Respiration Of Rat Heart Mitochondria Research Articles

Astaxanthin Prevents Mitochondrial Impairment Induced by Isoproterenol in Isolated Rat Heart Mitochondria.

Mitochondria are considered to be a power station of the cell. It is known that they play a major role in both normal and pathological heart function. Alterations in mitochondrial bioenergetics are one of the main causes of the origin and progression of heart failure since they have an inhibitory effect on the activity of respiratory complexes in the inner mitochondrial membrane. Astaxanthin (AST) is a xanthophyll carotenoid of mainly marine origin. It has both lipophilic and hydrophilic properties and may prevent mitochondrial dysfunction by permeating the cell membrane and co-localizing within mitochondria. The carotenoid suppresses oxidative stress-induced mitochondrial dysfunction and the development of diseases. In the present study, it was found that the preliminary oral administration of AST upregulated the activity of respiratory chain complexes and ATP synthase and the level of their main subunits, thereby improving the respiration of rat heart mitochondria (RHM) in the heart injured by isoproterenol (ISO). AST decreased the level of cyclophilin D (CyP-D) and increased the level of adenine nucleotide translocase (ANT) in this condition. It was concluded that AST could be considered as a potential mitochondrial-targeted agent in the therapy of pathological conditions associated with oxidative damage and mitochondrial dysfunction. AST, as a dietary supplement, has a potential in the prevention of cardiovascular diseases.

Protamine Sulfate Induces Mitochondrial Hyperpolarization and a Subsequent Increase in Reactive Oxygen Species Production.

Protamine sulfate (PS) is widely used in heart surgery as an antidote for heparin, albeit its pharmacological effects are not fully understood and applications are often accompanied by unwanted side effects. Here we show the effect of PS on mitochondrial bioenergetics profile resulting in mitochondrial reactive oxygen species (ROS) production. Polarographic measurements were performed in parallel to membrane potential and ROS measurements by FACS analyzer using tetramethylrhodamine ethyl ester and MitoSOX fluorescent dyes, respectively. PS inhibited intact rat heart mitochondrial respiration (stimulated by ADP) to 76% (P < 0.001) from the baseline of 51.6 ± 6.9 to 12.4 ± 2.3 nmol O2⋅min-1⋅ml-1 The same effect was found when respiration was inhibited by antimycin A (101.0 ± 8.9 vs. 38.0 ± 9.9 nmol O2 ⋅min-1⋅ml-1, P < 0.001) and later stimulated by substrates of cytochrome oxidase (CytOx) i.e., ascorbate and tetramethyl phenylene diamine, suggesting that PS exerted its effect through inhibition of CytOx activity. Furthermore, the inhibition of mitochondrial respiration by PS was concentration dependent and accompanied by hyperpolarization of the mitochondrial membrane potential (Δψ m), i.e., 18% increase at 50 µg/ml and an additional 3.3% increase at 250 µg/ml PS compared with control. This effect was associated with a strong consequent increase in the production of ROS, i.e., 85% and 88.6% compared with control respectively. We propose that this excessive increase in ROS concentrations results in mitochondrial dysfunction and thus might relate to the "protamine reaction," contributing to the development of various cardiovascular adverse effects.

Direct effects of (-)-epicatechin and procyanidin B2 on the respiration of rat heart mitochondria.

Flavonol (−)-epicatechin and its derived dimer procyanidin B2, present in high amounts in cocoa products, have been shown to exert beneficial effects on the heart and cardiovascular system; however, their mechanism of action has not been fully elucidated. We studied effects of (−)-epicatechin and procyanidin B2 on the oxidative phosphorylation of isolated rat heart mitochondria. (−)-Epicatechin and procyanidin B2 had stimulating effect (up to 30% compared to control) on substrate-driven (State 2) mitochondrial respiration. Their effect was dependent on the respiratory substrates used. (−)-Epicatechin at higher concentrations (from 0.27 µg/mL) significantly decreased (up to 15%) substrate- and ADP-driven (State 3) mitochondrial respiration in case of pyruvate and malate oxidation only. Procyanidin B2 (0.7–17.9 ng/mL) inhibited State 3 respiration rate up to 19%, the most profound effect being expressed with succinate as the substrate. (−)-Epicatechin at concentrations of 0.23 µg/mL and 0.46 µg/mL prevented loss of the cytochrome c from mitochondria when substrate was succinate, supporting the evidence of membrane stabilizing properties of this flavonol. Thus, both (−)-epicatechin and procyanidin B2 directly influenced mitochondrial functions and the observed effects could help to explain cardiometabolic risk reduction ascribed to the consumption of modest amounts of cocoa products.

