Heart Irradiation Research Articles

Heart ultrasound and biomechanical evaluation of radiation‐induced heart toxicity using transthoracic echocardiogram (TTE) and dynamic mechanical analysis (DMA)

AbstractRadiation‐induced heart disease (RIHD) is a serious complication but difficult to assess in patients undergoing thoracic radiotherapy (RT). We aim to analyze RIHD using heart ultrasound and elastic modulus, exploring relationships between functional, anatomical or biomechanical changes of the heart and radiation dose.Twenty BALB/c mice were divided into four groups (control, 10 Gy, 20 Gy and 25 Gy) with a single fraction of image‐guided volumetric modulated arc radiotherapy (VMAT) to murine heart on a linear accelerator. Transthoracic echocardiography (TTE) was performed on a small‐animal ultrasound imaging system with a handheld microscan transducer. E‐wave/A‐wave ratio (E/A) and myocardial performance index (MPI) for diastolic performance were noninvasively evaluated weekly, as well as ejection fraction (EF%), fractional shortening (FS%), left ventricle (LV) mass and heart wall thickening for systolic performance. At the end of the fifth week, all mice were sacrificed for elastic modulus measurement on a dynamic mechanical analyzer (DMA) and for histopathological staining. All experiments were conducted in accordance with the local institution's animal research committee guideline.Significant difference was observed in E/A ratio between the control and 25 Gy irradiated groups (1.8±0.5 and 0.7±0.9, respectively; p<0.05), indicating reduced diastolic performance and increased stiffness in left ventricle after high‐dose heart radiation. Diastolic dysfunction in irradiated groups was also observed with significantly increased MPI. In contrast, posterior wall thickness, aortic peak velocity, heart rate, EF and FS were not significantly different after RT. Heart elasticity was reduced substantially with the increased radiation dose. HE and Masson Trichrome staining confirmed more fibrosis deposition in irradiated hearts.RIHD evaluation with ultrasound imaging noninvasively and biomechanical modulus measurement invasively in the image guided, precision dose‐escalated murine heart irradiation is feasible. Increased myocardial stiffness, abnormal diastolic relaxation, more collagen deposition, and reduced tissue elasticity are observed in irradiated heart tissue. This study may facilitate our understanding of RIHD and facilitate improving patients’ quality of life in the future.

Abstract Mo024: Integrated analysis of ex vivo biomechanical ventricular remodeling and imaging-based in vivo strain changes in a pre-clinical murine model of radiation-induced cardiotoxicity

Introduction: Thoracic radiation therapy (RT) is commonly used for the treatment of many common malignancies, such as lung and breast cancer, but carries a high risk of radiation-induced cardiotoxicity (RIC). Existing diagnosis of RIC is challenged by subclinical ventricular dysfunction, not captured by global indices of cardiac function. Understanding RIC-driven biomechanical remodeling of the myocardium will provide novel insights into developing high-fidelity imaging approaches for early detection of RIC. Methods: A pre-clinical model of RIC was developed by administering whole heart irradiation under image guidance in mice (8 Gy x 5) over five consecutive days. A longitudinal study was carried out to investigate the biomechanical response of the left ventricle (LV). Cardiac magnetic resonance imaging was performed to estimate end-systolic strains. Mechanical testing was performed on the harvested LV free wall (LVFW) specimens to measure passive stiffness, calculated as the tangent to the ensemble stress-strain curve at 30% strain. Results: A significant increase in passive LVFW stiffness was observed at 3M post-RT (Figs. 1A and B). Significant declines in both global circumferential strain (GCS) and global radial strain (GRS) at 3M post-RT, compared to respective measurements at pre-RT, were observed (Figs. 1C and D). Suppressed contractile strains suggested biomechanical remodeling of the LV, including impaired contractility and passive stiffening of the LVFW, consistent with ex-vivo measurements. Conclusion: We have established a direct correlation between ex-vivo mechanical testing (elevated stiffness) and reduced contractile strains in in-vivo analysis in a mouse model of RIC. Such integrated analysis will pave the way towards developing highly sensitive imaging approaches that non-invasively recapitulate the ventricular remodeling events associated with RIC, thereby enabling early detection of RIC in patients receiving thoracic RT.

