Exercise intervention decreases acute and late doxorubicin‐induced cardiotoxicity

Figures

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  FIGURE 1. Doxorubicin (Dox)‐induced juvenile mouse cardiotoxicity model. (A) Schematic representation of the study design to assess acute Dox‐induced cardiotoxicity using nude or Balb/c mice (n = 8 in each group). Four‐week‐old mice were randomly divided into four groups: control, exercise alone, Dox alone, or Dox + Exer. All mice were treated by tail vein with phosphate‐buffered saline or Dox twice per week for 2 weeks. The exercise regimen was 45 min treadmill walking/day, 5 days/week. Echocardiography was conducted before treatment and 24 h after the final dose of Dox. Mice were killed 48 h after the final dose of Dox. (B) To assess Dox‐induced late cardiotoxicity, mice were treated for 2 weeks as in A and then followed them for 12 weeks after therapy. Echocardiography evaluation was done before and 24 h after therapy and then at 2, 4, 8, and 12 weeks following therapy (n = 10 in each group). (C) To assess the effect of initiating exercise after Dox therapy, mice were treated with or without Dox. The exercise protocol described in A was initiated 24 h after therapy. Echocardiography evaluation was done before and 24 h after therapy and then at 2, 4, 8, and 12 weeks after therapy (n = 10…

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  FIGURE 2. Exercise inhibited doxorubicin‐induced autophagy and abnormal mitochondria in nude mice treated with Dox for 2 weeks. (A) Representative transmission electron microscopy (TEM) images of autophagosomes (arrow) 48 h after Dox therapy. (B) Quantitative analysis of autophagosomes. (C) Representative TEM images of abnormal mitochondria (arrow) and vacuolization (asterisk). n = 8 mice per group. Control versus Dox, p = 0.0025; Dox versus Dox + Exer, p = 0.0034 by one‐way ANOVA followed by Bonferroni test

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  FIGURE 3. Effect of doxorubicin with and without exercise on cardiac vessel morphology and cardiac diastolic blood flow in nude mice following Dox therapy. (A) Quantitative analysis of the total area of pericytes (as defined by α‐SMA) in the heart tissues. (B) Ratio of α‐SMA/CD31 cells (mean ± SD for five random fields). (C) Representative images of CD31+ staining. Scale bar: 50 μm. (D) Quantitative analysis of number of open lumens in vessels >100 μm from each treatment group (mean ± SD; Control versus Dox, p = 0.0003; Dox versus Dox + Exer, p = 0.0007). Statistical analysis as described in Figure 2. (E) Representative transmission electron microscopy images of cardiac vessels. Vascular endothelial cells and pericytes are indicated by the arrow. E indicates endothelial. P indicates pericytes. (F, G) Change in mitral valve E and A velocity 2 weeks after therapy. (H) Representative echocardiography images of the E and A peak. (I) E/A ratio was measured for each mouse. Dox indicates doxorubicin and Exer indicates exercise

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  FIGURE 4. Exercise reduced doxorubicin (Dox)‐induced oxidative stress after 2 weeks in peripheral blood mononuclear cells (PBMCs) from immunodeficient and immunocompetent mice. (A) Reactive oxygen species production in PBMCs. Nude mice were treated with phosphate‐buffered saline (black line), Dox alone (red line), exercise alone (green line), or Dox + exercise (blue line). Peripheral blood was collected 24 h after therapy and PBMCs were isolated. Dihydroethidium staining was used for the measurement of intracellular superoxide using flow cytometry. Mean fluorescent intensity for dihydroethidium is shown for representative mice. (B) Balb/c mice were treated with phosphate‐buffered saline (black line), Dox alone (red line), exercise alone (green line), or Dox + exercise (blue line). Peripheral blood was collected 24 h after therapy. PBMCs were then isolated and stained for intracellular peroxides by flow cytometry using dichlorofluorescein. Mean fluorescent intensity is shown for representative mice

