Investigation of the Antiremodeling Effects of Losartan, Mirabegron and Their Combination on the Development of Doxorubicin-Induced Chronic Cardiotoxicity in a Rat Model

Figures

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  Figure 1. Experimental protocol. Male Wistar rats (n = 50, 350–400 g) were divided into one control and four doxorubicin (DOXO)-treated groups (ip. 1.5 mg/kg in 6 cycles; cumulative dose: 9 mg/kg). From the 5th week after the last cycle of DOXO administration, rats were treated via oral gavage daily until the end of the experiments as follows: (i) control group treated with tap water (n = 8, 2 mL/kg/day), (ii) DOXO-only group treated with tap water (n = 11, 2 mL/kg/day), (iii) DOXO group treated with losartan (L, per os 20 mg/kg/day, n = 10) dissolved in tap water, iv) DOXO group treated with mirabegron (M, per os 30 mg/kg/day, n = 10) dissolved in tap water, and (v) DOXO group treated with the combination of losartan (per os 20 mg/kg/day) and mirabegron (per os 30 mg/kg/day) dissolved in tap water (n = 11, 2 mL/kg/day). Cardiac morphology and function were assessed by transthoracic echocardiography (Echo) at weeks 4 and 8 under isoflurane anesthesia. At week 9, an invasive blood pressure (BP) measurement was performed under pentobarbital anesthesia, then blood was collected from the abdominal aorta to measure routine laboratory parameters, and hearts, lungs, and tibias were isolated. Left…

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  Figure 2. Effects of losartan, mirabegron, and their combination on morphological changes and systolic dysfunction in DOXO-induced chronic cardiotoxicity assessed by echocardiography at week 8. Values are presented as mean ± S.E.M., * p < 0.05 vs. control group, # p < 0.05 vs. DOXO-only group (n = 6–9, one-way ANOVA, Holm-Sidak post hoc test). (a) Representative M-mode images, (b) systolic septal wall thickness (SWTs), (c) systolic inferior wall thickness (IWTs), (d) left ventricular end-systolic diameter (LVESD), (e) left ventricular end-diastolic diameter (LVEDD), (f) fractional shortening (FS), and (g) ejection fraction (EF). DOXO: doxorubicin, L: losartan, M: mirabegron.

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  Figure 3. Effects of losartan, mirabegron, and their combination on diastolic dysfunction in DOXO-induced chronic cardiotoxicity assessed by echocardiography at week 8. Values are presented as mean ± S.E.M., * p < 0.05 vs. control group, # p < 0.05 vs. DOXO-only group (n = 6–9, one-way ANOVA or ANOVA on ranks in the case of E/e’, Holm-Sidak post hoc test). (a) representative pulse wave Doppler (PWD) and tissue Doppler (TD) images, (b) mitral valve early flow velocity (E) to septal mitral annulus velocity (e’) ratio (E/e’), (c) mitral valve early flow velocity (E), (d) septal mitral annulus velocity (e’), and (e) isovolumic relaxation time (IVRT). DOXO: doxorubicin, L: losartan, M: mirabegron.

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  Figure 4. Effects of losartan, mirabegron, and their combination on DOXO-induced histologic changes at week 9. Values are presented as mean ± S.E.M., * p < 0.05 vs. control group, # p < 0.05 vs. DOXO-only group (n = 6–9, one-way ANOVA in the case of cardiomyocyte cross-sectional area and ANOVA on ranks in the case of LV collagen, Holm-Sidak post hoc test). (a) Representative hematoxylin-eosin (HE)-stained sections at 40× and 100× magnifications, (b) cardiomyocyte cross-sectional area, (c) representative picrosirius red and fast green (PSFG)-stained sections at 20× magnification, (d) left ventricular (LV) collagen content. DOXO: doxorubicin, L: losartan, M: mirabegron.

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  Figure 5. The effects of losartan, mirabegron, and their combination on the left ventricular expression of several elements of the TGF-β/SMAD fibrotic pathway in our DOXO-induced chronic cardiotoxicity model at week 9. (a) Left ventricular mRNA expressions of connective tissue growth factor (Ctgf) and (b) transforming growth factor-beta (Tgfb); (c) left ventricular protein expression and cropped representative Western blot images of (c) transforming growth factor-beta receptor type II (TGFβRII); (d) left ventricular mRNA expressions of mothers against decapentaplegic homolog 2 (Smad2); (e) mothers against decapentaplegic homolog 3 (Smad3), and (f) collagen 1a1 (Col1a1). Values are presented as mean ± S.E.M., * p < 0.05 vs. control group, # p < 0.05 vs. DOXO-only group (n = 5–9 for qPCR and n = 5–6 for Western blot results, ANOVA on ranks in the cases of Tgfb and TGFβRII expressions, and one-way ANOVA for the other parameters, Holm-Sidak post hoc test). DOXO: doxorubicin, GAPDH: glyceraldehyde 3-phosphate dehydrogenase (loading control in Western blot measurements), L: losartan, M: mirabegron. Ppia: peptidylprolyl isomerase A was used as the housekeeping gene for normalization in qPCR…

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  Figure 6. Effects of losartan, mirabegron, or their combination on the left ventricular expression of SERCA2a, β3AR, eNOS and p-eNOS in DOXO-induced cardiotoxicity at week 9. Left ventricular expression and cropped representative images of (a) sarcoendoplasmic reticulum calcium ATPase 2a (SERCA2a); (b) beta-3 adrenoceptor (β3AR); (c) endothelial nitric oxide synthase (eNOS); (d) phospho-eNOS (p-eNOS), and (e) p-eNOS/eNOS ratio. Values are presented as mean ± S.E.M., * p < 0.05 vs. control group (n = 5–6, ANOVA on ranks in the cases of SERCA2a and p-eNOS expressions, and one-way ANOVA for the other parameters, Holm-Sidak post hoc test). Tubulin was used as a loading control. DOXO: doxorubicin, L: losartan, M: mirabegron. Images were captured with the Odyssey CLx machine and exported with Image Studio 5.2.5 software. The full-length Ponceau-stained membranes and Western blots are presented in Supplementary Figures S2–S5.

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  Figure 7. Effects of losartan, mirabegron, or their combination on the left ventricular expression of selected elements of the tissue renin-angiotensin-aldosterone system and inflammatory markers in DOXO-induced cardiotoxicity at week 9. Left ventricular expressions of (a) chymase (Cma); (b) angiotensinogen (Agt); (c) angiotensin II receptor type 1 (Agtr1); (d) interleukin-1 (Il1); (e) interleukin-6 (Il6), and (f) tumor necrosis factor-alpha (Tnf). Values are presented as mean ± S.E.M., * p < 0.05 vs. control group, # p < 0.05 vs. DOXO-only group (n = 5–6, ANOVA on ranks in the cases of Il6 and Tnf expressions, and one-way ANOVA for the other parameters, Holm-Sidak post hoc test). Peptidylprolyl isomerase A (Ppia) was used as the housekeeping gene for normalization DOXO: doxorubicin, L: losartan, M: mirabegron.

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  Figure 8. Summary figure; please see the conclusions for further explanations. Inflam: inflammatory, LV: left ventricular, SERCA2a: sarcoendoplasmic reticulum calcium ATPase.