Rat Heart Mitochondria Research Articles

Lipid peroxidation in cardiac mitochondrial fraction of rats exposed to different supplementation with polyunsaturated fatty acids

The effect of diet supplementation with polyunsaturated fatty acids (PUFAs) used at different ratios of w-6/w-3 was studied on the content of primary (diene conjugates, DC; triene conjugates, TC), secondary (ketodienes, CD; coupled trienes, CT; TBA-active products) and terminal (Schiff bases) lipid peroxidation products (LPO) and generation of superoxide anion-radical in rat heart mitochondrial fraction. It was shown that diet supplementation with high doses of w-6 or w-3 PUFAs increased the content of primary, secondary and terminal LPO in rat heart mitochondrial fraction. Llipid peroxidation was accompanied by the intensification of superoxide anion-radical generation in rat heart mitochondrial fraction. During diet consumption with the PUFAs leading factor affecting the intensity of lipoperoxidation in rat heart mitochondria is fatty acid composition, rather than the level of their saturation.

Ca2+ Accumulation in Isolated Rat Heart Mitochondria under Maintenance of Mitochondrial Membrane Potential

Ca2+ Accumulation in Isolated Rat Heart Mitochondria under Maintenance of Mitochondrial Membrane Potential

Mechanisms underlying Li+ effects on the myocardium of vertebrates

The inotropic Li+ effects on the myocardium of frog Rana temporaria, as well as its influence on ion transport in the rat heart mitochondria (RHM), have been studied. The addition of Li+ to the normal Ringer solution (Li+-R) was found to attenuate the myocardium tension, decrease the maximal rate of tension development and its half-relaxation time. A comparison of the cardiac muscle contractile parameters in Li+-R with the effects of voltage-gated Ca2+ channel (Cav 1.2) inhibitors, verapamil and CdCl2, has shown that the negative inotropic effect of Na+ replacement by Li+ in the limited intracellular intermembrane (“fuzzy”) space is based on the block of Ca2+ influx into myoplasm via the reverse Ca2+/Na+ exchanger in the plasma membrane. This, in turn, prevents massive Ca2+-induced Ca2+ release to the myoplasm via RYR2-channels in the sarcoplasmic reticulum finally leading to the suppression of Ca2+-dependent myocardial contractions. The experiments on the Li+ effects on RHM have established that Li+ only slightly increases the passive permeability of the mitochondrial inner membrane for K+ and H+ and decreases the intensity of proton pumping from the matrix of energized mitochondria to the external medium. This may indicate no relationship between the oxidative processes in mitochondria and the decreased myocardium contractility under Na+ replacement in the medium by Li+.

Myocardial ROS Production in Case of Stress is Decreased by Inhibition of the Mitochondrial Respiratory Complex IV

Objectives: The second mechanism of mitochondrial respiratory control was proposed to be physiologically most important for the regulation of complex IV (CytOx) activity, and also of mitochondrial respiration. According to this mechanism the mitochondrial ROS concentrations in myocardium is kept under low healthy values 1 by regulating the mitochondrial respiration and membrane potential 2. In an new experimental setting we show the correlation between 'kinetics' of CytOx activity and mitochondrial membrane potential together with ROS production in heart mitochondria. Methods: Rat heart mitochondria were isolated by standard procedure of isolation (n = 3 in each group). Protein estimation was performed by BCA method. In kinetics studies (w/wo inhibition of CytOx), oxygen consumption by CytOx in intact mitochondria was measured polarographically. Changes in mitochondrial membrane potential and ROS concentrations were detected by DIOC6(3) and MitoSOX method, respectively, in fluorescence-based assays. Results: For the first time, a direct correlation of CytOx kinetics studies to the concentrations of ROS in intact mitochondria is shown. We found a sharp decrease in the ROS concentrations when the activity of CytOx is measured in the presence of ATP and regenerating system (Phosphoenolpyruvate + Pyruvate Kinase). Moreover, in the presence of allosteric ATP inhibition of CytOx, lowest concentrations of ROS were measured in intact rat heart mitochondria. Conclusion: ATP dependent inhibition of CytOx activity is crucial for mitochondrial bioenergetics as it maintains the redox balance by keeping the ROS concentrations at low values. References

