Analysis of lncRNA-miRNA-mRNA Expression Pattern in Heart Tissue After Total Body Irradiation in a Mouse Model

Background: Radiation therapy is integral to effective thoracic cancer treatments, but its application is limited by sensitivity of critical organs such as the heart. The impacts of acute radiation-induced damage and its chronic effects on normal heart cells are highly relevant in radiotherapy with increasing lifespans of patients. Biomarkers for normal tissue damage after radiation exposure, whether accidental or therapeutic, are being studied as indicators of both acute and delayed effects. Recent research has highlighted the potential importance of RNAs, including messenger RNAs (mRNAs), microRNAs (miRNAs), and long non-coding RNAs (lncRNAs) as biomarkers to assess radiation damage. Understanding changes in mRNA and non-coding RNA expression will elucidate biological pathway changes after radiation. Methods: To identify signicant expression changes in mRNAs, lncRNAs, and miRNAs, we performed whole transcriptome microarray analysis of mouse heart tissue at 48h after whole-body irradiation with 1, 2, 4, 8, and 12 Gray (Gy). We also validated changes in specic lncRNAs through RT-qPCR. Ingenuity Pathway Analysis (IPA) was used to identify pathways associated with gene expression changes. Results: We observed sustained increases in lncRNAs and mRNAs, across all doses of radiation. Alas2, Aplnr, and Cxc3r1 were the most signicantly downregulated mRNAs across all doses. Among the signicantly upregulated mRNAs were cell-cycle arrest biomarkers Gdf15, Cdkn1a, and Ckap2. Additionally, IPA identied signicant changes in gene expression relevant to senescence, apoptosis, hemoglobin synthesis, inammation, and metabolism. LncRNAs Abhd11os, Pvt1, Trp53cor1, and Dino showed increased expression with increasing doses of radiation. We did not observe any miRNAs with sustained up- or downregulation across all doses, but miR-149-3p, miR-6538, miR-8101, miR-7118-5p, miR-211-3p, and miR-3960 were signicantly upregulated after 12 Gy. Conclusions: Radiation-induced RNA expression changes may be predictive of normal tissue toxicities and may indicate targetable pathways for radiation countermeasure development and improved radiotherapy treatment plans.


Introduction
Radiation therapy (RT) is a mainstay of cancer treatment as it reduces recurrence, improves survival, and enhances the e cacy of other treatments. Ionizing radiation (IR) exposure, whether from RT, diagnostic imaging, or accidental sources (e.g. a nuclear disaster), can cause a multitude of side effects, including secondary cancers and other iatrogenic diseases [1][2][3]. Accidental or radiotherapeutic normal tissue injury can cause many transient or permanent alterations in both cellular and extracellular components within the irradiated eld [4]. These are particularly harmful to critical organs such as the heart. Retrospective studies of atomic bomb survivors found evidence that excess relative risk of death due to heart disease increased by 14% per Gray (Gy) of radiation absorbed, linking radiation exposure with longterm cardiac effects [5][6][7]. Similarly, liquidators exposed to radiation in the Chernobyl exclusion zone displayed a statistically signi cant excess relative risk for developing cardiovascular disease [8].
pathway corepressor 1 (Trp53cor1) in a whole-body irradiation mouse model [45]. The results of these studies informed the approach we used in the current investigation.
With cancer and cardiovascular disease as the two leading causes of mortality in the world, understanding the effects of RIHD from RT or accidental exposure will be critical to minimizing health consequences [46]. In this study, we utilized whole transcriptome analysis on mouse heart tissues 48 h after whole-body doses of 1, 2, 4, 8, or 12 Gy. Understanding biological pathways that lead to RIHD development will allow for the identi cation of treatments to improve quality of life for individuals exposed to radiation, either therapeutically or accidentally, and provide diagnostic and/or prognostic markers of damage.

Total body irradiation of mice and sample collection
Six-to eight-week old female C57BL/6J mice were given total-body irradiation (TBI) with x-rays using the Small Animal Radiation Research Platform (SARRP Xstrahl Ltd.). Mice were placed in plastic containers and exposed to a single surface dose of 1, 2, 4, 8, or 12 Gy at a dose rate of 1.05 Gy/min. Control mice (0 Gy) were placed in the same type of plastic container and sham irradiated. Three animals per dose were included in the study. Hearts of irradiated and control animals were harvested 48 hours after TBI.