Action of oxidative reactions in mitochondria on contractivity of heart muscle. Effects of Ni2+

Inotropic effect of Ni2+ on mitochondrial oxidative reactions were studied on myocardium preparations excised from the left frog ventricle and the rat heart mitochondria (RHM), respectively. In the presence of 10–200 μM Ni2+, the cardiac contraction amplitude increased in the dose-dependent manner. It has been found that Ni2+ is not toxic for RHM. The state 4 by Chance in the KCl medium was stimulated by 100 μM Ni2+. At the same time, Ni2+ did not affect the state 3 and the 2,4-dinitrophenol-stimulated respiration of RHM. Our findings allow suggesting that the Ni2+-induced increase in the cardiac contraction amplitude is affected by energy state of the rat cardiomyocytic mitochondria.

Influence of propolis water solution on heart mitochondrial function

The effect of propolis water solution (PWS) on the respiration of rat heart mitochondria with NAD-linked (pyruvate + malate), FAD-linked (succinate) substrates and fatty acids (palmitoyl-L-carnitine) was investigated in this study. PWS at the lowest concentration of 4 microg mL(-1) of phenolic compounds (PC) had no effect on mitochondrial respiration with all investigated substrates. PWS at concentrations of 63 and 125 microg mL(-1) of PC caused a significant decrease of basal (24 and 54%) and maximal (58 and 70%) respiration rates with succinate as substrate. At these PWS concentrations the oxidation of pyruvate + malate and palmitoyl-L-carnitine was diminished to a lower degree: the basal respiration rate decreased by 13-18% and the maximal respiration rate by 15-28%. Succinate oxidation was affected, probably because of the inhibition of succinate dehydrogenase by the 1,2-benzenedicarboxylic acid esters found in PWS. The PWS-caused decrease in the mitochondrial respiration rate with pyruvate + malate and fatty acids could be due to diminished activities of respiratory chain complexes and/or ADP/ATP translocator.

Effects of N-acylethanolamines on the respiratory chain and production of reactive oxygen species in heart mitochondria

Long-chain N-acylethanolamines (NAEs) have been found to uncouple oxidative phosphorylation and to inhibit uncoupled respiration of rat heart mitochondria [Wasilewski, M., Więckowski, M.R., Dymkowska, D. and Wojtczak, L. (2004) Biochim. Biophys. Acta 1657, 151–163]. The aim of the present work was to investigate in more detail the mechanism of the inhibitory effects of NAEs on the respiratory chain. In connection with this, we also investigated a possible action of NAEs on the generation of reactive oxygen species (ROS) by respiring rat heart mitochondria. It was found that unsaturated NAEs, N-oleoylethanolamine ( N-Ole) and, to a greater extent, N-arachidonoylethanolamine ( N-Ara), inhibited predominantly complex I of the respiratory chain, with a much weaker effect on complexes II and III, and no effect on complex IV. Saturated N-palmitoylethanolamine had a much smaller effect compared to unsaturated NAEs. N-Ara and N-Ole were found to decrease ROS formation, apparently due to their uncoupling action. However, under specific conditions, N-Ara slightly but significantly stimulated ROS generation in uncoupled conditions, probably due to its inhibitory effect on complex I. These results may contribute to our better understanding of physiological roles of NAEs in protection against ischemia and in induction of programmed cell death.

Similar nature of inhibition of mitochondrial respiration of heart tissue and malignant cells by methylglyoxal. A vital clue to understand the biochemical basis of malignancy.

