A dose-dependent perturbation in cardiac energy metabolism is linked to radiation-induced ischemic heart disease in Mayak nuclear workers - PubMed (original) (raw)

A dose-dependent perturbation in cardiac energy metabolism is linked to radiation-induced ischemic heart disease in Mayak nuclear workers

Omid Azimzadeh et al. Oncotarget. 2017.

Abstract

Epidemiological studies show a significant increase in ischemic heart disease (IHD) incidence associated with total external gamma-ray dose among Mayak plutonium enrichment plant workers. Our previous studies using mouse models suggest that persistent alteration of heart metabolism due to the inhibition of peroxisome proliferator-activated receptor (PPAR) alpha accompanies cardiac damage after high doses of ionising radiation. The aim of the present study was to elucidate the mechanism of radiation-induced IHD in humans. The cardiac proteome response to irradiation was analysed in Mayak workers who were exposed only to external doses of gamma rays. All participants were diagnosed during their lifetime with IHD that also was the cause of death. Label-free quantitative proteomics analysis was performed on tissue samples from the cardiac left ventricles of individuals stratified into four radiation dose groups (0 Gy, < 100 mGy, 100-500 mGy, and > 500 mGy). The groups could be separated using principal component analysis based on all proteomics features. Proteome profiling showed a dose-dependent increase in the number of downregulated mitochondrial and structural proteins. Both proteomics and immunoblotting showed decreased expression of several oxidative stress responsive proteins in the irradiated hearts. The phosphorylation of transcription factor PPAR alpha was increased in a dose-dependent manner, which is indicative of a reduction in transcriptional activity with increased radiation dose. These data suggest that chronic external radiation enhances the risk for IHD by inhibiting PPAR alpha and altering the expression of mitochondrial, structural, and antioxidant components of the heart.

Keywords: PPAR alpha; heart disease; ionising radiation; mitochondrial dysfunction; proteomics.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1

Figure 1. Principal component analysis (PCA) based on all proteomic features in the left ventricle of sample donors in different dose groups

The PCA used features with charges +2 to +7 resulting in PC1 and PC2 as follows: PC1 23.65% and PC2 8.36%. The control samples with the corresponding donor number are represented as blue spots, the samples exposed to < 100 mGy in purple, the samples exposed to 100–500 mGy in orange and the samples exposed to > 500 mGy in green. Samples number 26 and 38 were run as 2 technical replicates and are indicated as 26, 26B and 38, 38B. Detailed information of the sample donors and the exact doses are given in Supplementary Table S10. The analysis was performed using the Progenesis QI software (

http://www.nonlinear.com

).

Figure 2

Figure 2. Pathway and network analysis of significantly differentially expressed mitochondrial proteins

A dose-dependent alteration is observed in the pathways involved in the energy production. The pathway scores are displayed using a purple colour gradient, where darker purple corresponds to higher scores (increased statistical significance). The score is the negative log of the _p_-value derived from the Fisher′s Exact test. By default, the rows (pathways) with the highest total score across the set of observations are sorted to the top (A). Heat map for the expression values of differentially expressed OXPHOS proteins between dose groups is displayed using a green colour gradient for downregulated proteins, where dark green corresponds to large downregulation. The numbers shows how many proteins were deregulated in each subunit (B) (

http://www.INGENUITY.com

). Protein-protein interaction analysis of the significantly differentially expressed proteins showing the networks of deregulated mitochondrial proteins in the dose groups < 100 mGy (**C**), 100–500 mGy (**D**) and > 500 mGy (E) (

http://string-db.org

).

Figure 3

Figure 3. Immunoblot validation of the proteomics data

The heart protein lysates from each individual sample were pooled within the dose groups and tested using anti-Troponin T (TNNT2), anti-Tropomyosin 2 (TPM2), anti- Myosin light chain (MYL2), anti-Mn superoxide dismutase (SOD2), and anti-Peroxiredoxin 5 (PRDX5) (A).The columns represent the average ratios of relative protein expression in control and irradiated samples. The amount of the total protein was measured by Ponceau S staining for accurate comparison between the groups. The error bars represent standard error of the mean (+ SEM) (B) (_t_-test; *p < 0.05, **p < 0.01; n = 3).

Figure 4

Figure 4. Analysis of the protein carbonyl levels and miR-21 and miR-146a in different dose groups

The total amount of carbonylated protein was measured in individual samples from each dose group. The samples in the control group were run in two technical replicates. Significantly increased level of carbonylated proteins was shown in the dose group > 500 mGy (A). Analysis of miR-21 and miR-146a from samples of all dose groups showed significant upregulation of both miRNAs in the dose group > 500 mGy (B) The error bars represent standard error of the mean (+ SEM) (_t_-test; * p < 0.05; **p < 0.01; *** p < 0.001).

Figure 5

Figure 5. Analysis of the activation status of PPAR alpha

IPA prediction of inactivation of PPAR alpha based on the deregulated proteins from proteomics analysis in the dose groups < 100 mGy (**A**), 100–500 mGy (**B**) and > 500 mGy (C). The upregulated proteins are marked in red and the down-regulated in green. The blue colour of the node (PPAR alpha) indicates inactivation. The list of proteins is available in Supplementary Tables S2–S4. Immunoblot analysis of total and phospho-PPAR alpha (Ser12) in pooled samples is shown (D). The columns represent the average ratios of relative protein expression in control and irradiated samples. Immunoblot analysis of total PPAR alpha (E) and phospho-PPAR alpha (F) in individual samples from each dose group is shown. The icons represent individual samples in different dose groups. The samples in the control group were run in two technical replicates. The amount of the total protein was confirmed by Ponceau S staining for accurate comparison between the groups (_t_-test; *p < 0.05).

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