Comparative Study of Functional Changes in Heart Mitochondria in Two Modes of Epinephrine Exposure Modeling Myocardial Injury in Rats.

The parameters of coupled respiration and transport of calcium ions in mitochondria isolated from the heart of rats were studied in two modes of exposure to epinephrine for modelling myocardial damage. In 24 h after injection of 1.5 mg/kg epinephrine to rats, we observed a decrease in the efficiency of oxidative phosphorylation in heart mitochondria in the presence of both NADH- and FADH-dependent respiratory substrates. Increasing the epinephrine dose and exposure (2 mg/kg, 72 h) led to a more pronounced decrease in the ADP/O coefficient when succinate was used as a substrate, which indicated a predominant decrease in the activity of complex II of the respiratory chain. The injection of epinephrine in the two modes resulted in a decrease in the rate of calcium entry in rat heart mitochondria, but had no effect on mitochondrial calcium retention capacity, which reflects the resistance of the organelles to the induction of the Са2+-dependent pore. These findings suggest that both cardiomyopathy models in rats can be used to study the effectiveness of pharmacological therapy using mitochondria-targeted agents.

Does the Operation of Mitochondrial ATP-Dependent Potassium Channels Affect the Structural Component of Mitochondrial and Endothelial Dysfunctions in Experimental Parkinsonism?

We have previously demonstrated that the development of oxidative stress in some pathologies can be prevented by activation of the mitochondrial ATP-dependent potassium channel (mitoKATP). Here we studied the effect of modulation of mitoKATP on the development of mitochondrial and endothelial dysfunction in the medulla oblongata and myocardium of rats with experimental parkinsonism. It is known that uridine-5'-diphosphate, activator of mitoKATP, does not penetrate the plasma membrane, but it can be synthesized in cells from exogenous uridine that is delivered into cells by special transport systems. Our results suggest that mitoKATP is involved in the development of mitochondrial and endothelial dysfunction in experimental parkinsonism and prove the cardio- and neuroprotective effects of uridine.

[Effect of per os administration of dihydroquercetin aqueous form on energy exchange in blood lymphocytes of rats with experimental cardiomyopathy].

Cardiomyopathies are among the most severe myocardial pathologies, which are characterized by resistance to therapy and high mortality due to increasing heart failure and arrhythmia. Cardiomyocyte pathological changes upon cardiomyopathies are associated with mitochondrial dysfunction, leading to excessive formation of reactive oxygen species and the development of oxidative stress. In this regard, the study of the therapeutic potential of antioxidants in cardiomyopathies, as well as the mechanisms of their action on the functioning of mitochondria, is relevant and of high practical importance. The aim of this study was to determine the effect of oral 14-day administration of dihydroquercetin in a water-soluble form (DHQWF) on the activity of the key marker of mitochondrial respiration [succinate dehydrogenase (SDH)] and the cytoplasmic marker of glycolysis [lactate dehydrogenase (LDH)] in blood lymphocytes, as well as on the serum level of lipid peroxidation (LPO) in control rats and rats with experimental cardiomyopathy. Material and methods. Adult male Wistar rats (body weight 220-240 g) were used for the study. Isoprenaline hydrochloride was used to induce cardiomyopathy (IIC) in animals (twice subcutaneous injection at a dose of 150 mg/kg body weight, with a break of 24 hours). DHQ-WF was added to the drinking water for 14 days at the dose of 15 or 30 mg/kg body weight. SDH and LDH activity in lymphocytes was measured using a highly sensitive cytobiochemical method on a blood smear according to the reduction of nitrotetrazolium blue chloride to diformazan of dark blue color. The content of malone dialdehyde (MDA) in the blood serum, heart and liver mitochondria was determined spectrophotometrically using thiobarbituric acid. Mitochondria were isolated from rat tissues by the conventional method of differential centrifugation. Mitochondrial respiration was recorded using a polarographic method. Results. Experimental cardiomyopathy in rats was accompanied by a twofold increase in blood serum MDA level, as well as by a significant increase in SDH and LDH activity in blood lymphocytes. The oral administration of DHQ-WF in cardiomyopathy at a dose of 15 mg/kg body weight led to a significant decrease in serum MDA level, but did not reduce the activity of SDH and LDH in blood lymphocytes, compared with animals with cardiomyopathy that did not receive DHQ-WF. In the control group of animals, the use of DHQ-WF at a dose of 15 mg/kg body weight significantly increased blood lymphocyte LDH activity, but did not have a statistically significant effect on SDH activity and the parameters of mitochondrial respiration and oxidative phosphorylation, the level of MDA in heart and liver mitochondria. Increasing the dose of DHQ-WF administered to 30 mg/kg had less effect on changes in these parameters in control animals. Conclusion. The data obtained indicate that in experimental cardiomyopathy in rats, the course application of DHQ-WF at a dose of 15 mg/kg of body weight acts as an effective antioxidant that prevents the development of lipid peroxidation in blood serum, and can modulate energy metabolism towards the enhancement of glycolysis in blood lymphocytes in control animals.