The effect of methylglyoxal on the oxygen consumption of mitochondria of heart and of several other organs of normal animals of different species has been tested. The results indicate that methylglyoxal (3.5 mM) strongly inhibits ADP-stimulated alpha-oxoglutarate and malate plus pyruvate-dependent respiration of exclusively heart mitochondria of normal animals of different species. Whereas, with the same substrates, but at a higher concentration of methylglyoxal (7.5 mM), the respiration of mitochondria of other organs of normal animals is not inhibited. Methylglyoxal also inhibits the respiration of slices of rat and toad hearts. But this inhibition is less pronounced. However, methylglyoxal (15 mM) fails to have any effect on perfused toad heart. Using rat heart mitochondria as a model, the effect of methylglyoxal on the oxygen consumption was also tested with different respiratory substrates, electron donors at different segments of the mitochondrial respiratory chain and site-specific inhibitors to identify the specific respiratory complex which might be involved in the inhibitory effect of methylglyoxal. The results strongly suggest that methylglyoxal inhibits the electron flow through complex I of rat heart mitochondrial respiratory chain. Moreover, lactaldehyde (0.6 mM), a catabolite of methylglyoxal, can exert a protective effect on the inhibition of rat heart mitochondrial respiration by methylglyoxal (2.5 mM). The effect of methylglyoxal on heart mitochondria as described in the present paper is strikingly similar to the results of our previous work with mitochondria of Ehrlich ascites carcinoma cells and leukemic leukocytes. We have recently proposed a new hypothesis on cancer which suggests that excessive ATP formation in cells may lead to malignancy. The above mentioned similarity apparently provides a solid experimental foundation for the proposed hypothesis which has been discussed.

Inhibition of rat heart mitochondrial respiration by cadmium chloride

Mitochondria were isolated from hearts obtained from adult male Sprague-Dawley rats by two-part differential centrifugation of heart homogenates. Time-dependent (0–120 sec) and concentration-dependent (0–10 μ m CdCl 2) effects of cadmium on pyruvate-malatesupported state 3 and state 4 respiration were measured in a constant temperature reaction chamber at 37°C, according to established procedures. The ID50 for cadmium chloride on state 3 respiration was determined to be 4.2 μ m. The inhibition produced by cadmium chloride in heart mitochondria was compared, using identical procedures, to the effects induced by two compounds, sodium atractyloside and potassium cyanide, which are known to alter mitochondrial respiration at specific sites. The calculated ID50 values for these agents in heart mitochondria were 1.8 and 16 μ m, respectively. The concentration-dependent inhibition of mitochondrial respiration induced by either cadmium chloride or potassium cyanide was maintained in the presence of 50 μ m carbonyl cyanide m-chlorophenylhydrazone (CCCP), a known uncoupling agent. In contrast, sodium atractyloside did not block the uncoupling effect of 50 μ m CCCP. In addition cadmium chloride was also shown to inhibit CCCP-uncoupled mitochondrial respiration. The cadmium-induced inhibition of mitochondrial respiration was reversed partially by cysteine and completely by 2,3-dimercaptopropanol. The results of the present study indicate that, at all concentrations, cadmium chloride acted solely as an inhibitor of rat heart pyruvatemalate-supported mitochondrial respiration. These findings suggest a possible mechanism for the reported disturbances in myocardial metabolism and function that occur in conjunction with acute and chronic cadmium exposure in humans and experimental animals.

Myocardial ischemia: Correlation of mitochondrial adenine nucleotide and respiratory function

State 3 respiration of rat heart mitochondria decreased approximately 60% after 20 min normothermic in vitro ischemia. After 20 min ischemia, the levels of intramitochondrial adenine nucleotides (ATP + ADP + AMP) decreased to approximately 20% of control values, with a rapid loss between 10 and 20 min. Also, the exchangeable adenine pool of the mitochondria decreased 60% after 20 min ischemia. State 4 respiration was not affected by the ischemic insult. The adenine nucleotide translocase activities of mitochondria from control and ischemic hearts were too high to measure accurately. Therefore, the effects of ischemia on adenine nucleotide translocase activity could not be established. However, 1 microM carboxyatractyloside did not impair state 3 respiration of control mitochondria, but did inhibit the adenine translocase activity by at least 80%. Moreover, titration of state 3 respiration with carboxyatractyloside produced sigmoidal curves for mitochondria from control and ischemic tissue. State 3 respiration correlated well with the total mitochondrial adenine nucleotides and the exchangeable adenine pool (r = 0.63 and 0.78, respectively). Data collected from isolated perfused rat hearts also showed a good correlation between state 3 respiration and the exchangeable adenine nucleotides (r = 0.92). In this study, mitochondria were isolated from hearts that were either perfused, made ischemic for 30 min by aortic cross-clamp, or reperfused for 10 min after the aortic cross-clamp. The slopes and y-intercepts of the regression lines were similar for the in vitro ischemic and the perfusion studies. There was no significant difference between the effects of ischemia on the state 3 and uncoupled respiratory rates.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of inorganic phosphate on creatine kinase in respiring rat heart mitochondria

