Radiation–Induced Signaling Results in Mitochondrial Impairment in Mouse Heart at 4 Weeks after Exposure to X-Rays

BACKGROUND: Radiation therapy treatment of breast cancer, Hodgkin's disease or childhood cancers expose the heart to high local radiation doses, causing an increased risk of cardiovascular disease in the survivors decades after the treatment. The mechanisms that underlie the radiation damage remain poorly understood so far. Previous data show that impairment of mitochondrial oxidative metabolism is directly linked to the development of cardiovascular disease. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the radiation-induced in vivo effects on cardiac mitochondrial proteome and function were investigated. C57BL/6N mice were exposed to local irradiation of the heart with doses of 0.2 Gy or 2 Gy (X-ray, 200 kV) at the age of eight weeks, the control mice were sham-irradiated. After four weeks the cardiac mitochondria were isolated and tested for proteomic and functional alterations. Two complementary proteomics approaches using both peptide and protein quantification strategies showed radiation-induced deregulation of 25 proteins in total. Three main biological categories were affected: the oxidative phophorylation, the pyruvate metabolism, and the cytoskeletal structure. The mitochondria exposed to high-dose irradiation showed functional impairment reflected as partial deactivation of Complex I (32%) and Complex III (11%), decreased succinate-driven respiratory capacity (13%), increased level of reactive oxygen species and enhanced oxidation of mitochondrial proteins. The changes in the pyruvate metabolism and structural proteins were seen with both low and high radiation doses. CONCLUSION/SIGNIFICANCE: This is the first study showing the biological alterations in the murine heart mitochondria several weeks after the exposure to low- and high-dose of ionizing radiation. Our results show that doses, equivalent to a single dose in radiotherapy, cause long-lasting changes in mitochondrial oxidative metabolism and mitochondria-associated cytoskeleton. This prompts us to propose that these first pathological changes lead to an increased risk of cardiovascular disease after radiation exposure

Hyperacetylation of Cardiac Mitochondrial Proteins Is Associated with Metabolic Impairment and Sirtuin Downregulation after Chronic Total Body Irradiation of ApoE -/- Mice

Chronic exposure to low-dose ionizing radiation is associated with an increased risk of cardiovascular disease. Alteration in energy metabolism has been suggested to contribute to radiation-induced heart pathology, mitochondrial dysfunction being a hallmark of this disease. The goal of this study was to investigate the regulatory role of acetylation in heart mitochondria in the long-term response to chronic radiation. ApoE-deficient C57Bl/6J mice were exposed to low-dose-rate (20 mGy/day) gamma radiation for 300 days, resulting in a cumulative total body dose of 6.0 Gy. Heart mitochondria were isolated and analyzed using quantitative proteomics. Radiation-induced proteome and acetylome alterations were further validated using immunoblotting, enzyme activity assays, and ELISA. In total, 71 proteins showed peptides with a changed acetylation status following irradiation. The great majority (94%) of the hyperacetylated proteins were involved in the TCA cycle, fatty acid oxidation, oxidative stress response and sirtuin pathway. The elevated acetylation patterns coincided with reduced activity of mitochondrial sirtuins, increased the level of Acetyl-CoA, and were accompanied by inactivation of major cardiac metabolic regulators PGC-1 alpha and PPAR alpha. These observations suggest that the changes in mitochondrial acetylation after irradiation is associated with impairment of heart metabolism. We propose a novel mechanism involved in the development of late cardiac damage following chronic irradiation

A mechanistic model for atherosclerosis and its application to the cohort of Mayak workers.

