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Fig. 5
Proliferation inhibition in transgenic zebrafish using SB431542 sensitizes them to LC cardiac toxicity lowering survival, increasing cardiac cell death and dysfunction. Representative images (left) from isolated control and AL-LC TG embryonic zebrafish hearts following 24-h SB431542 treatment display a decrease in EdU-labeled nuclei. Quantification of EdU-positive cells in zebrafish heart following 24-h SB431542 treatment. n = 5–7 zebrafish embryos/group; P = 0.03 for control embryos with DMSO vs. SB431542; P = 0.02 for TG DMSO vs. SB431542 (A). Dimensional analysis performed as (23) at 72 hpf following a 24-h SB431542 treatment revealed a loss of the cardiac functional compensation by stroke volume (P = 0.02) (B). Kaplan-Meier curve shows that survival is significantly decreased in AL-LC transgenic zebrafish embryos treated with 10 µM SB431542 for 96 h administered starting at 2 days postfertilization (*P = 0.002 for control embryos with SB431542 vs. TG SB431542; **P = 0.0003 for TG with DMSO vs. SB431542); n ≥ 40 zebrafish embryos/group (C). Representative images of isolated control and AL-LC TG zebrafish hearts following 24-h and 96-h SB431542 treatment revealed increased TUNEL-stained nuclei (red, left). Quantification of TUNEL-positive cells in isolated zebrafish hearts following 24-h and 96-h SB431542 treatment. Treatment for both durations resulted in a significant increase in cell death in the AL-LC TG fish hearts, n = 7–14 zebrafish embryos/group; P = 0.02 at 24 h; P = 0.008 at 96 h (D). Further evidence of apoptosis was observed with an increase in cleaved caspase-3 protein by immunoblotting of the AL-LC TG embryos treated with 10 μM SB431542 (P = 0.048) (E). Indication of abnormal authophagic flux was observed via increased LC3-II protein levels as measured by immunoblotting of the AL-LC TG embryos treated with 10 μM SB431542 (P = 0.045). n = 3 separate experiments with 7–14 zebrafish embryos/group (F). Con, control; EdU, 5-ethynyl-2′-deoxyuridine; hpf, hours postfertilization; SB, SB431542; TG, transgenic.