Investigation of potential sex-based differences in trastuzumab-induced chronic cardiotoxicity in a rat model

Figures

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  FIGURE 1. Protocol figure. Male and female Wistar-Hannover rats were divided into 2-2 groups: control treated with physiological saline (6 × 1 mL/kg, i.p.), and TZB (on the first day 2 mg/kg, then on days 4, 7, 10, 13, and 16, 1 mg/kg, i.p.) groups. At weeks 12 and 19, echocardiography was performed to assess cardiac function and morphology. Then, animals were placed in metabolic cages for 24 h to collect urine. At week 20, rats were anesthetized with sodium pentobarbital, and the abdominal cavity was opened to collect blood from the aorta. Then, under anesthesia, the hearts, lungs, and tibias were excised and washed in a calcium-free Krebs-Henseleit solution to remove the residual blood. The mid-papillary parts of the left ventricles were fixed in 4% buffered formalin for histological analysis, and the apical parts of the left ventricles were freshly frozen in liquid nitrogen and stored at −80 °C until further biochemical measurements, including RT-qPCR and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).

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  FIGURE 2. Left ventricular morphologic alterations in response to trastuzumab (TZB) treatment assessed by echocardiography in both sexes. (A) Representative M-mode images, (B) septal wall thickness in diastole (SWTd), (C) septal wall thickness in systole (SWTs), (D) posterior wall thickness in diastole (PWTd), (E) posterior wall thickness in systole (PWTs), (F) left ventricular end-diastolic diameter (LVEDD), and (G) left ventricular end-systolic diameter (LVESD). Values are presented as mean ± S.E.M., *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 6-7, Two-Way ANOVA, Holm-Sidak post hoc test. CT: Control and TZB: trastuzumab. Each circle corresponds to one individual data point.

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  FIGURE 3. Left ventricular functional alterations in response to trastuzumab (TZB) treatment assessed by echocardiography in both sexes. (A) Left ventricular end-diastolic volume (LVEDV), (B) left ventricular end-systolic volume (LVESV), (C) stroke volume (SV), (D) ejection fraction (EF), (E) heart rate (HR), (F) cardiac output (CO), (G) representative Doppler and tissue Doppler images, (H) peak early diastolic mitral inflow velocity (E), (I) early diastolic mitral annular velocity (e’), and (J) E/e’. Values are presented as mean ± S.E.M., *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 6-7, Two-Way ANOVA, Holm-Sidak post hoc test. CT: Control and TZB: trastuzumab. Each circle corresponds to one individual data point.

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  FIGURE 4. The effects of TZB and sex on cardiomyocyte hypertrophy, interstitial fibrosis, and related gene expressions and metabolite levels. (A) Representative histologic sections stained with hematoxylin-eosin (HE, 100x) and picrosirius red and fast green (PSFG, 20x), (B) cardiomyocyte (CM) cross-sectional area, (C) beta-myosin heavy chain to alpha-myosin heavy chain ratio (Myh7/Myh6), (D) left ventricular (LV) collagen content, LV expression of (E) connective tissue growth factor (Ctgf), (F) transforming growth factor beta (Tgfb), and (G) LV concentration of p-cresyl sulfate. Values are presented as mean ± S.E.M., *p < 0.05, **p < 0.01, ***p < 0.001, n = 6-7, Two-Way ANOVA, Holm-Sidak post hoc test. CT: Control and TZB: trastuzumab. Each circle corresponds to one individual data point.

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  FIGURE 5. Changes in the left ventricular gene expressions and metabolite levels related to nitro-oxidative stress and inflammation in response to TZB treatment in both sexes. (A) Interleukin-1 (Il1b), (B) interleukin-6 (Il6), (C), inducible nitric oxide synthase (Nos2), (D) endothelial nitric oxide synthase (Nos3), (E) asymmetric dimethylarginine (ADMA), (F) symmetric dimethylarginine (SDMA), (G) cyclic guanosine monophosphate (cGMP), (H) reduced glutathione (GSH), (I) oxidized glutathione (GSSG) and (J) reduced glutathione to oxidized glutathione ratio (GSH/GSSG). Values are presented as mean ± S.E.M., *p < 0.05, **p < 0.01, ***p < 0.001, n = 5-7, One-Way ANOVA, Holm-Sidak post hoc test. CT: Control and TZB: trastuzumab. Each circle corresponds to one individual data point.

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  FIGURE 6. Changes in the left ventricular gene expression and metabolite concentrations related to glucose and fatty acid metabolism in response to TZB treatment in both sexes. (A) Glucose transporter-1 (Slc2a1), (B) glucose transporter 4 (Slc2a4), (C) glyceraldehyde-3-phosphate dehydrogenase (Gapdh), (D) pyruvate kinase (Pkm), (E) lactate dehydrogenase-A (Ldha), (F) lactate/pyruvate ratio, (G) carnitine O-palmitoyltransferase-1α (Cpt1α), and (H) peroxisomal acyl-coenzyme A oxidase-1 (Acox1). Values are presented as mean ± S.E.M., *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 5-7, Two-Way ANOVA, Holm-Sidak post hoc test. CT: Control and TZB: trastuzumab. Each circle corresponds to one individual data point.

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  FIGURE 7. Changes in the left ventricular levels of tryptophan and its metabolites in the kynurenine pathway in response to TZB treatment in both sexes. (A) Tryptophan (Trp), (B) kynurenine, (C) kynurenic acid, (D) anthranilic acid, (E) 3-hydroxykynurenine (3-OH kynurenine), (F) xanthurenic acid, (G) quinolinic acid, and (H) nicotinamide-adenine-dinucleotide (NAD+). Values are presented as mean ± S.E.M., *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 5-7, Two-Way ANOVA, Holm-Sidak post hoc test. CT: Control and TZB: trastuzumab. Each circle corresponds to one individual data point.