Organs were snap frozen in liquid nitrogen and stored at -80 °C until processed for RNA isolation. All animal experiments were performed at the Department of Pathology at New York University (NYU) Langone Medical Center under an approved IACUC protocol as part of a collaborative study.

RNA isolation
Samples were bathed in liquid nitrogen and pulverized into a ne powder using a mortar and pestle. Approximately 100 µg of powdered sample was lysed with 700 µl of QIAzol lysis buffer (Cat # 79306, QIAGEN) and homogenized by passing the solution through QIAshredder spin columns (Cat # 79654, QIAGEN). RNA isolation was performed using standard miRNeasy mini kit (Cat # 217004, QIAGEN) according to the manufacturer's protocol. Quality and quantity of the RNA samples were assessed using a DeNovix DS-11 nanodrop spectrophotometer (DeNovix, DE, US) and Agilent Bioanalyzer with the RNA6000 Nano Lab Chip (Agilent Technologies, Santa Clara, CA).

Microarray analysis
Microarray analysis was performed for sham animals (0 Gy) and 1 Gy, 2 Gy, 4 Gy, 8 Gy, and 12 Gy irradiated animals. Quality assessments and microarray experiments were completed as previously reported [47]. Samples were hybridized to Agilent Mouse GE 8 × 60K v2 arrays for mRNA expression analysis and to Agilent Mouse miRNA 8 × 60K v21.0 arrays (Design ID 070155) for miRNA expression analysis. Slides were washed and scanned on an Agilent SureScan Microarray Scanner. Expression values were extracted using Agilent Feature Extraction software and data were analyzed with GeneSpring GX software (Agilent Technologies).

Statistical analysis
Analysis of mRNA and miRNA data was performed using R statistical software and the Bioconductor Linear Model for Microarray Analysis (LIMMA) package in R [50]. Background correction and normalization were performed in R using the normal-exponential correction method and quantile normalization between arrays [51]. Only probes with intensities above background on at least one array were kept in the dataset for analysis. Transcripts with multiple probes were averaged such that the nal set re ected best estimates of transcript level expression. A linear model was t to each probe to assess differential expression for pair-wise dose comparisons within the heart-tissue samples. This method employed an empirical Bayes smoothing approach that results in more stable model estimates by using information on variance from the whole probe set, despite the small number of arrays. Models were developed for each of the pair-wise comparisons between each dose (1, 2, 4, 8, and 12 Gy) and the control probes (0 Gy), and resulting probes were ltered using log 2 fold change and adjusted p-value thresholds (|log 2 FC| > 1, adjusted p-value < 0.05) [52]. Additionally, a nested interaction model was t for each probe to examine dose within tissue as a linear (continuous) trend. Each model yielded main effects for the heart tissue and dose within the heart tissue. Probes were ltered using the nested dose coe cients with log fold change and adjusted p-value thresholds (|log 2 FC| > 1, adjusted p-value < 0.05). Finally, gene ontology analysis was utilized to identify affected pathways from the differentially expressed probes.
To identify potential interactions, paired analysis was conducted to evaluate correlative relationships between pairs of differentially expressed mRNA and miRNA probes. mRNA and miRNA probes were paired using shared target transcript Ensembl IDs [53]. Probes that could not be mapped or paired were excluded. Transcripts for miRNA probes were identi ed using an Agilent microarray gene dataset and the TargetScan database; transcripts for mRNA probes were identi ed using an Agilent microarray gene dataset [54]. Transcript-miRNA pairs with a TargetScan context + + score above − 1 were excluded. Probe pairs with differentially expressed miRNA and mRNA probes were identi ed within the heart tissue for continuous dose contrast models. Pearson correlation coe cients of miRNA and mRNA expression across all experiments were calculated and plotted for the differentially expressed probe pairs.

Ingenuity Pathway Analysis
Both core and comparison analyses were performed in IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis). Pathways and function terms that satis ed an absolute z-score > 2 and p-value < 0.01 were predicted to be altered based on the gene expression data.