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  FIGURE 5. Effect of doxorubicin with or without exercise on diastolic and systolic function in nude mice 12 weeks after therapy. Diastolic (A) and systolic (B) functions were assessed after therapy and 12 weeks later using echocardiography by measuring left ventricular posterior wall thickness in diastole (LVPW; d) and systole (LVPW; s). At 2 weeks post‐Dox (n = 8 mice per group), control versus Dox, p = 0.11; Dox versus Dox + Exer, p = 0.07; at 12 weeks post‐Dox (n = 9 mice per group), control versus Dox, p = 0.03; Dox versus Dox + Exer, p = 0.04. Statistical analysis as described in Figure 2

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  FIGURE 6. Exercise during doxorubicin (Dox) therapy prevented late Dox‐induced cardiotoxicity in nude mice 12 weeks after therapy. (A) Quantitative analysis of autophagosomes in the heart tissue. Twelve weeks after therapy, control versus Dox, p = 0.0014; Dox versus Dox + Exer, p = 0.0281. Statistical analysis as described in Figure 2. (B) Representative transmission electron microscopy (TEM) images of cardiac vessels. P indicates pericytes and E, endothelial cells. (C) Representative TEM images of abnormal mitochondria (arrow) and vacuolization (asterisk). (D, E) Collagen deposition in the left ventricle was determined using Masson trichrome staining after therapy completion (D) and 12 weeks after therapy (E). wks indicates weeks. Scale bar: 100 μm. (F) Quantification of collagen deposition by percentage collagen volume fraction (CVF%). Control versus Dox, ****p < 0.0001; Dox versus Dox + Exer, p = 0.0007 by two‐factor repeated‐measures ANOVA. (G, H) Change in mitral valve E and A velocity 12 weeks after therapy. (I) Representative echocardiography images of the E and A peak. (J) The E/A ratio was measured for each mouse (n = 9 in each group)

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  FIGURE 7. Exercise after doxorubicin (Dox) therapy inhibited long‐term Dox‐induced cardiotoxicity in nude mice. The data in Figure 7 were analyzed from the same experiment as that in Figure 6. There were six total groups in the experiment (Figure S1). The data were then presented to focus on the effect of exercise during therapy versus exercise initiated after therapy. The six individual groups in Figure S1 are Contro–l, Dox for 2 weeks and then no therapy; exercise for 2 weeks and then no therapy; and Dox + Ex for 2 weeks and. then no therapy in Figure 6. In Figure 7, the groups were from the same experiment but show the Control, Dox for 2 weeks and then no therapy; exercise for 14 weeks; and Dox for 2 weeks and then Exercise for 12 weeks. (A) Ejection fraction (EF) and fractional shortening (FS) were quantified by echocardiography 24 h after Dox therapy and then 2, 4, 8, and 12 weeks later. *p < 0.05. **p < 0.01. ****p < 0.0001. (B) EF and FS were quantified as in A in mice that exercised after Dox therapy. ND indicates no difference compared to control. *p < 0.05. (C) Left ventricular posterior wall thickness was measured in the diastole (LVPW, d) and systole (LVPW, s). In LVPW, d,…

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  FIGURE 8. Recovery following left anterior descending artery ligation (myocardial infarction). Myocardial infarction (MI) surgery was performed 90 days after treatment with doxorubicin (Dox), Dox + exercise (Exer), or no therapy (control). (A, B) Fractional shortening (FS) and ejection fraction (EF) were assessed before surgery and then 14 and 45 days after surgery. ****p < 0.0001. Statistical analysis as described in Figure 2. (C) Survival rates were quantified following MI for each group. Kaplan–Meier survival curves of mice at day 45 and day 100 (arrows) after MI. n indicates number of mice. *p < 0.05. (D) Collagen deposition in the left ventricle was assessed using Masson trichrome staining 100 days after MI. Mice treated with Dox alone had a thinned left ventricle with collagen deposition in the left ventricular wall. This was not observed in the mice treated with Dox + Exer. Scale bar: 0.5 mm. (n = 9 in each group). (E) Left ventricular wall heart tissue following myocardial infarction (MI) for 100 days in nude mice. LV indicates left ventricle. Dox indicates doxorubicin. Exer indicates exercise