We propose a stochastic model for use in epidemiological analysis, describing the age-dependent development of atherosclerosis with adequate simplification. The model features the uptake of monocytes into the arterial wall, their proliferation and transition into foam cells. The number of foam cells is assumed to determine the health risk for clinically relevant events such as stroke. In a simulation study, the model was checked against the age-dependent prevalence of atherosclerotic lesions. Next, the model was applied to incidence of atherosclerotic stroke in the cohort of male workers from the Mayak nuclear facility in the Southern Urals. It describes the data as well as standard epidemiological models. Based on goodness-of-fit criteria the risk factors smoking, hypertension and radiation exposure were tested for their effect on disease development. Hypertension was identified to affect disease progression mainly in the late stage of atherosclerosis. Fitting mechanistic models to incidence data allows to integrate biological evidence on disease progression into epidemiological studies. The mechanistic approach adds to an understanding of pathogenic processes, whereas standard epidemiological methods mainly explore the statistical association between risk factors and disease outcome. Due to a more comprehensive scientific foundation, risk estimates from mechanistic models can be deemed more reliable. To the best of our knowledge, such models are applied to epidemiological data on cardiovascular diseases for the first time

Ingenuity pathway analysis of proteins deregulated 12 h after irradiation in the absence of miR-525-3p.

<p>(A) IPA of direct and indirect miR-525-3p target proteins. The most significant network ”Cell Death and Survival, Free Radical Scavenging, Cancer” (score 26) is shown. (B) IPA of direct miR-525-3p target proteins. The most significant network “Cell Death and Survival, Organismal Injury and Abnormalities, Respiratory Disease” (score 14) is shown. Molecules in grey represent miR-525-3p target proteins. direct interaction, ------ indirect interaction.</p

Radiation response after depletion of ARRB1, TXN1, hnRNP

<p><b>K and HSPA9</b>. (<b>A</b>) siRNA-mediated knockdown of ARRB1 and TXN1. EA.hy926 cells were transfected with siARRB1 or with an unspecific control (siControl). ARRB1 and TXN1 were quantified 24 h after transfection by western blot. (B) siRNA-mediated knockdown of hnRNP K and HSPA9. (C) Proliferation activity after IR in ARRB1 and TXN1 knockdown cells. Depletion of ARRB1 and TXN1 results in increased radiation resistance after irradiation up to 7.5 Gy. Endothelial EAhy926 cells were transfected with siARRB1, siTXN1 or scrambled control RNA (siControl), reseeded and the cell proliferation assay was performed 5d after ionizing radiation. The mean ± s.e.m. of two independent experiments is shown. (D) Proliferation activity after IR in hnRNP K and HSPA9 knockdown cells. Depletion of hnRNP K did not change the proliferative activity and depletion of HSPA9 led to decreased proliferative activity. The mean ± s.e.m. of two independent experiments is shown. (E) Apoptosis induction in knockdown cells after IR. Apoptosis induction was quantified by sub-G1 analysis 48 h after IR. Depletion of ARRB1 and TXN1 led to decreased apoptosis, while depletion of HSPA9 increased apoptosis. * indicate significant difference to the respective siControl transfected cells (** p < 0.05, * p < 0.01). The mean ± s.e.m. of three independent experiments is shown. </p

Immunoblot analysis of control and anti-miR-525-3p transfected EA.hy whole cell extracts (12 h after irradiation).

<p>(A) Representative images of the blots. (B) Fold differences between irradiated and non-irradiated samples normalized to PCNA. * indicate significant differences between irradiated and non-irradiated samples (p< 0.05). The mean ± s.e.m. of three independent experiments is shown.</p

Expression of miR-525-3p is up-regulated after ionizing radiation and modulation of the miR-525-3p expression effects cell survival.

<p>(A) <i>left</i> miR-525-3p expression was examined 0, 2, 4, 8, 12, 24 and 48 h after 2.5 Gy IR in the endothelial cell line EA.hy926 by quantitative real time PCR. <i>right</i> modulation of miR-525-3p results in a change in endothelial cell proliferation after IR. Endothelial cells were transfected with pre-miR-525-3p, miR-525-3p-inhibitor or scrambled control RNA, reseeded and the cell proliferation assay was performed 5d after IR. (B) HeLa cells, (C) RPE cells, (D) U2-OS cells. The mean ± s.e.m. of three independent experiments is shown. * mark significant differences between samples harvested at the 0 h timepoint compared with the indicated time point (* p<0.05, ** p< 0.01).</p