The function of ATP/ADP translocator in the regulation of mitochondrial respiration during development of heart ischemic injury.

Inhibitor titration studies were carried out in order to quantify the amount of control exerted by ATP/ADP translocator on the rate of succinate, palmitoylcarnitine + malate and pyruvate + malate oxidation in ischemia-damaged heart mitochondria. It was shown that after 30 min of total ischemia in vitro the maximal value of the control coefficient of the translocator was as high as in the control: 0.5-0.72 (succinate), 0.8-0.87 (palmitoylcarnitine + malate), 0.83-0.95 (pyruvate+malate). However, the translocator-controlled range of respiratory rates of ischemic mitochondria was narrower than that of normal mitochondria. The control coefficient of the translocator close to State 3 and State 4 was equal to 0-0.15. After 45 min ischemia the maximal value of the translocator control coefficient decreased by 25-30% in comparison with normal mitochondria with all substrates investigated. This value was preserved within a wide range of mitochondrial respiratory rates including the maximal rate in State 3. It was found that the amount of ATP/ADP translocator in mitochondria decreased by 20% after only 45 min ischemia. Our data show that the ATP/ADP translocator is one of the most important steps in regulation of oxidative phosphorylation in isolated mitochondria during development of heart ischemic injury.

Control and kinetic analysis of ischemia-damaged heart mitochondria: which parts of the oxidative phosphorylation system are affected by ischemia?

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.

Heart tissue viability monitoring in vivo by using combined fluorescence, thermography and electrical activity measurements.

A prototype system for in vivo monitoring of the heart tissue viability by using combined measurements of fluorescence, thermography and electrical activity has been elaborated for cardiac surgery. The fluorescence imaging of nicotinamide adenine dinucleotide NAD(P)H in the blue light range (lambda=467 nm) by using UV light (lambda=347 nm) excitation was used to detect metabolic disturbances. The method of the principal component analysis was used for the processing of the fluorescence image sequences. Far infrared (lambda=7.5-13 microm) imaging was used to evaluate temperature dynamics of the tissue surface during circulation disturbances. Evaluation of the epicardial electrogram shape by using continuous wavelet transform was used to detect and evaluate ischemia-caused disturbances of the electrical activity of the tissue. The combination of temperature, fluorescence and electrical activity estimates obtained from synchronically registered parameters during the experiments on model systems and experimental animals yielded qualitatively new results for the evaluation of cardiac tissue viability and enabled to achieve a versatile evaluation of the heart tissue viability.

The role of long-chain acyl-CoA in the damage of oxidative phosphorylation in heart mitochondria.

The aim of this investigation was to study the effect of intramitochondrial acyl-CoA on the respiration of rabbit heart mitochondria over the whole range of stationary respiratory rates between States 4 and 3. The creatine phosphokinase system was used for stabilization of extramitochondrial adenine nucleotide concentration. It was shown that acyl-CoA depressed respiration more effectively in the intermediate range of respiration between States 4 and 3. The effect of acyl-CoA was negligible near State 4 and in State 3. These data are in line with our previous results concerning the dependence of the adenine nucleotide translocator control coefficient on the rate of mitochondrial respiration. Thus, our data suggest that long-chain acyl-CoA may regulate oxidative phosphorylation in heart mitochondria in vivo.

Contribution of ATP synthase to stimulation of respiration by Ca2+ in heart mitochondria.

A variety of experimental conditions were applied with the aim to estimate the correlation between the contribution of ATP synthase to the respiratory flux control and the calcium-induced activation of succinate oxidation in heart mitochondria isolated from rat, rabbit and guinea pig. The sensitivity of respiration in heart mitochondria to the decrease in temperature from 37 degrees C to 28 degrees C decreases in the order rabbit > guinea pig > rat. Ca2+ effect on succinate oxidation rate in state 3 respiration was species- and temperature-dependent and ranged from 0 (rat, 37 degrees C) to +44% (rabbit, 28 degrees C). For mitochondria from all experimental animals, the increase of Ca2+ in physiological range of concentration did not change state 2 respiration rate, and the stimulatory effect of Ca2+ on state 3 respiration was more pronounced at 28 degrees C than at 37 degrees C. The respiratory subsystem was sensitive to Ca2+ ions only in rabbit heart mitochondria. A high positive correlation between Ca2+ ability to stimulate succinate oxidation in state 3 and the control exerted by ATP synthase over the respiratory flux provides argument confirming stimulation of ATP synthase by Ca2+ ions.

The targets of 2,2',5,5'-tetrachlorobiphenyl in the respiratory chain of rat liver mitochondria revealed by modular kinetic analysis.

The response of the respiratory subsystem of oxidative phosphorylation to the environmental pollutant, 2,2',5,5'-tetrachlorobiphenyl (2,2',5,5'-TCB) was investigated by modular kinetic approach. The effects of 20 microM 2,2',5,5'-TCB on the activity of the respiratory chain modules in rat liver mitochondria oxidizing succinate (+ rotenone) in state 3 were assessed. The toxin inhibited the rate of respiration by 23%. Analysis around cytochrome c revealed that 2,2',5,5'-TCB inhibited both cytochrome c-oxidizing and reducing modules. The toxin inhibited also CoQ-oxidizing module, however it did not affect the kinetics of CoQ-reducing module. Taken together, these data indicated that 2,2',5,5'-TCB inhibited cytochrome bc1 but had no effect on succinate dehydrogenase.