Induction of Oxidative Stress in Heart Mitochondria under Focal Cerebral Ischemia-Reperfusion and Protective Effect of Ecdysterone

Based on the fact that the acute phase of ischemic stroke is accompanied by the development of cerebrocardial syndrome, oxidative stress in the rat heart mitochondria and possible mechanisms of cardioprotective action of ecdysterone were examined using the model of focal cerebral ischemia-reperfusion. When focal cerebral ischemia-reperfusion, the rate of reactive oxygen species generation, namely superoxide (. O2− ) and hydroxyl radical (. OH), pools of stable hydrogen peroxide (H2O2) increases, and products of lipid peroxidation (diene conjugates and malonic dialdehyde) are accumulated due to the activation of xanthine oxidase (uric acid as a marker), lipoxygenase (leukotriene C4 as marker), and cyclooxygenase (thromboxane B2 as a marker) ways of·O2- generating. In animals treated with ecdysterone for 18 days, the rate of reactive oxygen species generation decreases, pools of lipid peroxidation products are reduced, the ways of·O2-generating are inhibited, mortality of animals decreases. The results obtained confirm the development of oxidative stress in the rat heart mitochondria, strong antiradical properties of ecdysterone, cardioprotective effect of the latter under focal cerebral ischemia-reperfusion.

Remote ischemic preconditioning of the heart: protective responses in functional and biophysical properties of cardiac mitochondria.

Remote ischemic preconditioning (RIP)-induced protection of myocardial energetics was well documented on the level of tissue, but data concerning the involvement of mitochondria were missing. We aimed at the identification of changes in membrane properties and respiratory functions induced in rat heart mitochondria by RIP. Experiments were performed on 46 male Wistar rats divided into control and RIP-treated groups of 21 animals each. Blood flow in the occluded area was recorded by MRI angiography in four animals. RIP protocol comprised of three successive 5-min occlusions each followed by 5-min reperfusions of descending branches of the right hind limb femoral artery. The efficacy of RIP was evaluated as the extent of RIP-induced protection against damage to the functions of mitochondria isolated by differential centrifugation after 30-min global ischemia followed by 40-min reperfusion of the hearts in Langendorff mode. mitochondrial membrane fluidity with a fluorescent probe DPH, CoQ(9) and CoQ(10) with HPLC, mitochondrial respiration with the Oxygraph-2k (Oroboros). Results revealed that RIP was affecting the mitochondria. The immediate protection conferred by RIP involves beneficial and prognostically significant effects: a total elimination of ischemia/reperfusion-induced depression of mitochondrial membrane fluidity and a trend for better preservation of mitochondrial state 3 respiration.

Characterization of Two Novel Pharmacological ATP-Sensitive Potassium Channels Modulators in Isolated Rat Heart Mitochondria

Characterization of Two Novel Pharmacological ATP-Sensitive Potassium Channels Modulators in Isolated Rat Heart Mitochondria

Effects of standardized extract of Ginkgo biloba leaves EGb761 on mitochondrial functions: mechanism(s) of action and dependence on the source of mitochondria and respiratory substrate.

In this work, the effects of standardized dry extract of Ginkgo biloba leaves, EGb761, on the respiration of rat heart and liver mitochondria were investigated. We revealed uncoupling of oxidative phosphorylation in rat heart mitochondria by EGb761 which was not observed in liver mitochondria respiring on pyruvate + malate; oxidation of succinate in heart mitochondria was inhibited by EGb761, concentration-dependently, almost completely at 1.00mg/mL. Uncoupling effect of EGb761 was found to be due to increase in H(+) and K(+) permeability of inner membrane of mitochondria which is most likely to be mediated by the ATP/ADP-translocator and uncoupling proteins. EGb761 depressed State 3 respiration with pyruvate + malate (similarly in heart and liver mitochondria) and succinate (stronger than with pyruvate + malate) but not respiratory chain Complex IV; inhibition of respiration was not restored by uncoupler indicating the inhibitory action of EGb761 on the respiratory complexes preceding to Complex IV and/or on the substrate transport. Moreover, EGb761 rapidly reduced pure cytochrome c. This property of EGb761 together with the observed uncoupling of oxidative phosphorylation and reduction of H2O2 accumulation may be beneficial for the cell in the prevention of apoptosis and protection of cellular functions in pathological situations.

