Figure 2
Inhibitors of MAP4K4 confer protection from DOX in human PSC-derived cardiomyocytes. (A,B) Protection of hPSC-CM viability (CellTiter-Glo). (A) Human vCorv.4U ventricular myocytes were treated for 72 h with 3 μM DOX, 2 μM Idarubicin, or 30 μM menadione, a proven MAP4K4-dependent death signal11, ± 10 μM F1386-0,303. Data were normalised to untreated and 0.1% Triton X-100-treated cells (100% and 0% viability, respectively). Data are triplicates, plotted as the mean ± SEM, and are representative of 3 independent experiments. **P ≤ 0.01; ***P ≤ 0.001. (B) Human IMR-90 cardiomyocytes were treated for 24 h with 0–75 μM DOX ± 10 μM DMX-5804. **P ≤ 0.01. (C–F) Suppression of hPSC-CM apoptosis. (C,D) Human vCor.4U cells were treated for 24 h with 0–10 μM DOX ± 10 μM DMX-5804 and were analyzed by TUNEL staining. (C) Representative images. Bar, 50 μm. (D) Data are the mean ± SEM of ≥ 3 replicates and are representative of 2 independent experiments. ***P ≤ 0.001. (E,F) IMR-90 cardiomyocytes, treated for 24 h as shown, were assayed by Western blotting for cleaved PARP1. (E) Representative Western blot (See also Supplementary Fig. S1). (F) Data are the mean ± SEM of 2 independent experiments. **P = 0.0016. (G) No protection from the Bcl-2 family inhibitor, ABT-737. IMR-90 cardiomyocytes were treated for 24 h as shown and analysed by the CellTiter-Glo viability assay. Data are the mean ± SEM of 4 replicates in each of two independent experiments. (H) Partial protection of ∆Ψm. IMR-90 cardiomyocytes were treated for 24 h as shown and analysed by JC-10 fluorescence. Data are the mean ± SEM of 3 replicates in each of two independent experiments. *P ≤ 0.05. (I) No interference with DOX-induced expression of BAX or the indicated death domain receptor genes. IMR-90 cardiomyocytes were treated for 24 h as shown and analysed by by QRT-PCR. Data are the mean of 2 replicates in each of 2 independent experiments. (J,K) Preservation of Ca2+ cycling (FLIPR). vCor.4U cells were treated with sub-maximal (500 nM) DOX for 24 h, after 1 h pre-incubation with 10 μM F1386-0303 (blue) or DMX-5804 (red). Cardiomyocytes were monitored for 100 s and the first 40 s are illustrated. (J) Representative signals. RFU, relative fluorescence units. (K) Data are duplicates, plotted as the mean ± SEM from 3 independent experiments. By contrast to the loss of beat frequency and total peak area, only small changes occurred in median peak height and width (not shown). *P < 0.05; **P < 0.01; ****P ≤ 0.0001 versus DOX alone.
