Figure 3.

Protection of Human Stem Cell-Derived Ventricular Myocytes by Small-Molecule Inhibitors of MAP4K4

(A and B) vCor.4U ventricular myocytes were assayed 24 h after oxidative stress conferred by H₂O₂ (left) or menadione (right) at the indicated concentrations, ±DMX-5804 or the parent compound F1386-0303 (10 μM) 1 h prior to insult. Cardiomyocyte protection was confirmed in three independent experiments, using three different lots of vCor.4U cells (CV98CL V, CV99CL V, and CV102CL V). A representative dose-response curve is shown in each panel (2 replicate wells per condition).

(A) CellTiter-Glo assay. Results (% viability) are normalized to the difference between untreated control cells (no death signal and no inhibitor) versus 100% cell death (0.1% Triton X-100 2 h before CellTiter-Glo [CTG]).

(B) Human cardiac troponin I release (AlphaLISA).

(C) Cross-titration of DMX-5804 and H₂O₂. (Left) CellTiter Glo is shown; (right) troponin release is shown; n = 10. Arrows illustrate the loss of viability at 500 μM H₂O₂ and rescue by 10 μM DMX-5804. The half-maximal concentration for protection shifts systematically rightward as oxidative stress increases. To minimize inter-experimental variation, results at a fixed concentration of death signal (D–F: CellTiter Glo; % rescue) are normalized to the difference between untreated control cells versus stress-induced death in the absence of inhibitor.

(D) Partial protection by MAP4K4 inhibitors given 1 h before versus 1 h after 400 μM H₂O₂; N ≥ 3.

(E) vCor.4U cells were stressed with 400 μM H₂O₂ 1 h after treatment with the compounds shown. Nine-point dose-response curves were obtained; for clarity, rescue of viability is shown as a bar graph at the highest concentration (10 μM). Fidelity to pathways driving infarct size in human trials is suggested by the positive result for β1-adrenergic blockade and weak result for p38. Inhibition of the MAP4K4 target TAK1 was likewise effective; N ≥ 3; ^∗p = 0.0225; ^∗∗p = 0.0011.

(F) (Left) Dose-response relations for in-cell activity, measured as protection from 400 μM H₂O₂ (CellTiter-Glo). (Right) Potency for human cardiomyocyte protection correlates with the potency against recombinant human MAP4K4, across three orders of magnitude.

Results are shown as the mean ± SE.