Cardiac Troponins are Among Targets of Doxorubicin-Induced Cardiotoxicity in hiPCS-CMs

Figures

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  Figure 1. Time-lapse videomicroscopy of induced pluripotent stem cells (hiPCS-CMs) exposed to a range of doxorubicin (DOX) concentrations (10, 1 and 0.3 µM) during 192 h. Control and DOX-exposed hiPCS-CMs cultures maintained according to the manufacturer’s recommendation were continuously screened with enhanced phase contract microscopy using BioStation IM system over the mentioned time intervals. Resulting time-lapse sequences were adjusted and frame by frame analyzed using specialized software. Shown are representative morphologies of cardiomyocytes at individual time intervals. Phase contrast 400×. Bar 30 µm.

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  Figure 2. Effects of DOX (10, 1 and 0.3 µM) on viability, metabolism and membrane permeability of hiPCS-CMs during 192 h has measured by WST-1, Neutral red (NR) and ethidium bromide assays as described in Materials and methods section. (A) WST-1 assay (B) NR assay (C) ethidium bromide assay. Values represent means ± SD of at least three 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 3. Effects of 1 and 0.3 µM DOX on appearance of select morphologies in hiPCS-CMs cardiomyocytes during 192 h. Cells were exposed to DOX and at individual time intervals, their morphology was examined and quantified by the Cell Scoring module of MetaXpress® Image Acquisition and Analysis Software. (A,B) Cells with vacuoles. Phase contrast 400×. Bar 10 µm. p < 0.05 * Significantly higher than control at the same treatment interval (C,D) Blebbing cells. Phase contrast 400×. Bar 10 µm. p < 0.05 * Significantly higher than control at the same treatment interval (E,F) Extended cells. Phase contrast 400×. Bar 10 µm. p < 0.05 * Significantly higher than control at the same treatment interval 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 the expression of cardiac troponin T and troponin I genes in hiPCS-CMs during 192 h. Control and DOX-exposed cardiomyocytes were harvested at individual time intervals, mRNA was isolated and its quantity determined by qPCR as described in Materials and Methods. (A) Expression of troponin T. p < 0.05 * Significantly lower than control at the same treatment interval one way-Anova test and Dunnett’s post test for multiple comparisons. (B) Expression of troponin I. p < 0.05 * Significantly lower than control at the same treatment interval one way-Anova test and Dunnett’s post test for multiple comparisons.

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  Figure 5. Effects of 1 µM DOX on the localization and arrangement of troponin T and troponin I in hiPCS-CMs during 72 h. Control and DOX-exposed cardiomyocytes were at individual time intervals rinsed with PBS, fixed, permeabilized and incubated with particular antibodies as specified in Materials and Methods section. Specimens were mounted and troponin T and troponin I-specific fluorescence were examined by the fluorescent microscopy and cell analysis as described in Materials and Methods. Fluorescence 400×. Bar 10 µm.

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  Figure 6. Effects of 1 and 0.3 µM DOX on mitochondrial topography and activity in hiPCS-CMs during 192 h. Mitochondrial morphology and arrangement in (A) control and (B) 1 µM DOX-treated cells (72 h) was examined by microscopic evaluation of MitoTracker Green FM fluorescence. Mitochondrial membrane potential (ψ) in (C) control and (D) 1 µM DOX-treated cells (72 h) was examined by microscopic measurement of potential-sensitive JC-1 dye. Software-aided cell quantitation of ψ in 0.3 and 1 µM DOX-treated cells during 192 h (E) as described in Materials and methods section. Fluorescence 400× (A,B) and 200× (C,D). Bars 10 µm (A,B), 30 µm (C,D). * 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.