Abstract Mo073: Elucidating the Role of Cardiac Radiotherapy on the Sympathetic Nervous System as a Treatment for Ventricular Tachycardia

Ventricular tachycardia (VT) is a dangerous arrhythmia that occurs in structurally and electrically heterogenous regions of the diseased heart, leading to sudden cardiac arrest and death if left untreated. One of the main drivers underlying the arrhythmogenic substrate is abnormal remodeling of the autonomic nervous system after cardiac injury, resulting in a heterogenous distribution of sympathetic innervation and subsequent ventricular arrhythmia. In patients with VT refractory to conventional therapy, noninvasive cardiac stereotactic body radiotherapy (SBRT) has emerged as a promising new treatment. Here, we investigated whether cardiac radiotherapy homogenizes the arrhythmogenic substrate through induction of cardiac sympathetic nerve innervation and activity in a closed-chest ischemia reperfusion murine model of ischemic heart failure. Using single nucleus-RNA sequencing, we identified global transcriptomic changes in the left ventricle 6 weeks after 25 Gray (Gy) cardiac X-ray radiation. Notably, these changes included increased expression of neurotrophins such as brain derived neurotrophic factor, a growth factor essential for neuronal survival and development, in irradiated hearts compared to sham controls. Consequently, Western blot and immunofluorescence showed increased expression of the sympathetic neuronal markers tyrosine hydroxylase and neuropeptide Y at the scar border zone of irradiated mice. Similar immunofluorescence results were observed at the irradiated border zone from the explanted heart of a patient previously targeted with 25 Gy compared to a nontargeted remote region within the same heart. These findings suggest a novel role for radiation-induced remodeling of the sympathetic nervous system in the anti-arrhythmic response to SBRT.

Sparing of the Heart Facilitates Recovery From Cardiopulmonary Side Effects After Thoracic Irradiation

IntroductionWhen irradiating thoracic tumors, dose to the heart or lung has been associated with survival. We previously showed in a rat model that in addition to known side-effects such as pericarditis, pneumonitis and fibrosis, heart and/or lung irradiation also impaired diastolic function and increased pulmonary artery pressure. Simultaneous irradiation of both organs strongly intensified these effects. However, the long-term consequences of this interactions are not yet known. Therefore, here we investigate the long-term effects of combined heart and lung irradiation. Material and MethodsDifferent regions of the rat thorax containing the heart and/or 50% of the lungs were irradiated with protons. Respiratory rate (RR) was measured bi-weekly as an overall parameter for cardiopulmonary function. Echocardiography of the heart was performed at 8, 26 and 42 weeks after irradiation. Tissue remodeling and vascular changes were assessed using Masson Trichrome and Verhoeff-stained lung and LV tissue collected at 8 and 42 weeks after irradiation. ResultsDuring the entire experimental period RR was consistently increased after combined heart/lung irradiation. This coincided with persistent effects on lung vasculature and reduced right ventricle contraction. In contrast, recovery of RR, pulmonary remodeling and right ventricle contraction was observed after sparing of the heart. These corresponding temporal patterns suggest that the reduction of right ventricle (RV) function is related to vascular remodeling in the lung. ConclusionCombined irradiation of lung and heart leads to an intensified, persistent reduction of cardiopulmonary function. Recovery of the pulmonary vasculature and right ventricle function requires heart-sparing.

PD-1 Inhibitor Aggravate Irradiation-Induced Myocardial Fibrosis by Regulating TGF-β1/Smads Signaling Pathway via GSDMD-Mediated Pyroptosis.

Cancer therapy has entered a new era with the use of programmed cell death protein 1 (PD-1) immune checkpoint inhibitors. When combined with thoracic radiotherapy, it demonstrates synergistic anti-tumor effects and potentially worsens radiation-induced myocardial fibrosis (RIMF). RIMF is the final stage of radiation-induced heart disease (RIHD) and a potentially fatal clinical complication of chest radiotherapy. It is characterized by decreased ventricular elasticity and distensibility, which can result in decreased ejection fraction, heart failure, and even sudden cardiac death. Pyroptosis, a type of programmed cell death, is mediated by members of the gasdermin (GSDM) family and has been associated with numerous cardiac disorders. The effect of pyroptosis on myocardial fibrosis caused by a combination of radiotherapy and PD-1 inhibitors remains uncertain. In this study, a 6MV X-ray of 20Gy for local heart irradiation was used in the RIHD mouse model. We noticed that PD-1 inhibitors aggravated radiation-induced cardiac dysfunction and RIMF, concurrently enhancing the presence of CD8+ T lymphocytes in the cardiac tissue. Additionally, our findings indicated that the combination of PD-1 inhibitor and thoracic radiation can stimulate caspase-1 to cleave GSDMD, thereby regulating pyroptosis and liberating interleukin-8 (IL-18). In the myocardium of mice, the manifestation of pyroptosis mediated by GSDMD is accompanied by the buildup of proteins associated with fibrosis, such as collagen I, transforming growth factor β1 (TGF-β1), interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and tumornecrosisfactorα (TNF-α). Moreover, it was discovered that TFG-β1 induced the phosphorylation of Smad2/Smad3 when the cardiac underwent PD-1 inhibitor in conjunction with thoracic irradiation (IR). The findings of this research indicate that PD-1 inhibitor worsen RIMF in mice by triggering GSDMD-induced pyroptosis and influencing the TGF-β1/Smads pathway. While using the caspase-1 inhibitor Z-YVAD-FMK, RIMF can be alleviated. Blocking GSDMD may be a viable strategy for managing myocardial fibrosis caused by the combination of PD-1 inhibitors and radiotherapy.

