Astaxanthin Reduces Н2O2- and Doxorubicin-Induced Cardiotoxicity in H9c2 Cardiomyocyte Cells

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Abstract

Cardiovascular diseases are one of the most challenging problems in clinical practice. Astaxanthin (AST) is a keto-carotenoid (xanthophyll) mainly of marine origin, which is able to penetrate the cell membrane, localize in the mitochondria and prevent mitochondrial dysfunction. The present study examined the effect of astaxanthin on the death of H9c2 cardiomyocytes caused by the cytotoxic effect of hydrogen peroxide (H2O2) and doxorubicin. Using the methods of spectrophotometry, spectrofluorimetry, and Western blotting analysis, it was shown that treatment of cells with AST contributed to an increase in the number of H9c2 cells resistant to H2O2 and doxorubicin, while maintaining the value of their mitochondrial transmembrane potential, reducing the intracellular production of reactive oxygen species and an increase in the intracellular content of mitophagy markers PINK1, Parkin and prohibitin 2. The obtained results suggest that the use of AST may be a highly effective way to prevent and treat cardiovascular diseases.

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About the authors

R. R. Krestinin

Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences

Author for correspondence.
Email: ovkres@mail.ru
Rwanda, 142290, Pushchino, Moscow Region

M. I. Kobyakova

Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences

Email: ovkres@mail.ru
Russian Federation, 142290, Pushchino, Moscow Region

Yu. L. Baburina

Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences

Email: ovkres@mail.ru
Russian Federation, 142290, Pushchino, Moscow Region

L. D. Sotnikova

Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences

Email: ovkres@mail.ru
Russian Federation, 142290, Pushchino, Moscow Region

O. V. Krestinina

Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences

Email: ovkres@mail.ru
Russian Federation, 142290, Pushchino, Moscow Region

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2. Fig. 1. Viability of H9c2 cardiomyocytes after 24 h incubation with AST(a), H2O2(b) and Dox(c). Cells were seeded into a 96-well plate at a density of 2.5× 103 cells per well and treated with the indicated concentrations of AST(a), H2O2(b) and Dox(c) in H9c2 for 24 hours . The data is presented as the average ± standard deviation (SD) from ten separate experiments

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3. Fig. 2. The dependence of the viability of H9c2 cardiomyocyte cells on the concentration of AST, H2O2, Dox and on the incubation time with AST. Cells with AST were incubated for 1 (a, d), 4 (b, e) and 6 (c, e) hours. The number of living cells in an intact culture (control, without drug treatment) was assumed to be 100%. The data is presented as an average ± SD from six separate experiments. The control consisted of untreated cells

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4. Fig. 3. Cytotoxic effect of AST and its combined effect with H2O2 and Dox in H9c2 cardiomyocyte cells. As a positive control, cells treated with 1% Triton X-100 were used, which were mistaken for 100%. a is the number of dead cells in the presence of various concentrations of AST; b is the number of dead cells when using AST with H2O2 after 4 hours of incubation with AST; c is the number of dead cells when using AST with Dox after 6 hours of incubation with AST. The data is presented as the mean ± standard deviation (n = 8); * p < 0.05 is a significant change compared to the control (intact culture); # p < 0.05 is a significant change compared to the corresponding values of H2O2 or Dox

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5. Fig. 4. The effect of AST, H2O2 and Dox on the change in ΔΨm in H9c2 cardiomyocyte cells. The fluorescence intensity of intact cells was used as a control (without drug treatment). a – Change in fluorescence intensity in the presence of AST (5 and 10 microns) and H2O2; b – change in fluorescence intensity in the presence of AST (5 and 10 microns) and Dox. Valinomycin (250 nM) was used as a positive control. The data are presented as the average value ± standard deviation (n = 6); * p < 0.05 is a significant change compared to the corresponding control (untreated cells); # p < 0.05 is a significant change compared to H2O2 or Dox

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6. Fig. 5. Effect of AST, H2O2 and Dox on changes in intracellular ROS production in H9c2 cardiomyocyte cells. a – Change in fluorescence intensity in the presence of AST (5 and 10 microns) and H2O2; b – change in fluorescence intensity in the presence of AST (5 and 10 microns) and Dox. H2O2 (100 microns) was used as a positive control. The data are presented as mean ± standard deviation (n = 6); * p < 0.05 – significant change compared to the control (untreated cells); # p < 0.05 – significant change compared to H2O2 or Dox

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7. Fig. 6. The effect of AST (10 µm), H2O2, and Dox on changes in PINK1 (a), Parkin(b), and RNB2 (c) levels in H9c2 cardiomyocyte cells. The upper part was immunostaining with antibodies to PINK1 (a), Parkin(b) and RNB2 (c); GAPDH was used as a protein load control. The lower part contains diagrams that quantify changes in the content of proteins normalized by GAPDH. The protein level in the cell lysate (without additives) served as a control (100%). The data are presented as the mean ± standard deviation (n = 4); * p < 0.05 is a significant change compared to the control; # p < 0.05 is a significant change compared to H2O2 or Dox

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