Prospects for the use of regulators of oxidative stress in the comprehensive treatment of the novel Coronavirus Disease 2019 (COVID-19) and its complications.

Some surface proteins of the newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can bind to the hemoglobin molecule of an erythrocyte, which leads to the destruction of the structure of the heme and the release of harmful iron ions to the bloodstream. The degradation of hemoglobin results in the impairment of oxygen-carrying capacity of the blood, and the accumulation of free iron enhances the production of reactive oxygen species. Both events can lead to the development of oxidative stress. In this case, oxidative damage to the lungs leads then to the injuries of all other tissues and organs. The use of uridine, which preserves the structure of pulmonary alveoli and the air-blood barrier of the lungs in the course of experimental severe hypoxia, and dihydroquercetin, an effective free radical scavenger, is promising for the treatment of COVID-19. These drugs can also be used for the recovery of the body after the severe disease.

Structural and Functional Features of Ca2+ Transport Systems in Liver Mitochondria of Rats with Experimental Hyperthyroidism.

We analyzed structural and functional features of the main mitochondrial Ca2+-transporting systems, mitochondrial Ca2+ uniporter complex (MCUC) and Ca2+-dependent cyclosporin A-sensitive mitochondrial permeability transition pore (MPT pore), in rats with hyperthyroid state. It was found that, the rate of Ca2+ accumulation by rat liver mitochondria in this pathology increases by 1.3 times, which can be associated with higher level of the channel-forming subunit of the uniporter MCU and lower content of dominant-negative subunit of this complex MCUb. At the same time, the level of the regulatory subunit MICU1 remained unchanged. It was shown that calcium retention capacity of liver mitochondria in rats with experimental hyperthyroidism decreased by 2 times in comparison with the control, which attested to reduced resistance of liver mitochondria of hyperthyroid rats to induction of the MPT pore. The observed changes are consistent with the data on increased amount of cyclophilin D, a mitochondrial matrix peptidyl-prolyl isomerase that is known to modulate the MPT pore opening and expression of the Ppif gene that encodes mitochondrial cyclophilin D in rats with experimental hyperthyroidism.

Mitochondrial Ca2+ Transport: Mechanisms, Molecular Structures, and Role in Cells.

Mitochondria are among the most important cell organelles involved in the regulation of intracellular calcium homeostasis. During the last decade, a number of molecular structures responsible for the mitochondrial calcium transport have been identified including the mitochondrial Ca2+ uniporter (MCU), Na+/Ca2+ exchanger (NCLX), and Ca2+/H+ antiporter (Letm1). The review summarizes the data on the structure, regulation, and physiological role of such structures. The pathophysiological mechanism of Ca2+ transport through the cyclosporine A-sensitive mitochondrial permeability transition pore is discussed. An alternative mechanism for the mitochondrial pore opening, namely, formation of the lipid pore induced by saturated fatty acids, and its role in Ca2+ transport are described in detail.