2147: Entresto as a novel radioprotectant in a partial heart irradiation mouse model

2147: Entresto as a novel radioprotectant in a partial heart irradiation mouse model

Proteomic and phosphoproteomic characterization of cardiovascular tissues after long term exposure to simulated space radiation.

Introduction: It may take decades to develop cardiovascular dysfunction following exposure to high doses of ionizing radiation from medical therapy or from nuclear accidents. Since astronauts may be exposed continually to a complex space radiation environment unlike that experienced on Earth, it is unresolved whether there is a risk to cardiovascular health during long-term space exploration missions. Previously, we have described that mice exposed to a single dose of simplified Galactic Cosmic Ray (GCR5-ion) develop cardiovascular dysfunction by 12months post-radiation. Methods: To investigate the biological basis of this dysfunction, here we performed a quantitative mass spectrometry-based proteomics analysis of heart tissue (proteome and phosphoproteome) and plasma (proteome only) from these mice at 8months post-radiation. Results: Differentially expressed proteins (DEPs) for irradiated versus sham irradiated samples (fold-change ≥1.2 and an adjusted p-value of ≤0.05) were identified for each proteomics data set. For the heart proteome, there were 87 significant DEPs (11 upregulated and 76 downregulated); for the heart phosphoproteome, there were 60 significant differentially phosphorylated peptides (17 upregulated and 43 downregulated); and for the plasma proteome, there was only one upregulated protein. A Gene Set Enrichment Analysis (GSEA) technique that assesses canonical pathways from BIOCARTA, KEGG, PID, REACTOME, and WikiPathways revealed significant perturbation in pathways in each data set. For the heart proteome, 166 pathways were significantly altered (36 upregulated and 130 downregulated); for the plasma proteome, there were 73 pathways significantly altered (25 upregulated and 48 downregulated); and for the phosphoproteome, there were 223 pathways significantly affected at 0.1 adjusted p-value cutoff. Pathways related to inflammation were the most highly perturbed in the heart and plasma. In line with sustained inflammation, neutrophil extracellular traps (NETs) were demonstrated to be increased in GCR5-ion irradiated hearts at 12-month post irradiation. NETs play a fundamental role in combating bacterial pathogens, modulating inflammatory responses, inflicting damage on healthy tissues, and escalating vascular thrombosis. Discussion: These findings suggest that a single exposure to GCR5-ion results in long-lasting changes in the proteome and that these proteomic changes can potentiate acute and chronic health issues for astronauts, such as what we have previously described with late cardiac dysfunction in these mice.

Late Effects of Ionizing Radiation on the Ultrastructure of Hepatocytes and Activity of Lysosomal Enzymes in Mouse Liver Irradiated In Vivo.

The study aimed to investigate late radiation-induced changes in the histology, ultrastructure, and activity of lysosomal enzymes in mouse liver exposed to ionizing radiation. The experiment was conducted on C57BL/6J male mice whose distal part of the liver was exposed occasionally to single doses of radiation (6 MV photons) during targeted heart irradiation; estimated doses delivered to analyzed tissue were 0.025 Gy, 0.25 Gy, 1 Gy, and 2 Gy. Tissues were collected 40 weeks after irradiation. We have observed that late effects of radiation have an adaptive nature and their intensity was dose-dependent. Morphological changes in hepatocytes included an increased number of primary lysosomes and autophagic vacuoles, which were visible in tissues irradiated with 0.25 Gy and higher doses. On the other hand, a significant increase in the activity of lysosomal hydrolases was observed only in tissues exposed to 2 Gy. The etiology of these changes may be multifactorial and result, among others, from unintentional irradiation of the distal part of the liver and/or functional interaction of the liver with an irradiated heart. In conclusion, we confirmed the presence of late dose-dependent ultrastructural and biochemical changes in mouse hepatocytes after liver irradiation in vivo.

Heart and Lung Dose as Predictors of Overall Survival in Patients With Locally Advanced Lung Cancer. A National Multicenter Study

IntroductionIt is an ongoing debate how much lung and heart irradiation impact overall survival (OS) after definitive radiotherapy for lung cancer. This study uses a large national cohort of patients with locally advanced NSCLC to investigate the association between OS and irradiation of lung and heart. MethodsTreatment plans were acquired from six Danish radiotherapy centers, and patient characteristics were obtained from national registries. A hybrid segmentation tool automatically delineated the heart and substructures. Dose-volume histograms for all structures were extracted and analyzed using principal component analyses (PCAs). Parameter selection for a multivariable Cox model for OS prediction was performed using cross-validation based on bootstrapping. ResultsThe population consisted of 644 patients with a median survival of 26 months (95% confidence interval [CI]: 24–29). The cross-validation selected two PCA variables to be included in the multivariable model. PCA1 represented irradiation of the heart and affected OS negatively (hazard ratio, 1.14; 95% CI: 1.04–1.26). PCA2 characterized the left-right balance (right atrium and left ventricle) irradiation, showing better survival for tumors near the right side (hazard ratio, 0.92; 95% CI: 0.84–1.00). Besides the two PCA variables, the multivariable model included age, sex, body-mass index, performance status, tumor dose, and tumor volume. ConclusionsBesides the classic noncardiac risk factors, lung and heart doses had a negative impact on survival, while it is suggested that the left side of the heart is a more radiation dose–sensitive region. The data indicate that overall heart irradiation should be reduced to improve the OS if possible.

