Immune and stromal remodeling underlies radiation-induced heart injury: insights from single-cell transcriptomics.

Background: Radiation-induced heart injury (RIHI) is a major late toxicity of thoracic radiotherapy, yet the cellular and molecular mechanisms driving its progression remain poorly defined. Methods: Single-cell RNA sequencing (scRNA-seq) was performed on rat hearts and matched peripheral blood mononuclear cells (PBMCs) 12 weeks after whole-heart irradiation (20 Gy) or sham control, profiling 38,941 cardiac cells across 15 types and 41,097 PBMCs across 9 types. Differential expression, pathway enrichment, pseudotime, and ligand-receptor interaction analyses were conducted. Key findings in endothelial cells and fibroblasts were validated by Western blotting and flow cytometry. Results: Major cardiac populations, including cardiomyocytes, endothelial cells (ECs), fibroblasts, neutrophils, macrophages, T cells, NK cells, and B cells, were defined in control and RIHI hearts. Following irradiation, ECs showed distinct subtype shifts with marked MHC-II upregulation, while fibroblasts exhibited iron accumulation, pro-inflammatory activation, and antigen-presenting properties. These stromal alterations coincided with myeloid activation (macrophage and IL-1β⁺ neutrophil programs) and T/NK cell polarization toward cytotoxic yet partially exhausted states, together with enhanced B-cell antigen presentation. Collectively, these findings delineate a stromal-immune cascade linking radiation injury to chronic cardiac inflammation. Conclusion: RIHI progresses through a stromal-immune cascade where EC and fibroblast immunogenic reprogramming initiates sustained myeloid and lymphoid activation, creating a pro-inflammatory cardiac microenvironment. These findings highlight non-hematopoietic antigen presentation as a therapeutic target in thoracic radiotherapy, particularly when combined with immune checkpoint inhibitors.