NOTE: End date changed to 5/11/2021 per NSSC information (Ed., 8/12/20)

Reference: Yan, X., et al., Cardiovascular risks associated with low dose ionizing particle radiation. PLoS One, 2014. 9(10): p. e110269.

Cardiac function -- Functionally, in contrast to other forms of radiation, a single exposure to 5 ion Galactic Cosmic Ray Simulation (GCRSim) appears to have significant effects on clinically relevant parameters of cardiac function.

Tissue repository-- a large tissue repository covering a range of organs has been established. Tissues have been shared with other investigators as well as used in our proteomics studies.

Proteomics-- Bioinformatics analyses reveals both commonly as well as uniquely affected biological pathways across radiation types and doses. Notably, GCR exhibits highly perturbed cornification and keratinization pathways as well as mitochondrial pathways modulated by phosphorylation. In agreement with numerous other studies, perturbation of mitochondrial processes and cellular components was affected across most of the radiation conditions evaluated.

We are currently working on four papers based on our findings. The first paper is an evaluation of cardiac function focusing on GCR mice. This paper is in revision at iscience. The second paper is focusing on our GCR proteomics findings. This paper is still in draft format but relevant findings are noted in this report. The third paper is an evaluation of cardiac function looking across all doses of gamma, oxygen, and iron. The 4th paper focuses on proteomics findings across gamma, oxygen, and iron.

NOTE: End date changed to 5/11/2021 per NSSC information (Ed., 8/12/20)

Reference: Yan, X., et al., Cardiovascular risks associated with low dose ionizing particle radiation. PLoS One, 2014. 9(10): p. e110269.

Five month old male C57Bl6 mice (Jackson Laboratories) underwent transthoracic echocardiograms at Duke University Medical Center to establish baseline cardiac function. Subsequently, mice were subjected to single fully body irradiation at Brookhaven National Laboratories (BNL) under the following conditions: a) gamma (50-400cGy), b) 16O (15-150cGy/ 600 MeV/n), c) 56Fe (15-150cGy 1 GeV/n), d) Galactic Cosmic Radiation (GCR) (150cGy). All radiation groups included sham irradiated control animals. A subset of these animals underwent comprehensive cardiac structural and functional evaluation at 9-12 months post radiation including a) serial transthoracic echocardiograms capturing systolic and diastolic parameters, b) terminal pressure volume PV loop hemodynamic assessments, c) cardiac MRI, (d) blood pressure assessment, and e) cardiac and aortic tissue histology and immunohistochemistry. All echocardiograms were performed on awake animals, and were independently interpreted by two clinicians in a blinded fashion. Cardiac MRIs were performed using a 7.0 T Bruker Biospec small animal MRI scanner. Approximately 1000 mice were irradiated during five different BNL campaigns.

Non-invasive echocardiography and cardiac MRI did not demonstrate gross differences on systolic and diastolic function in GCR treated mice in comparison to controls. However, invasive pressure-volume loop hemodynamic analyses demonstrated that GCR treated mice, in comparison to controls, have a significant reduction in load independent (Preload Recruitable Stroke Work) and load dependent measures of cardiac systolic function (Cardiac Output and Stroke Volume). The differences in systolic function are partly explained by GCR induced increases in arterial elastance, suggesting that GCR exposure increases vascular resistance and afterload. The rise in afterload is associated with elastic fiber thickening, degeneration and disruption in aortic tissue, identified by histology that is present in GCR irradiated mice and absent in age-matched controls.

Quantitative mass spectrometry was performed on peptides (total proteome) as well as phosphopeptides (phosphoproteome) obtained from digested protein homogenates from the hearts of male C57B6 mice that had undergone radiation exposure at the NASA Radiation Science Laboratory at Brookhaven National Laboratories. These mice were subjected to single fully body irradiation at 6 months of age under the following conditions: a) 56Fe (50 cGy; n=3; obtained 3 month post radiation, b) 16O (50 cGy 600 MeV/n; n=3; obtained 7 months post radiation), c) 56Fe (50 cGy 1 GeV/n; n=3; obtained 7 months post radiation), and d) GCR (150 cGy, n=6; obtained 8 months post radiation). All radiation groups included sham irradiated control animals. Bioinformatics analysis of mass spectrometry data sets include: a) identification of significantly expressed proteins for each does and radiation types compared to corresponding control samples, (b) pathway enrichment analysis using functional annotation data obtained from Gene Ontology, KEGG (Kyoto Encyclopedia of Genes and Genomes), Reactome, and Molecular Signature Database (MSigDB), and c) identification of protein and pathway interaction modules that distinguish molecular signature of the various radiation types.

