CCL8-mediated recruitment of cytotoxic lymphocytes by P16-positive cells promotes adverse post-ischemic cardiac remodeling

BACKGROUND: Ischemic heart disease remains a leading cause of mortality worldwide, with adverse remodeling post-myocardial infarction (MI) driven by inflammation and cardiomyocyte loss. While cytotoxic lymphocytes exacerbate injury, and p16 marks cellular senescence in diseased hearts, the cell-type-specific roles of P16+ populations remain undefined. METHODS: Using p16-CreER;R26-tdT reporter mice, we mapped P16+ cellular heterogeneity post-MI. Senolytic effects were assessed via combination of dasatinib and quercetin (DQ) administration. Transcriptomic profiling (bulk/scRNA-seq) of sorted P16+ cells identified secreted factors, validated by in silico predictions and qPCR. Intercellular communication was analyzed using CellChat. Functional roles were tested via CCL8 neutralization, genetic Ccl8 ablation in P16+ cells, lymphocyte depletion, and intersectional genetic ablation of P16+ fibroblasts/macrophages using dual-recombinase systems (p16-DreER;Pdgfra-CreER;R26-lr-tdT-DTR and p16-DreER;Cx3cr1-CreER;R26-lr-tdT-DTR). RESULTS: P16 was induced in fibroblasts, macrophages, coronary endothelial cells, and cardiomyocytes post-MI. DQ treatment selectively eliminated P16+ macrophages and fibroblasts, improving cardiac function. Transcriptomics revealed P16+ macrophages and fibroblasts as primary CCL8 sources. CCL8 blockade reduced infiltration of cytotoxic lymphocytes (CD8+ T cells and NK cells), decreased cardiomyocyte apoptosis, and improved repair. Genetic Ccl8 ablation in P16+ cells replicated these benefits. Crucially, intersectional genetic ablation of P16+ fibroblasts, but not macrophages, reduced fibrosis and enhanced function, whereas CD8+ T cell (not NK cell) depletion attenuated remodeling. CONCLUSIONS: P16+ cells orchestrate adverse post-MI remodeling via CCL8-dependent recruitment of cytotoxic lymphocytes, expecially CD8+ T cells, driving cardiomyocyte apoptosis. Precision targeting of P16+ fibroblasts or CCL8 blockade represents a promising therapeutic strategy for ischemic heart disease. Overall design: p16-2A-CreER;R26-mTmG mice underwent myocardial infarction (MI) surgery, received tamoxifen via oral gavage 5 days post-MI. Non-cardiomyocyte cells were harvested from the cardiac infarct zone on day 7 to prepare a single-cell suspension. GFPhi and GFPlo non-cardiomyocyte cells were subsequently isolated by flow cytometry sorting (FACS) and analyzed using single-cell RNA sequencing (scRNA-seq).