Molecular Changes In Cardiac Tissue As A New Marker To Predict Cardiac Dysfunction Induced By Radiotherapy

Figures

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  Figure 1. CT simulation and dose distribution planning preceded thoracic radiation therapy in Wistar rats. (A) After echocardiography, radiopaque fiducial markers were placed over the shaved skin of the animal (left panel) to correlate the CT images to the external anatomical references used during animal setup prior to RT (right panel). (B) Coronal (upper panel) and axial view (lower panel) from CT-based radiation therapy planning dose distribution. Isodose curves are shown in a range of 100% (dark blue), to 10% (dark pink), being the most internal ones: 90% (yellow), 85% (light blue) and 80% (light pink), for the prescribed doses of 0.04, 0.3, and 1.2 Gy. Target heart structures (red) and organs at risk such as the lungs (blue) and medulla (purple) are also shown. (C) Twelve to fourteen week old Wistar rats were cumulatively irradiated with 0.92, 6.9, or 27.6 Gy, administered in a fractionated scheme of 23 daily doses of 0.04, 0.3, or 1.2 Gy, respectively.

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  Figure 2. GLS is significantly decreased by the cumulative doses of 0.92, 6.9, and 27.6 Gy. Rat hearts were sham-irradiated or irradiated with 0.04, 0.3, and 1.2 Gy for 23 days. Rats, neither irradiated nor anesthetized, were used as controls. Echocardiography was performed at 9 and 12 months after the end of IR exposure. Measurements of the GLS show a significant decrease only at 12 months, with the daily doses of 0.04, 0.3, and 1.2 Gy. Individual data and mean ± SD (in red) are shown from n = 9 rats per group. Between-group changes were assessed by two-way repeated measurements ANOVA followed by a Bonferroni post-hoc test with a between-subject factor of irradiation, relative to the 0.0 Gy experimental group; significant P-values are displayed.

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  Figure 3. Cardiac microvasculature density is significantly decreased by the cumulative dose of 27.6 Gy. (A) Microvascular density in the cardiac apex of rats, sham-irradiated (n = 8) or irradiated with 23 daily fractions of 0.04 (n = 5), 0.3 (n = 5), or 1.2 Gy (n = 7), was evaluated by SEM, 12 months after IR exposure. Quantitative analysis of microvascular density is shown as a percentage of average capillary coverage relative to several successive ×100x magnification fields, demonstrating a significant decrease by the 1.2 Gy daily dose. One-way ANOVA combined with Bonferroni correction was used for statistical analysis. (B, C) Seven months after the end of IR exposure, (B) cardiac ECs were analyzed by flow cytometry based on cell percentage, in relation to total live single cells. (C) Cell senescence was assessed as the percentage of C12FDG positive ECs. (B, C) No significant differences were observed between groups n = 10 (0.0 Gy), n = 8 (0.04 Gy), n = 9 (0.3 Gy), and n = 9 (1.2 Gy). Kruskal–Wallis test with Dunn’s correction was used for statistical analysis. (A–C) Individual data and mean ± SD (in red) are shown. Significant P-values are displayed. EC, endothelial cell.

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  Figure 4. Cumulative doses of 6.9 and 27.6 Gy upregulate the mRNA expression of pro-inflammatory, pro-fibrotic, and oxidative stress markers in cardiac tissue, prior to myocardial strain dysfunction. The mRNA expression of (A) Il6, (B) Vcam1, (C) Tnc, (D) Tgfb2, (E) Fgf2, and (F) Sod2 from the cardiac tissue of rats, sham-irradiated or irradiated with 0.04, 0.3, or 1.2 Gy for 23 daily fractions, was quantified by qRT-PCR. Cycle threshold values were normalized to 18S to obtain relative gene expression values. Data (mean ± SD) represents the Log2 of fold change in gene expression for each animal, in triplicate measurements relative to the fold change average of the sham-irradiated (0.0 Gy) group; n = 6–9 rats per group. Results show (A) an increasing trend of Il6 expression, exclusively in response to 1.2 Gy. (B–F) The levels of Vcam1, Tnc, Tgfb2, Fgf2, and Sod2 present a significant augmentation or increasing trend in response to the daily doses of 0.3 and 1.2 Gy. A Kruskal–Wallis test with Dunn’s correction was used for statistical analysis; significant P-values are displayed. Il6, Interleukin6; Vcam1, Vascular cell adhesion molecule1; Sod2, Superoxide dismutase2; Fgf2, Fibroblast growth…

