Single-Cell RNA Sequencing Reveals a Subset of cMAS can Aggravate RIHD through CXCL1-CXCR2 Axis

Abstract

Radiation induced heart disease (RIHD) is any form of cardiac toxicity induced by radiation therapy (RT) for thoracic cancers. Our previous studies have shown that RT obviously contributed to cardiovascular diseases-specific death over 3 years while RT became protective in the short term within 2 years survival in non-small cell lung cancer patients. Here, single cell RNA sequencing (scRNA-seq) was performed to identify various cell subsets and investigate their functions and dynamics in RIHD which offered several targets for early clinical interventions to alleviate RIHD. Based on evaluation of histopathological characteristics, ejection fraction and serum levels of cardiac injury biomarkers, we have established mouse models during different stages to simulate clinical RIHD progression. Hence, we performed single cell RNA-sequencing of RIHD models to characterize the diversity within specific cell types and obtain basic information of differently expressed genes (DEGs). We investigated the role of several cell clusters and DEGs in RIHD through bioinformatics analysis and experimental verification. In vivo, mouse models were given intraperitoneal injection of CXCR2 inhibitor. Bone marrow macrophages and primary cardiac fibroblasts were extracted for in vitro experiments. RIHD processes were divided into acute injury, compensation and decompensation stage. Transcriptomes of 31769 single cells from cardiac suspension have been profiled. Analysis of scRNA-seq revealed that there were 30 cell clusters participating in RIHD. The fraction of cell populations varied greatly at three stages which indicated RIHD was a dynamic process and each cell cluster functioned differently at different stages. Notably, we observed cardiac resident macrophages (cMAS) subset accounted for the highest fraction during the compensatory period and decreased in decompensation period. Pseudotime analysis showed cMAS had a different developmental trajectory compared to myeloid derived cells. Moreover, CXCR2 was significantly expressed in cMAS cluster. Ligand-receptor interaction results suggested that CXCL1 secreted by cardiac fibroblasts bind primarily to CXCR2+ cMAS and participated in the formation of the extracellular matrix (ECM) related to cardiac fibrosis. Moreover, cardiac fibrosis of RIHD models were relieved after CXCR2 inhibitor treatment. CXCL1 expression in primary cardiac fibroblast elevated after RT. The identification of main cell clusters provided a new insight to investigate RIHD through dynamics of cell phenotypes and cell-cell communications during RIHD processes. In compensation stage, CXCR2+ cMAS could be activated by CXCL1 secreted by cardiac fibroblasts. Both were associated with ECM and contribute to the decompensation stage.