Radiation induces widespread transcriptional changes
Microarray analysis performed on all mouse heart samples revealed 2041 differentially expressed genes (|log2FC| > 1; p-value < 0.05) that distinguished unirradiated control samples from samples of at least one dose of TBI mice. Overall, most genes had relatively low to no basal expression in control samples and showed increased expression levels after TBI; however, a cluster of genes showed relatively pronounced high expression in control samples that decreased to low expression after irradiation (Fig. 1A). For each dose, more genes were differentially upregulated than downregulated (Fig. 1B). Across all doses, 99 genes were commonly expressed in response to radiation and 128, 55, 390, 322, and 316 genes were expressed exclusively after 1, 2, 4, 8, and 12 Gy of TBI, respectively (Fig. 1C). Supplemental Table S1 lists fold changes and p-values of all differentially expressed genes by dose. While there was no systemic dose-response in terms of the number of genes expressed, we did observe more differentially expressed genes in the higher doses (4,8,12 Gy) than in the lower doses (1, 2 Gy). When dose-response of each gene was analyzed by tting a linear model to each probe, 596 probes were found to have signi cant dose-responsive up-or down-regulation across all doses (Supplemental Table S2). Cdkn1a, Ckap2, and Gdf15 were among the top 20 probes with the strongest upward linear trend, and Alas2, Aplnr, and Cx3cr1 were among the top 20 probes with the strongest downward linear trend (Fig. 1D). All six genes have previously been reported in the context of radiation or DNA damage response and fall into three main biological roles: cell cycle arrest, hemoglobin metabolism, and in ammatory response (Table 1).
Low basal expression levels of long non-coding RNAs in heart tissue showed increased expression after TBI In order to understand the response of heart-based lncRNAs to TBI, we ltered whole genome microarray data to include only probes that correspond to transcripts of lncRNAs. Of the 87 lncRNA transcripts in the microarray data that passed the background intensity cutoff in at least one condition, 46 were differentially expressed in response to radiation, irrespective of the TBI dose ( Fig. 2A). Most lncRNAs showed relatively low expression in unirradiated control samples with increased expression after radiation. More probes showed upregulation than downregulation in all doses except 2 Gy, which had 4 downregulated lncRNAs and 3 upregulated lncRNAs ( Fig. 2A, B). Two lncRNAs were signi cantly altered at all doses after radiation, systematic names are: chr10:69819062-69871640_F and chr17:29183003-29217681_R (Trp53cor1-up) (Supplemental Table S3) (Fig. 2C). Additional lncRNAs were altered only at speci c doses, with 1, 1, 7, 7, and 10 lncRNAs expressed exclusively in 1, 2, 4, 8, and 12 Gy, respectively (Fig. 2C). Supplemental Table S3 lists the fold changes and p-values for the differentially expressed lncRNAs at each dose. Twenty probes showed signi cant linear upward or downward trends as the dose of TBI increased, demonstrating a linear dose response (Supplemental Table S4). Abhd11os, Trp53cor1, Pvt1, and Kalrn were among the most signi cant annotated lncRNAs that became upregulated as radiation dose increased, while the linc-RAM (Malrn) transcript had the most signi cant dose-responsive downregulation (Fig. 2D). Trp53cor1 was the most sensitive to radiation, showing signi cant increase in the relative intensity in comparison to the unirradiated control even after 1 Gy of TBI. The basal level expression of Trp53cor1 lncRNA expression was below detection threshold levels in unirradiated heart tissue. Due to this reason, we used relative intensity to describe expression of Trp53cor1 after radiation.
In contrast, basal expression of Abhd11os was at a much higher threshold across all doses, including control samples, with signi cantly higher expression levels after 8 and 12 Gy TBI. Pvt1 showed signi cance after 2 Gy and Abhd11os showed signi cance after 8 Gy, while Kalrn and linc-RAM showed signi cance only after 12 Gy TBI. We con rmed the expression of Dino, Pvt1, and Trp53cor1 in heart samples through RT-qPCR (Fig. 2E). In concordance with the microarray data, Trp53cor1 showed very low expression in control samples but signi cant dose-responsive upregulation after radiation. Pvt1 also showed consistent results with the microarray in terms of the dose response; however, it showed signi cance in expression change only after 12 Gy TBI. Damage induced noncoding lncRNA (Dino) was not present in our microarray data due to lack of a probe, but prior data from our lab led us to validate its expression in the heart via RT-qPCR. We found that Dino is also signi cantly expressed in the heart after every dose of TBI. Like Trp53cor1, Dino showed very low expression levels in control samples but increased signi cantly after radiation (Fig. 2E).
