Effect of VBIT-4 on the functional activity of isolated mitochondria and cell viability.

VBIT-4 is a new inhibitor of the oligomerization of VDAC proteins of the outer mitochondrial membrane preventing the development of oxidative stress, mitochondrial dysfunction, and cell death in various pathologies. However, as a VDAC inhibitor, VBIT-4 may itself cause mitochondrial dysfunction in healthy cells. The article examines the effect of VBIT-4 on the functional activity of rat liver mitochondria and cell cultures. We have demonstrated that high concentrations of VBIT-4 (15-30 µM) suppressed mitochondrial respiration in state 3 and 3UDNP driven by substrates of complex I and II. VBIT-4 induced depolarization of organelles fueled by substrates of complex I but not complex II of the respiratory chain. VBIT-4 has been found to inhibit the activity of complexes I, III, and IV of the respiratory chain. Molecular docking demonstrated that VBIT-4 interacts with the rotenone-binding site in complex I with similar affinity. 15-30 µM VBIT-4 caused an increase in H2O2 production in mitochondria, decreased the Ca2+ retention capacity, but increased the time of Ca2+-dependent mitochondrial swelling. We have found that the incubation of breast adenocarcinoma (MCF-7) with 30 µM VBIT-4 for 48 h led to the decrease of the mitochondrial membrane potential, an increase in ROS production and death of MCF-7 cells. The mechanism of action of VBIT-4 on mitochondria and cells is discussed.

Effect of 2-aminoethoxydiphenyl borate on the functions of mouse skeletal muscle mitochondria.

This work examined the effect of 2-aminoethoxydiphenyl borate (2-APB) on the functioning of isolated mouse skeletal muscle mitochondria and modeled its putative interaction with mitochondrial proteins. We have shown that 2-APB is able to dose-dependently suppress mitochondrial respiration in state 3 and 3UDNP driven by substrates of complex I and II. This effect of 2-APB was accompanied by a slight dose-dependent decrease in mitochondrial membrane potential and appears to be due to inhibition of complex I and complex III of the electron transport chain (ETC) with IC50 values of 200 and 120 µM, respectively. The results of molecular docking identified putative 2-APB interaction sites in these ETC complexes. 2-APB was shown to dose-dependently inhibit both mitochondrial Ca2+ uptake and Ca2+ efflux, which seems to be caused by a decrease in the membrane potential of the organelles. We have found that 2-APB has no significant effect on mitochondrial calcium retention capacity. On the other hand, 2-APB exhibited antioxidant effect by reducing mitochondrial hydrogen peroxide production but without affecting superoxide generation. It is concluded that the effect of 2-APB on mitochondrial targets should be taken into account when interpreting the results of cell and in vivo experiments.

Mitochondrial Transplantation Therapy Ameliorates Muscular Dystrophy in mdx Mouse Model.

Duchenne muscular dystrophy is caused by loss of the dystrophin protein. This pathology is accompanied by mitochondrial dysfunction contributing to muscle fiber instability. It is known that mitochondria-targeted in vivo therapy mitigates pathology and improves the quality of life of model animals. In the present work, we applied mitochondrial transplantation therapy (MTT) to correct the pathology in dystrophin-deficient mdx mice. Intramuscular injections of allogeneic mitochondria obtained from healthy animals into the hind limbs of mdx mice alleviated skeletal muscle injury, reduced calcium deposits in muscles and serum creatine kinase levels, and improved the grip strength of the hind limbs and motor activity of recipient mdx mice. We noted normalization of the mitochondrial ultrastructure and sarcoplasmic reticulum/mitochondria interactions in mdx muscles. At the same time, we revealed a decrease in the efficiency of oxidative phosphorylation in the skeletal muscle mitochondria of recipient mdx mice accompanied by a reduction in lipid peroxidation products (MDA products) and reduced calcium overloading. We found no effect of MTT on the expression of mitochondrial signature genes (Drp1, Mfn2, Ppargc1a, Pink1, Parkin) and on the level of mtDNA. Our results show that systemic MTT mitigates the development of destructive processes in the quadriceps muscle of mdx mice.

