Identification of Pathways and Genetic Variants Important for Radiation-Induced Cardiotoxicity Using Genetic Mapping

Abstract

Over 50% of cancer patients use radiation therapy (RT), but doses can be limited by normal tissue side effects. While RT can improve cancer-specific survival, cardiac morbidity can be increased in patients receiving RT to the heart. Mounting evidence suggests complex genetic modifiers contribute to the risk of RT-induced toxicity, but these genetic modifiers remain largely unknown and poorly understood. Thus, we have developed the first genetic mapping model of radiation-induced cardiotoxicity. Salt-sensitive (SS) and Brown Norway (BN) rats were selectively bred to produce SS.BN3 consomic rats genetically identical to SS rats, except chromosome 3 is inherited from the BN strain. SS and SS.BN3 rats were bred to produce 4 congenic rats that break up chromosome 3: CG1, CG2, CG3 and CG4. Adult female rats received image-guided whole-heart RT (24 Gy x1 or 9 Gy x5, AP:2 laterals). Echocardiograms with strain analysis, RNA-sequencing with pathway analysis, and IHC staining for immune infiltrates were performed. Student’s t-test and Fisher exact test were used for comparisons. SS rats exhibited enhanced cardiac toxicity vs. SS.BN3 rats. Increased cardiac mortality was observed after 24Gy (p<0.01). At 5 months, SS rats had increased pleural effusions (12.9 vs. 3.1 ml, p<0.01). Ejection fraction was lower in SS rats (81% vs. 94%, p<0.01), and other echocardiogram parameters indicated worse cardiac function in the SS rats. Similar results were seen with 9 Gy x5 and in male rats. IHC revealed increased mast cells in the SS.BN3 rat hearts (p<0.01), but decreased macrophages in SS.BN3 rat hearts at 10 weeks post-RT (p<0.05). RNA-seq demonstrated mitochondrial dysfunction pathways were differentially expressed in SS vs. SS.BN3 hearts. Only the CG4 congenic had heart damage similar to SS rats, with post-RT cardiac hypertrophy only seen in SS and CG.4, pleural effusions largest in SS and CG.4, and the lowest cardiac strain seen in SS and CG.4 (p<0.01). CG.4 contains a 25 MB unique region of SS chromosome 3 (0.9% of genome), containing 93 differentially expressed genes. SS rats are more sensitive to cardiac RT than SS.BN3 rats, demonstrating the role of heritable factors in determining cardiac radiosensitivity. Genetic mapping narrowed the region to a 25 MB region with <100 differentially expressed genes. RNA-seq identified potentially important pathways for cardiotoxicity. Immune cells differentially infiltrated the hearts post-RT, with increased mast cells in the more resistant SS.BN3 hearts, consistent with the protective role of mast cells post-cardiac RT in previous studies. Macrophage increases in the SS heart may be due to cardiac damage. Further genetic mapping and targeted studies of the genes/pathways and immune responses important in this model are ongoing. This project has the potential to enhance the effectiveness and toxicity profile of RT.