Low dose of whole-body irradiation induces the most signi cant and pronounced changes of microRNA expression in mouse heart tissue A separate whole genome microarray analysis of miRNA expression revealed 102 signi cantly altered miRNAs in mouse heart tissue in response to radiation. Surprisingly, the largest and most signi cant changes in expression occurred after 1 Gy of TBI, with 86 differentially expressed miRNAs identi ed at this dose (Fig. 3A). Furthermore, there were no commonly expressed miRNAs across all doses and no miRNAs signi cantly expressed in 4 or 8 Gy TBI samples (Fig. 3B, C). We did observe signi cant regulation of miRNAs at 2 and 12 Gy; however, the numbers were relatively low, with 1 and 19 miRNAs in 2 and 12 Gy, respectively. Fold changes and p-values for all signi cantly altered miRNAs at each dose are listed in Supplemental Table S5. Linear trends were t to all miRNA probes to identify dose response across all doses and found 21 probes that showed signi cant upward linear trend (Supplemental Table   S6). No miRNAs showed a signi cant downward linear trend. Among the top linear probes were miR-149-3p, miR-6538, miR-3960, miR-8101, miR-7118-5p, and miR-211-3p, all of which showed a statistically signi cant increase in expression only after 12 Gy of TBI (Fig. 3D).
Pathway analysis of mRNA targets reveals biological role of inversely correlated mRNA-miRNA pairs and their potential as part of an integrated TBI response signature Since we are ultimately interested in developing integrated signatures of coding and non-coding RNA response to radiation, we sought to identify potential interactions of the miRNA and mRNA signatures and their biological signi cance. Using IPA, we rst conducted miRNA target lter analysis of the signi cant differentially expressed miRNAs to identify experimentally veri ed mRNA targets in our dataset. Canonical pathway analysis of the identi ed targets revealed signi cant activation of pathways relevant to cell cycle checkpoint activation and senescence, including p53 signaling and numerous apoptosis signaling pathways, among others (Fig. 4A). Interestingly, most pathways activated across all doses were predicted to have the highest activation after 4 Gy TBI, followed by lower activation in 8 and 12 Gy. One exception to this was the senescence pathway, which showed the lowest activation at 4 Gy. A concurrent higher activation of various apoptosis pathways after 4 Gy TBI may suggest that miRNA-mRNA pairs at this dose exhibit a different response to stress and DNA damage than both lower and higher doses at this time-point after irradiation. After pathway analysis, we identi ed predicted miRNA-mRNA pairs with inverse expression patterns, demonstrating their potential for inclusion in an integrated RNA marker signature to improve clinical decision making. We found three miRNAs that showed signi cant expression in at least one dose and had predicted targets with inverse expression patterns, each miRNA with two targets. Radiation decreased expression of both miR-128-3p and miR-122-5p relative to control, while their targets-Tgfbr1 and Wee1, and Fam117b and Slc7a11, respectively-showed a statistically signi cant increase in expression as radiation increased (Fig. 4B). The third miRNA, miR-18-5p, showed increased expression after radiation, most signi cantly after 1 Gy. Its targets, E2f1 and E2f2, showed signi cant down regulation across all doses.
Pathway analysis of all mRNAs predicts activation of immune and cell-cycle related pathways and deactivation of metabolic pathways after TBI While understanding the interactions and biological implications of the miRNAs and mRNAs is critical for developing an integrated biomarker signature, we hypothesized that because the relatively low number of differentially expressed miRNAs would limit the number of mRNAs included in the pathway analysis, we could potentially miss genes that play a signi cant role in pathway regulation. Therefore, we also conducted a canonical pathway analysis using all differentially expressed mRNAs, irrespective of interactions with miRNAs in our dataset. A similar overall pattern of pathway regulation exists between the target mRNAs and target/non-target mRNAs, with signi cant deactivation of most of the pathways involved (Fig. 5A). Several pathways related to coagulation, including both ex-and intrinsic prothrombin activation and the coagulation system pathway, were downregulated across all doses. Changes in immune-related pathways were less consistent in terms of activation or deactivation. While natural killer cell signaling was activated signi cantly after 4 and 12 Gy TBI, the complement system was inhibited across all doses.
