Exercise Inhibits Doxorubicin-Induced Damage to Cardiac Vessels and Activation of Hippo/YAP-Mediated Apoptosis

Figures

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  Figure 1. Exercise prevents Dox-induced decreases in cardiac function and blood flow. (A) Schematic representation of experiment design and mouse treatment. (B,E,G) Representative Echo images of M-mode of parasternal short axis (PSAX) view of hearts (B), PW Doppler waveform of trans-mitral blood flow in hearts (E), and PW Doppler waveform of transverse aorta blood flow in hearts (G) from Ctr, Dox, and Dox+Ex group mice. (C,D,F,H) Echo data show changes in LVEF (C), LVFS (D), mean velocity at LVMV (F), and mean velocity at LVOT (H) after treatment in Ctr, Dox, and Dox+Ex group mice. N = 10 mice/group. P values are indicated by the GraphPad t test. Ctr vs Dox+Ex, not significant statistically. (I–K) Echo data show changes in LVEF (I), LVFS (J), and mean velocity at LVMV (K) after treatment in Ctr and Ex group mice. N = 8 mice/group. P values are indicated by the GraphPad t test. Ctr vs Ex, not significant statistically. LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVMV, left ventricular mitral valve; LVOT, left ventricular outflow tract in the ascending aorta; PW Doppler, pulse wave Doppler; Ctr, control; M, mock; Ex/E, exercise; Dox/D, doxorubicin;…

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  Figure 2. Bone marrow transplantation does not affect cardiac function and blood flow. (A) Schematic representation of bone marrow transplantation (BMT) procedure and subsequent mouse treatment. (B) Schematic representation of BMT experiment design. (C–H) Echo data show the difference between Ctr and BMT group mice in LVEF, LVFS, MV E/A Ratio, MV ET, LVAW;d, and LVPW;d. N = 5 mice/group. n.s. indicates not statistically significant by the GraphPad t test. LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; MV E/A Ratio, mitral valve peak E and A velocity ratio; MV ET, mitral valve ejection time; LVAW;d, left ventricular anterior wall thickness in diastole; LVPW;d, left ventricular posterior wall thickness in diastole; Ctr, control; BMT, bone marrow transplantation.

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  Figure 3. Exercise inhibits Dox-induced cardiomyocyte apoptosis. (A) Schematic representation of experiment design of BMT and mouse treatment. (B,C) Echo data show changes in LVEF and LVFS before and after treatment in Ctr, Ex, Dox, and Dox+Ex group mice. N = 6 mice/group. P values are indicated by the GraphPad t test. Ctr vs Dox+Ex, not statistically significant. (D) Representative immunofluorescence images of heart sections stained for DAPI (blue) and cTn I (red) from Ctr, Ex, Dox, and Dox+Ex group mice. Magnification, 40×; Scale bar, 50 µm. (E) Representative immunofluorescence images of heart sections stained for DAPI (blue) and cleaved caspase-3 (red) from Ctr, Ex, Dox, and Dox+Ex group mice. Magnification, 40×; Scale bar, 50 µm. (F) Western blot analysis for cleaved caspase-3, cleaved PARP, and histone H3 expression in hearts from Ctr, Ex, Dox, and Dox+Ex group mice. LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; Ctr, control; M, mock; Ex/E, exercise; Dox/D, doxorubicin; Echo, echocardiography; Eu, euthanize; BMT, bone marrow transplant.

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  Figure 4. Exercise promotes migration of bone marrow (stem) cells into the heart after Dox treatment. (A) Representative immunofluorescence images of heart sections stained for DAPI (blue) and GFP (green) from Ctr, Ex, Dox, and Dox+Ex group mice. Magnification, 40×; Scale bar, 50 µm. (B) Quantification of GFP-positive cells in heart tissues per 10× field from Ctr, Ex, Dox, and Dox+Ex group mice. N = 5 mice/group, 3 slides/mouse. Values represent mean ± SEM. P values are indicated by the Student’s t test. (C) Representative immunofluorescence images of heart sections stained for DAPI (blue), GFP (green), and c-Kit (red) from Ctr, Ex, Dox, and Dox+Ex group mice. Magnification, 400×; Scale bar, 5 µm. (D,E) Quantification of the number and percentage of c-Kit and GFP double-positive cells in heart tissues per 10× field and per total c-Kit-positive cells from Ctr, Ex, Dox, and Dox+Ex group mice. N = 5 mice/group, 3 slides/mouse. Values represent mean ± SEM. p values are indicated by the Student’s t test.