Modulators reducing the efficiency of oxidative ATP synthesis in mitochondria: protonophore uncouplers, cyclic redox agents, and decouplers.

This work considers the main indicators of the oxidative phosphorylation efficiency in mitochondria: the ADP/O and H+/O ratios. Three groups of modulators that reduce the efficiency of oxidative phosphorylation are compared: protonophore uncouplers, cyclic redox compounds, and decouplers. It is noted that some of them are considered effective therapeutic agents. The paper analyzes the authors' original data on the mechanism of action of natural decouplers, represented by long-chain α,ω-dioic acids, as antioxidants. In conclusion, we discuss the hypothesis of their participation in the rescue of hepatocytes in various disorders of carbohydrate and lipid metabolism.

Effect of Fusidic Acid and Some Nitrogen-Containing Derivatives on Liposomal and Mitochondrial Membranes.

The paper assesses the membranotropic action of the natural antibiotic fusidic acid (FA) and its derivatives. It was found that a FA analogue with ethylenediamine moiety (derivative 2), in contrast to native FA and 3,11-dioxime analogue (derivative 1), is able to increase the mobility of the lipid bilayer in the zone of lipid headgroups, as well as to induce permeabilization of lecithin liposome membranes. A similar effect of derivative 2 is also observed in the case of rat liver mitochondrial membranes. We noted a decrease in the microviscosity of the mitochondrial membrane and nonspecific permeabilization of organelle membranes in the presence of this agent, which was accompanied by a decrease in mitochondrial Δψ and OXPHOS efficiency. This led to a reduction in mitochondrial calcium retention capacity. The derivatives also reduced the production of H2O2 by mitochondria. The paper considers the relationship between the structure of the tested compounds and the observed effects.

Pharmacological and Genetic Suppression of VDAC1 Alleviates the Development of Mitochondrial Dysfunction in Endothelial and Fibroblast Cell Cultures upon Hyperglycemic Conditions.

Prolonged hyperglycemia related to diabetes and its complications leads to multiple cellular disorders, the central one being the dysfunction of mitochondria. Voltage-dependent anion channels (VDAC) of the outer mitochondrial membrane control the metabolic, ionic, and energy cross-talk between mitochondria and the rest of the cell and serve as the master regulators of mitochondrial functions. Here, we have investigated the effect of pharmacological suppression of VDAC1 by the newly developed inhibitor of its oligomerization, VBIT-4, in the primary culture of mouse lung endotheliocytes and downregulated expression of VDAC1 in human skin fibroblasts on the progression of mitochondrial dysfunction upon hyperglycemic stress. The cells were grown in high-glucose media (30 mM) for 36 h. In response to hyperglycemia, the mRNA level of VDAC1 increased in endotheliocytes and decreased in human skin fibroblasts. Hyperglycemia induced overproduction of mitochondrial ROS, an increase in the susceptibility of the organelles to mitochondrial permeability transition (MPT) pore opening and a drop in mitochondrial membrane potential, which was accompanied by a decrease in cell viability in both cultures. Treatment of endotheliocytes with 5 µM VBIT-4 abolished the hyperglycemia-induced increase in susceptibility to spontaneous opening of the MPT pore and ROS generation in mitochondria. Silencing of VDAC1 expression in human skin fibroblasts exposed to high glucose led to a less pronounced manifestation of all the signs of damage to mitochondria. Our data identify a mitochondria-related response to pharmacological and genetic suppression of VDAC activity in vascular cells in hyperglycemia and suggest the potential therapeutic value of targeting these channels for the treatment of diabetic vasculopathies.

Conjugation of Triterpenic Acids of Ursane and Oleanane Types with Mitochondria-Targeting Cation F16 Synergistically Enhanced Their Cytotoxicity against Tumor Cells.

