset of contracture during global ischemia. Myocardial ATP was depleted at a faster rate during the prolonged ischemia in preconditioned than in sham-treated hearts, which is consistent with the reduced time to contracture. ATP during reperfusion was repleted more rapidly in preconditioned hearts, which is consistent with their enhanced contractile function. Preconditioning significantly reduced lactate accumulation during the prolonged ischemia. We were not able to demonstrate that mitochondrial F1F0 ATPase (measured in submitochondrial particles) was inhibited by preconditioning before or during the prolonged ischemia. The mitochondrial ATPase inhibitor oligomycin significantly conserved ATP during ischemia and increased the time to the onset of contracture, which is consistent with inhibition of the mitochondrial ATPase. Our results show that preconditioning in rat hearts can be independent of mitochondrial ATPase inhibition as well as ATP conservation.

Topics: Adenosine Triphosphate; Animals; Coronary Circulation; Heart; Heart Rate; In Vitro Techniques; Ischemic Preconditioning, Myocardial; Male; Mitochondria, Heart; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion; Oligomycins; Proton-Translocating ATPases; Rats; Rats, Sprague-Dawley; Time Factors; Ventricular Function, Left

The mitochondrial ATPase enzyme accounts for roughly 35-50% of the overall energy demand that leads to ATP depletion under conditions of severe myocardial ischemia. In larger mammalian hearts, this energy squandering action of the ATPase is modulated by an endogenous inhibitor protein. The present studies were undertaken to characterize the time course of inhibition of the mitochondrial ATPase in canine myocardium under conditions of severe regional ischemia in vivo. In addition, we determined if the energy sparing effects of ischemic preconditioning (PC) can be explained by persistent inhibition of the mitochondrial ATPase enzyme. The circumflex coronary artery was ligated for 1.5 min (n = 4), 5 min (n = 6), or 15 min (n = 5). In a separate group (n = 7), hearts were preconditioned by four 5-min periods of ischemia each followed by 5 min of reperfusion. Sub-mitochondrial particles were prepared from the sub-endocardial zone of the ischemic and non-ischemic regions and were assayed for oligomycin-sensitive ATPase activity. ATPase activity was reduced to about 79% at 1.5 min and to approximately 55% at 5 and 15 min of ischemia, relative to non-ischemic tissue from the same heart. The rate of HEP utilization slowed concurrently with the development of ATPase inhibition. In preconditioned myocardium, ATPase activity was not significantly different from control myocardium from the same heart. We conclude that the early inhibition of the mitochondrial ATPase activity slows the utilization of high energy phosphate and thereby serves as an important endogenous cardioprotective mechanism. Nevertheless, altered activity of the ATPase is not the explanation of the energy sparing effect of ischemic preconditioning.

Topics: Adenosine Diphosphate; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Cell Fractionation; Coronary Circulation; Coronary Vessels; Dogs; Energy Metabolism; Kinetics; Microscopy, Electron, Scanning; Mitochondria, Heart; Myocardial Ischemia; Myocardium; Oligomycins; Phosphocreatine; Reference Values; Regional Blood Flow; Submitochondrial Particles; Time Factors

Cultured cardiac myocytes were depleted of ATP by incubation with oligomycin (1 mg/ml). Then the ability of the cells to oxidize various substrates and to restore ATP levels was studied. Following ATP depletion, the cells were found to be able to oxidize glucose given alone, but not palmitate. However, with both substrates, palmitate was oxidized in the presence of glucose and ATP recovery was enhanced. Pyruvate had a minor effect on palmitate oxidation, while acetate given alone was oxidized, but did not enhance cellular ATP content. These results show that glucose is essential for restoration of mitochondrial function and the coupling between oxidation and ATP synthesis.

Topics: Adenosine Triphosphate; Animals; Carbon Dioxide; Cells, Cultured; Energy Metabolism; Fatty Acids; Glucose; Heart Ventricles; Myocardial Ischemia; Myocardium; Oligomycins; Oxidation-Reduction; Palmitic Acid; Rats

We investigated the effects of ischemia on the kinetics and control of mitochondria isolated from normal and ischemic heart. The dependence of the respiratory chain, phosphorylation system and proton leak on the mitochondrial membrane potential were measured in mitochondria from hearts after 0, 30 min and 45 min of in vitro ischemia. Data showed that during the development of ischemia from the reversible (30 min) to the irreversible (45 min) phase, a progressive decrease in activity of the respiratory chain occurs. At the same time an increase in proton leak across the mitochondrial inner membrane was observed. Phosphorylation is inhibited but seems to be less affected by ischemia than respiratory chain or proton leak. Control coefficients of the 3 blocks of reactions over respiration rate were determined in different respiratory states between state 4 and state 3. Ischemia caused the control exerted by the proton leak to increase in state 3 and the intermediate state and caused the control by the phosphorylation system to decrease in the intermediate state. Taken together, these results indicate that the main effects of ischemia on mitochondrial respiration are an inhibition of the respiratory chain and an increase of the proton leak.

Topics: Animals; Electron Transport; In Vitro Techniques; Kinetics; Membrane Potentials; Mitochondria, Heart; Myocardial Ischemia; Oligomycins; Onium Compounds; Oxidative Phosphorylation; Oxygen Consumption; Phosphorylation; Protons; Rats; Rubidium; Trityl Compounds