Chronic Occupational Exposure to Ionizing Radiation Induces Alterations in the Structure and Metabolism of the Heart: A Proteomic Analysis of Human Formalin-Fixed Paraffin-Embedded (FFPE) Cardiac Tissue

Figure 1. The principal component analysis (PCA) based on all proteomics features. The PCA used all features resulting in PC1 and PC2 as follows: PC1 24.8% and PC2 12.6%. The distribution of proteomics features is illustrated based on the dose (mGy) (A), the age (regular font) and the years of exposure (italics) (B). The control samples are presented as blue spots and the irradiated samples with the corresponding individual dose in orange. The analysis was performed using the Proteome Discoverer 2.3 software (Thermo Scientific, Waltham, MA, USA). Detailed information of the sample donors and the exact doses are given in Table S1.

Figure 2. The features of protein identifications in formalin-fixed paraffin-embedded (FFPE) proteomics. The number of identified proteins was assigned based on the number of unique peptides (A), protein coverage (B), molecular weight (MW kDa) (C), and isoelectric point (pI) (D). The analysis was performed using the Proteome Discoverer 2.3 software (Thermo Scientific, Waltham, MA, USA). Detailed information of the proteomics features is given in Table S2.

Figure 3. Comparison of protein abundance between heart samples. The box-and-whisker plot shows the abundance of the not-normalized intensity values for each individual of irradiated (IR) and control (Ctrl) groups. The analysis was performed using the Proteome Discoverer 2.3 software (Thermo Scientific, Waltham, MA, USA). Detailed information of the sample donors and the exact doses are given in Table S4.

Figure 4. Graphical representation of quantitative proteomics data of human FFPE cardiac tissue after chronic irradiation. Proteins are ranked in a volcano plot according to the −log10 of their statistical p-value (y-axis) and log2 fold change (x-axis). The green and red points represent the significantly downregulated and upregulated proteins, respectively (A). The heat map shows hierarchical clustering (average linkage, Spearman ranked correlation) of significantly deregulated proteins in 5 controls (Ctrl) and 15 irradiated (IR) samples (B). The green bars indicate downregulation and the red bars upregulation. The analysis was performed using Heatmapper web server [34]. Detailed information of the proteomics features and individuals is given in Tables S1 and S2.

Figure 5. Pathway analysis of differentially regulated proteins from human FFPE heart tissues. The most significant canonical pathways altered by irradiation are shown. The analyses were generated using Ingenuity Pathway Analysis (IPA) (QIAGEN Inc., Hilden, Germany, https://www.qiagenbio-informatics.com/products/ingenuity-pathway-analysis) (Table S5). The bars indicate canonical pathways and the y-axis displays the −(log p) enrichment significance. The tall bars are more significant than the short ones (A). Graphical representation of the deregulated protein networks with their upstream transcriptional regulators peroxisome proliferator-activated receptor (PPAR) alpha (B) and transforming growth factor beta (TGFB) (C) are shown. The upregulated proteins are marked in red and the downregulated in green. The nodes in blue (inhibition) and orange (activation) represent transcription factors. The full protein names are given in Table 1.

Figure 6. The detection of fibrosis in the heart samples. The sections were stained with Mason’s Trichrome for visualization of connective tissue. The fibrotic area was shown in representative sections of the myocardium in control (A) and irradiated samples (B). Myocytes (m) are stained in red, fibrous connective tissue (f) in blue. The arrows indicate degenerating cardiomyocytes entrapped by fibrosis. The areas of fibrotic tissue within the myocardium were quantified and presented as percentage of region of interest (ROI) (C). The icons represent individual samples in control (Ctrl) and irradiated (IR) groups. The details are given in Table S6.

Figure 7. The comparison of proteomics data using FFPE or fresh-frozen cardiac material The enriched protein clusters of different affected pathways (A) and cardiac pathologies (B) were compared using data from the FFPE and fresh frozen cardiac tissue. The analyses were performed using IPA (QIAGEN Inc., Hilden, Germany, https://www.qiagenbio-informatics.com/products/ingenuity-pathway-analysis). The bars indicate affected pathways (A) or predicted diseases (B) and the y-axis displays the −(log p) enrichment significance. The tall bars are more significant than the short ones.