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  Figure 5. Proteomic analysis reveals a significant change in the expression of cTnT, Calsequestrin2, and Myosin6, in response to the cumulative doses of 0.92, 6.9, and 27.6 Gy. Rat hearts were sham-irradiated (n = 10) or irradiated with 0.04 (n = 8), 0.3 (n = 9), or 1.2 Gy (n = 9) for 23 daily fractions and analyzed by mass spectrometry at 7 months. (A) Venn diagram indicates the non- and overlapping number of proteins. (B) Heatmap represents the significantly up (green) and downregulated (red) proteins (adjusted P-value <0.05) from each group vs. sham-irradiated. (C) Volcano plots based on Log2 fold change against −Log10 (P-value) show the significantly up (upper right corner) and downregulated (upper left corner) proteins in each group vs. 0.0 Gy. (D) Quantitative Log2(iBAQ + 1) expression values show the significant upregulation of cTnT and Calsequestrin2 by 1.2 Gy, and downregulation of Myosin6 by 0.3 and 1.2 Gy. Individual data and mean ± SD (in red) are shown. A Kruskal–Wallis test with Dunn’s correction was used; significant P-values are displayed. (E) Heatmap of KEGG pathway enrichment analysis, for significantly dysregulated proteins, revealed “Tight junctions,” “Cardiac muscle…

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  Figure 6. cTnT, Calsequestrin2, and Myosin6 expression levels are modulated in the cardiac tissue by the cumulative doses of 6.9 and/or 27.6 Gy. The cardiac tissue samples used in proteomics were analyzed by western-blot for (A) cTnT, (B) Calsequestrin2, and (C) Myosin6. Protein expression levels are shown as the relative densitometric intensity of the labeled proteins in relation to total protein in each lane. A Kruskal–Wallis test with Dunn’s correction was used for statistical analysis. Quantitative analysis shows (A) an increasing trend in the expression level of cTnT and (B) a significant augmentation of Calsequestrin2 in response to the daily dose of 1.2 Gy. (C) Myosin6 expression levels decrease by both daily doses of 0.3 and 1.2 Gy. (D) Plasma levels of circulating cTnT were assessed at 7 (left) and 12 months (right) after the end of IR exposure. Although no augmentation was detected at 7 months, an increasing trend was found for circulating cTnT in response to 0.3 and 1.2 Gy at 12 months. (A–D) Individual data and mean ± SD (in red) are shown for n = 3–9. cTnT, cardiac muscle troponin T.

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  Figure 7. GLS is decreased in a dose-dependent manner 18 months after the end of IR exposure. Rat hearts were sham-irradiated (n = 4) or irradiated with 0.04 Gy (n = 3), 0.3 Gy (n = 5), and 1.2 Gy (n = 4) for 23 days. Rats neither irradiated nor anesthetized were used as controls (n = 5). Then, echocardiography was performed 18 months after the end of IR exposure and the GLS was measured. Observations show a decrease in a dose-dependent manner, with a significant difference associated with the 1.2 Gy daily dose. Individual data and mean ± SD (in red) are shown. A Kruskal–Wallis test with Dunn’s correction was used for statistical analysis; significant P-values are displayed.

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  Figure 8. Molecular changes in cardiac tissue precede microvasculature density decrease and GLS impairment, affecting its severity in a dose-dependent manner. The illustrative figure summarizes the main conclusions of this study. Rat hearts were exposed to daily doses of 0.04, 0.3, and 1.2 Gy, achieving cumulative doses of 0.92, 6.9, and 27.6 Gy, respectively to evaluate the contribution of radiotherapy, per se, in late cardiotoxicity. At 7 months after the end of IR exposure, the cardiac tissue was modulated as demonstrated by the number/expression of several genes and proteins related to inflammation, fibrosis, and cardiac muscle contraction, differently expressed according to the cumulative dose. Importantly, the molecular changes in cardiac tissue precede microvascular density decrease and myocardial deformation, which are not observed until 12 months after the end of IR exposure. Later, at 18 months, the severity of GLS impairment is increased, demonstrating a myocardial deformation in a dose-dependent manner.