Single‐Cell RNA Sequencing Reveals Macrophage–Endothelial Cell Crosstalk in Viral Myocarditis

Viral myocarditis is characterized by inflammatory cell infiltration and myocardial damage. However, the involvement of immune cells and the interaction between immune cells and stromal cells remain poorly understood. We successfully established a mouse model of viral myocarditis induced by Coxsackievirus B3 (CVB3) and systematically analyzed immune cell infiltration and myocardial injury at different time points. Single-cell RNA sequencing (scRNA-seq) was performed at the peak of immune cell infiltration to characterize the immune landscape of infected cardiac tissue and peripheral blood mononuclear cells (PBMCs). Macrophage depletion and vascular endothelial growth factor receptor (VEGFR) inhibition were performed to validate the immune-stromal crosstalk. Peak immune cell infiltration and myocardial injury occurred on the 7th day of infection. scRNA-seq revealed that endothelial cells and mononuclear phagocytes (MNPs) were the most substantially expanded cell populations in the hearts of mice with viral infection. Trem2 macrophage, characterized by tissue repair gene signatures, was the predominant MNP subcluster in the infected heart, while tip cells and capillaries were the most expanded endothelial cell clusters. Cell-cell communication analysis identified increased macrophage-endothelial cell interactions during CVB3 infection. Macrophage-derived VEGFA secretion, partially induced by CVB3 infection and apoptotic cardiomyocyte debris, promoted angiogenesis, while macrophage depletion resulted in reduced VEGFA secretion and endothelial proliferation. Moreover, inhibition of VEGFR exacerbated cardiac dysfunction, highlighting the protective role of angiogenesis in myocarditis progression. In summary, these results elucidated a cardioprotective role of macrophage-driven angiogenesis via vascular endothelial growth factor signaling during viral myocarditis, providing new insights into therapeutic strategies for inflammatory heart diseases.