Metabolite signatures of doxorubicin induced toxicity in human induced pluripotent stem cell-derived cardiomyocytes

Figures

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  Fig. 1. A schematic view of experimental design with timeline of the study. Synchronously beating monolayer of hiPSC-CMs was exposed to DOX for 2 days or for 6 days in a repeated way (DOX-supplemented culture media was refreshed every 2 days). Following 2 and 6 days exposure to DOX, hiPSC-CMs were further cultured in drug free culture medium until day 14 (after the start of drug exposure). Culture media was refreshed every 2 days. For metabolic profiling, culture media samples were collected on day 2, 6 and 14. Experimental design was also used in cytotoxicity and ATP content determination studies

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  Fig. 2. An extracellular metabolic profile of hiPSC-CMs model. The regions of the spectrum shown in the figure are a typical full resolution 1D 600 MHz 1H CPMG spin-echo NMR spectrum from the control media samples after 2 days of incubation. 1 3-methyl-2-oxovalerate, 2 isoleucine, 3 Leucine, 4 valine, 5 3-hydroxyisobutyrate, 6 ethanol, 7 lactate, 8 threonine, 9 2-hydroxyisobutyrate, 10 alanine, 11 lysine, 12 acetate, 13 pyroglutamate, 14 glutamine, 15 methionine, 16 pyruvate, 17 T-methylhistidine, 18 phenylalanine, 19 galactose, 20 tyrosine, 21 formate

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  Fig. 3. NMR spectroscopy detected significant alterations in extracellular metabolites levels of hiPSC-CMs. DOX reduced uptake of a pyruvate, b acetate, and c reduced efflux of formate. During drug washout pyruvate showed a reversible effect, while acetate and formate showed a irreversible effect. Error bars in the bar graphs represents standard deviation (SD). n = 3 biological replicates, *p value 0.05

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  Fig. 4. Response of extracellular metabolites- valine, 3-methyl-2-oxovalerate, isoleucine, tyrosine, 3-hydroxyisobutyrate, methionine and 2-hydroxybutyrate to DOX exposure in hiPSC-CMs model. 1H NMR spectroscopy was used to calculate the concentration (mM) of the metabolites in the culture media at day 2, 6 and 14 of DOX study. The levels of the metabolite are the net accumulation of influx and efflux over the previous 2 day period

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  Fig. 5. Determination of DOX induced cytotoxicity and energy levels in hiPSC-CMs. a DOX on repeated exposure induced cytotoxicity, as evidenced by increased LDH activity in the culture media. b DOX exposure declined energy level in cardiomyocytes with depletion of high energy ATP molecules. Data in bar graphs presented as mean ± SD. n = 3 technical replicates, *p value 0.05