Ponatinib confers adult human cardiomyocyte toxicity via inhibition of AKT signaling

Figures

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  FIGURE 1. Ponatinib (PON) induces death in hPCMs and hiPSC‐CMs. (a) Concentration–response curve of hPCMs treated with ponatinib for 48 h. (b) Representative Western blots of cleaved caspase‐3 expression in hPCMs (left) and hiPSC‐CMs (right) treated with 1 μM PON for 2 h, with GAPDH as the loading control. (c) Quantification of (b), n = 3 independent experiments. Data are mean ± SEM; significance was calculated using unpaired Student's t‐test.

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  FIGURE 2. PON suppresses the PI3Kα‐AKT pathway. (a) Representative Western blots of p‐AKTS473 and p‐P38 MAPK in hPCMs (left) and hiPSC‐CMs (right) treated with 1 μM PON for 2 h, with GAPDH as the loading control. (b‐c) Quantification of p‐AKTS473 and p‐P38 in hPCMs (n = 4, b) and hiPSC‐CMs (n = 5 for p‐AKTS473 and n = 3 for p‐P38, c). (d) Representative Western blots of p‐ERK1/2 in hPCMs (left) and hiPSC‐CMs (right). (e, f) Quantification of p‐ERK1/2 in hPCMs (n = 4, e) and hiPSC‐CMs (n = 4, f). Data are mean ± SEM, significance was calculated using paired Student's t‐test.

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  FIGURE 3. MK‐2206 induces cell death in hPCMs. (a) Western blotting of AKT phosphorylation (S473 and T308) in hPCMs treated with 5 μM PON or 10 μM MK‐2206 for 48 h, with GAPDH as the loading control. (b, c) Quantification of p‐AKTS473 (b) and p‐AKTT308 (c) in (a), n = 5. (d) Live/dead staining of hPCMs. Green (Calcein‐AM) indicates live cells, red (EthD‐1) indicates dead cells, and the DMSO group served as the control. Scale bar = 100 μm. (e) Quantification of (d), n = 6 in each group. (f) Cell viability assay of hPCMs (n = 5). Data are presented as mean ± SEM, data in b, c, e, and f were analyzed by repeated measures one‐way ANOVA, followed by Dunnett's multiple comparison test.

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  FIGURE 4. Activation of PI3Kα‐AKT pathway attenuates PON‐induced cardiomyocyte death. (a) Western blotting of AKT phosphorylation (S473 and T308) in hPCMs pretreated with 0.1 μM UCL‐TRO‐1938 or 1 μM insulin (INS) for 1 h, followed by exposure to 5 μM PON for 48 h, with GAPDH as the loading control. (b, c) Quantification of p‐AKTS473 (b) and p‐AKTT308 (c) in (a), n = 6. D, Live/dead staining of hPCMs. Green (Calcein‐AM) indicates live cells, red (EthD‐1) indicates dead cells. Scale bar = 100 μm. (e) Quantification of (d), n = 4. (f), Cell viability assay of hPCMs (n = 4). Data are presented as mean ± SEM. Data in b, c, e, and f were analyzed by repeated measures one‐way ANOVA, followed by Dunnett's multiple comparison test.