Early Impairment of Paracrine and Phenotypic Features in Resident Cardiac Mesenchymal Stromal Cells after Thoracic Radiotherapy.

Radiotherapy-induced cardiac toxicity and consequent diseases still represent potential severe late complications for many cancer survivors who undergo therapeutic thoracic irradiation. We aimed to assess the phenotypic and paracrine features of resident cardiac mesenchymal stromal cells (CMSCs) at early follow-up after the end of thoracic irradiation of the heart as an early sign and/or mechanism of cardiac toxicity anticipating late organ dysfunction. Resident CMSCs were isolated from a rat model of fractionated thoracic irradiation with accurate and clinically relevant heart dosimetry that developed delayed dose-dependent cardiac dysfunction after 1 year. Cells were isolated 6 and 12 weeks after the end of radiotherapy and fully characterized at the transcriptional, paracrine, and functional levels. CMSCs displayed several altered features in a dose- and time-dependent trend, with the most impaired characteristics observed in those exposed in situ to the highest radiation dose with time. In particular, altered features included impaired cell migration and 3D growth and a and significant association of transcriptomic data with GO terms related to altered cytokine and growth factor signaling. Indeed, the altered paracrine profile of CMSCs derived from the group at the highest dose at the 12-week follow-up gave significantly reduced angiogenic support to endothelial cells and polarized macrophages toward a pro-inflammatory profile. Data collected in a clinically relevant rat model of heart irradiation simulating thoracic radiotherapy suggest that early paracrine and transcriptional alterations of the cardiac stroma may represent a dose- and time-dependent biological substrate for the delayed cardiac dysfunction phenotype observed in vivo.

Chest Wall to Heart Distance Reproducibility in Postoperative Deep Inspiration Breath-Hold Radiotherapy for Left-Sided Breast Cancer Using an Anzai Laser Sensor With Visual Feedback.

Background Left-sided breast cancer radiotherapy may increase the risk of cardiovascular death due to possible heart irradiation. Thereproducibility of the chest wall to heart distance in deep inspiration breath-hold (DIBH) was studied using a laser sensor with visual feedback. Methodology A total of 10 consecutive postoperative left-sided breast cancer cases receiving DIBH radiotherapy between December 2022 and September 2023 were retrospectively investigated. The prescribed dose was 50 Gy in 25 fractions. An Anzai respiratory gating system, AZ-733VI (Anzai, Tokyo, Japan), was employed that has a laser displacement sensor and a visual feedback device. An Elekta linac with a cone-beam CT unit, Axesse (Elekta AB, Stockholm, Sweden), was used in this study. The interfractional changes in the chest wall to heart distance among 25 fractions were analyzed for each of the 10 patients in each coordinate axis. In addition, the median with the 95% confidence interval (CI) and interquartile range (IQR) for all 250 fractions were calculated in each axis to assess the reproducibility of our DIBH technique. Results The medians of the interfractional changes in the chest wall to heart distance in each of the 10 patients ranged from -2 mm to 3 mm, -1 mm to 3 mm, and -2 mm to 1 mm in the lateral (X), superior-inferior (Y), and anterior-posterior (Z) directions, respectively. For all 10 cases, the medians were 1 mm (95% CI = 0.72 to 1.28 mm) in X, 1 mm (95% CI = 0.76 to 1.24 mm) in Y, and 0 mm (95% CI = -0.20 to 0.20 mm) in Z directions, whereas the IQRs were 4 mm in X, 2 mm in Y and 2 mm in Z directions. The measured IQRs were two to three times smaller than those shown in a previous report without visual feedback, suggesting a clinical advantage of the visual feedback in DIBH for left-sided breast cancer radiotherapy. The DIBH solution shown in this study required approximately 10 minutes from room-in to room-out, thereby not reducing the daily number of patients. Conclusions Our DIBH approach with visual feedback achieved better distance reproducibility between the chest wall and heart by a factor of two to three in terms of IQR compared to the previously reported data without visual feedback. Patient throughput was also favorable. To our knowledge, this is the first report demonstrating the chest wall to heart distance reproducibility in DIBH with visual feedback.