From the hearts of gamma, 56Fe, and 16O irradiated mice, 4,381 proteins and 5435 phosphopeptides were quantified. Similarly, from hearts from GCR irradiated mice, 4,650 proteins and 7,002 phosphopeptides (mapping to 303 phosphoproteins) were quantified. Pathways commonly influenced by all radiation types evaluated include mitochondrial gene expression and translation and fatty acid catabolic processes and oxidation. Pathways uniquely modulated by GCR involve cornification and keratinization.

Our data suggests that, in contrast to other forms of radiation, a single exposure to GCR increases afterload by injuring aortic architecture, and causes pronounced defects in load dependent cardiac systolic function. The cornification and keratinization processes observed in heart tissue from GCR irradiated animals may be contributing to the cardiovascular functional decline observed by hemodynamic and MRI analyses of these mice. Further characterization of the proteins involved in these processes may serve as targets of GCR countermeasures.

Reference: Yan, X., et al., Cardiovascular risks associated with low dose ionizing particle radiation. PLoS One, 2014. 9(10): p. e110269.

Mice were shipped to the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratories where they were subjected to single full body irradiation at 6 months of age under the following conditions: a) gamma (50cGy, 100cGy, 200cGy), b) 16O (15cGy, 25cGy, 50cGy/ 600 MeV/n), c) 56Fe (15cGy, 25cGy, 50cGy 1 GeV/n). There were a total of 20 sham irradiated control animals who traveled to NSRL. Evaluations include: a) serial transthoracic echocardiograms capturing all above parameters, b) terminal pressure volume loop hemodynamic assessments. Overall conclusions from the Fall 2016 experiment were that no significant changes in any echo measurement at any dose or rad type compared to control at any time point were observed. However across all groups including controls aging related decrements in function were observed as expected. Caveats were that this was essentially a pilot study (n=10 per group). There was also excessive early fighting among mice. The study was terminated at 9M post rad with the exception of a small group of animals which were held to 1 year post radiation when pressure volume (PV) loops were obtained. There was no significant difference in invasive hemodynamic measurements at 1 year post radiation. Again, small # of animals was examined in this manner.

A larger group of mice were evaluated in Spring 2018. Energies and doses are the following: Gamma (50cGy, 100cGy, 200cGy), 16Ox 600 MeV/n (15cGy, 25cGy, 50cGy), 56Fe 1GeV/n (15cGy, 25cGy, 50cGy). There was a larger number of animals per group in this experiment. Echocardiographic images have been obtained for each of the animals (if they were alive at the time of Echo acquisition) at the following time points: Pre-irradiation, 1 week post, 1M post, 2M post, 3M post, 4M post, 7M post, 9M post. Pressure volume (PV) loops were acquired on 30 animals 9 M post radiation. At the time of experiment termination, multiple organs were processed and biobanked for ‘omics analyses and future collaboration.

Overall, the echo reads for Spring 2017 are ongoing. We had a computer hard drive malfunction and lost a large amount of analyzed data. Therefore, no definite conclusions can be made yet with this larger cohort of animals. The PV loop analysis focused on 56Fe 50cGy, 1 year post IR with 10-15 per group. No statistically significant differences were observed in these measurements. However, a trend in several measurements was observed which suggests the possibility of diastolic dysfunction development. Additional PV loops have been done from animals in Fall 2017 experiment but have yet to be analyzed. Between Fall 2016 and Spring 2017 experiments we have developed an extensive biorepository of tissues for molecular analyses and for collaborations.

