Cardiac miRNA expression during the development of chronic anthracycline-induced cardiomyopathy using an experimental rabbit model

Figures

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  FIGURE 1. Levels of cardiac troponin T (cTnT), a biomarker of cardiac damage, and examination of systolic function in rabbits after 5 and 10 weekly doses of DAU. (A) Plasma concentrations of cardiac troponin T as a marker of cardiac damage. (B) Echocardiography-assessed left ventricular systolic function (FS - fractional shortening). (C) Catheterization-assessed index of the left ventricular systolic function (dP/dtmax). The results are shown as median values, with boxes and whiskers representing the interquartile range and the 5th–95th percentile, respectively (A), or as the mean value ± SD (B,C). Significant differences are displayed as follows: p < 0.05 (*); p < 0.01 (**); p < 0.001 (***). 5 weeks corresponds to 5x DAU 3 mg/kg/week (cumulative dose ∼ 250 mg/m2); 10 weeks corresponds to 10x DAU 3 mg/kg/week (cumulative dose ∼ 500 mg/m2).

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  FIGURE 2. Histological analysis of LV myocardium. Comparison of the structure of the LV myocardium after five (B) and 10 weeks of daunorubicin (DAU) administration, (D) confirmation of two markedly different stages of chronic anthracycline-induced cardiomyopathy development, while normal structure of the myocardium was found in both age-matched controls (A,C). After 5 weeks of ANT administration (B), only initial stages of developing cardiomyopathy were seen, namely, a single cardiomyocyte with vacuolized cytoplasm and loss of myofibrils (*) could be found inside otherwise mostly intact myocardium. In contrast, 10 weeks ANT treatment (D) resulted in the development of distinct focal damage of the myocardium, manifested mainly by advanced stages of cell degeneration (necrosis)—i.e., gradually increasing loss of myofibrils and vacuolization of the cytoplasm (*), resulting in cell death. Necrotic cardiomyocytes were found to be gradually replaced by fibrotic scar tissue, more specifically, blue-stained collagen fibres (collagen type I) that form a fine mesh at first (x), but gradually progress to thick bundles (xx). Masson’s blue trichrome staining. Bar 50 μm.

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  FIGURE 3. Deregulation of miRNA expression after DAU treatment (A) miRNAs which showed deregulation after 5 weeks of DAU treatment. (B) miRNAs which showed deregulation after 10 weeks of DAU treatment. Results are given as the fold change relative to the control group. Data are presented as the mean value ± SD. The statistical significance of changes as compared with the control group is displayed as follows: p < 0.05 (*); p < 0.01 (**); p < 0.001 (***).

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  FIGURE 4. Deregulation of miRNA expression after DAU treatment (the scatter plot showing all the individual values) (A) miRNAs which showed deregulation after 5 weeks of DAU treatment. (B) miRNAs which showed deregulation after 10 weeks of DAU treatment. Results are given as the fold change relative to the control group. Data are presented as the mean value ± SD. The statistical significance of changes as compared with the control group is displayed as follows: p < 0.05 (*); p < 0.01 (**); p < 0.001 (***).

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  FIGURE 5. Correlations between the expression levels of miR-21-5p, miR-34a-5p, miR-155-5p and cTnT levels (a marker of cardiac damage) after 5 weeks of DAU treatment R: the coefficient of correlation. The statistical significance of changes as compared to the control group is displayed as follows: p < 0.05 (*); p < 0.01 (**); p < 0.001 (***).

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  FIGURE 6. Correlations between the expression of miRNAs and cTnT levels, and between the expression of miRNAs and functional parameters of LV after 10 weeks of DAU treatment cTnT - plasma concentrations of cardiac troponin T, a marker of cardiac damage. FS - fractional shortening - echocardiography-assessed left ventricular systolic function. dP/dtmax - catheterization-assessed index of left ventricular systolic function. R: the coefficient of correlation. The statistical significance of changes as compared to the controls is displayed as follows: p < 0.05 (*); p < 0.01 (**); p < 0.001 (***).

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  FIGURE 7. Venn diagram of the target genes of miRNAs with differential expression relative to the control in a model of chronic DAU-induced cardiomyopathy (A) The Venn diagram shows the number of genes shared between two selected miRNAs (B) Venn diagram of the target genes of miRNA-21-5p, miR-34a-5p and miR-155-5p, all of which were shown to be involved in the development of chronic DAU-induced cardiomyopathy in rabbits. The diagram shows the number of genes identified for each miRNA and the number of genes shared between the key miRNAs.

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  FIGURE 8. Analysis of p53 levels in the LV myocardium by Western blotting Myocardial p53 (molecular weight about 53 kDa) was analysed by WB, and total protein levels on the PVDF membrane were visualized by Stain-free imaging technology (Bio-Rad, Hercules, CA). Data are presented as the mean value ± SD. Significant changes are displayed as follows: p < 0.05 (*); p < 0.01 (**); p < 0.001 (***). 5 weeks corresponds to 5x DAU 3 mg/kg/week (cumulative dose ∼ 250 mg/m2); 10 weeks corresponds to 10x DAU 3 mg/kg/week (cumulative dose ∼ 500 mg/m2).

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  FIGURE 9. Plasma levels of miR-34a-5p, the correlations with expression in LV, and quantitative indices of toxic damage to the heart in rabbits after 10 weekly doses of DAU. miR-34a-5p expression in plasma vs. miR-34a-5p expression in the left ventricular myocardium. miR-34a-5p expression in plasma vs. plasma concentrations of cardiac troponin T, a marker of cardiac damage. miR-34a-5p expression in plasma vs. echocardiography-assessed left ventricular systolic function (FS - fractional shortening). miR-34a-5p expression in plasma vs. catheterization-assessed index of the left ventricular systolic function (dP/dtmax). The statistical significance of changes is displayed as follows: p < 0.05 (*); p < 0.01 (**); p < 0.001 (***).