A multimodality assessment of the protective capacity of statin therapy in a mouse model of radiation cardiotoxicity

PurposeDespite technological advances in radiotherapy (RT), cardiotoxicity remains a common complication in patients with lung, oesophageal and breast cancers. Statin therapy has been shown to have pleiotropic properties beyond its lipid-lowering effects. Previous murine models have shown statin therapy can reduce short-term functional effects of whole-heart irradiation. In this study, we assessed the efficacy of atorvastatin in protecting against the late effects of radiation exposure on systolic function, cardiac conduction, and atrial natriuretic peptide (ANP) following a clinically relevant partial-heart radiation exposure. Materials and MethodsFemale, 12-week old, C57BL/6j mice received an image-guided 16 Gy X-ray field to the base of the heart using a small animal radiotherapy research platform (SARRP), with or without atorvastatin from 1 week prior to irradiation until the end of the experiment. The animals were followed for 50 weeks with longitudinal transthoracic echocardiography (TTE) and electrocardiography (ECG) every 10 weeks, and plasma ANP every 20 weeks. ResultsAt 30–50 weeks, mild left ventricular systolic function impairment observed in the RT control group was less apparent in animals receiving atorvastatin. ECG analysis demonstrated prolongation of components of cardiac conduction related to the heart base at 10 and 30 weeks in the RT control group but not in animals treated with atorvastatin. In contrast to systolic function, conduction disturbances resolved at later time-points with radiation alone. ANP reductions were lower in irradiated animals receiving atorvastatin at 30 and 50 weeks. ConclusionsAtorvastatin prevents left ventricular systolic dysfunction, and the perturbation of cardiac conduction following partial heart irradiation. If confirmed in clinical studies, these data would support the use of statin therapy for cardioprotection during thoracic radiotherapy.

TFAM Modulates Cardiomyocytes Pyroptosis Induced by Ionizing Radiation through mtDNA/TLR9/NF-kB Pathway

Mitochondrial transcription factor A (TFAM) is a pivotal factor for regulating mitochondrial DNA (mtDNA) replication, transcription and biogenesis. Previous studies have reported that cytosolic mtDNA stress can lead to cardiomyocytes pyroptosis, which is characterized by inflammasome formation. In this study, we attempted to investigate the mechanism of TFAM regulate cardiomyocytes pyroptosis induced by ionizing radiation. The peripheral blood serum of patients with esophageal cancer before and after definitive chemoradiotherapy was collected for Luminex multiplex cytokine assays. C57BL/6 mice were irradiated with the whole heart using small animal radiation research platform (SARRP) to construct a radiation-induced myocardial damage (RIMD) mouse model, and the ventricular function was evaluated using 9.4T Bruker magnetic resonance (MR) scanner. The function changes of cardiomyocytes exposed to radiation were observed in vitro and in vivo after knocking out GSDMD. Furthermore, the changes of mitochondrial function, the levels of cytosolic mtDNA, and the protein levels of NF-kB and pyroptosis pathway in irradiated cardiomyocytes were analyzed by knockdown and overexpression of TFAM in vitro and in vivo. By multifactor cytokine assays we found that pyroptosis related IL-1β and IL-18 were significantly increased in patients with high mean heart dose (MHD) after radiotherapy, while those with low MHD were not significantly increased after radiotherapy. Next, we successfully constructed the RIMD mouse model using a single heart irradiation of 20 Gy. We found that the gene expression of pyroptosis pathway was significantly up-regulated after cardiac irradiation by myocardial tissue transcriptomic sequencing. Compared with wild-type (WT) mice, cardiac systolic function of Gsdmd-/- mice was significantly improved at 1, 2, 6, 12, and 24 weeks after heart irradiation. In vitro, we also demonstrated increased viability of irradiated cardiomyocytes by knocking out GSDMD. In vitro and in vivo experiments confirmed the expression of TFAM decreased after radiation. By overexpression of TFAM, we found that irradiated cardiomyocytes showed improved mitochondrial function, decreased release of mtDNA into cytoplasm through mitochondrial permeability transition pores (mPTPs), decreased binding of cytosolic mtDNA to TLR9, and decreased expression of NF-kB and pyroptosis pathway proteins. Dual luciferase gene reporter assays and Chromatin immunoprecipitation (CHIP) assay confirmed that p65 could bind the NLRP3 promoter region. In addition, we found that ventricular function deteriorated and improved in mice with knockdown and overexpression of TFAM through adeno-associated virus serotype 9 (AAV9), respectively. Our study indicated that TFAM regulate irradiated cardiomyocytes pyroptosis through mtDNA/TLR9/NF-kB pathway. We provide a novel mechanism of RIMD, revealing an underappreciated intervention target for RIMD.