The ability of creatine to stimulate the respiration of rat heart mitochondria in vitro is reversibly affected by the concentration of inorganic phosphate. The rate of oxygen consumption due to post-ADP state-4 respiration in the presence of 20 m m creatine is reduced significantly when the potassium phosphate concentration is raised from 5 to 20 m m. State-3 respiration is reduced only by potassium phosphate concentrations higher than 20 m m. The rate of synthesis of creatine phosphate is also affected by phosphate concentration, and the apparent K m of the coupled reactions for ADP is significantly higher at 25 m m phosphate as compared to that at 5 m m phosphate. These observations are consistent with the hypothesis that inorganic phosphate acts as an effector molecule, regulating creatine phosphate synthesis by favoring the dissociation of mitochondrial creatine kinase from the mitochondrial membrane. Such regulation may be important in the case of cells undergoing partial or severe ischemia, where changes in phosphate concentration within this range have been reported.

Effect of superoxide generation on rat heart mitochondrial pyruvate utilization

Previous research has shown that heart mitochondria are able to produce reactive species of oxygen such as superoxide radicals, hydrogen peroxide and hydroxyl radicals [ 10, 11]. When these compounds are formed beyond a certain level they are not completely removed by the enzymatic and metabolic processes which neutralize their toxicity, and as a result they are able to produce structural and functional damages that impair mitochondrial function [ 5, 10]. In order to study the molecular mechanism/s by which the oxygen radicals may function as mediators of cellular injury a flow of these radicals by chemical, enzymatic or photochemical methods has been generated in vitro in the presence of cellular preparations. For example, the exposure of isolated subcellular particles to the enzymatic flow of oxygen radicals produced by the reaction of xanthine oxidase upon xanthine reduced both calcium uptake velocity and Ca 2+-ATPase activity in sarcoplasmic reticulum [ 7], while it reduced Ca 2+-stimulated ATPase activity in myofibrillar preparations [ 4]. In addition, incubation with the xanthine oxidase reaction produced an impairment of the respiratory functions associated with an increased lipid peroxidation in the isolated mitochondria [ 5, 10]. These negative effects were augmented in α-tocopherol-deficient mitochondria [ 3], but were opposed by the exogenous addition of superoxide dismutase [ 10]. This report shows that the superoxide radicals generated by the xanthine oxidase reaction reduced rat heart mitochondrial respiration induced by pyruvate. This negative effect was partially prevented by superoxide dismutase and catalase and by thiol protecting agents. Moreover, the generation of free radicals caused a significant reduction in the rate of (1- 14C)-pyruvate decarboxylation, while it did not change the transport of pyruvate into mitochondria.

Inhibition of mitochondrial carnitine-acylcarnitine translocase by sulfobetaines.

Sulfobetaines (N-alkyl-N,N-dimethyl-3-ammonio-1-propanesulfonates) have been identified as relatively specific and selective inhibitors of mitochondrial carnitine-acylcarnitine translocase. Thus, sublytic concentrations of sulfobetaines (alkyl = octyl to tetradecyl) inhibit the respiration of rat heart mitochondria supported by added acylcarnitines or pyruvate plus malonate and carnitine. Both exchange efflux and unidirectional net efflux of mitochondrial carnitine are also inhibited; the half-maximal inhibition of the former occurs at micromolar concentrations of sulfobetaines and the inhibitory effect is reversible and competitive with respect to carnitine. As a stop-inhibitor, 20 mM sulfobetaine, (alkyl = octyl), is useable at near 0 degrees C but is less effective than 2 mM mersalyl when transport rates are very rapid as at higher temperatures especially with liver mitochondria. The loss of mitochondrial carnitine that normally occurs owing to the progress of net efflux during the isolation of mitochondria is prevented by the inclusion of 20 mM sulfobetaine in the isolation medium and this enables a better estimate of the mitochondrial carnitine content. Sulfobetaines inhibit the activities of mitochondrial carnitine acetyltransferase and carnitine palmitoyltransferase but only at concentrations severalfold higher than those inhibitory for the translocase. This observation supports the belief that carnitine-acylcarnitine translocase is an entity distinct from that of carnitine acyltransferases.