The present work shows the cytotoxic effects of novel conjugates of ursolic, oleanolic, maslinic, and corosolic acids with the penetrating cation F16 on cancer cells (lung adenocarcinoma A549 and H1299, breast cancer cell lines MCF-7 and BT474) and non-tumor human fibroblasts. It has been established that the conjugates have a significantly enhanced toxicity against tumor-derived cells compared to native acids and also demonstrate selectivity to some cancer cells. The toxic effect of the conjugates is shown to be due to ROS hyperproduction in cells, induced by the effect on mitochondria. The conjugates caused dysfunction of isolated rat liver mitochondria and, in particular, a decrease in the efficiency of oxidative phosphorylation, a decrease in the membrane potential, and also an overproduction of ROS by organelles. The paper discusses how the membranotropic- and mitochondria-targeted effects of the conjugates may be related to their toxic effects.

Bile Acids as Inducers of Protonophore and Ionophore Permeability of Biological and Artificial Membranes.

It is now generally accepted that the role of bile acids in the organism is not limited to their participation in the process of food digestion. Indeed, bile acids are signaling molecules and being amphiphilic compounds, are also capable of modifying the properties of cell membranes and their organelles. This review is devoted to the analysis of data on the interaction of bile acids with biological and artificial membranes, in particular, their protonophore and ionophore effects. The effects of bile acids were analyzed depending on their physicochemical properties: namely the structure of their molecules, indicators of the hydrophobic-hydrophilic balance, and the critical micelle concentration. Particular attention is paid to the interaction of bile acids with the powerhouse of cells, the mitochondria. It is of note that bile acids, in addition to their protonophore and ionophore actions, can also induce Ca2+-dependent nonspecific permeability of the inner mitochondrial membrane. We consider the unique action of ursodeoxycholic acid as an inducer of potassium conductivity of the inner mitochondrial membrane. We also discuss a possible relationship between this K+ ionophore action of ursodeoxycholic acid and its therapeutic effects.

Effect of Large-Conductance Calcium-Dependent K+ Channel Activator NS1619 on Function of Mitochondria in the Heart of Dystrophin-Deficient Mice.

Dystrophin-deficient muscular dystrophy (Duchenne dystrophy) is characterized by impaired ion homeostasis, in which mitochondria play an important role. In the present work, using a model of dystrophin-deficient mdx mice, we revealed decrease in the efficiency of potassium ion transport and total content of this ion in the heart mitochondria. We evaluated the effect of chronic administration of the benzimidazole derivative NS1619, which is an activator of the large-conductance Ca2+-dependent K+ channel (mitoBKCa), on the structure and function of organelles and the state of the heart muscle. It was shown that NS1619 improves K+ transport and increases content of the ion in the heart mitochondria of mdx mice, but this is not associated with the changes in the level of mitoBKCa protein and expression of the gene encoding this protein. The effect of NS1619 was accompanied by the decrease in the intensity of oxidative stress, assessed by the level of lipid peroxidation products (MDA products), and normalization of the mitochondrial ultrastructure in the heart of mdx mice. In addition, we found positive changes in the tissue manifested by the decrease in the level of fibrosis in the heart of dystrophin-deficient animals treated with NS1619. It was noted that NS1619 had no significant effect on the structure and function of heart mitochondria in the wild-type animals. The paper discusses mechanisms of influence of NS1619 on the function of mouse heart mitochondria in Duchenne muscular dystrophy and prospects for applying this approach to correct pathology.

Ion Channels of the Sarcolemma and Intracellular Organelles in Duchenne Muscular Dystrophy: A Role in the Dysregulation of Ion Homeostasis and a Possible Target for Therapy.