Індукція оксидативного стресу в мітохондріях серця за фокальної ішемії−реперфузії головного мозку та протекторна дія екдистерону

Based on the fact that the acute phase of ischemic stroke is accompanied by the development of heart damage, manifestations of which are oxidative stress, morphological changes in the myocardium, in the model of brain focal ischemia-reperfusion, we investigated the oxidative stress in rat heart mitochondria and possible mechanisms of cardioprotective effect of ecdysterone. Under the conditions of brain focal ischemia-reperfusion, there is an increase rate of the generation of reactive oxygen species: superoxide (*O2-) and hydroxyl radicals (*OH), pools of stable hydrogen peroxide (H2O2), accumulate products of lipid peroxidation (diene conjugates and malonic dialdehyde), as a result of activation xanthine oxidase (marker uric acid), lipooxygenase (marker leukotriene C4) and cyclooxygenase (marker tromboksane B2) ways of *O2-(generating). In animals that received ecdysterone for 18 days, under conditions of brain focal ischemia-reperfusion, the rate of reactive oxygen species generation and the pools of lipid peroxidation products were decreased, and the survival of animals was increased. The obtained results support the development of oxidative stress in heart mitochondria of rats, powerful antiradical properties ofecdysterone, its cardioprotective effect, in conditions of brain focal ischemia-reperfusion.

Effect of yttrium on calcium-dependent processes in vertebrate myocardium

Inoptopic effect of yttrium acetate (Y3+) on myocardium of the marsh frog Rana ridibunda and its effect on ion transport across the inner mitochondrial membrane (IMM) of rat heart was studied. Y3+ was found to decrease the rate of heart contractions and to stimulate ion transport in the rat heart mitochondria in media with 10 mM glutamate and 2 mM malate. Presence of Y3+ induced inhibition of energy-dependent Ca2+ transport into mitochondria, which was expressed as a marked decrease of their swelling in the media containing 125 mM NH4NO3 and Ca2+ or 25 mM potassium acetate, 100 mM sucrose and Ca2+. It is suggested that the Y3+-induced decrease in rat muscle contractions is determined not only by direct suppressing effect of Y3+ on potential-modulated Ca2+-channels of pacemaker and contractile cardiomyocytes (CM), but also by its indirect effect on Ca2+-carrier in IMM. The data confirming that Y3+ activates energy-dependent K+ transport catalyzed by mitochondrial uniporter and blocks Ca2+-channels in the mitochondrial membrane are important for more complete understanding of mechanisms of the Y3+ action on vertebrates and human CM.

BCNU-induced gR2 DEFECT mediates S-glutathionylation of Complex I and respiratory uncoupling in myocardium

A deficiency of mitochondrial glutathione reductase (or GR2) is capable of adversely affecting the reduction of GSSG and increasing mitochondrial oxidative stress. BCNU [1,3-bis (2-chloroethyl)-1-nitrosourea] is an anticancer agent and known inhibitor of cytosolic GR ex vivo and in vivo. Here we tested the hypothesis that a BCNU-induced GR2 defect contributes to mitochondrial dysfunction and subsequent impairment of heart function. Intraperitoneal administration of BCNU (40mg/kg) specifically inhibited GR2 activity by 79.8±2.7% in the mitochondria of rat heart. However, BCNU treatment modestly enhanced the activities of mitochondrial Complex I and other ETC components. The cardiac function of BCNU-treated rats was analyzed by echocardiography, revealing a systolic dysfunction associated with decreased ejection fraction, decreased cardiac output, and an increase in left ventricular internal dimension and left ventricular volume in systole. The respiratory control index of isolated mitochondria from the myocardium was moderately decreased after BCNU treatment, whereas NADH-linked uncoupling of oxygen consumption was significantly enhanced. Extracellular flux analysis to measure the fatty acid oxidation of myocytes indicated a 20% enhancement after BCNU treatment. When the mitochondria were immunoblotted with antibodies against GSH and UCP3, both protein S-glutathionylation of Complex I and expression of UCP3 were significantly up-regulated. Overexpression of SOD2 in the myocardium significantly reversed BCNU-induced GR2 inhibition and mitochondrial impairment. In conclusion, BCNU-mediated cardiotoxicity is characterized by the GR2 deficiency that negatively regulates heart function by impairing mitochondrial integrity, increasing oxidative stress with Complex I S-glutathionylation, and enhancing uncoupling of mitochondrial respiration.

