Astaxanthin Protects Against H2O2- and Doxorubicin-Induced Cardiotoxicity in H9c2 Rat Myocardial Cells

Figure 1. Dependence of the viability of H9c2 cells on the concentration of AST (10 µM), H2O2 (100 µM), and DOX (100 µM). (a) Cytotoxic effect of AST, H2O2, and AST+H2O2; (b) cytotoxic effect of AST, DOX, and AST+DOX. The proportion of living cells was determined one, four, and six hours after the addition of substances using the resazurin reduction method. The number of living cells in the intact culture (control, without drug treatment) was taken as 100%. Data are presented as the mean ± standard deviation (n = 10). * p < 0.05 indicates significant changes compared to the control (intact culture); # p < 0.05 indicates significant change compared to the corresponding values of H2O2 or DOX.

Figure 2. Effect of AST and its combined action with H2O2 and DOX on the death of H9c2 cardiomyocytes. (a) Cell death in control (untreated cells), with AST, H2O2, and AST+H2O2; (b) cell death in control (untreated cells), with AST, DOX, and AST+DOX. Cells treated with 1% Triton X-100 were considered 100% dead and were taken as a positive control. Data are presented as the mean ± standard deviation (n = 8). * p < 0.05 indicates significant change compared to the control (intact culture); # p < 0.05 indicates significant changes compared to the corresponding values of H2O2 or DOX.

Figure 3. Effect of AST (10 μM) and H2O2 (100 μM) on the area of mitochondrial mass in H9c2 cells. (a) Mitochondria stained with MitoTracker Green FM; the cell nuclei stained with Hoechst 33342; (b) mitochondrial area calculated using the ImageJ software. The data are presented as the mean ± standard deviation (n = 5). * p < 0.05 indicates significant change compared to the control (intact culture); # p < 0.05 indicates significant changes compared to the corresponding values of H2O2.

Figure 4. Effect of AST (10 μM) and DOX (100 μM) on the area of mitochondrial mass in H9c2 cells. (a) Mitochondria stained with MitoTracker Green FM; cell nuclei stained with Hoechst 33342; (b) the mitochondrial area calculated using the software ImageJ. Data are presented as the mean ± standard deviation (n = 5). * p < 0.05 indicates a significant change compared to the control (intact culture); # p < 0.05 indicates significant changes compared to the corresponding values of DOX.

Figure 5. Effect of AST on the content of cytosolic Ca2+ in H9c2 cardiomyocytes during H2O2- and DOX-induced cytotoxicity. (a) the content of cytosolic Ca2+ in the presence of AST and H2O2; (b) the content of cytosolic Ca2+ in the presence of AST and DOX. Fluorescence intensity of intact cells was used as a control (without drug treatment), standardized to 2.5 × 105 cells. Data are presented as the mean fluorescence intensity ± standard deviation (n = 6). * p <0.05 significant changes compared to the corresponding control, # p <0.05 significant change compared to H2O2 or DOX.

Figure 6. Effect of AST on the content of VDAC1 and CHOP in H9c2 cardiomyocytes during H2O2 and DOX-induced cytotoxicity. (a) Upper part: Western blot stained by VDAC1 antibody; low part: graphs quantifying changes in the protein content normalized to GAPDH; (b)—upper part: Western blot stained by CHOP antibody; low part: graphs quantifying changes in the protein content normalized to GAPDH. Protein levels in cell lysate (without additives) served as control (100%). Data are presented as the mean ± standard deviation (n = 4), * p < 0.05 indicates significant changes compared to the control. # p < 0.05 indicates significant changes compared to H2O2 or DOX.