Duchenne muscular dystrophy (DMD) is caused by the absence of the dystrophin protein and a properly functioning dystrophin-associated protein complex (DAPC) in muscle cells. DAPC components act as molecular scaffolds coordinating the assembly of various signaling molecules including ion channels. DMD shows a significant change in the functioning of the ion channels of the sarcolemma and intracellular organelles and, above all, the sarcoplasmic reticulum and mitochondria regulating ion homeostasis, which is necessary for the correct excitation and relaxation of muscles. This review is devoted to the analysis of current data on changes in the structure, functioning, and regulation of the activity of ion channels in striated muscles in DMD and their contribution to the disruption of muscle function and the development of pathology. We note the prospects of therapy based on targeting the channels of the sarcolemma and organelles for the correction and alleviation of pathology, and the problems that arise in the interpretation of data obtained on model dystrophin-deficient objects.

Comparative Study of Cytotoxic and Membranotropic Properties of Betulinic Acid-F16 Conjugate on Breast Adenocarcinoma Cells (MCF-7) and Primary Human Fibroblasts.

The present study evaluates the cytotoxicity of a previously synthesized conjugate of betulinic acid (BA) with the penetrating cation F16 on breast adenocarcinoma (MCF-7) and human fibroblast (HF) cell lines, and also shows the mechanism underlying its membranotropic action. It was confirmed that the conjugate exhibits higher cytotoxicity compared to native BA at low doses also blocking the proliferation of both cell lines and causing cell cycle arrest in the G0/G1 phase. We show that the conjugate indeed has a high potential for accumulation in mitochondria, being visualized in these organelles, which is most pronounced in cancer cells. The effect of the conjugate was observed to be accompanied by ROS hyperproduction in both cancerous and healthy cells, despite the lower base level of ROS in the latter. Along with this, using artificial liposomes, we determined that the conjugate is able to influence the phase state of lipid membranes, make them more fluid, and induce nonspecific permeabilization contributing to the overall cytotoxicity of the tested agent. We conclude that the studied BA-F16 conjugate does not have significant selective cytotoxicity, at least against the studied breast cancer cell line MCF-7.

BKCa Activator NS1619 Improves the Structure and Function of Skeletal Muscle Mitochondria in Duchenne Dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive hereditary disease caused by the absence of the dystrophin protein. This is secondarily accompanied by a dysregulation of ion homeostasis, in which mitochondria play an important role. In the present work, we show that mitochondrial dysfunction in the skeletal muscles of dystrophin-deficient mdx mice is accompanied by a reduction in K+ transport and a decrease in its content in the matrix. This is associated with a decrease in the expression of the mitochondrial large-conductance calcium-activated potassium channel (mitoBKCa) in the muscles of mdx mice, which play an important role in cytoprotection. We observed that the BKCa activator NS1619 caused a normalization of mitoBKCa expression and potassium homeostasis in the muscle mitochondria of these animals, which was accompanied by an increase in the calcium retention capacity, mitigation of oxidative stress, and improvement in mitochondrial ultrastructure. This effect of NS1619 contributed to the reduction of degeneration/regeneration cycles and fibrosis in the skeletal muscles of mdx mice as well as a normalization of sarcomere size, but had no effect on the leakage of muscle enzymes and muscle strength loss. In the case of wild-type mice, we noted the negative effect of NS1619 manifested in the inhibition of the functional activity of mitochondria and disruption of their structure, which, however, did not significantly affect the state of the skeletal muscles of the animals. This article discusses the role of mitoBKCa in the development of DMD and the prospects of the approach associated with the correction of its function in treatments of this secondary channelopathy.

Effect of Chronic Treatment with Uridine on Cardiac Mitochondrial Dysfunction in the C57BL/6 Mouse Model of High-Fat Diet-Streptozotocin-Induced Diabetes.