Inhibition of Gene Expression of Carnitine Palmitoyltransferase I and Heart Fatty Acid Binding Protein in Cyclophosphamide and Ifosfamide-Induced Acute Cardiotoxic Rat Models

This study investigated whether cyclophosphamide (CP) and ifosfamide (IFO) therapy alters the expression of the key genes engaged in long-chain fatty acid (LCFA) oxidation outside rat heart mitochondria, and if so, whether these alterations should be viewed as a mechanism during CP- and IFO-induced cardiotoxicity. Adult male Wistar albino rats were assigned to one of the six treatment groups: Rats in group 1 (control) and group 2 (L-carnitine) were injected intraperitoneal (i.p.) with normal saline and L-carnitine (200mg/kg/day), respectively, for 10 successive days. Animals in group 3 (CP group) were injected i.p. with normal saline for 5days before and 5days after a single dose of CP (200mg/kg, i.p.). Rats in group 4 (IFO group) received normal saline for 5 successive days followed by IFO (50mg/kg/day, i.p.) for 5 successive days. Rats in group 5 (CP-carnitine supplemented) were given the same doses of L-carnitine as group 2 for 5days before and 5days after a single dose of CP as group 3. Rats in group 6 (IFO-carnitine supplemented) were given the same doses of L-carnitine as group 2 for 5days before and 5days concomitant with IFO as group 4. Immediately, after the last dose of the treatment protocol, blood samples were withdrawn and animals were killed for biochemical, histopathological and gene expression studies. Treatment with CP and IFO significantly decreased expression of heart fatty acid binding protein (H-FABP) and carnitine palmitoyltransferase I (CPT I) genes in cardiac tissues. Moreover, CP but not IFO significantly increased acetyl-CoA carboxylase2 mRNA expression. Conversely, IFO but not CP significantly decreased mRNA expression of malonyl-CoA decarboxylase. Both CP and IFO significantly increased serum lactate dehydrogenase, creatine kinase isoenzyme MB and malonyl-CoA content and histopathological lesions in cardiac tissues. Interestingly, carnitine supplementation completely reversed all the biochemical, histopathological and gene expression changes induced by CP and IFO to the control values, except CPT I mRNA, and protein expression remained inhibited by IFO. Data from the current study suggest, for the first time, that (1) CP and IFO therapy is associated with the inhibition of the expression of H-FABP and CPT I genes in cardiac tissues with the consequent inhibition of mitochondrial transport and oxidation of LCFA. (2) The progressive increase in cardiotoxicity enzymatic indices and the decrease in H-FABP and CPT I expression may point to the possible contribution of these genes to CP- and IFO-induced cardiotoxicity.

Effect of NaHS, Hydrogen Sulfide Donor, on the Functional State of the Respiratory Chain in the Rat Heart Mitochondria

Effect of NaHS, Hydrogen Sulfide Donor, on the Functional State of the Respiratory Chain in the Rat Heart Mitochondria

Independent and additive effects of atenolol and methionine restriction on lowering rat heart mitochondria oxidative stress

A low rate of mitochondrial ROS production (mitROSp) and a low degree of fatty acid unsaturation are characteristic traits of long-lived animals and can be obtained in a single species by methionine restriction (MetR) or atenolol (AT) treatments. However, simultaneous application of both treatments has never been performed. In the present investigation it is shown that MetR lowers mitROSp and complex I content. Both the MetR and the AT treatments lower protein oxidative modification and oxidative damage to mtDNA and the fatty acid unsaturation degree in rat heart mitochondria. The decrease in fatty acid unsaturation seems to be due, at least in part, to decreases in desaturase and elongase activities or peroxisomal β-oxidation. Furthermore, the phosphorylation of extracellular signal-regulated kinase (ERK) was stimulated by MetR and AT. The decrease in membrane fatty acid unsaturation and protein oxidation, and the changes in fatty acids and p-ERK showed additive effects of both treatments. In addition, the increase in mitROSp induced by AT observed in the present investigation was totally avoided with the combined MetR + AT treatment. It is concluded that the simultaneous treatment with MetR plus atenolol is more beneficial than either single treatment alone to lower oxidative stress in rat heart mitochondria, analogously to what has been reported in long-lived animal species.