Dependence of H2O2 formation by rat heart mitochondria on substrate availability and donor age.

We have examined the substrate specificity and inhibitor sensitivity of H2O2 formation by rat heart mitochondria. Active H2O2 production requires both a high fractional reduction of Complex I (indexed by NADH/NAD+ + NADH ratio) and a high membrane potential, delta psi. These conditions are achieved with supraphysiological concentrations of succinate. With physiological concentrations of NAD-linked substrates, rates of H2O2 formation are much lower (less than 0.1% of respiratory chain electron flux) but may be stimulated by the Complex III inhibitor antimycin A, but not by myxothiazol. Addition of Mn2+ to give 10 nmol/mg of mitochondrial protein enhances H2O2 production with all substrate combinations, possibly by repleting mitochondrial superoxide dismutase with this cation. Contrary to previously published work, no increased activity of H2O2 production was found with heart mitochondria from senescent (24 month) rats, relative to young adults (6 month).

Tetraphenylphosphonium inhibits oxidation of physiological substrates in heart mitochondria.

We show that tetraphenylphosphonium inhibits oxidation of palmitoylcarnitine, pyruvate, malate, 2-oxoglutarate and glutamate in heart mitochondria in the range of concentration (1-5 microM) commonly used for the determination of mitochondrial membrane potential. The inhibition of 2-oxoglutarate (but not other substrate) oxidation by tetraphenylphosphonium is dependent on the concentration of 2-oxoglutarate and on extramitochondrial free calcium, and the kinetic plots are consistent with a mixed type of inhibition. Our results indicate that tetraphenylphosphonium interacts with enzymes, specifically involved in the oxidation of 2-oxoglutarate, most possibly, 2-oxoglutarate dehydrogenase.

Kinetics and control of oxidative phosphorylation in rat liver mitochondria after chronic ethanol feeding.

Changes in the kinetics and regulation of oxidative phosphorylation were characterized in isolated rat liver mitochondria after 2 months of ethanol consumption. Mitochondrial energy metabolism was conceptually divided into three groups of reactions, either producing protonmotive force (Deltap) (the respiratory subsystem) or consuming it (the phosphorylation subsystem and the proton leak). Manifestation of ethanol-induced mitochondrial malfunctioning of the respiratory subsystem was observed with various substrates; the respiration rate in State 3 was inhibited by 27+/-4% with succinate plus amytal, by 20+/-4% with glutamate plus malate, and by 17+/-2% with N,N,N',N'-tetramethyl-p-phenylenediamine/ascorbate. The inhibition of the respiratory activity correlated with the lower activities of cytochrome c oxidase, the bc(1) complex, and the ATP synthase in mitochondria of ethanol-fed rats. The block of reactions consuming the Deltap to produce ATP (the phosphorylating subsystem) was suppressed after 2 months of ethanol feeding, whereas the mitochondrial proton leak was not affected. The contributions of Deltap supply (the respiratory subsystem) and Deltap demand (the phosphorylation and the proton leak) to the control of the respiratory flux were quantified as the control coefficients of these subsystems. In State 3, the distribution of control exerted by different reaction blocks over respiratory flux was not significantly affected by ethanol diet, despite the marked changes in the kinetics of individual functional units of mitochondrial oxidative phosphorylation. This suggests the operation of compensatory mechanisms, when control redistributes among the different components within the same subsystem.

Calcium indirectly increases the control exerted by the adenine nucleotide translocator over 2-oxoglutarate oxidation in rat heart mitochondria.

The effect of calcium on the control exerted by the adenine nucleotide translocator over respiration in isolated heart mitochondria was investigated in order to determine whether calcium directly stimulates the translocator. At respiration rates intermediate between states 3 and 4, Ca2+ is shown to increase the control over 2-oxoglutarate oxidation exerted by the adenine nucleotide translocator in rat heart mitochondria. This did not occur when succinate was the respiratory substrate, even though the control exerted by the translocator was substantial, indicating that Ca2+ does not have a direct effect on the adenine nucleotide translocator. Ca2+ increased the uncoupled oxidation rate of 2-oxoglutarate, but not succinate. Using the summation theorem for flux control, the effect of Ca2+ is explained by a shift of the control over respiration rate toward the adenine nucleotide translocator, from the respiratory chain, presumably as the result of the activation of the 2-oxoglutarate dehydrogenase complex.

Ca2+ stimulates both the respiratory and phosphorylation subsystems in rat heart mitochondria.

Stimulation of mitochondrial respiration by physiological concentrations of Ca2+ was studied to determine which components of oxidative phosphorylation are affected by Ca2+. The kinetic dependence of the respiratory chain, phosphorylation subsystem and proton leak on the mitochondrial membrane potential in isolated rat heart mitochondria respiring on 2-oxoglutarate or succinate was measured at two different concentrations of external free Ca2+. The results show that proton leak is not directly affected by Ca2+, but that both the respiratory and phosphorylation systems can be directly stimulated by Ca2+ depending on conditions. Although Ca2+ directly stimulates the phosphorylation system, this has relatively little effect on respiration rate with 2-oxoglutarate in States 3 and 4 because the subsystem has little control over respiration. However, in intermediate states, the phosphorylation system has greater control and Ca2+ stimulation of this system contributes substantially to the stimulation of respiration and phosphorylation. In the case of succinate oxidation neither the respiratory subsystem nor the phosphorylation system is stimulated by Ca2+.