Long-term hyperglycemia in diabetes mellitus is associated with complex damage to cardiomyocytes and the development of mitochondrial dysfunction in the myocardium. Uridine, a pyrimidine nucleoside, plays an important role in cellular metabolism and is used to improve cardiac function. Herein, the antidiabetic potential of uridine (30 mg/kg/day for 21 days, i.p.) and its effect on mitochondrial homeostasis in the heart tissue were examined in a high-fat diet-streptozotocin-induced model of diabetes in C57BL/6 mice. We found that chronic administration of uridine to diabetic mice normalized plasma glucose and triglyceride levels and the heart weight/body weight ratio and increased the rate of glucose utilization during the intraperitoneal glucose tolerance test. Analysis of TEM revealed that uridine prevented diabetes-induced ultrastructural abnormalities in mitochondria and sarcomeres in ventricular cardiomyocytes. In diabetic heart tissue, the mRNA level of Ppargc1a decreased and Drp1 and Parkin gene expression increased, suggesting the disturbances of mitochondrial biogenesis, fission, and mitophagy, respectively. Uridine treatment of diabetic mice restored the mRNA level of Ppargc1a and enhanced Pink1 gene expression, which may indicate an increase in the intensity of mitochondrial biogenesis and mitophagy, and as a consequence, mitochondrial turnover. Uridine also reduced oxidative phosphorylation dysfunction and suppressed lipid peroxidation, but it had no significant effect on the impaired calcium retention capacity and potassium transport in the heart mitochondria of diabetic mice. Altogether, these findings suggest that, along with its hypoglycemic effect, uridine has a protective action against diabetes-mediated functional and structural damage to cardiac mitochondria and disruption of mitochondrial quality-control systems in the diabetic heart.

The Effect of Uridine on the State of Skeletal Muscles and the Functioning of Mitochondria in Duchenne Dystrophy.

Duchenne muscular dystrophy is caused by the loss of functional dystrophin that secondarily causes systemic metabolic impairment in skeletal muscles and cardiomyocytes. The nutraceutical approach is considered as a possible complementary therapy for this pathology. In this work, we have studied the effect of pyrimidine nucleoside uridine (30 mg/kg/day for 28 days, i.p.), which plays an important role in cellular metabolism, on the development of DMD in the skeletal muscles of dystrophin deficient mdx mice, as well as its effect on the mitochondrial dysfunction that accompanies this pathology. We found that chronic uridine administration reduced fibrosis in the skeletal muscles of mdx mice, but it had no effect on the intensity of degeneration/regeneration cycles and inflammation, pseudohypetrophy, and muscle strength of the animals. Analysis of TEM micrographs showed that uridine also had no effect on the impaired mitochondrial ultrastructure of mdx mouse skeletal muscle. The administration of uridine was found to lead to an increase in the expression of the Drp1 and Parkin genes, which may indicate an increase in the intensity of organelle fission and the normalization of mitophagy. Uridine had little effect on OXPHOS dysfunction in mdx mouse mitochondria, and moreover, it was suppressed in the mitochondria of wild type animals. At the same time, uridine restored the transport of potassium ions and reduced the production of reactive oxygen species; however, this had no effect on the impaired calcium retention capacity of mdx mouse mitochondria. The obtained results demonstrate that the used dose of uridine only partially prevents mitochondrial dysfunction in skeletal muscles during Duchenne dystrophy, though it mitigates the development of destructive processes in skeletal muscles.

Alisporivir Normalizes Mitochondrial Function of Primary Mouse Lung Endothelial Cells Under Conditions of Hyperglycemia.