Dynamic Restructuring of the Myocardial Mitochondria in Response to Uridine Modulation of the Activity of Mitochondrial ATP-Dependent Potassium Channel under Conditions of Acute Hypoxic Hypoxia.

We studied the effects of in vivo modulation of activity of mitochondrial ATP-dependent potassium channel (mitoKATP) by uridine on the morphofunctional state of mitochondria in rat cardiomyocytes under conditions of acute hypoxia. Preinjection of uridine to animals reduced the number of structurally modified mitochondria, but had practically no effect on their morphogenesis after hypoxia. Uridine in vivo stimulated the formation of micromitochondria and their release into the cytoplasm. The number of "maternal" mitochondria containing three and more new micromitochondria, increased as well. The use of mitoKATP blocker 5-hydroxydecanoate in parallel with uridine abolished its protective effect, as it significantly inhibited the formation of micromitochondria in rat cardiomyocytes after acute hypoxic exposure.

Study of Uridine Effect on the Development of Audiogenic Tonic Seizures in Krushinsky-Molodkina Strain Rats.

The latency of tonic seizure in response to loud sound (in rats of the Krushinsky-Molodkina strain with audiogenic epilepsy) had been slightly (although statistically significantly) longer after chronic uridine injections (100 mg/kg, i.p., three times a day during 9 or 12 days). The recovery time from the tonic seizure was shorter after 12 days of injections in comparison to the 9-day injection period. At the same time, the intensity of tonic seizures provoked by loud sound did not change after chronic uridine injections. The lack of uridine anticonvulsive effect demonstrated in the audiogenic epilepsy model contradicts the anticonvulsant effects of uridine in experiments with other seizure models, in which the epileptic foci were localized in the forebrain structures.

Specific Features of Immediate Ultrastructural Changes in Brain Cortex Mitochondria of Rats with Different Tolerance to Hypoxia under Various Modes of Hypoxic Exposures.

We performed ultrastructural study of cerebral cortex mitochondria in rats with different tolerance to oxygen deficiency (low resistant and highly resistant specimens). Low resistant rats were characterized by the prevalence of mitochondria with lightened matrix due to the nondense packing of cristae. By contrast, mitochondria of highly resistant animals had the dense packing of cristae. The structure of mitochondria underwent adaptive changes at 14-10% O2 in the inspired air. Under these conditions, structural characteristics of the cerebral cortex in hypoxia-sensitive rats resembled those in resistant animals. The decrease in O2 concentration to 8% was accompanied by ultrastructural signs of mitochondrial damage, which correlated with de-energization of the cell and dysfunction of adaptive signaling systems. Ultrastructural features of cerebral cortex mitochondria in animals with low and high tolerance to acute oxygen deficiency confirm the hypothesis that they are associated with two different "functionaland-metabolic portraits".

Structural and Dynamic Changes in Mitochondria of Rat Myocardium under Acute Hypoxic Hypoxia: Role of Mitochondrial ATP-Dependent Potassium Channel.

The ultrastructure and spatial localization of mitochondria (MC) in the myocardium of rats exposed to a 30-min hypoxic hypoxia were investigated. The mitochondrial structure was found to undergo changes; however, marked necrotic injuries were not observed. Changes occurring in the myocardium are aimed at the intensification of energy processes. This shows up as an increase in the number of MC in the subsarcolemmal zone of the myocardium and changes in the surface of the sublemmal membrane due to its bending around mitochondria, which improves the diffusion of oxygen into MC. In addition, the division of MC is enhanced, which partially explains the increase in their total number. In structurally altered MC with intact membrane, electron dense formations with small diameter appear, which probably represent newly formed organelles (microMC). In normoxia, changes of this kind do not occur. It was found that the ATP-dependent K+ channel is involved in the regulation of the morphological state of MC under hypoxic hypoxia. The activator of the channel diazoxide increases the number of newly formed microMC, and the channel inhibitor 5HD significantly prevents their formation. Possible mechanisms of structural and dynamic changes in rat myocardial MC under acute hypoxic hypoxia are discussed.