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  Figure 5. Exercise promotes differentiation of BM stem cells into endothelial cells after Dox treatment. (A) Representative immunofluorescence images of heart sections stained for DAPI (blue) and CD31 (red) from Ctr, Ex, Dox, and Dox+Ex group mice. Magnification, 40×; Scale bar, 50 µm. (B) Quantification of CD31-positive cells normalized to DAPI (density) in heart tissues from Ctr, Ex, Dox, and Dox+Ex group mice. N = 5 mice/group, 3 slides/mouse. Values represent mean ± SEM. P values are indicated by the Student’s t test. Ctr vs Dox+Ex, not statistically significant. (C) Representative immunofluorescence images of heart sections stained for DAPI (blue), GFP (green), and CD31 (red) from Ctr, Ex, Dox, and Dox+Ex group mice. Arrows indicate colocalization (yellow) of GFP (green) and CD31 (red). Magnification, 40×; Scale bar, 50 µm. (D) Quantification of CD31 and GFP double-positive cells in heart tissues per 10× field from Ctr, Ex, Dox, and Dox+Ex group mice. N = 5 mice/group, 3 slides/mouse. Values represent mean ± SEM. P values are indicated by the Student’s t test. (E) Quantification of percentage of CD31 and GFP double-positive cells to total CD31-positive cells in heart tissues from Ctr,…

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  Figure 6. Exercise promotes differentiation of BM stem cells into pericytes after Dox treatment. (A) Representative immunofluorescence images of heart sections stained for DAPI (blue) and NG2 (red) from Ctr, Ex, Dox, and Dox+Ex group mice. Magnification, 40×; Scale bar, 50 µm. (B) Quantification of NG2-positive cells normalized to DAPI (density) in heart tissues from Ctr, Ex, Dox, and Dox+Ex group mice. N = 5 mice/group, 3 slides/mouse. Values represent mean ± SEM. P values are indicated by the Student’s t test. Ctr vs Dox+Ex, not statistically significant. (C) Representative immunofluorescence images of heart sections stained for DAPI (blue), GFP (green), and NG2 (red) from Ctr, Ex, Dox, and Dox+Ex group mice. Arrows indicate colocalization (yellow) of GFP (green), and NG2 (red). Magnification, 40×; Scale bar, 50 µm. (D) Quantification of NG2 and GFP double-positive cells in heart tissues per 10× field from Ctr, Ex, Dox, and Dox+Ex group mice. N = 5 mice/group, 3 slides/mouse. Values represent mean ± SEM. p values are indicated by the Student’s t test. (E) Quantification of percentage of NG2 and GFP double-positive cells to total NG2-positive cells in heart tissues from Ctr, Ex, Dox, and…

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  Figure 7. Exercise prevents Dox-induced increase in fibroblasts. (A) Representative immunofluorescence images of heart sections stained for DAPI (blue) and Vimentin (red) from Ctr, Ex, Dox, and Dox+Ex group mice. Magnification, 40×; Scale bar, 50 µm. (B) Representative immunofluorescence images of heart sections stained for DAPI (blue), GFP (green), and Vimentin (red) from Ctr, Ex, Dox, and Dox+Ex group mice. Magnification, 40×; Scale bar, 50 µm.

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  Figure 8. Activation of Hippo-YAP signaling is induced by Dox therapy. (A) MTT assay analysis for viability of human cardiac myocytes (HCMs) treated with various concentrations of Dox as indicated for 24 or 48 h. Values represent mean ± SEM of three independent experiments. (B) Western blot analysis for cleaved caspase-3, cleaved PARP, and histone H3 expression in HCMs treated with Dox at 1.25 µM for 0, 6, or 12 h. (C) Western blot analysis of phospho-MST1/2, MST1, phospho-LATS1, LATS1, phospho-YAP, YAP, and histone H3 expression in HCMs treated with Dox at 1.25 µM for 0, 6, or 12 h. (D) Western blot analysis of phospho-YAP, cleaved caspase-3, cleaved PARP, and histone H3 expression in HCMs treated with mock (control), verteporfin (VP) at 2.5 µM, Dox at 1.25 µM, or combination of VP with Dox for 8 h. (E,F) Western blot analysis of phospho-MST1/2, MST1, phospho-LATS1, LATS1, phospho-YAP, YAP, histone H3 (E), CTGF, CYR61, and Histone H3 (F) expression in hearts from Ctr, Ex, Dox, and Dox+Ex group mice. (G) Representative immunofluorescence images of heart sections stained for DAPI (blue) and p-YAP (red) from Ctr, Ex, Dox, and Dox+Ex group mice. Magnification, 40×; Scale bar, 50 µm. (H,I)…