Effect of alisporivir (a mitochondrial permeability transition pore inhibitor) on the development of mitochondrial dysfunction under hyperglycemic conditions in the primary culture of mouse lung endothelial cells was investigated in this work. We demonstrated that hyperglycemia (30 mM glucose for 24 h) leads to the decrease in viability of the pulmonary endotheliocytes, causes mitochondrial dysfunction manifested by the drop in membrane potential and increase in superoxide anion generation as well as facilitates opening of the mitochondrial permeability transition pore (MPT pore). Incubation of endothelial cells with 5 µM alisporivir under hyperglycemic conditions leads to the increase in cell viability, restoration of the membrane potential level and of the MPT pore opening activity to control values. Hyperglycemia causes increased mitophagy in the lung endothelial cells: we observed increase in the degree of colocalization of mitochondria and lysosomes and upregulation of the Parkin gene expression. Alisporivir restores these parameters back to the levels observed in the control cells. Hyperglycemia results in the increase in the expression of the Drp1 gene in endotheliocytes responsible for synthesis of the protein involved in the process of mitochondria fission. Alisporivir does not significantly alter expression of the genes. The paper discusses mechanisms of the effect of alisporivir on mitochondrial dysfunction in murine pulmonary endotheliocytes under conditions of hyperglycemia.

Effect of Modified Levopimaric Acid Diene Adducts on Mitochondrial and Liposome Membranes.

This paper demonstrates the membranotropic effect of modified levopimaric acid diene adducts on liver mitochondria and lecithin liposomes. We found that the derivatives dose-dependently reduced the efficiency of oxidative phosphorylation of mitochondria due to inhibition of the activity of complexes III and IV of the respiratory chain and protonophore action. This was accompanied by a decrease in the membrane potential in the case of organelle energization both by glutamate/malate (complex I substrates) and succinate (complex II substrate). Compounds 1 and 2 reduced the generation of H2O2 by mitochondria, while compound 3 exhibited a pronounced antioxidant effect on glutamate/malate-driven respiration and, on the other hand, caused ROS overproduction when organelles are energized with succinate. All tested compounds exhibited surface-active properties, reducing the fluidity of mitochondrial membranes and contributing to nonspecific permeabilization of the lipid bilayer of mitochondrial membranes and swelling of the organelles. Modified levopimaric acid diene adducts also induced nonspecific permeabilization of unilamellar lecithin liposomes, which confirmed their membranotropic properties. We discuss the mechanisms of action of the tested compounds on the mitochondrial OXPHOS system and the state of the lipid bilayer of membranes, as well as the prospects for the use of new modified levopimaric acid diene adducts in medicine.

An in vivo study of the toxic effects of triclosan on Xenopus laevis (Daudin, 1802) frog: Assessment of viability, tissue damage and mitochondrial dysfunction.

The present study describes the in vivo effect of triclosan on the frog Xenopus laevis (Daudin, 1802). We have found a dose-dependence of the effect of triclosan on the survival of frogs. At a dose of 2 mg/L, the death of frogs was observed already on the 4th day of the experiment, while at a concentration of 0.5 mg/L, the frogs remained viable for 11 days. Triclosan caused damage to the liver tissue, which was expressed in an increase in the area of hemorrhage and the number of melanomacrophage centers. 0.5 mg/L of this agent did not affect the number of frog red blood cells, but reduced their osmotic resistance. Keeping animals in water containing triclosan (0.5 mg/L for 96 h) led to the suppression of the state 3 respiration rate of frog liver mitochondria. This effect was accompanied by suppression of the combined activity of complexes II and III of the mitochondrial respiratory chain. In parallel with this, we observed a reduction in the Ca2+ retention capacity of frog liver mitochondria, indicating a decrease in the resistance of organelles to mitochondrial permeability transition pore opening. The paper discusses the effects of triclosan on aquatic organisms.

Comparison of structural properties of cyclosporin A and its analogue alisporivir and their effects on mitochondrial bioenergetics and membrane behavior.

