Simvastatin Reduces Doxorubicin-Induced Cardiotoxicity: Effects beyond Its Antioxidant Activity

Figures

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  Figure 1. Effect of Simvastatin on Doxorubicin-induced cytosolic and mitochondrial ROS production. H9c2 cells were pre-treated with Sim (10 µM) for 4 h and then co-exposed to Sim and Doxo (1 µM) for 20 h. The fluorescent probe 2′,7′-dichlorofluorescein diacetate (H2DCF-DA) was used to evaluate cytosolic ROS generation by flow cytometry analysis. Results are reported as mean ± S.E.M. of DCF-positive cells’ percentage of at least three independent experiments, each performed in duplicate (A). The fluorescent probe MitoSOX was used to evaluate mitochondrial superoxide production by flow cytometry analysis. Results are reported as mean ± S.E.M. of MitoSOX-positive cells’ percentage of at least three independent experiments, each performed in duplicate (C). Representative histograms for the flow cytometry analysis are reported in Panels (B,D), respectively. Data were analyzed by variance test analysis, and multiple comparisons were made by Bonferroni’s test. ** p < 0.005, *** p < 0.001, and **** p < 0.0001 vs. untreated cells; ° p < 0.05 vs. Doxo-treated cells.

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  Figure 2. Effect of Simvastatin on Doxorubicin-induced calcium homeostasis dysregulation. H9c2 cells were pre-treated with Sim (10 µM) for 4 h and then co-exposed to Sim and Doxo (1 µM) for 20 h. FCCP (50 nM) in calcium-free medium was used to evaluate mitochondrial calcium levels (A). Ionomycin (1 μM) in calcium-free medium was used to evaluate intracellular calcium levels (B). Results are reported as mean ± S.E.M. of percentage of delta (% Δ) increase in FURA2 ratio fluorescence (340/380 nm) from at least three independent experiments, each performed in duplicate. Data were analyzed by variance test analysis, and multiple comparisons were made by Bonferroni’s test. * p < 0.05 vs. untreated cells; ° p < 0.05 vs. Doxo-treated cells.

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  Figure 3. Effect of Simvastatin on Doxorubicin-induced apoptosis. H9c2 cells were pre-treated with Sim (10 µM) for 4 h and then co-exposed to Sim and Doxo (1 µM) for 20 h. Cells were stained by propidium iodide, and fluorescence of individual nuclei was measured by flow cytometry. Results are reported as mean ± S.E.M. of percentage of hypodiploid nuclei from at least three independent experiments, each performed in duplicate (A). Flow cytometry analysis was used to evaluate cytosolic cytochrome c level (C). Results are reported as mean ± S.E.M. of cytochrome c-positive cells’ percentage from at least three independent experiments, each performed in duplicate. Representative histograms for the flow cytometry analysis are reported in panels (B,D), respectively. Data were analyzed by variance test analysis, and multiple comparisons were made by Bonferroni’s test. ** p < 0.005 vs. untreated cells; ° p < 0.05 vs. Doxo-treated cells.

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  Figure 4. Effect of Simvastatin on intracellular Connexin43 expression. H9c2 cells were pre-treated with Sim (10 µM) for 4 h and then co-exposed to Sim and Doxo (1 µM) for 20 h. Intracellular Cx43 level was detected by flow cytometry analysis. Results are reported as mean ± S.E.M. of Cx43-positive cells’ percentage from at least three independent experiments, each performed in duplicate (A). Representative histograms for the flow cytometry analysis (B). Mitochondrial Cx43 expression was detected by Western blot analysis, and TOM20 expression was used as the loading control. Results are expressed as mean ± S.E.M from at least three independent experiments, each performed in duplicate (panel (C). Cells were stained with Cx43 (red) (panels a,e,i,m), VDAC1 (green) (panels b,f,j,n), and nucleus with DAPI (blue) (panels c,g,k,o) and were determined by immunofluorescence analysis (merge in panels d,h,l,p). Scale bar, 50 μm. A representative of three experiments is shown (D). Data were analyzed by variance test analysis, and multiple comparisons were made by Bonferroni’s test. * p < 0.05, *** p < 0.001, and **** p < 0.0001 vs. untreated cells; ° p < 0.05 vs. Doxo-treated cells.

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  Figure 5. Effect of Simvastatin on membrane levels of Connexin43 and Connexin43 phosphorylated on Ser368. H9c2 cells were pre-treated with Sim (10 µM) for 4 h and then co-exposed to Sim and Doxo (1 µM) for 20 h. Cx43 (A) and pCx43 (C) levels were detected by flow cytometry analysis. Results are reported as mean ± S.E.M. of Cx43- and pCx43-positive cells’ percentage from at least three independent experiments, each performed in duplicate. Representative histograms for the flow cytometry analysis are reported in Panels (B,D), respectively. Data were analyzed by variance test analysis, and multiple comparisons were made by Bonferroni’s test. ** p < 0.005 and *** p < 0.001 vs. untreated cells; °° p < 0.005 vs. Doxo-treated cells.