Fig. 1. Modulation of microtubule density and myofibrillar structural damage induced by paclitaxel or nocodazole. (A–C) Cells were fixed and immunostained for beta-tubulin. (D–F) Cells were immunostained for myomesin and the percentage of cells showing myofibrillar damage was counted. (A) Untreated cells; (B) Cells treated with paclitaxel 6 μM for 48 h; (C) Cells treated with nocodazole 10 μM for 48 h. (D) Untreated cardiomyocytes. (E) Cardiomyocytes were incubated for 48 h with paclitaxel 6 μM. (F) Analysis of myofibrillar structural damage with increasing dose of paclitaxel (TX), p < 0.01 vs. CTL, one-way ANOVA p < 0.0001, n = 6 independent experiments.

Fig. 2. Cell viability assays. ARVM were either cultured for 10 days in the presence of 10% FCS and treated for 48 h (A, C, E) or cultured serum-free for 2 days and treated for 18 h (B, D, F). (A, B) MTT assay (mitochondrial activity) for increasing doses of paclitaxel (Tx) or MEK1/2 inhibitors in the FCS-containing culture, PD: PD98059 50 μM, U126: U126 5 μM, anti-ErbB2: mAb clone 7.16.4 3.4 μg/mL. (C, D) LDH release after paclitaxel treatment at different concentrations. (E, F) TUNEL-assay for DNA-degradation, p < 0.01 vs. CTL, n = 3.

Fig. 3. Downstream signaling kinases Erk1/2 or Akt and ErbB2 receptors were tested by Western blotting. (A, B) Cardiomyocytes were cultured for 10 days and then treated for 48 h with either paclitaxel 6 μM (Tx) or anti-ErbB2 (mAb clone 7.16.4 3.4 μg/mL). Densitometric analysis is presented on the left and representative Western blots on the right. (C) Cardiomyocytes were treated with increasing doses of paclitaxel and lysates were tested for ErbB2 receptor protein. (A, B) p < 0.01 vs. CTL; p < 0.001 vs. CTL; (A) one-way ANOVA p < 0.001, n = 3. (B) One-way ANOVA p < 0.01, n = 3. (C) No statistically significant differences, n = 3.

Fig. 4. Myofibrillar structural damage induced by the inhibition of Erk1/2 with PD98059 (PD). (A–D) Cells were immunostained for myomesin (green) and actin (red) and images were recorded by confocal microscopy. (A) CTL: vehicle treated cells; (B) Cells treated with PD 0.1 μM for 48 h. (C) Cells treated with PD 1 μM for 48 h. (D) Cells treated with PD 10 μM for 48 h. (E) Phosphorylation of Erk1/2 after PD treatment tested by Western blot and densitometry. (F) Cells showing altered morphology were counted after immunostaining for myomesin. (E, F) p < 0.001 vs. CTL; one-way ANOVA p < 0.01, n = 3.

Fig. 5. Myofibrillar structural damage induced by PD98059, anti-ErbB2 alone or in combination with paclitaxel after treatment for 48 h. Cells showing altered morphology were counted after immunostaining for myomesin. (A) PD: PD98059 50 μM, Tx: paclitaxel 6 μM. p < 0.001 vs. CTL; p < 0.0001 vs. CTL; one-way ANOVA p < 0.0001. (B) Tx: paclitaxel 6 μM, anti-ErbB2: mAb clone 7.16.4 3.4 μg/mL. p < 0.01, anti-ErbB2 vs. anti-ErbB2 in combination with paclitaxel; p < 0.001 vs. CTL; p < 0.0001 vs. CTL; one-way ANOVA p < 0.0001, n = 6.

Fig. 6. Effects of paclitaxel and Erb2 inhibition on oxidative stress. (A) Average DCF-fluorescence intensity was measured in isolated cardiomyocytes cultured for 18 h in serum free media. H₂O₂ was used as a positive control at 100 μM for 15 min. NAC at 50 μM was applied 2 h before the other treatments, paclitaxel was used at 3, 6, 50 μM. (B) anti-ErbB2: mAb clone 7.16.4 3.4 μg/mL. (C) Images of representative cells treated as indicated and photographed after incubation with DCF. Each condition was tested in 33 cells from 3 hearts. p < 0.05 vs. CTL; p < 0.001 vs. CTL; p < 0.0001 vs. CTL; one-way ANOVA < 0.005. Data were normalized to CTL level (0%) and H₂O₂ (100%).

Fig. 7. Effects of paclitaxel on EC-coupling. Isolated cardiomyocytes were cultured for 18 h in paclitaxel-containing serum free medium. Cell shortening was measured by video-edge detection and calcium transients by Fura-2 ratios. (A) Fractional shortening. (B) Calcium amplitude. (C) Diastolic calcium. (D) Time to 50% of relaxation. Each condition was tested in 45 cells from 3 hearts. One-way ANOVA < 0.005, p < 0.05 vs. 0 μM paclitaxel (CTL).

Fig. 8. Effects of an anti-ErB2 antibody and paclitaxel on EC-coupling. Isolated cardiomyocytes were cultured for 18 h in serum free media, with paclitaxel 6 μM (Tx) or anti-ErbB2 3.4 μg/mL. (A) Fractional shortening, (B) Calcium amplitude, (C) diastolic calcium, (D) time to 50% of relaxation. Each condition was tested in 45 cells from 3 hearts. One-way ANOVA < 0.005, p < 0.05 vs. 0 μM (CTL). Data were normalized to CTL level (100%).

Fig. 9. Proposed mechanisms for paclitaxel- and anti-ErbB2-associated cardiotoxicity. Both paclitaxel and anti-ErbB2 downregulate the MAPK/Erk1/2 signaling pathway. Myofibrillar structural damage occurs by downregulation of sarcomeric and protective genes on one side and increasing oxidative stress and changes in calcium handling on the other side.