The paper considers the effect of the MPT pore inhibitor cyclosporin A (CsA) and its non-immunosuppressive analogue alisporivir (Ali) on the functioning of rat skeletal muscle mitochondria. We have shown that both agents at a standard in vitro concentration of 1 µM increase the calcium capacity of organelles and have no effect on the parameters of oxidative phosphorylation. However, an increase in their concentration to 5 µM leads to the suppression of oxygen consumption by mitochondria, which is more pronounced in the case of Ali. This effect is accompanied by a decrease in the membrane potential of organelles and, apparently, is based on the inhibition of electron transport along the mitochondrial respiratory chain due to limited mobility of coenzyme Q. We have noted that both agents do not affect the production of hydrogen peroxide by isolated mitochondria. NMR spectroscopy and molecular dynamics simulation did not reveal significant differences in the structure and backbone flexibility of CsA and Ali. Both agents decrease the overall fluidity of the membrane of DPPC liposomes, inducing an increase in laurdan generalized polarization parameter. A similar effect was also found in the case of mitochondrial membranes. We suggested that these effects of CsA and Ali, associated with their lipophilic nature and the ability to accumulate in the lipid phase of membranes, may cause a decrease in the efficiency of electron transport in the respiratory chain of mitochondria and suppression of the bioenergetics of these organelles.

Comparative study of free respiration in liver mitochondria during oxidation of various electron donors and under conditions of shutdown of complex III of the respiratory chain.

The present work shows that the rate of free respiration of liver mitochondria (in the absence of ATP synthesis (state 4) during the oxidation of succinate is 1.7 times higher than during the oxidation of glutamate with malate. In turn, in the case of oxidation of ferrocyanide with ascorbate, this value is 3.1 times greater than in the case of succinate oxidation. A similar pattern is also observed upon stimulation of free respiration by low concentrations (5 and 10 µM) of the protonophore uncoupler 2,4-dinitrophenol (DNP). It is found that the passive leakage rate of protons in state 4 is the same if the H+/O ratios are 10, 6, and 2 upon the oxidation of glutamate with malate, succinate, and ferrocyanide with ascorbate, respectively. At these values of the H+/O ratio, low concentrations of DNP stimulate passive proton leakage equally during the oxidation of these respiration substrates. In the case of succinate oxidation, bypassing complex III by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) to the maximum degree, as well as switching this complex completely to idle mode by α,ω-hexadecanedioic acid (HDA) cause a 3-fold stimulation of respiration in state 4. We conclude that at mitochondrial free respiration the values of the H+/2e- ratio for complexes I, III, and IV of the respiratory chain are 4, 4, and 2, respectively. It is assumed that the free respiration of mitochondria is carried out by simple diffusion of protons through the inner membrane, and the rate of this diffusion depends on the total number of protons released by the complexes of the electron transport chain into the intermembrane space.

S-15176 Difumarate Salt Can Impair Mitochondrial Function through Inhibition of the Respiratory Complex III and Permeabilization of the Inner Mitochondrial Membrane.

S-15176 difumarate salt, a derivative of the anti-ischemic metabolic drug trimetazidine, has been intensively studied for its impact on cellular metabolism in animal models of ischemia-reperfusion injury of the liver, heart, spinal cord, and other organs. Despite evidence of some reduction in oxidative damage to cells, the results of therapy with S-15176 have been mostly disappointing, possibly because of the lack of data on its underlying mechanisms. Here, we aimed to investigate in more detail the role of complexes I-IV of the electron transport chain and membrane permeability transition in mitochondrial toxicity associated with S-15176. Using rat thymocyte and liver mitochondria, we demonstrated that: (1) acute exposure to S-15176 (10 to 50 µM) dose-dependently decreased the mitochondrial membrane potential; (2) S-15176 suppressed the ADP-stimulated (State 3) and uncoupled (State 3UDNP) respiration of mitochondria energized with succinate or malate/glutamate, but not ascorbate/TMPD, and increased the resting respiration (State 4) when using all the substrate combinations; (3) S-15176 directly inhibited the activity of the respiratory complex III; (4) low doses of S-15176 diminished the rate of H2O2 production by mitochondria; (5) at concentrations of above 30 µM, S-15176 reduced calcium retention capacity and contributed to mitochondrial membrane permeabilization. Taken together, these findings suggest that S-15176 at tissue concentrations reached in animals can impair mitochondrial function through suppression of the cytochrome bc1 complex and an increase in the nonspecific membrane permeability.