Chronic inflammatory effects of in vivo irradiation of the murine heart on endothelial cells mimic mechanisms involved in atherosclerosis

PurposeRadiotherapy is a major pillar in the treatment of solid tumors including breast cancer. However, epidemiological studies have revealed an increase in cardiac diseases approximately a decade after exposure of the thorax to ionizing irradiation, which might be related to vascular inflammation. Therefore, chronic inflammatory effects were examined in primary heart and lung endothelial cells (ECs) of mice after local heart irradiation.MethodsLong-lasting effects on primary ECs of the heart and lung were studied 20–50 weeks after local irradiation of the heart of mice (8 and 16 Gy) in vivo by multiparameter flow cytometry using antibodies directed against cell surface markers related to proliferation, stemness, lipid metabolism, and inflammation, and compared to those induced by occlusion of the left anterior descending coronary artery.ResultsIn vivo irradiation of the complete heart caused long-lasting persistent upregulation of inflammatory (HCAM, ICAM‑1, VCAM-1), proliferation (CD105), and lipid (CD36) markers on primary heart ECs and an upregulation of ICAM‑1 and VCAM‑1 on primary ECs of the partially irradiated lung lobe. An artificially induced heart infarction induces similar effects with respect to inflammatory markers, albeit in a shorter time period.ConclusionThe long-lasting upregulation of prominent inflammatory markers on primary heart and lung ECs suggests that local heart irradiation induces chronic inflammation in the microvasculature of the heart and partially irradiated lung that leads to cardiac injury which might be related to altered lipid metabolism in the heart.

A pooled analysis of nine studies in one institution to assess effects of whole heart irradiation in rat models

Purpose Over the years, animal models of local heart irradiation have provided insight into mechanisms of and treatments for radiation-induced heart disease in human populations. However, it is not completely clear which manifestations of radiation injury are most commonly seen after whole heart irradiation, and whether certain biological factors impact experimental results. Combining 9 homogeneous studies in rat models of whole heart irradiation from one laboratory, we sought to identify experimental and/or biological factors that impact heart outcomes. We evaluated the usefulness of including (1) heart rate and (2) bodyweight as covariates when analyzing biological parameters, and (3) we determined which echocardiography, histological, and immunohistochemistry parameters are most susceptible to radiation effects. Finally, (4) as an educational example, we illustrate a hypothetical sample size calculation for a study design commonly used in evaluating radiation modifiers, using the pooled estimates from the 9 rat studies only for context. The results may assist investigators in the design and analyses of pre-clinical studies of whole heart irradiation. Materials and Methods We made use of data from 9 rat studies from our labs, 8 published elsewhere in 2008–2017, and one unpublished study. Echocardiography, histological, and immunohistochemical parameters were collected from these studies. Using mixed effects analysis of covariance models, we estimated slopes for heart rate and bodyweight and estimated the radiation effect on each of the parameters. Results Bodyweight was related to most echocardiography parameters, and heart rate had an effect on echocardiography parameters related to the diameter of the left ventricle. For some parameters, there was evidence that heart rate and bodyweight relationships with the parameter depended on whether the rats were irradiated. Radiation effects were found in systolic measures of echocardiography parameters related to the diameter of the left ventricle, with ejection fraction and fractional shortening, with atrial wall thickness, and with histological measures of capillary density, collagen deposition, and mast cells infiltration in the heart. Conclusion Accounting for bodyweight, as well as heart rate, in analyses of echocardiography parameters should reduce variability in estimated radiation effects. Several echocardiography and histological parameters were particularly susceptible to whole heart irradiation, showing robust effects compared to sham-irradiation. Lastly, we provide an example approach for a sample size calculation that will contribute to a rigorous study design and reproducibility in experiments studying radiation modifiers.

Effects of low-to-moderate doses of X-rays on the cardiac rhythm via the sympathetic nervous system in female mice

Radiotherapy is one of the common therapeutic approaches in the treatment of breast cancer, with the heart inevitably being exposed to ionizing radiation which may cause late cardiac pathologies. Our recent study revealed that right atrium irradiation of right-sided breast cancer patients is associated with the development of cardiac diseases such as heart failure, arrhythmias, and conduction disorders. The aim of this project is to examine the effect of low to moderate doses of X-rays on the development of late cardiac pathologies. We specifically studied the alterations in the cardiac sympathetic nervous system which is located in potentially arrhythmogenic structures of the sinoatrial and atrioventricular nodes. Female C57Bl/6J mice were exposed to X-rays irradiation targeted to top, bottom or whole heart, such that different cardiac nodes were exposed. To examine the presence of a threshold in a dose-response for cardiac rhythm pathology, as assessed by ultrasound examinations, mice were exposed to doses ranging from 0.25 to 2 Gy. Molecular and cellular mechanisms involved in these cardiac disorders at early (24-hours) and late (60-weeks) time points post-irradiation were studied. At the functional level, our Doppler echography data show the presence of an alteration of cardiac function with the occurrence of arrhythmic events at 60-weeks post-irradiation in all irradiated animals, specifically in top heart irradiation. To confirm these results, we will also use transesophageal atrial stimulation (10 Hz during 3 sec) to assess supraventricular arrhythmias. At the molecular level, we focused on proteins involved in cardiac conduction system and in sympathetic innervation patterning 24-hours post-irradiation. Our Western Blot results show a significant increase of Connexin 40 (P = 0.02), Connexin 43 (P = 0.02), Tyrosine Hydroxylase (P = 0.04), and b3-Tubulin (P = 0.03) protein levels in whole heart irradiated animals. Altogether, our preliminary results showed modifications of cardiac function 60-weeks post-irradiation with the development of cardiac rhythm disorders and molecular alterations of cardiac conduction and sympathetic nervous system already present 24-hours post-irradiation.