For the NSRL Fall 2017 experiment the same energies and doses from the two previous experiments were used. Echocardiographic images have been obtained for each of the animals at the following time points: Pre-irradiation, 1M post, 2M post, 3M post, 4M post, 7M post, 9M post, 16M post. PV loops were acquired on 97 animals 16M post radiation. All of these echos and PV loops are yet to be analyzed. At the time of animal sacrifice, multiple organs were processed and biobanked for ‘omics analyses and future collaboration. A subset of animals were AHA labeled for mass spec proteomics analyses. Brain regions from a subset of animals (59) were processed by Andrew Wryobek for funded tissue sharing FLAGSHIP grant in March 2019. NSRL Summer 2018 study examined GCR (galactic comic radiation) sim 5 ion (150 cGy). Echocardiographic images have been obtained for each of the animals at the following time points: Pre-irradiation, 1M post, 3M post, 7M post, 8M post. Echos are planned for 11 and 17M post irradiation in May 2019 and Nov 2019, respectively. PV loops are planned for November 2019. 12 animals have been labeled with AHA and submitted to Duke Proteomics Core for dynamic, abundance, and phosphoproteomics analyses. Echos are currently being read for this cohort. Data is still preliminary.

Outline of the study design for the Group 5 mice irradiated at NSRL Fall 2018. Energies and doses are the following: Gamma (300cGy, 400cGy), 16Ox (75cGy, 150cGy) 600 MeV/n, 56Fe (75cGy, 150cGy) 1 GeV/n, GCR sim 5 ion (150 cGy). Echocardiographic images have been obtained for each of the animals (if they were alive at the time of Echo acquisition) at the following time points: Pre-irradiation, 2M post, 3M post, 4M post. Echos are planned 7M, 9M, 12M, and 18M post irradiation. PV loops are planned for May 2019.

Yan, X., et al., Cardiovascular risks associated with low dose ionizing particle radiation. PLoS One, 2014. 9(10): p. e110269.

Male C57B6 mice are purchased from Jackson Laboratories. Mice are shipped to Duke University Medical Center where at 5 months of age they undergo transthoracic echocardiograms to establish baseline cardiac function (pre IR echo). Parameters evaluated included (a) M-mode (done in both long and short axis), (b) Septal and posterior wall width in diastole, (c) End diastolic dimension and end systolic dimension, (d) aortic valve velocity, and (e) aortic ejection time (all measured and averaged over 3 consecutive beats). Echocardiogram images acquired are also evaluated for measurement of diastolic dysfunction and strain.

Mice are then shipped to the NASA Space Radiation Laboratory (NSRL) at BNL where they are subjected to single full body irradiation at 6 months of age under the following conditions: a) gamma (50cGy, 100cGy, 200cGy), b) 16O (15cGy, 25cGy, 50cGy/ 600 MeV/n), c) 56Fe (15cGy, 25cGy, 50cGy 1 GeV/n). Control (sham irradiated) animals also travel back and forth from Duke to NRSL and back to Duke in order to experience identical stressors as the irradiated mice. Evaluations include: a) serial transthoracic echocardiograms capturing all above parameters. These are done at 1, 2, 3, 7, and 9 months post irradiation; b) terminal pressure volume loop hemodynamic assessments; and c) mass spectrometry based proteomics assessments (quantitative, dynamic, and post-translational modification proteomics) of the cardiac proteome.

All studies are ongoing. Therefore, the data set is still preliminary and conclusions presented in this report should be considered preliminary.

To date, we have not observed a significant difference in any parameter evaluated that allows us to make a statement that cardiovascular function is affected at any time point, radiation type, or dose post irradiation compared to control sham irradiated control mice.

Yan, X., et al., Cardiovascular risks associated with low dose ionizing particle radiation. PLoS One, 2014. 9(10): p. e110269.

A second trip to BNL will occur in April 2017. A larger group of mice will be administered the same radiation exposures. The exposures will be single fully body irradiation at 6 months of age under the following conditions: a) gamma (50cGy, 100cGy, 200cGy), b) 16O (15cGy, 25cGy, 50cGy/ 600 MeV/n), c) 56Fe (15cGy, 25cGy, 50cGy 1 GeV/n). All radiation groups include sham irradiated control animals. All animals will be monitored as described above. Evaluations include: a) serial transthoracic echocardiograms capturing all above parameters, b) terminal pressure volume loop hemodynamic assessments, and c) mass spectrometry based proteomics assessments (quantitative, dynamic, and post-translational modification proteomics) of the cardiac proteome.

Yan, X., et al., Cardiovascular risks associated with low dose ionizing particle radiation. PLoS One, 2014. 9(10): p. e110269.