Antiarrhythmic effect of uridine and uridine-5'-monophosphate in acute myocardial ischemia.

Experiments on rats with acute myocardial ischemia accompanied by early postocclusive arrhythmias have shown normalizing, energy-stabilizing, and antiarrhythmic effects of uridine and uridine-5'-monophosphate. The drugs decreased lactate and restored reserves of glycogen and creatine phosphate depleted by ischemia. Uridine and uridine-5'-monophosphate significantly decreased the severity of ventricular arrhythmias. Both drugs reduced the incidence and duration of fibrillation. Uridine -5'-monophosphate demonstrated most pronounced antifibrillatory effectiveness. We hypothesize that the antiarrhythmic effect of the drugs is determined by their capacity to activate energy metabolism.

[A role for calreticulin in functioning of mitochondrial ATP-dependent potassium channel].

Homology of the amino acid sequence of the mitochondrial potassium-transporting protein (MW 57kDa), having the properties of a channel subunit of the mitochondrial ATP-dependent potassium channel, and calreticulin (MW 55kDa) was detected by MALDI-TOF-TOF analysis method. Inhibitory analysis of ATP-dependent potassium transport in mitochondria with polyclonal antibodies to calreticulin was carried out. A dose-dependent inhibition of potassium transport in mitochondria by these antibodies was shown. The maximum value of inhibition was 55-60%. Based on these data it is hypothesized that at least two types of ATP-sensitive potassium channels are localized in mitochdndrial membrane. It is expected that the type of mitochondrial ATP-dependent potassium channel, which includes homologous calreticulin protein is localized mainly at mitochondrial and reticulum membrane contact sites.

[The effect of uridine on the endurance of animals with different resistance to physical stress: the role of mitochondrial ATP-dependent potassium channel].

The effect of a metabolic precursor of natural activator of mitochondrial ATP-dependent potassium channel (mitochondrial K+(ATP))--uridine on animal's endurance to physical stress was studied. The endurance was determined by recording the time period during which the rat loaded with a plummet of 20% of body weight can swim until physical exhaustion at 32 degrees C. It was found that highly resistant animals swam until exhaustion for 7.40 ± 0.35 min, whereas low resistant rats hold out 2.07 ± 0.10 min only. The injection of uridine influenced the swimming time of the animals, increasing it twofold in low-resistant rats. The effect of uridine was decreased by injection of inhibitors of mitochondrial K+(ATP) channel. It was found that the injection of uridine into low resistant rats increased the rate of potassium transport in mitochondria isolated from liver of these rats, and inhibitors of the channel prevent the channel activating effect of uridine. The role of mitochondrial K+(ATP) cannel in the formation of animal's resistance to physical stress and protection of tissues from hypoxia is discussed.

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.

[Determination of the antioxidant properties of activators of mitochondrial ATP-dependent potassium channels with the Amplex Red fluorescent indicator].

The effect of adaptogens-antihypoxants that participate in the activation of mitochondrial ATP-dependent potassium channels (mitoK(ATP)) at the oxidation of the Amplex Red (AR) fluorescent indicator in a peroxidase system was tested. It was shown that Extralife, Hypoxen, taurine, and synthetic antioxidant ionol can be arranged in the following row, according to the fluorescence inhibition activity: Extralife > Hypoxen > > ionol > taurine; their effect was shown to be concentration-dependent. The calculated K(i) value of fluorescence indicators demonstrate fast and slow phases of inhibition of the AR oxidation by Extralife and Hypoxen. The fast phase occurs in the presence of microdoses (0.05-3 microg/mL) of adaptogens and is related to the competition for H2O2, which is in agreement with our previous data on the mitoK(ATP) activation by doses of adaptogens related to the H2O2 consumption. The slow phase is characteristic of high adaptogen and ionol concentrations and is related to the competition for phenoxyl radicals of resorufin formed during AR oxidation. The obtained results allow one to suggest the application of a highly sensitive model peroxidase system with AR for the preliminary testing of compounds activating mitoK(ATP) channels.