Comparative Studies on the Effects of Linoleate and Methyl Linoleate and Their Hydroperoxides on the Respiration and Reactivities of Rat Heart Mitochondria1

Linoleate hydroperoxide and linoleate at concentrations of 100-140 nmol-mg protein activated state 4 respiration of rat heart mitochondria 4.2-fold, increased the apparent enthalpy change of the respiration per gram atom of oxygen consumed from -148 to -226 kJ/O and completely inhibited oxidative phosphorylation. Methyl linoleate hydroperoxide or methyl linoleate did not show the same effects. Further addition of linoleate hydroperoxide or linoleate induced oligomycin-insensitive Mg-ATPase to a level 5 or 2 times, respectively, that obtained with 120 muM dinitrophenol, accompanied by clearing of the mitochondrial suspension and release of malate dehydrogenase from the matrix. Methyl linoleate hydroperoxide had the same effects except that the induced Mg-ATPase activity retained oligomycin sensitivity. Methyl linoleate did not show either effect.

The effects of β-adrenergic receptor blockers on heart mitochondrial metabolism

1. The effects of propranolol, acebutolol and practolol on rat heart mitochondrial respiration and oxidative phosphorylation were examined. 2. Propranolol (0.2–0.5 mM) produced dose dependent decreases in state 3 oxygen consumption ( QO 2(3)), respiratory control index (RCI), ADP: O ratio and phosphorylation rate (PR) and increased state 4 oxygen consumption ( QO 2(4)). 3. The effects of propranolol were apparent regardless of the substrate used and were not reversed by the addition of 10–100 μM adrenaline. 4. Changes in mitochondrial activities due to acebutolol or practolol were observed only at high (3 mM) drug concentrations and the effects were qualitatively similar to those induced by propranolol. 5. The data support a possible role for drug-induced mitochondrial alterations, independent of β-blockade, in the negative inotropic action of propranolol.

The effect of Ca 2+ on the oxidation of β-hydroxybutyric acid by heart mitochondria

The respiration of rat heart mitochondria incubated with EGTA fails to respond to the addition of uncouplers when β-hydroxybutyric acid is the substrate. By contrast, the addition of ADP and phosphate is followed by the normal State 4/State 3 transition. The phenomenon is due to the complete loss of Ca 2+ from mitochondria induced by uncouplers and EGTA, and can indeed be duplicated by incubation in the presence of the specific Ca 2+ ionophore A23187 and ruthenium red (the latter prevents the re-uptake of the lost Ca 2+). Since the loss of Ca 2+ has no effect on the oxidation of other NAD-dependent substrates, it is concluded that Ca 2+ is essential for the interaction of β-hydroxybutyric acid dehydrogenase with a specific intramembrane NAD + NADH pool.

Effect of plague murine toxin on the uptake of calcium and inorganic phosphate ions by heart mitochondria

Plague murine toxin can inhibit to a significant extent the uptake of Ca ++ and inorganic phosphate (P i) by rat heart mitochondria in the presence of succinate, α-ketoglutarate, malate, or β-hydroxybutyrate as the respiratory substrate. When these mitochondrial suspensions are incubated with ATP, but without a respiratory substrate, the uptake of these ions is also inhibited by the toxin. Although the toxin does not alter rat heart mitochondrial respiration supported by the oxidation of reduced cytochrome c, it does inhibit the uptake of Ca ++ and P i supported by this segment of the electron transport chain. When toxin is inactivated by treatment with formalin, it has little or no effect on the uptake of Ca ++ or P i by rat heart mitochondria in any of the systems investigated. The uptake of these ions by heart mitochondria isolated from the toxin-resistant rabbit is either not inhibited at all by the toxin or is inhibited to a much lesser extent as compared with rat heart mitochondria. Ethylenediaminetetraacetic acid, at a concentration which does not inhibit Ca ++ or P i uptake but does prevent swelling, prevents to a large extent the inhibitory effect of the toxin on the accumulation of these ions by rat heart mitochondria.