Late Effects of Chronic Low Dose Rate Total Body Irradiation on the Heart Proteome of ApoE−/− Mice Resemble Premature Cardiac Ageing

Figure 1. Changes in the proteome of hearts after chronic irradiation. The volcano plot represents the distribution of all quantified proteins in cardiac tissue after chronic exposure to accumulated doses of 0.3 Gy (A) and 6 Gy (B). Deregulated proteins (±1.3-fold; p < 0.05) are highlighted in green (downregulated) and red (upregulated) (A,B). The full protein names are provided in Table S1. The Venn diagram illustrates the set of significantly deregulated proteins shared between both radiation doses (C). The most significant canonical pathways altered in proteome profiles (D). The analysis was generated by the toolset of g: Profiler. Graphical representation of different predicted upstream regulators (Supplementary Table S6) (E). Graphical representation of predicted cardiac toxicity (F). The analyses were performed by Ingenuity® Pathway Analysis and are displayed using a green colour gradient (representative of −log p-value), where a darker colour corresponds to a higher statistical significance. A positive z-score implies potential activation (orange) and a negative z-score indicates potential inhibition (blue) of the pathway. The white colour indicates pathways where no prediction can…

Figure 2. Analysis of the cardiac PPARα and metabolic enzymes after chronic irradiation. Graphical representation of the deregulated protein networks with their upstream transcriptional regulators PPARα after exposure to an accumulated dose of 0.3 Gy (A) and 6 Gy (B). The upregulated proteins are marked in red and the downregulated are marked in green. The nodes in blue represent transcription factors. A negative z-score indicates potential inhibition (blue) of the transcription factor. The proteins in red are upregulated and in green are downregulated. The analyses were generated using Ingenuity® Pathway Analysis. The full protein names are given in Tables S1–S3. Immunoblot analysis of total and p-PPARα (at Ser12), FASN, and ACOT1 in heart lysate samples is shown (C). The columns represent the average ratios of relative protein expression in control and irradiated samples after background correction and normalisation to VDAC expression (D) (one-way ANOVA followed by Tukey’s multiple comparisons test; * adjusted p value < 0.05; n = 3). The error bars represent the standard error of the mean (±SEM).

Figure 3. Analysis of the oxidative stress response. The activities of GLXR and GPX were compared in mice hearts (A,B). The ratio of GSSG/GSH was measured in hearts (C). Immunoblot analysis of protein s-glutathionylation is shown (one-way ANOVA followed by Tukey’s multiple comparisons test; * adjusted p value < 0.05; n = 5) (D). The columns represent the average ratios of relative protein s-glutathionylation in the control and irradiated samples after background correction and normalisation to VDAC expression (one-way ANOVA followed by Tukey’s multiple comparisons test; * adjusted p value < 0.05; n = 3) (E). The amount of lipid peroxidation as a marker of oxidative stress was measured in mice hearts using ELISA (one-way ANOVA and Tukey test for multiple comparisons; * adjusted p value < 0.05; n = 5) (F). The error bars represent the standard error of the mean (±SEM).

Figure 4. Analysis of the α-SMA and TGF-β signalling pathways. Immunoblot analysis of α-SMA is shown (A). The amount of the total protein was measured by Ponceau S staining. The columns represent the average ratios of relative protein expression in control and irradiated samples after background correction and normalisation to VDAC expression (one-way ANOVA followed by Tukey’s multiple comparisons test; * adjusted p value < 0.05; n = 3) (B). The expression levels of SMAD 2/3 and p-SMAD 2/3 were compared in mice hearts using ELISA (one-way ANOVA followed by Tukey’s multiple comparisons test; * adjusted p value < 0.05; n = 3) (C). The error bars represent the standard error of the mean (±SEM). Collagen deposition was evaluated in the RV using the HistoQuest 2.0.2.0249 software (TissueGnostics, Vienna, Austria) and expressed as a percentage of the total RV area (D). The red lines indicate the mean and the error bars represent the standard deviations (±SD).

Figure 5. Analysis of the sirtuin proteins activity and expression. Immunoblotting analysis of SIRT1 and SIRT3 was performed in the hearts (A,B). The columns represent the average ratios of relative protein expression in the control and irradiated samples after background correction and normalisation to VDAC expression (one-way ANOVA and Tukey test for multiple comparisons t-test; * adjusted p value < 0.05; n = 3) (B). The activity of total SIRTs was compared between irradiated and control samples (one-way ANOVA followed by Tukey’s multiple comparisons test; * adjusted p value < 0.05; n = 5) (C). The error bars represent the standard error of the mean (±SEM).

Figure 6. Analysis of the AMPK, Akt, and mTOR. The expression levels of AMPK and phosphorylated -AMPK (T172) (A), and Akt and phosphorylated -Akt (B) were compared in mice hearts using immunoblotting (one-way ANOVA followed by Tukey’s multiple comparisons test; * adjusted p value < 0.05; n = 3). The expression levels of AMPK and phosphorylated -AMPK (S487) (C), and mTOR and phosphorylated -mTOR (D) were compared in mice hearts using ELISA (one-way ANOVA followed by Tukey’s multiple comparisons test; * adjusted p value < 0.05; n = 5). The error bars represent the standard error of the mean (±SEM).

Figure 7. Senescence-associated mRNA expression profiling. The heat map shows hierarchical clustering (average linkage, Spearman ranked correlation) of 84 mRNA relevant to senescence (A). The green bars indicate downregulation and the red bars upregulation. The analysis was performed using the Heatmapper web server [27]. The volcano plot represents the distribution of all quantified mRNA in cardiac tissue after chronic exposure to accumulated doses of 0.3 and 6 Gy (B,C). Data were analysed using the ΔΔCT method with normalization of the raw data to the housekeeping genes. They shared significantly deregulated mRNA in the irradiated hearts (D). The error bars represent the standard error of the mean (±SEM) (t-test; * p < 0.05; n = 4).

Figure 8. A proposed model for the role of the main ageing-associated signalling pathways in cardiac injury after chronic irradiation. Created with BioRender.com.