Effect of uridine on energy metabolism, LPO, and antioxidant system in the myocardium under conditions of acute coronary insufficiency.

Experiments on rats have shown that preventive treatment with uridine stabilizes energy metabolism in the heart under conditions of 60-min left coronary artery occlusion. The preparation also prevented antioxidant system dysfunction and LPO hyperactivation. 5-Hydroxydecanoate, a selective blocker of mitochondrial ATP-dependent K(+)-channels, abolished the protective effect of uridine, which attested to the involvement of these channels into mechanisms of the cardioprotective effect of the preparation. The elimination of intravenously administered uridine from the blood of animals with acute ischemia was accelerated in comparison with that in intact animals, which could suggest the participation of this nucleoside in the processes of activation of intracellular anti-ischemic defense mechanisms.

[Isolation and purification of human blood plasma proteins able to form potassium channels in artificial bilayer lipid membrane].

Protein fraction able to induce K(+)-selective transport across bilayer lipid membrane was isolated from human blood plasma with the use of the detergent and proteolytic enzyme-free method developed at our laboratory. After addition of the studied sample to the artificial membrane in the presence of 100 mM KCl, a discrete current change was observed. No channel activity was recorded in the presence of calcium and sodium ions. Channel forming activity of fraction was observed only in the presence of K+. Using a threefold gradient of KCl in the presence of studied proteins the potassium-selective potential balanced by voltage of -29 mV was registered. This value is very close to the theoretical Nernst potential in this case. This means that the examined ion channel is cation-selective. According to data obtained with MS-MALDI-TOF/TOF and database NCBI three protein components were identified in isolated researched sample.

[The mirochondrial lipid palmitate/Ca2+-induced pore and its possible role in a degradation of nervous cells].

The subject of the review is a new type of mitochondrial pore--a pore which has lipid nature and is induced by palmitic acid and Ca2+. The review considers molecular mechanisms of its formation and regulation, conditions of its opening in biological membranes and the role in physiological and pathophysiological processes. Also discussed is involvement of the lipid pore in glutamate-induced degradation of nervous cells.

Activity of mitochondrial ATP-dependent potassium channel in animals with different resistance to hypoxia before and after the course of hypoxic training.

Activity of mitochondrial ATP-dependent potassium channel in rats with high genetically determined resistance to hypoxia was higher than in sensitive animals. Adaptation of low resistant rats to hypoxia was accompanied by activation of the channel, facilitation of potassium recycling in mitochondria, and a decrease in the rate of H2O2 formation. Our results indicate that mitochondrial ATP-dependent potassium channel plays an important role in the delayed mechanisms of animal's adaptation to hypoxia.

[Localization in a cell of a protein forming the ATP-dependent potassium-selective channels in the bilayer lipid membrane. An ultrastructural study].

The localization in the cell of the protein forming the ATP-dependent potassium-selective channels in the bilayer lipid membrane has been studied. The electron microscope investigation of rat liver and heart tissue sections after their incubation with Abs against this protein and the visualization of the protein with secondary Abs conjugated with colloid gold were carried out. Colloid gold particles were observed both in mitochondrial membranes and in membranes of endoplasmic and sarcoplasmic reticulum. In heart mitochondria, these particles were significantly greater than in liver mitochondria. The localization of the channel protein both in mitochondria and reticulum, as well as the structural similarity between the mitochondrial channel and the precursor of calreticulin suggest that the channel protein belongs to the family of calreticulins. The possible function of the protein as a channel subunit of the mitochondrial ATP-dependent potassium channel is discussed.