Abstract 3584: Early and non-invasive detection of radiation-induced cardiotoxicity in pre-clinical and clinical models

Abstract Background: Nearly half of all patients receive radiation therapy as a component of their cancer care. Despite the efficacy of radiation therapy as a cancer treatment, cardiotoxicity is a major concern in patients receiving chest radiotherapy. There are currently no standardized approaches for early detection of radiation-induced cardiotoxicity as a stage that offers potential for early intervention. In this study, we employ hyperpolarized 13C- pyruvate magnetic resonance spectroscopy to non-invasively characterize early metabolic changes in the heart in response to radiation. Methods: We established a pre-clinical model of radiation induced heart disease (RIHD) by performing whole heart irradiation (8Gy x 5) in rats. Echocardiography was used characterize mechanical changes in the heart following radiation. To non-invasively detect myocardial mitochondrial dysfunction following cardiac irradiation in vivo, we employed hyperpolarized 13C- pyruvate magnetic resonance spectroscopy (MRS) to track the fate of pyruvate, an intermediate of glucose metabolism, as it is metabolized in the heart. Hyperpolarized 13C- pyruvate MRS was also employed in a patient who received thoracic radiation for treatment of thymoma to non-invasively study changes in glucose metabolism in response to radiation. Results: We identified evidence of early cardiac mitochondrial dysfunction prior to onset of mechanical changes in the heart. Following cardiac irradiation, due to mitochondrial dysfunction, pyruvate was preferentially metabolized to lactate in the cytoplasm (as opposed to bicarbonate in the mitochondria in non-irradiated hearts). The ratio of products of 13C- pyruvate metabolism in the mitochondria and cytoplasm as ascertained by hyperpolarized 13C- pyruvate MRS served as a biomarker for metabolic dysfunction in the heart both in a pre-clinical rat model and in a patient who received thoracic radiation. Conclusions: We have developed a non-invasive approach for detection of cardiac mitochondrial dysfunction, which may serve as a biomarker for early detection of radiation-induced cardiotoxicity, and shown feasibility in a human patient. Clinical adoption of this approach may enable early identification of patients who are at high risk for future cardiac complications and may benefit from mitigation strategies. Citation Format: Taylor-Jade Higgins, Jayesh Sharma, Elizabeth R. Zhang-Velten, Sarah Elliott, Junjie Ma, Jun Chen, Gabriele Schiattarella, Chantal Vidal, Nan Jiang, Daniel Daou, Joseph Hill, Thomas Gillette, Craig Malloy, Vlad Zaha, Jae Mo Park, Prasanna Alluri. Early and non-invasive detection of radiation-induced cardiotoxicity in pre-clinical and clinical models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3584.

Abstract 3209: Metabolic characterization of radiation-induced cardiotoxicity

Abstract Background: Approximately 50% of all cancer patients receive radiation therapy as a component of their cancer care. While radiation therapy has demonstrable benefit in improving survival in patients, radiation-induced heart disease (RIHD) is a major contributor to morbidity and mortality in cancer survivors with history of thoracic radiation. Understanding metabolic changes underlying RIHD may enable development of novel diagnostic and therapeutic approaches for early detection and treatment of RIHD. The high energetic demands of the heart require properly functioning mitochondria to meet the ATP production needs. Fatty acids are the primary metabolic substrates for energy production in a healthy heart. However, under conditions of stress, the cardiac metabolism shifts towards increased glucose utilization. In this study, we characterize the impact of radiation of cardiac substrate utilization and energy production. Methods: Pre-clinical RIHD models were established by administering whole heart radiation (8Gy x 5) in mice and rats followed by structural and biochemical characterization. Transmission electron microscopy and western blot analyses were used to characterize structural and biochemical changes in the heart in response to radiation. Spectrophotometric assays and Western blot analyses were used to characterize changes in pyruvate dehydrogenase (PDH) activity and function in the heart in response to radiation. Results: Our studies identified myocardial mitochondrial dysfunction as an early event following exposure of heart to radiation. Cardiac irradiation resulted in increased mitochondrial fusion and higher expression of MFN1, a key protein that mediates mitochondrial fusion. The pathological structural and biochemical changes in the myocardial mitochondria are associated with reduced glucose metabolism due to impaired PDH activity. This decline was mediated by increased phosphorylation of PDH in irradiated hearts, which inhibits its activity. Conclusion: Cardiac irradiation results in early metabolic dysregulation, especially in glucose metabolism, which may be exploited for early detection and mitigation of radiation-induced cardiotoxicity. Citation Format: Sarah C. Elliott, Jayesh Sharma, Elizabeth R. Zhang-Velten, Abdallah Elnwasany, Chantal Vidal, Luke Szweda, Prasanna Alluri. Metabolic characterization of radiation-induced cardiotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3209.

