Fig 1.
TKI effect on EHT contractility.
(A) Depiction of concentration-effect curves (curve-fitted) of 9 TKIs after 96 hours of TKI incubation (▲), normalized to vehicle control (Δ). The toxic threshold (black dashed line) is defined as a decline in contractile force of >40% vs. baseline (BL). Mean values ± SEM; n = 4; *p<0.05 vs. baseline, two-way ANOVA and Bonferroni's multiple comparison post-test. (B) Total therapeutic plasma concentration (TPC), toxic threshold concentration (TTC: TKI concentration leading to ≥40% reduction in EHT contractile force) and safety margin (SM: TTC/TPC), n/a: not applicable.
Fig 2.
(A) α-sarcomeric actin staining of longitudinal EHT sections in the presence of TKIs and controls after 96 hours of incubation. Cross-striation (CS), extracellular matrix (ECM), nucleus (N). (B-F) Representative images of EM alterations in EHTs in the presence of TKIs. (B) Regularly structured sarcomere with Z-band (Z, 0.1% DMSO); (C) sarcomere with sarcomeric disarray, widened Z-band, (Zw, imatinib 10 μM); (D) mutilamellar bodies (MB, imatinib 10 μM); (E) mitochondria with structural defects (Md), vacuoles and inclusion bodies (IB, sunitinib 1 μM); (F) autophagy (A, vandetanib 10 μM), scale bar 1 μm.
Fig 3.
(A) LC3-II/I ratio Western blot of EHTs after 96 hours incubation: (vehicle) control or TKIs at two concentrations as indicated. LC3-I and -II band densities were quantified and LC3-II/I ratios were calculated. (B) Quantitative analysis of LC3-II/I ratios. Results are presented as means±SEM, n = 13 (controls), n = 3–5 (TKIs). One-way ANOVA and Dunnett's post-test (compared to vehicle control) were used. P-values <0.05 were considered statistically significant and indicated in the graphs (*).
Table 1.
Correlation of biochemical (LDH, CK) or autophagic (LC3-II/I) markers with contractility of EHTs.
Significant increase or decline at any time point is marked by +. Correlation of LC3-II/I ratio with contractility is higher (Phi coefficient φ = +0.894) than LDH with contractility (φ = +0.798) or CK with contractility (φ = +0.707).
Fig 4.
Quantification of cross-sectional area of included region (%) for disarray/autophagy (A), mitochondria (B), cytoplasma (C) and myofibrils (D) in mosaic transmission electron microscopy images after 24, 48, 72 and 96 hours of incubation under (vehicle) control conditions and with sorafenib (100 μM), imatinib (100 μM) and sunitinib (10 μM). Mosaic TEM images are shown in S4–S7 Figs. EM alterations are described in S2–S6 Tables.
Fig 5.
LC3 Western blot analysis of heart cell EHT and cardiac fibroblast EHT after 24–96 hours of TKI incubation in the absence and presence of bafilomycin A (B) (100 nM, 2 hours incubation). LC3 band densities were quantified and LC3–II/I ratios were calculated. (A) Representative Western blot for heart cell EHT analysis, (B-G) Quantitative analysis of LC3-II/I ratio in heart cell EHTs (B-G) and fibroblast EHT (H-M). (G, M) Comparison of LC3-II/I ratio of all time points for heart cell EHTs (G) and cardiac fibroblast EHTs (M). Results are presented as means±SEM. (B-F), (H-L): n = 3–4; (G, M): n = 16–20.One-way ANOVA and Bonferroni's post-test was used to analyze statistical significance. P-values <0.05 were considered statistically significant and indicated in the graphs (*), ns: not significant.
Fig 6.
Active caspase-3 immunohistochemistry of time series (24 h-96 h) for vehicle control DMSO 0.1% (DMSO), sorafenib (100 μM), imatinib (100 μM), and sunitinib (10 μM) on heart cell EHTs.
Arrows indicate cytoplasmatic caspase-3 staining. Please note nuclear caspase staining for vehicle control interpreted as fixation artifact. Scale bar 100 μm.