MKT-077 suppresses the functional activity of isolated mouse skeletal muscle mitochondria

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Abstract

This study demonstrates the effect of the rhodocyanine derivative MKT-077 on the function of isolated mitochondria from mouse skeletal muscle. MKT-077 was shown to dose-dependently inhibit mitochondrial respiration fueled by glutamate/malate (complex I substrates) or succinate (complex II substrate). This effect of MKT-077 was accompanied by a decrease in the membrane potential of organelles and was associated with both inhibition of the activity of complexes I and II of the mitochondrial respiratory chain and an increase in the proton permeability of the inner mitochondrial membrane. Molecular docking revealed sites in mitochondrial respiratory chain complex I that have an affinity for MKT-077 comparable to that of the specific inhibitor rotenone. At a concentration of 5 μM, MKT-077 caused a significant increase in hydrogen peroxide production by skeletal muscle mitochondria. However, 1 μM MKT-077 reduced the pro-oxidant effect of antimycin A. In addition, MKT-077 dose-dependently reduced the ability of mitochondria to uptake and retain calcium ions in the matrix. The article discusses the mechanisms of possible action of MKT-077 on the functioning of skeletal muscle mitochondria and their contribution to the side effects observed during the therapy of pathological conditions in vivo using this rhodocyanine derivative.

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About the authors

A. D. Igoshkina

Mari State University

Email: dubinin1989@gmail.com
Russian Federation, pl. Lenina, 1, Yoshkar-Ola

N. V. Mikina

Mari State University

Email: dubinin1989@gmail.com
Russian Federation, pl. Lenina, 1, Yoshkar-Ola

A. V. Chulkov

Mari State University

Email: dubinin1989@gmail.com
Russian Federation, pl. Lenina, 1, Yoshkar-Ola

E. I. Khoroshavina

Mari State University

Email: dubinin1989@gmail.com
Russian Federation, pl. Lenina, 1, Yoshkar-Ola

M. V. Dubinin

Mari State University

Author for correspondence.
Email: dubinin1989@gmail.com
Russian Federation, pl. Lenina, 1, Yoshkar-Ola

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Supplementary files

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2. Fig. 1. Chemical structure of MKT-077

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3. Fig. 2. Respiration of isolated skeletal muscle mitochondria from mice energized with glutamate and malate (a and b) or succinate (c and d) in the absence (a and c) and presence (b and d) of 20 μM MKT-077. Medium composition: 120 mM KCl, 5 mM NaH2PO4, and 10 mM HEPES-KOH (pH 7.4). Substrates and reagents: 2.5 mM potassium malate and 2.5 mM potassium glutamate, 5 mM succinic acid, 1 μM rotenone, 200 μM ADP, 50 μM DNP. C.2 – state 2, C.3 – state 3, C.4 – state 4, C.3UDNF – state 3UDNF, M – mitochondria.

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4. Fig. 3. Molecular docking of the interaction of MKT-077 and rotenone with complex I of the mitochondrial respiratory chain. a – Internal and external binding sites of decylubiquinone in complex I of the mitochondrial respiratory chain. b – Comparison of crystallographic data with the calculated position of rotenone for two binding sites (rotenone1 – crystallographic data, rotenone2 – docking result), the subunits forming the binding sites are indicated: 49 kDa (PDB chain 4), PSST (PDB chain 6), ND1 (PDB chain H). c – Comparison of the position in the internal binding site of MKT-077 and rotenone. d – Comparison of the position in the external binding site of MKT-077 and rotenone.

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5. Fig. 4. Effect of MKT-077 on the membrane potential of mouse skeletal muscle mitochondria energized with glutamate and malate (a) or succinate (b). Substrates and reagents: 2.5 mM potassium malate, 2.5 mM potassium glutamate, 1 μM TPP+, 5 mM succinic acid, 1 μM rotenone, 50 μM DNP. Each figure shows the data of a typical experiment obtained on one mitochondrial preparation. In each case, similar results were obtained in two more independent experiments. The dotted lines show the results of experiments in the absence of MKT-077 additives.

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6. Fig. 5. Rate of H2O2 formation by mouse skeletal muscle mitochondria energized by glutamate and malate (Glu/mal) or succinate (Succinate+rot): a – in the presence of different concentrations of MKT-077; b – under the action of 1 μM antimycin A (AntA) in the absence and presence of 1 μM MKT-077. Control – absence of AntA and MKT-077. Mean values ​​± standard error of the mean (n = 4) are shown. * – p < 0.05 versus control; # – p < 0.05 versus 1 μM AntA.

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7. Fig. 6. Effect of MKT-077 on calcium transport in mouse skeletal muscle mitochondria. a – Uptake of Ca2+ supplements (10 μM pulses) by skeletal muscle mitochondria energized with glutamate and malate in the absence of supplements (1) and in the presence of 1 μM (2), 5 μM (3), and 10 μM (4) MKT-077. b – Uptake of Ca2+ supplements (10 μM pulses) by skeletal muscle mitochondria energized with succinate in the absence of supplements (1) and in the presence of 1 μM (2), 5 μM (3), and 10 μM (4) MKT-077. c – Calcium capacity of skeletal muscle mitochondria energized by glutamate and malate (Glu/mal) or succinate (Succinate+rot) in the absence (control) and presence of different concentrations of MKT-077. Substrates and reagents: 2.5 mM potassium malate, 2.5 mM potassium glutamate (a, c), 5 mM succinic acid, 1 μM rotenone (b, c). The concentration of mitochondrial protein in the cuvette is 0.25 mg/ml. Mean values ​​± standard error of the mean (n = 4) are shown. * – p < 0.05 versus control.

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