Effects of Whole and Partial Heart Irradiation on Collagen, Mast Cells, and Toll-like Receptor 4 in the Mouse Heart.

In radiation therapy of tumors in the chest, such as in lung or esophageal cancer, part of the heart may be situated in the radiation field. This can lead to the development of radiation-induced heart disease. The mechanisms by which radiation causes long-term injury to the heart are not fully understood, but investigations in pre-clinical research models can contribute to mechanistic insights. Recent developments in X-ray technology have enabled partial heart irradiation in mouse models. In this study, adult male and female C57BL/6J mice were exposed to whole heart (a single dose of 8 or 16 Gy) and partial heart irradiation (16 Gy to 40% of the heart). Plasma samples were collected at 5 days and 2 weeks after the irradiation for metabolomics analysis, and the cardiac collagen deposition, mast cell numbers, and left ventricular expression of Toll-like receptor 4 (TLR4) were examined in the irradiated and unirradiated parts of the heart at 6 months after the irradiation. Small differences were found in the plasma metabolite profiles between the groups. However, the collagen deposition did not differ between the irradiated and unirradiated parts of the heart, and radiation did not upregulate the mast cell numbers in either part of the heart. Lastly, an increase in the expression of TLR4 was seen only after a single dose of 8 Gy to the whole heart. These results suggest that adverse tissue remodeling was not different between the irradiated and unirradiated portions of the mouse heart. While there were no clear differences between male and female animals, additional work in larger cohorts may be required to confirm this result, and to test the inhibition of TLR4 as an intervention strategy in radiation-induced heart disease.

Radiation-Associated Cardiotoxicity during Breast Cancer Treatment with Ionizing Radiation

Introduction: Breast cancer is the most common cancer in women. The treatment of breast carcinoma has advanced in the last decade and nowadays there are treatment protocols for all stages of the disease. Depending on the histopathology and stage breast cancer is treated with surgery, chemotherapy and radiotherapy. Regarding radiation, the field of irradiation includes the chest wall in patients with mastectomy, or the breast glandular tissue in patients with conserving surgical approaches. It is often treated with radiation therapy with two opposing tangential fields, and when indicated supraclavicular lymph nodes have to be irradiated. In this case an additional anterior field is applied. The tangential as well as the other radiation beams have a potential damaging effect on the healthy surrounding tissues, particularly over the heart in the left breast irradiation and in the lungs as well. Material and Methods: The study included 25 patients with left breast carcinoma, all post surgery, treated with radiation therapy, with the Elekta accelerator at our department. For academic purpose the treatment plans were generated following two methods. The first one with two tangential opposite beams plus a supraclavicular beam. In this method the angles of the tangential internal and external create an angle that is equal to 180&#730 {310&#730& 130&#730}; no further changes were made to the beam geometry. Even though this is not the best option from the dose distribution point of view, it is still the most applied method, probably because of the semplicity of it. For each patient, a second plan was generated using two opposite tangential beams plus the supraclavicular beam. The angles of the internal and external beam were changed from 1&#730to 3&#730, depending on the surface of the body, so that the resulting angle was 180&#730± 3&#730 {310&#730± 3&#730& 130&#730± 3&#730} with the aim to adapt the beam geometry as much as possible to the shape of the thoracic wall and to spare the OAR-s. Results and Discussion: The data show that the dose in the organs at risk, in terms of dose percentage, is lower when the angles of the beams are changed with 1&#730-3&#730, compared to the classic method where the internal and external angles equal 180&#730. This dose is not only non-negligible but significant; for every angle change from 1&#730to 3&#730, there is a significant reduction in the integral dose in the radiated volume, expressed in percentage, up to 5%. Conclusion: In most centers, the radiation treatment of breast is realized with two tangential opposite beams, which usually are mirror beams, or in other words, the internal and external beam angles create an angle of 180&#730 {α + β = 180&#730}. This is a simple method, which provides a good dose distribution, but leaves a relatively high dose in the organs at risk. This study shows the difference in the dose percentage in the heart and lung when the beam angles are changed adapting to the anatomy of the patient. Reducing these doses allows for better overall treatment and less longtime toxicity, particularly for the heart tissues.