Effects of Flavonoid-Containing Preparation Extralife on Hydrogen Peroxide Production and Functioning of Mitochondrial ATP-Dependent Potassium Channel

Extralife, a Pentaphylloides fruticos extract, in concentrations of 0.005-10 μg/ml dose-dependently increased H2O2 production in rat heart mitochondria in the presence of respiration substrates. Extralife decreased ATP-induced accumulation of H2O2 related to inhibition of mitochondrial ATP-dependent potassium channel. This effect was observed only at low doses of the adaptogen (0.05-3 μg/ml). High doses of the substance (5-10 μg/ml) did not abolish ATP-dependent production of H2O2 and increased the rate of H2O2 generation by the mitochondria. We concluded that Extralife in trace concentrations could activate mitochondrial ATP-dependent potassium channel and decrease H2O2 accumulation in the mitochondria.

Dietary macronutrients modulate the fatty acyl composition of rat liver mitochondrial cardiolipins

The interaction of dietary fats and carbohydrates on liver mitochondria were examined in male FBNF1 rats fed 20 different low-fat isocaloric diets. Animal growth rates and mitochondrial respiratory parameters were essentially unaffected, but mass spectrometry-based mitochondrial lipidomics profiling revealed increased levels of cardiolipins (CLs), a family of phospholipids essential for mitochondrial structure and function, in rats fed saturated or trans fat-based diets with a high glycemic index. These mitochondria showed elevated monolysocardiolipins (a CL precursor/product of CL degradation), elevated ratio of trans-phosphocholine (PC) (18:1/18:1) to cis-PC (18:1/18:1) (a marker of thiyl radical stress), and decreased ubiquinone Q9; the latter two of which imply a low-grade mitochondrial redox abnormality. Extended analysis demonstrated: i) dietary fats and, to a lesser extent, carbohydrates induce changes in the relative abundance of specific CL species; ii) fatty acid (FA) incorporation into mature CLs undergoes both positive (>400-fold) and negative (2.5-fold) regulation; and iii) dietary lipid abundance and incorporation of FAs into both the CL pool and specific mature tetra-acyl CLs are inversely related, suggesting previously unobserved compensatory regulation. This study reveals previously unobserved complexity/regulation of the central lipid in mitochondrial metabolism.

Tl+ induces both cationic and transition pore permeability in the inner membrane of rat heart mitochondria

Effects of Tl(+) were studied in experiments with isolated rat heart mitochondria (RHM) injected into 400mOsm medium containing TlNO3 and a nitrate salt (KNO3 or NH4NO3) or TlNO3 and sucrose. Tl(+) increased permeability of the inner membrane of the RHM to K(+) and H(+). This manifested as an increase of the non-energized RHM swelling, in the order of sucrose < K(+) < NH4 (+), respectively. After succinate administration, the swollen RHM contracted. The Tl(+)-induced opening of the mitochondrial permeability pore (MPTP) in Ca(2+)-loaded rat heart mitochondria increased both the swelling and the inner membrane potential dissipation, as well as decreased basal state and 2,4-dinitrophenol-stimulated respiration. These effects of Tl(+) were suppressed by the MPTP inhibitors (cyclosporine A, ADP, bongkrekic acid, and n-ethylmaleimide), activated in the presence of the MPTP inducer (carboxyatractyloside) or mitoKATP inhibitor (5-hydroxydecanoate), but were not altered in the presence of mitoKATP agonists (diazoxide or pinacidil). We suggest that the greater sensitivity of heart and striated muscles, versus liver, to thallium salts in vivo can result in more vigorous Tl(+) effects on muscle cell mitochondria.