Two clusters of pathways showed the most pronounced activation and deactivation. Five pathways were predicted to be activated across all doses, and all were involved in immune response or cell cycle regulation. There were 78 genes differentially expressed in these pathways, a majority of which were downregulated with respect to the control (Fig. 5B). A full list of genes can be found in Supplemental Table 7. In contrast to the pathway analysis of the mRNA targets, which showed the highest activation of several pathways after 4 Gy TBI, all activated pathways except natural killer cell signaling were consistently activated as the dose increased. We observed that radiation inhibits pathways relevant to xenobiotic metabolism and biosynthesis of lipids, including hormones. This is demonstrated by activation of LPS/IL-1 mediated inhibition of RXR function and inhibition of the super-pathway of melatonin degradation, among others. There were 79 genes involved in deactivation of these seven pathways, most of which were downregulated after TBI (Fig. 5C, Supplemental Table S7). TBI deactivates metabolic pathways and alters the expression of metabolism-related genes in heart tissue IPA analysis predicted signi cant deactivation of several metabolic pathways, such as triacylglycerol degradation and type I diabetes mellitus signaling, among others (Fig. 5A). To further understand how radiation alters metabolism, we used IPA to lter the microarray gene expression data to include only the signi cantly differentially expressed genes with involvement in metabolic energy production pathways, with emphasis on fatty acid oxidation (Fig. 6A). Of note, there was a signi cant downregulation of solute carrier 2a2 (Slc2a2) and concurrent upregulation of pyruvate dehydrogenase kinase 4 (Pdk4), especially after 4 Gy TBI. Similarly, as dose increased, we observed upregulation of glutamine synthetase (Glul). which encourages conversion of glutamate to glutamine. We also observed downregulation of glutaminase (Gls2), which converts glutamine to glutamate.
Gene expression data for fatty acid metabolism at 48 h post radiation was contradictory. We observed a downregulation of solute carrier 27a5 (Slc27a5), which is associated with fatty acid entry into the cell; an upregulation of sestrin 2, which inhibits lipid catabolism; and an upregulation of hormone sensitive lipase (Lipe) and adipose triglyceride lipase (Atgl or Pnpla2), which cleave lipid droplets to allow use of fatty acids in FAO. However, there was a downregulation of arylacetamide deacetylase (Aadac) which shares homology with Lipe and is also thought to control triglyceride levels. Additionally, acyl-CoA synthetase medium chain family members 1 (Acsm1) was downregulated, while acyl-CoA thioesterases 1 (Acot1) and acyl-coA dehydrogenase 10 (Acad10) were notably upregulated as radiation doses increased.

