Mitochondrial Protection by Astaxanthin Reduces Toxicity Caused by H2O2 and Doxorubicin in Human Cardiomyocytes

Figure 1. Dose–response effects of astaxanthin, hydrogen peroxide, and doxorubicin on AC16 cell viability. Viability quantification was performed 24 h after compound exposure using resazurin metabolism assay. (a) Upper panel: Chemical structure of astaxanthin (AST). Lower panel: AC16 cardiomyocytes following treatment with AST (0–100 μM). (b) Upper panel: Structural formula of hydrogen peroxide (H2O2). Lower panel: Cellular response to H2O2 exposure (0–400 μM). (c) Upper panel: Molecular structure of doxorubicin (DOX). Lower panel: Morphological assessment of cells treated with DOX (0–400 μM). Results are expressed as percentage of untreated control (defined as 100%) and shown as mean ± SD (n = 10 independent experiments).

Figure 2. Viability of AC16 cardiomyocytes treated with AST, H2O2, and DOX. Dependence of AC16 cardiomyocyte viability on the concentration of AST (5 and 10 µM), H2O2 (100 µM), and DOX (5 µM). (a) Cytotoxic effect of AST and H2O2 during 1, 4, and 6 h incubation with AST. (b) Cytotoxic effect of AST and DOX during 1, 4, and 6 h incubation with AST. The number of viable cells in the untreated control culture was set as 100%. Data are presented as mean ± SD (n = 10). * p < 0.05 indicates a significant effect compared to the control (untreated culture); # p < 0.05 indicates a significant effect compared to H2O2 or DOX treatment alone.

Figure 3. Modulation of cytosolic Ca2+ levels by astaxanthin in H2O2- and doxorubicin-treated AC16 cardiomyocytes. Cells were pre-treated with AST (5 or 10 µM) for 1 or 4 h prior to exposure to cytotoxic concentrations of H2O2 (100 µM) or DOX (5 µM). (a) Intracellular Ca2+ concentrations following AST and H2O2 co-treatment. (b) Intracellular Ca2+ levels after AST and DOX administration. Fluorescence intensity values were normalized to untreated controls. Data represent mean ± SD from six independent experiments (n = 6). Asterisks denote significant differences compared to control (* p < 0.05), while hash symbols indicate significant effects relative to H2O2 or DOX treatment alone (# p < 0.05).

Figure 4. AST modulation of caspase-8 and caspase-3 expression in H2O2- and doxorubicin-stressed AC16 cardiomyocytes. Cells were pre-treated with 10 µM AST for 4 h prior to induction of cytotoxicity with either 100 µM H2O2 or 5 µM DOX. (a) Upper panel: representative immunoblot depicting caspase-8 protein expression. Lower panel: densitometric quantification of caspase-8 levels normalized to GAPDH. (b) Upper panel: representative immunoblot showing caspase-3 expression. Lower panel: quantitative analysis of caspase-3 protein levels relative to GAPDH. Untreated control values were set as 100%. Data represent mean ± standard deviation from three independent experiments (n = 3). Asterisks indicate statistically significant differences compared to control (* p < 0.05), while hash symbols denote significant effects relative to H2O2 or DOX treatment alone (# p < 0.05).

Figure 5. Effect of AST on BIP, CHOP, and ERO1α levels under H2O2- and DOX-induced cytotoxicity in AC16 cardiomyocytes. Cells were pre-incubated with 10 µM AST for 4 h. Cytotoxicity was induced with 100 µM H2O2 or 5 µM DOX. (a) Upper panel: representative immunoblot for BIP. Lower panel: quantitative analysis of protein levels normalized to GAPDH. (b) Upper panel: representative immunoblots for CHOP and ERO1α. Lower panel: quantitative analysis of protein levels normalized to GAPDH. The protein level in the untreated cell lysate was set as 100%. Data are presented as mean ± SD (n = 3). * p < 0.05 indicates a significant effect compared to the control; # p < 0.05 indicates a significant effect compared to H2O2 or DOX treatment alone.

Figure 6. Effect of AST on mitochondrial membrane potential (ΔΨm) under H2O2- and DOX-induced cytotoxicity in AC16 cardiomyocytes. Cells were pre-incubated with 5 and 10 µM AST for 1 or 4 h. Cytotoxicity was induced with 100 µM H2O2 or 5 µM DOX. (a) Changes in ΔΨm in the presence of AST and H2O2. (b) Changes in ΔΨm in the presence of AST and DOX. The fluorescence intensity of the untreated cell culture was used as a control. Data are presented as mean ± SD (n = 6). * p < 0.05 indicates a significant effect compared to the control; # p < 0.05 indicates a significant effect compared to H2O2 or DOX treatment alone.

Figure 7. Effect of AST on intracellular ROS production under H2O2- and DOX-induced cytotoxicity in AC16 cardiomyocytes. Cells were pre-incubated with 5 and 10 µM AST for 1 or 4 h. Cytotoxicity was induced with 100 µM H2O2 or 5 µM DOX. (a) Changes in intracellular ROS production in the presence of AST and H2O2. (b) Changes in intracellular ROS production in the presence of AST and DOX. The fluorescence intensity of the untreated culture was used as a control. Data are presented as mean ± SD (n = 6). * p < 0.05 indicates a significant effect compared to the control; # p < 0.05 indicates a significant effect compared to H2O2 or DOX treatment alone.

Figure 8. Effect of AST on the levels of NDUFB8 and SDHB subunits under H2O2- and DOX-induced cytotoxicity in AC16 cardiomyocytes. Cells were pre-incubated with 10 µM AST for 4 h. Cytotoxicity was induced with 100 µM H2O2 or 5 µM DOX. (a) Representative immunoblots for the NDUFB8 (Complex I) and SDHB (Complex II) subunits. β-tubulin was used as a loading control. (b) Quantitative assessment of immunoblots performed using computer densitometry. The protein level in the untreated cell lysate was set as 100%. Data are presented as mean ± SD (n = 3). * p < 0.05 indicates a significant effect compared to the control; # p < 0.05 indicates a significant effect compared to H2O2 treatment alone.