Activation of PPARα by Fenofibrate Attenuates the Effect of Local Heart High Dose Irradiation on the Mouse Cardiac Proteome

Figures

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  Figure 1. Changes in the cardiac proteome after treatment with fenofibrate. Volcano plot represents the distribution of all quantified proteins in cardiac tissue after treatment with fenofibrate. Deregulated proteins (+1.3-fold; p < 0.05) are highlighted in green (downregulated) and red (upregulated) (A). Protein–protein interactions were analyzed by the STRING software tool. Available online: (http://string-db.org) (accessed on 14 October 2021). indicating the most affected protein clusters (B). Prediction of activation of PPARα (orange colour) based on deregulated proteins. The upregulated proteins are marked in red (C). The analyses were generated using Ingenuity® Pathway Analysis. Available online: (https://www.qiagenbio-informatics.com/products/ingenuity-pathway-analysis) (accessed on 14 October 2021). The full protein names are provided in Tables S1 and S2.

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  Figure 2. Changes in the proteome of irradiated heart with and without fenofibrate treatment. Volcano plots represent the distribution of all quantified proteins in irradiated cardiac tissue without (A) or with (B) fenofibrate. Deregulated proteins (±1.3-fold; p < 0.05) are highlighted in green (downregulated) and red (upregulated). The full protein names are provided in Table S1. A Venn diagram illustrating the shared deregulated proteins between the two experimental groups (C). The most significant canonical pathways altered in both proteome profiles (D). The analyses were generated by Ingenuity® Pathway Analysis. Available online: https://www.qiagenbio-informatics.com/products/ingenuity-pathway-analysis) (accessed on 14 October 2021). Bars indicate canonical pathways with the enrichment significance shown on the horizontal axis (-log p-value). Longer bars indicate a higher significance of the pathway.

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  Figure 3. Analysis of the cardiac PPARα and serum FFA. Graphical representations of the deregulated protein networks with their upstream transcriptional regulators PPARα in the irradiated heart after treatment with placebo (A) and fenofibrate (B). The upregulated proteins are marked in red and the downregulated are marked in green. The nodes in blue (inhibition) and orange (activation) represent transcription factors. The analyses were generated using Ingenuity® Pathway Analysis. Available online: https://www.qiagenbio-informatics.com/products/ingenuity-pathway-analysis) (accessed on 14 October 2021). The full protein names are given in Table S1. Immunoblot analysis of PPARα was performed in mouse hearts (C). The amount of 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 (±SEM) (t-test; * p < 0.05; n = 3) (D). The amount of the FFA was measured in 100 µg of serum using ELISA (+SEM) (t-test; * p < 0.05; n = 5) (E).

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  Figure 4. The effect of fenofibrate on mitochondria of irradiated heart. The cluster of deregulated mitochondrial proteins in the irradiated heart without (A) and with (B) fenofibrate treatment. Analysed by STRING software. Available online: (http://string-db.org) (accessed on 14 October 2021). The activity of complex I (C) and the level of cardiac ATP (D) were measured and expressed as a percentage compared to sham-irradiated, placebo-treated mice. The error bars represent standard error of the mean (±SEM) (t-test; * p < 0.05; n = 5). Immunoblot analysis of PGC1 and SIRT3 (E). The amount of the total protein was measured by Ponceau S staining for an accurate comparison between the groups. The columns represent the average ratios of relative protein expression in control and irradiated samples (F). The error bars represent standard error of the mean (±SEM) (t-test; * p < 0.05; n = 3).

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  Figure 5. Analysis of the oxidative stress response. Immunoblot analysis of NRF2 and KEAP1 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 (t-test; * p < 0.05; n = 3) (B). The activity of SOD2 was compared in (C). The amount of lipid peroxidation as a marker of oxidative stress was measured in mice hearts (D). The error bars represent standard error of the mean (±SEM) (t-test; * p < 0.05; n = 3).

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  Figure 6. The analysis of serum cytokines. Graphical representations of the potential upstream transcriptional regulators predicted to be affected in the irradiated heart after treatment with placebo and fenofibrate (A). The predictions were generated based on the deregulated proteins using Ingenuity® Pathway Analysis. Available online: (https://www.qiagenbio-informatics.com/products/ingenuity-pathway-analysis) (accessed on 14 October 2021). The scores of activities are displayed using an orange colour gradient where a darker colour corresponds to a high score (high statistical significance) (A). The level of cytokines was measured in 100 µg of serum using ELISA. The error bars represent standard error of the mean (±SEM) (t-test; * p < 0.05; n = 5) (B).

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  Figure 7. Analysis of the ECM markers and TGF-β signalling pathways. Immunoblot analysis of ECM markers 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 (t-test; * p < 0.05; n = 3) (B). The expression levels of SMAD 2/3 and p-SMAD 2/3 were compared in mice hearts using ELISA (C). The expression levels of ERK, p38, JNK and their phosphorylated forms were compared in mice hearts using ELISA (D,E). The error bars represent standard error of the mean (±SEM) (t-test; * p < 0.05; n = 3).

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  Figure 8. Analysis of the cardiac eNOS and serum NO. Immunoblot analysis of eNOS was performed in mouse hearts (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 (±SEM) (t-test; * p < 0.05; n = 3) (B). The activity of eNOS was compared in mice hearts (C). The levels of NO were compared in mice serum (D). The error bars represent standard error of the mean (±SEM) (t-test; * p < 0.05; n = 3).