Discussion
Normal tissue damage of the heart is a clinically relevant problem in both therapeutic and accidental exposure to radiation. A previous study highlighted the long term impact of radiation on male macaques, which showed signi cant myocardial brosis and smaller cardiac dimensions at 5.6-9.7 years post radiation exposure with 6.5-8.4 Gy [55]. Historical data for macaques receiving TBI, LD50/30 post x-ray irradiation varies between 4.92-7.18 Gy [56]. In contrast, LD50/30 is approximately 7.2 Gy in female mice receiving TBI x-ray [57]. Our study focused on short term (48 h) changes in gene expression after TBI doses in mice. Understanding mechanisms and markers of radiation injury at an early time-point following exposure can improve methods to mitigate long-term damage and death. Recent research in the radiation biodosimetry eld indicates the importance of looking for alterations in multiple biomarkers rather than relying on a single marker [58,59]. To this end, we have identi ed changes in multiple mRNAs, miRNAs, and lncRNAs across doses to provide potential markers of tissue damage. TBI dysregulates pathways relevant to cell cycle arrest, hemoglobin synthesis and immune response in the heart The observed early changes in gene expression led to signi cant dysregulation of pathways commonly associated with radiation exposure, including cell cycle arrest, apoptosis, and senescence. The most signi cantly altered genes, Cdkn1a and Ckap2, are capable of inducing cell cycle arrest or apoptosis under stress conditions [60,61]. The observed upregulation of Gdf15 and downregulation of Aplnr are associated with stress induced senescence and have previously been reported as biomarkers of radiation exposure [62][63][64]. The inversely correlated expression of miRNAs and their mRNA targets that are associated with these pathways provides insight into the potential mechanisms of acute effects of TBI. and targets to mitigate acute and delayed effects on heart tissue. Prior research demonstrated miR-128 negatively regulated Wee1 and Tgfbr1, genes involved in mitotic inhibition [65][66][67]. Additionally, miR-122 is known to increase radiation sensitivity, and targets Slca11 and FAM117b [68]. While little is known about the function of Fam117b, Slc7a11 downregulation is associated with RT-induced ferroptosis in tumor cells [69,70]. The miR-17-92 cluster represses E2F1-3 to regulate cell proliferation and apoptosis, and includes miR-18a [71]. A negative feedback loop has been observed within this cluster, as E2F1 upregulates miR-17-92 which causes increased repression of the genes E2F1-3 [72]. Mechanistic studies are required to con rm the miRNA-mRNA pair functions in our data; however, their expression patterns suggest potential targets for mitigation of acute and delayed effects of radiation on heart tissue.
Anemia and decreased hemoglobin levels are a known side effect of RT [73]. We observed decreased expression levels of genes relevant to hemoglobin synthesis. Alas2, the rate limiting enzyme in heme synthesis, became increasingly downregulated as radiation dose increased. Similar downregulation of Alas2 was recently cited as a potential predictive marker for radiation induced hematological toxicity in cancer patients [74]. Other genes associated with hemoglobin synthesis, including Hbb-bt, Hbb-b1, and Hba-a1, were also downregulated.
Our pathway analysis also indicated in ammatory pathways are downregulated at higher dose levels at 48 h post radiation. Of note, TBI induced signi cant downregulation of Cx3cr1, which is known to induce recruitment of immune cells and an in ammatory response in smooth muscle and endothelial cells [75].
Short-term data contrasts long-term in vivo data that showed upregulation of in ammatory markers in the heart of a mouse model 40 days post-irradiation [17]. Additional studies that include intermediate timepoints are needed to clarify when the heart transitions from an anti-in ammatory to a proin ammatory response to radiation to enable improved treatment options.
Consistent with previous research [76], our ndings indicated inhibition of extrinsic and intrinsic prothrombic pathways that couple with inhibition of coagulation pathway after radiation exposure. Radiation is known to increase likelihood of coagulopathy, which can lead to death when untreated [77]. We also observed downregulation of hemoglobin subunit beta (Hbb) and hemoglobin subunit alpha (Hba). In combination with anemia, failure to clot produces hemostatic dysfunction and potential death, though the pathogenesis is poorly understood [78]. Our research highlights these alterations in gene expression to provide insight into potential mechanisms of and therapeutic targets for acute radiation syndrome (ARS).
Retinoid X receptor and liver X receptor (RXR/LXR) activation is associated with protection against heart failure due to their role in improving glucose tolerance, decreasing lipid accumulation, and decreasing in ammation [79]. RXR signaling has previously been shown to increase estradiol synthesis from pregnenolone [80]. We observed that the estrogen and pregnenolone biosynthesis pathways are also inhibited. Inhibition of RXR/LXR and its downstream pathways coupled with inhibition of triacylglycerol degradation may indicate a deleterious increase in lipid accumulation within the heart. Radiation induced lncRNA and miRNA provide potential insight into RIHD through signaling pathways Previously, we reported the concomitant differential expression of p53-related lncRNAs such as Pvt1, Dino, Trp3cor1 after TBI in a mouse model [45]. In the current study, we observed signi cant alterations in abhydrolase domain containing 11, opposite strand (Abhd11os), Pvt1, Trp3cor1, Kalrn, linc-RNA activator of myogenesis (Linc-RAM), and Dino. Increased expression of Abhd11os has previously been shown to decrease lesion size in a Huntington's disease mouse model and the authors reported its crucial roles in neurodegenerative diseases [81], but the exact mechanism is still unclear. Linc-RAM encourages adult skeletal muscle stem cells to differentiate into skeletal muscle through myogenic differentiation (MyoD), which aids in muscle repair after injury [82,83]. While Linc-RAM has not been directly associated with cardiomyocytes, MyoD-null dystrophin-null transgenic mice develop severe cardiomyopathy [84]. Pvt1 has been associated with radiation resistance in cancer cells and cardiac hypertrophy in cardiomyocytes [85][86][87].
