Doxorubicin-Induced and Trastuzumab-Induced Cardiotoxicity in Mice is Not Prevented by Metoprolol

Figures

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  Figure 1. Plan of the experimental protocol for a chronic chemotherapy‐induced cardiomyopathy with beta‐blockers. Ten‐week‐old C57Bl6 male mice were evaluated 2 days before the onset of chemotherapy, with measurements of body weight (BW) and cardiac parameters by transthoracic echocardiography (TTE). This evaluation was repeated on Days 21 and 38. Doxorubicin (Dox, n = 11) or saline (n = 9) was IP injected every 2 days (from Days 0 to 14), and 1 week later, mice received IP of either trastuzumab (Trast) or saline again for 2 weeks. In addition, 14 mice were treated with metoprolol (100 mg/kg) in the drinking water from Day 10 prior to the onset of the protocol with Dox + Trast (n = 8) or saline (n = 6) to the end. BW, body weight; TTE, transthoracic echocardiography.

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  Figure 2. Effects of metoprolol on chemotherapy‐induced cardiac dysfunction. (A) Typical echo image of LV dysfunction without any LV dilation at Day 38 after chemotherapies were initiated. (B) The measure of LVEF with echocardiography from baseline to Day 38 indicates a rapid decline in LVEF, which is not prevented by metoprolol treatment (Ctrl with and without Meto n = 15, Dox + Trast n = 11, Dox + Trast + Meto n = 8, **P < 0.01, ***P < 0.001 by two‐way using ANOVA test followed by Bonferroni post hoc test). (C–D) Quantification of natriuretic peptides (NPPs) (C) and SERCA2a (D) mRNAs in ventricular tissues. Note the significant increase of NPPs after chemotherapy. The decrease in SERCA2a mRNA level is a sign of heart failure. Values are means ± SEM, *P < 0.05, **P < 0.01 by two‐way using ANOVA test followed by Bonferroni post hoc test. (Ctrl n = 8; Ctrl + Meto n = 5, Dox + Trast n = 10, Dox + Trast + Meto n = 7).

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  Figure 3. Chemotherapy‐induced cardiac injury and cardiac atrophy. (A) Plasma Troponin I at Day 42 (ng/mL, Ctrl n = 8, Ctrl + Meto n = 6, Dox + Trast n = 11, Dox + Trast + Meto n = 6). (B) Plasma caspase 3 activity (pmol/min/mL, Ctrl n = 8, Ctrl + Meto n = 6, Dox + Trast n = 11, Dox + Trast + Meto n = 6). (C) Vinculin (red) and caveolin (green) immunostaining. Analysis of myocyte cross sectional areas at Day 42 in control and chemotherapy‐treated mice (×20, Ctrl n = 5, Ctrl + Meto n = 5, Dox + Trast n = 5, Dox + Trast + Meto n = 5). Values are means ± SEM. **P < 0.01, ***P < 0.001 using two‐way ANOVA test followed by Bonferroni post hoc test.

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  Figure 4. Chemotherapy‐induced cardiac oxidative stress. (A) Intracardiac DHE staining (×20) and marked nuclei per area (Ctrl n = 5, Ctrl + Meto n = 5, Dox + Trast n = 5, Dox + Trast + Meto n = 5). (B) Plasma myeloperoxidase (ng/mL, Ctrl n = 8, Ctrl + Meto n = 6, Dox + Trast n = 11, Dox + Trast + Meto n = 6). Values are means ± SEM. **P < 0.01 using two‐way ANOVA test followed by Bonferroni post hoc test.

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  Figure 5. Chemotherapy‐induced cardiac fibrosis. (A) Representative cardiac section stained with Sirius Red (×20), and score graph of cardiac fibrosis (Ctrl n = 5, Ctrl + Meto n = 5, Dox + Trast n = 5, Dox + Trast + Meto n = 5). (B) Intracardiac CTGF (Connective tissue growth factor) expression (Ctrl n = 9, Ctrl + Meto n = 4, Dox + Trast n = 10, Dox + Trast + Meto n = 6). Values are means ± SEM. **P < 0.01 using two‐way ANOVA test followed by Bonferroni post hoc test.

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  Figure 6. Metoprolol is ineffective to prevent chronic cardiotoxicity induced by Dox + Trast. Dox, Doxorubicin; LVEF, left ventricular ejection fraction; PKA, Protein Kinase A; Trast, Trastuzumab.