Female C57BL6J mice, 12–16 weeks-old (Charles River Laboratories, Oxford, UK) were housed under controlled conditions (12hr light-dark cycle, 21°C) in standard caging with 3–5 littermates and received a standard laboratory diet (Teklad, Envigo, UK) with water ad libitum. Mice were randomly assigned to experimental groups prior to irradiation. Animal numbers were chosen to give power to detect differences in myocardial performance index (MPI) of 25% with a power of 80% for a 2-sided equality test with a significance threshold of 0.05 which required at least six mice per group. In this study we used 8 mice per group for each timepoint, with aged matched controls at week 50.

Mouse weight was monitored throughout the experiment, with no deviation outside the tolerated weight loss of <15%. All animal research and experiments carried out at Queen’s University Belfast were approved by the local Animal Welfare Ethical Review Board (AWERB) and a Department of Health Project License (PPL 2935). All experimental procedures were carried out in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986, published by His Majesty’s Stationary Office, London. Animal studies are reported in compliance with the ARRIVE guidelines and suggested reporting requirements for preclinical cardiac irradiation studies (25, 26).

Prior to irradiation mice were anaesthetised with ketamine (100 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection. Mice were randomized prior to irradiation to either a control non-irradiated group, heart base irradiation with 16 Gy, right lung irradiation with 10 Gy or a combined heart base (16 Gy) and right lung (10 Gy) irradiation. Mice were irradiated with 220 kVp X-rays under cone beam computed tomography (CBCT) image guidance using a SARRP (Xstrahl Life Sciences) calibrated using the Institute of Physics and Engineering in Medicine and Biology (IPEMB) code of practice (27). For heart base irradiation, the cranial third of the heart was localised using CBCT imaging and an anterior-posterior field arrangement was created using a 3 x 9 mm collimator with a dose rate of 2.67 ± 0.11 Gy/min as previously described (22). Dose to the upper region of the right lung was delivered using a 5 x 5 mm collimator at a dose rate of 2.75 ± 0.23 Gy/min in a similar anterior-posterior beam geometry. Dose-volume histograms (DVHs) were retrospectively calculated for each animal (Muriplan, Xstrahl Inc, Suwannee, GA, USA). A schematic overview of the study including the DVHs and endpoints is shown in Figure 1 .

TTE was performed at baseline and 50-weeks after irradiation using Vevo770^® ultrasound system, with a 45 MHz RMV707B scan head (FUJIFILM, VisualSonics Inc. ON, Canada). Mice were anaesthetised using inhalant isoflurane (3% for induction, and 1.5% during imaging). Left ventricular (LV) systolic and diastolic metrics were measured in triplicate for each mouse. LV ejection fraction (EF) and fractional shortening (FS) were calculated from the M-mode parasternal short-axis view, as previously described (28). Pulse-wave Doppler imaging was used to measure the early and late filling ratio (E/A ratio). MPI was calculated as previously described (28). Observers were blinded to experimental groups during data collection and analysis.

Lung were harvested at 10 and 50-weeks after irradiation. After fixation, samples were processed, paraffin embedded and cut into 5 µm sections for histological staining and evaluation. Pulmonary perivascular and interstitial oedema were assessed using the semi-quantitative scoring previously reported (17). Perivascular oedema was scored between 0-2 (2 indicating severe perivascular oedema) for at least 5 large vessels and 20 small vessels across the right lung. For interstitial oedema, scores from 0-3 (3 severe interstitial oedema) were given according to the level damage severity across 4 different fields of view (400 x 400 µm) across the right lung (17). Pulmonary vessel wall thickness was measured for 30 randomly chosen vessels across in the upper right lung as previously described (14). Fibrosis was measured by type I collagen staining using Masson’s Trichrome (Abcam, ab150686) as outlined in Supplementary Material S1 . Scores were recorded from 6 different regions within or outside of the irradiated field of the heart or right lung for 4 mice per experimental group and ≥2 age matched control animals. Lung fibrosis was quantified using Matlab R2022a as previously described (29).

Mouse XL Proteome Profiler Cytokine arrays (ARY028, R&D Systems, UK) were performed to simultaneously characterise the expression of 111 cytokine targets. Each cytokine was present in duplicate on the membrane. Serum used for analysis was isolated from blood samples at weeks 10 and 50 from irradiated mice alongside age-matched controls. Cytokine arrays were performed according to the manufacturer’s instructions using pooled serum samples for each treatment group (control n=2, heart n=6, lung n=6, heart and lung n=6). Membranes were developed using the GBox Imager and GeneSys software (V1.4.60) (Synotics Ltd, Cambridge, UK). Quantification of signal intensities was performed with GeneTools software (V4.3.14) (Synotics Ltd, Cambridge, UK), and densitometry values for spots were normalised to that of the age-matched controls. Cytokine arrays were completed in one independent experiment. Proteins which satisfied a threshold of > 2- and < 0.5-fold change were considered differentially expressed. These proteins were transformed into their corresponding gene names for pathway enrichment analysis using Enrichr (30).

Data are presented as the average for the entire experimental arm ± standard error. Error bars represent the standard error of the mean (SEM). Statistical analysis was performed using student’s t-test unless otherwise stated. Statistical significances are represented as p < 0.05 = *; p <0.01 = **; p <0.001 = ***; and p <0.0001 = ****. All bar graphs, heatmaps, and principal component analysis (PCA) plots were created using GraphPad Prism (V9.2.0).