Prior research demonstrated the integral role that miRNAs play in cardiac brosis and proliferation as well as response to radiation injury [88,89]. We therefore anticipated changes in miRNA expression in heart tissue after TBI. Surprisingly, and possibly due to the early time point and stringent statistical analysis, we only observed signi cant upregulation of miRNAs at 1 Gy and 12 Gy. Among the up-regulated miRNAs, miR-149-3p has previously been implicated in multiple functions, including cell migration repression and metabolic modi cations in A549 cells, a non-small cell lung carcinoma (NSCLC) model [90]. Additionally, miR-211 has been demonstrated to decrease cell proliferation and metastasis in vitro in breast and renal cell carcinoma models [91,92], while miR-3960 has been implicated in calci cation33, decreased elasticity, and cardiac dysfunction in vascular smooth muscle cells of male C57BL/6 mouse aortas [93].
Arterial calci cation and valvular, ventricular, and diastolic dysfunction are well-known complications of RIHD disease [94]. While the functions of certain miRNAs are not well understood, previous studies indicate that miR-8101 and miR-6538 are associated with heart failure [95,96].
TBI causes miRNA and mRNA expression changes that may indicate similar pathogenesis of end-stage heart failure Upregulation of miR-149-3p is associated with inhibition of glucose metabolism. Its role as a therapeutic target to protect against diet-induced obesity and metabolic dysfunctions was shown previously in both colorectal cancer patients, tumors taken from colorectal cancer patients and male C57BL/6J [97,98]. In general, the observed changes in metabolism-related gene expression suggest that fatty acids are not being used for catabolism (Fig. 6B). Increasing doses of radiation appear to inhibit glucose oxidation through increased expression of Pdk4, which uncouples glycolysis from oxidative phosphorylation by blocking pyruvate dehydrogenase (Pdh), and decreased expression of Acsm1 [99,100]. However, we also observe a signi cant downregulation of the transporter Slc27a5, which would inhibit entry of fatty acids into the cell for anabolic or catabolic use. This could be a fatal side effect of IR exposure because the adult heart relies on fatty acid oxidation as its main source of energy production [101]. Since Lipe and Pnpla2 were upregulated, the heart may be relying on internal stores of triacylglycerol to produce energy. Aside from Acsm1, no overall changes in acyl-CoA synthetases were observed; these enzymes combine fatty acids with Coenzyme A for use in FAO or lipogenesis. We observed upregulation of Acot1 which plays contradictory roles in FAO as it can separate long chain fatty acids from coenzyme A to decrease the available substrate pool [102]. However, prior research indicates Acot1 also increases FAO through activation of peroxisome proliferator-activated receptor α (Ppar-α), which upregulates Acad variants [103][104][105]. We only observed an upregulation of Acad10 and there was no signi cant change to Ppar-α or other Acad variants at 48 h after radiation. Furthermore, there was no change in expression of other genes within the FAO pathway. This inhibition of glucose oxidation paired with an apparent reliance on FAO and an increase in free fatty acids matches what is seen in some forms of end stage heart failure [106,107].