Abstract 104: Effects of Pinacidil and Ca 2 + on Sodium-loaded Rat Heart Mitochondria

Deterioration of the contractile parameters of the heart muscle caused by ischemia and followed reperfusion is known as the main postoperative complication which is related to Ca 2+ and Na + overload in cardiomyocytes and mitochondria. Pinacidil reduced the overload in ischemia/reperfusion experiments. The mechanism of this phenomenon is still not clear. We hypothesized that increased ion permeability of the inner mitochondrial membrane (IMM) followed drop of electrochemical potential (ΔΨ mito ) can reduce the calcium. The aim of the study was to elucidate the effect of pinacidil (100 μM) and Ca 2+ (100 μM ) on swelling, oxygen consumption and ΔΨ mito of isolated sodium-loaded rat heart mitochondria (RHM(Na)) energized glutamate and malate. Pinacidil significantly enchanced the permeability of IMM to protons in ammonium nitrate medium. Also increased swelling of RHM(Na) energized with substrates in potassium acetate medium revealed that pinacidil increased potassium transport into matrix. Pinacidil stimulated oxygen consumption of RHM(Na) in State 4 and detained Ca 2+ -induced dissipation of ΔΨ mito . Under condition of Ca 2+ and Na + overload simulating ischemia/reperfusion, RHM(Na) oxygen consumption was not affected with pinacidil in State 3 and in the presence of 2,4-dinitrophenol. Cyclosporin A and ADP, the inhibitors of mitochondrial permeability transition pore (MPTP), markedly decreased Ca 2+ - induced swelling of RHM(Na) in nitrate ammonium or potassium acetate medium in the presence of pinacidil. Carboxyatractyloside, an inhibitor of cytosolic side-specific adenine nucleotide translocase, eliminated a pinacidil-stimulated oxygen consumption of succinate-energized RHMNa in State 4 regardless of the presence of Ca 2+ . Pinacidil was also concluded to accelerat potassium flux into energized RHM(Na) and promot MPTP opening in the low conduction state. Based on our data we suggested that the effect of pharmacological preconditioning induced by pinacidil could be due to it’s direct effect on mitochondria which is connected with above stimulation of the potassium permeability of the inner mitochondrial membrane and following reduce of the ΔΨ mito that thus prevent calcium overload of cardiomyocytes after ischemia/reperfusion in turn.

Effect of aging on formation of reactive oxygen species by mitochondria of rat heart

Mitochondrial electron transport chain is thought to be a major source of reactive oxygen species (ROS) during aging. However, this view is supported mainly by accumulation of mitochondrial oxidative damage with age and the exact sites of ROS formation remains unknown. In the present study, we measured rate of ROS formation using 2',7'-dichlorofluorescein (DCF) probe in cardiac mitochondria from adult (6-month-old), old (15-month-old) and senescent (26-month-old) rats. In mitochondria oxidizing complex II substrate, succinate, the rate of ROS formation progressively increased with age. In the presence of complex I inhibitor rotenone or complex III inhibitor antimycin A, the rate ROS formation significantly decreased, but even the combination of inhibitors could not fully prevent generation of ROS. Age-dependent increase of ROS formation was accompanied by a loss of thiol groups, tryptophan degradation and increased lipid peroxidation. These data suggest that in addition to complex I and complex II other mitochondrial sites can contribute to accelerated ROS generation and oxidative damage during aging.

Action of La3+ on the systems providing contractility of vertebrate myocardium

The inotropic action of La3+ on frog myocardium was studied with taking into account of its effect on mitochondria of cardiomyocytes (CM). It has been established that in the range of studied concentrations (0.2-6 mM), La3+ decreases dose-dependently the strength of cardiac contractions (by 3.3-92.2 %). In parallel experiments on isolated rat heart mitochondria (RHM), La3+ at a concentration of 25 microM has been shown to cause swelling of non-energized and energized mitochondria in isotonic medium with 125 mM NH4NO3 and in hypotonic medium with 25 mM CH3COOK. The study of oxidative processes in mitochondria with aid of polarographic method of measurement of oxygen concentration has shown that La3+ at concentrations of 50 and 100 microM increases the oxygen consumption rate by mitochondria in the state 2. However, La3+ does not decrease the respiration rate of isolated mitochondria in the state 3, as this takes place in the case of use of Cd2+ or at the Ca(2+)-overloading of mitochondria. The rate of endogenous respiration of isolated mitochondria in the medium with La3+ was higher than in control, which implies its effect on ion permeability of the inner membrane. The data obtained in this work indicate that the La(3+)-produced decrease of the contractility of cardiac muscle is not only due to the direct blocking effect on the potential-controlled Ca2+ channels, but is also mediated by its unspecific action on the CM mitochondria. This action is manifested in acceleration of the energy-dependent K+ transport in matrix and in an increase of ion permeability of the inner mitochondrial membrane (IMM).