Increased Intracellular Free Zinc Has Pleiotropic Effects on Doxorubicin-Induced Cytotoxicity in hiPCS-CMs Cells

Figures

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  Figure 1. Effects of DOX (0.3 and 1 µM) on the viability, autophagy, cell death, and senescence of hiPCS-CMs during 192 h, as measured by particular assays described in Section 4. (A) Viability, (B,C) autophagy, (D) cell death, and (E) senescence. Values represent means ± SD of N = 3 (viability), N = 4 (autophagy), N = 3 (cell death), and N = 3 (senescence) experiments. * p < 0.05 compared to untreated control cells at the same treatment interval; # p < 0.05 compared to 1 µM DOX treated cells at the same treatment interval; + p < 0.05 compared to 0.3 µM DOX treated cells at the same treatment interval with one-way ANOVA test and Dunnett’s post test for multiple comparisons.

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  Figure 2. Effects of 0.3 µM and 1 µM DOX on various intracellular targets in hiPCS-CMs during 192 h. Control and DOX-exposed cardiomyocytes were harvested at individual time intervals, and (A) DNA damage, (B) mitochondrial production of ATP, (C) generation of superoxide ion, and (D) lysosomal membrane permeabilization, were determined by particular assays and procedures, as described in Section 4. Values represent means ± SD of N = 3 (DNA damage), N = 3 (ATP production), N = 3 (superoxide), and N = 3 (lysosomal permabilization) experiments. * p < 0.05 compared to untreated control cells at the same treatment interval; # p < 0.05 compared to untreated control cells at the same treatment interval; and + p < 0.05 compared to 1 µM DOX treated cells at the same treatment interval with one-way ANOVA test and Dunnett’s post test for multiple comparisons.

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  Figure 3. Effects of 0.3 µM and 1 µM DOX on intracellular signaling in hiPCS-CMs during 192 h. Control and DOX-exposed cardiomyocytes were harvested at individual time intervals, and (A) p53 activity, (B) NF-κB activity, (C) MAP kinases activity upon treatment with 0.3 µM DOX, and (D) MAP kinases activity upon treatment with 1 µM DOX, were determined by particular assays and procedures, as described in Section 4. Values represent means ± SD of N = 3 (p53 activity), N = 3 (NF-κB activity), N = 3 (MAP kinases activity at 0.3 µM DOX), and N = 3 (MAP kinases activity at 1 µM DOX) experiments. * p < 0.05 compared to untreated control cells at the same treatment interval; and + p < 0.05 compared to 1 µM DOX treated cells at the same treatment interval with one-way ANOVA test and Dunnett’s post test for multiple comparisons.

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  Figure 4. Effects of 0.3 µM and 1 µM DOX on intracellular free Zn levels in hiPCS-CMs during 192 h. Free zinc content in control and DOX-treated cells was measured by microfluorometry of the zinc-specific dye Newport Green diacetate, as described in Section 4. Values represent means ± SD of N = 4 experiments. # p < 0.05 compared to untreated control cells at the same treatment interval with one-way ANOVA test and Dunnett’s post test for multiple comparisons.

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  Figure 5. Effects of zinc pyrithione (ZnPyr) on viability and on free Zn levels in hiPCS-CMs over 192 h. Cardiomyocytes were exposed to a range of ZnPyr concentrations (in µM) and their viability was measured by neutral red uptake assay (NRU) (Section 4). Free zinc content in control and 0.3 µM and 1 µM DOX-treated cells was measured by microfluorometry of the zinc-specific dye Newport Green diacetate, as described in Section 4. Values represent means ± SD of N = 4 experiments. # p < 0.05 compared to untreated control cells at the same treatment interval with one-way ANOVA test and Dunnett’s post test for multiple comparisons.

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  Figure 6. Effects of enhanced intracellular free Zn levels on viability and cell phenotypes in hiPCS-CMs exposed to 0.3 µM and 1 µM DOX at 120 h. Cardiomyocytes were treated with 0.1 µM zinc pyrthione (ZnPyr) alone or together with 0.3 µM and 1 µM DOX concentrations, and their (A) viability, (B,C) autophagy, (D) cell death, and (E) senescence were measured by particular assays (Section 4). Values represent means ± SD of N = 3 (viability), N = 4 (autophagy), N = 3 (cell death), and N = 3 (senescence) experiments. #,* p < 0.05 compared to untreated cells with enhanced free Zn content at the same treatment interval with one-way ANOVA test and Dunnett’s post test for multiple comparisons.

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  Figure 7. Effects of enhanced intracellular free Zn content on DNA damage, oxidative stress, and mitochondrial and lysosomal status in hiPCS-CMs exposed to 0.3 µM and 1 µM DOX at 120 h. Cardiomyocytes were treated with 0.1 µM zinc pyrthione (ZnPyr) alone or together with 0.3 µM and 1 µM DOX concentrations, and (A) DNA damage, (B) mitochondrial production of ATP, (C) generation of superoxide ion, and (D) lysosomal membrane permeabilization were determined by particular assays and procedures, as described in Section 4. Values represent means ± SD of N = 3 (DNA damage), N = 3 (ATP production), N = 3 (superoxide), and N = 3 (lysosomal permabilization) experiments. #, * p < 0.05 compared to untreated cells with enhanced free Zn content at the same treatment interval with one-way ANOVA test and Dunnett’s post test for multiple comparisons.

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  Figure 8. Effects of enhanced intracellular free Zn levels on intracellular signaling in hiPCS-CMs exposed to 0.3 µM and 1 µM DOX at 120 h. Cardiomyocytes were treated with 0.1 µM zinc pyrthione (ZnPyr) alone or together with 0.3 µM and 1 µM DOX concentrations, and (A) p53 activity, (B) NF-κB activity, (C) MAP kinases activity upon treatment with 0.3 µM DOX, and (D) MAP kinases activity upon treatment with 1 µM DOX were determined by particular assays and procedures, as described in Section 4. Values represent means ± SD of N = 3 (p53 activity), N = 3 (NF-κB activity), N = 3 (MAP kinases activity at 0.3 µM DOX), and N = 3 (MAP kinases activity at 1 µM DOX) experiments. # p < 0.05 compared to DOX treated cells at the same treatment interval with one-way ANOVA test and Dunnett’s post test for multiple comparisons.