Gene expression changes in TBI C57BL/6 match those identi ed in previous study of TBI Gottingen minipigs A recent study from our lab reported survival predictive signatures inherent to heart, lung and liver in TBI Gottingen minipigs [108]. Increased expression of Pdk4 was signi cantly upregulated in the hearts of non-surviving (survived < 7d post-TBI) minipigs. We also observed increased expression of Pdk4 at 4 to 12 Gy radiation. As previously mentioned, the upregulation of Pdk4 is associated with decreased glucose oxidation and potential failure of energy production. Hearts from these non-surviving minipigs showed a decrease in the Apelin signaling pathway when compared to survivors and sham animals. In the present study, we also observed inhibition of the Apelin signaling pathway starting at 2 Gy. Apelin signaling is important for endothelial cell proliferation and migration and has been shown to inhibit TGF-β induced cardiac brosis and senescence [109]. Conversely, downregulation of Apelin has been linked to heart failure and ventricular dysfunction [110]. Both these ndings implicate centrality of the endothelial cell damage in radiation induced heart injury. Finding consistency between mouse and minipig studies indicates conservation of the radiation response across species, suggesting that rescuing the function of this pathway may prevent RIHD in humans as well.
Similarities in TBI-induced gene expression between heart and blood samples indicates potential biomarkers for effective triaging with radiation biodosimetry Finding normal tissue injury markers in a less invasive way is warranted for clinical applications. From this perspective, we looked at the commonality of gene and lncRNA expression changes between heart tissue of the current study and mouse whole blood after TBI from a study previously published by our group [45]. Interestingly, we detected few genes in common between both mouse heart tissue and whole blood. We focused on genes altered at 48 h after 8 Gy in whole blood and compared these alterations to changes found in our current study of mouse heart tissue. Signi cantly altered genes included Cdkn1a, Pmaip1, H2Aa, H2Ba1, Cx3cr1, Snca, and Gm9992 (Supplemental Table 8). Another previous study from our lab indicated that Pvt1 was signi cantly expressed in whole blood as early as 16 h after at least 2 Gy TBI, and was sustained until 48 h after TBI [49]. Similarly, at 48 h post-TBI, Trp53cor1 and Dino were signi cantly upregulated in the whole blood of 12 Gy and 8 Gy TBI mice, respectively. We observed similar expression patterns of these lncRNAs in heart samples after 48 h.
We also compared the heart data to a previously reported in vitro study of gene expression changes in human coronary artery endothelial cells (HCAEC) at 24 h after 10 Gy of single dose radiation [110]. The genes Cdkn1a, growth differentiation factor 15 (Gdf15), and DNA damage-inducible transcript 4 (Ddit4/Redd1) have previously been reported as radiation markers [61,111,112]. They showed concomitant upregulation in mouse heart tissue (Supplemental Table S8) and in HCAEC [110].
Additionally, we noted the upregulation of hypoxia-inducible factor 3a (Hif3a) and insulin-like growth factor 1 (Igf1) in mouse heart tissue, whereas these two genes showed signi cant downregulation in HCAEC in vitro study. In rat cardiomyocytes, Hif3a silencing led to increased cell viability and decreased necrosis after hypoxia challenge, suggesting decreased Hif3a expression is cardioprotective [113]. In a population study of elderly individuals, decreased serum Igf1 expression was shown to be a risk factor for mortality after ischemic heart disease [114]. These regulation inconsistencies may stem from differences between models, time points, and radiation dose rate between in vivo and in vitro experiments.

Future Directions
With the use of thoracic RT and the continued risk of a large-scale nuclear exposure incident inadvertently causing potential damage to the heart, understanding the effects of IR exposure on critical organs such as the heart will improve patient outcomes. For clinical management, early biomarkers could be predictive of later damage, enabling alteration of the dose to the organs at risk, use of medical countermeasures, or implementation of an appropriate long-term medical management strategy. For a nuclear exposure incident, the dose will have been delivered such that the injury falls within delayed effects of acute radiation exposure (DEARE), but the mitigator and medical-management approach would still be relevant.
In addition to identifying blood-based signatures for rapid triaging, we are also working on identifying expression changes within organs (e.g. heart, lungs, liver) affected by radiation to predict both short-and long-term organ injury. Recognizing that heart biopsies would not be a suitable method to triage patients in an exposure scenario, we are also currently investigating the short-and long-term circulating RNA response of non-human primates exposed to whole thorax irradiation. This would not only enable monitoring of organ-speci c damage sustained during accidental exposures but would also have applications in predicting normal tissue toxicity as a side-effect of RT for the treatment of cancer.
Disclosure of potential con icts of interest: The authors report no con icting interests.