Cardiac systolic and diastolic parameters were assessed following the irradiation of different thoracic volumes. No statistically significant changes in cardiac function parameters were observed between control and irradiated mice at 10 weeks after irradiation ( Supplementary Figure S1 ). At 50 weeks after irradiation, several changes in cardiac function were observed between control and irradiated animals ( Figure 2 ). Systolic function measured by EF was significantly lower after irradiation of the heart base and co-irradiation of the heart base and right lung (p<0.004) compared to control animals. A small but significant decrease in FS was observed only for heart base irradiation compared to control animals (p=0.036). Diastolic function measured by the E/A ratio was significantly lower after heart base and co-irradiation of the heart base and right lung (p =0.038). MPI is a measure of the overall performance of the heart integrating both systolic and diastolic function that increases with cardiac dysfunction. MPI was significantly increased in all irradiated groups with much higher increases in the heart base and co-irradiation groups compared to the control group (p<0.001). Overall, these data show a significant effect of heart base irradiation on cardiac function with co-irradiation of the right lung having minimal impact on the observed responses at 50 weeks after irradiation.

Histological changes were assessed in the irradiated right lung at 10 and 50 weeks after the irradiation of different thoracic target volumes ( Figure 3A-F ). At 10 weeks after irradiation, all groups showed a significant increase in pulmonary oedema compared to control animals (p<0.05) with the largest increase observed in the co-irradiation group. Irradiation of the heart and lungs as individual targets showed comparable levels of response that were not significantly different (p > 0.05, Panel A). A small but significant increase in pulmonary vessel wall thickness was only detected following heart base irradiation. Heart and heart lung co-irradiation showed small but significant increases in fibrosis that was higher than irradiation of the lung as a single target (Panel C). Similar trends were observed at 50 weeks after irradiation showing significant increases in pulmonary oedema compared to control animals (p<0.01) with similar increases in the heart and co-irradiation groups. A small but significant increase in pulmonary vessel wall thickness was only detected following co-irradiation whilst the levels of fibrosis were significantly higher in the heart and co-irradiation groups. These data suggest small but significant histological changes in lung that are dependent on the irradiation of the heart. Represantative images from histology analysis are shown in Supplementary Figure 2 .

Cytokine analysis was performed on serum samples obtained at 10 and 50-weeks after irradiation ( Figure 4 ). Heatmaps show the fold changes in the expression of 111 cytokines compared to age-matched controls at the respective timepoints for each target volume ( Figures 4A, B ). These data highlight distinct changes for each of the irradiated volumes and at each time point with an overall trend showing decreased cytokine expression following irradiation of the lungs compared to the other volumes. PCA was used to identify similarities in modulated cytokine expression between the different irradiated volumes ( Figure 4C ). The PCA plot shows clustering of the individual heart and lung volumes within each timepoint suggesting similar cytokine modulatory effects. The observed changes occurred predominantly across PC2. In contrast, combined heart and lung irradiation has a unique effect on cytokine expression at both timepoints, reflected by the lack of clustering and the individuality of these data points. These changes occurred across PC1.

Changes in specific cytokine levels demonstrated differential responses following irradiation of the different target volumes ( Figure 4D ). In particular, a clear difference was observed in the levels of CD14 for the different irradiated volumes. At 10 weeks, CD14 showed a >2-fold upregulation following irradiation of the heart yet the level was several orders of magnitude lower following co-irradiation or irradiation of the lung only. However, at 50 weeks, no major changes in CD14 were observed. Platelet-derived endothelial cell growth factor (PD-ECGF) also showed a pronounced volume dependent response. At 10 weeks, PD-ECGF showed > 2-fold upregulation following co-irradiation yet the levels were down regulated following irradiation of the heart or lung. At 50 weeks, the levels of PD-ECGF were lower in the co-irradiation group, elevated in the heart group and unchanged in the lung group compared to the level at 10 weeks. At 10 weeks, the levels of leptin remained unchanged compared to controls in the heart and co-irradiation groups but were lower in the lung irradiated group. At 50 weeks, the levels increase close to 2-fold following heart and lung irradiation as single targets but decreased in the co-irradiation group. Overall, these data highlight distinct changes in the circulating inflammatory proteome that are dependent on the irradiated target volume and time after irradiation.

Differential cytokine analysis was performed by applying a threshold of > 2-fold changes at 10 and 50 weeks ( Figure 5A ). At 10 weeks, 3, 15 and 26 cytokines were found to be volume-specific for the heart, heart and lung, and lung irradiations, respectively. At 50 weeks, 9, 9 and 13, cytokines were found to be volume-specific for the heart, heart and lung, and lung, respectively. The identified volume specific cytokines were then used for pathway enrichment analysis ( Figures 5B, C ). For all of the irradiated volumes at 10 and 50 weeks, the most enriched pathway was proinflammatory and profibrotic mediators. At 10 weeks, lung fibrosis was enriched following lung only and co-irradiation but not for heart irradiation, however, at 50 weeks, lung fibrosis was enriched in the heart and lung irradiation groups. These data support the underlying biological pathways involved in the inflammatory response of the respective tissues and suggest a potential interaction between the heart and lung that acts to suppress fibrosis when the organs are co-irradiated.