Cardiac Macrophages Exhibit Dynamic Heterogeneity and Functional Specialization During Experimental Autoimmune...

Autoimmune myocarditis frequently progresses to inflammatory cardiomyopathy through dysregulated immune-stromal interactions. This study employs single-nuclei RNA-sequencing (snRNA-seq) to profile 46,233 cardiac nuclei from the experimental autoimmune myocarditis (EAM) mouse model at four timepoints: day 0 (healthy), day 14 (inflammation), day 21 (acute inflammation), and day 40 (late cardiac remodelling). Single-nuclei RNA profiling identified 18 transcriptionally distinct cell populations. Global cell-cell communication analysis revealed a dramatic peak of intercellular signalling at day 14 (5907 interactions), with fibroblast subpopulations and macrophages as dominant hubs, followed by partial resolution at day 21 (2264 interactions) and renewed remodelling at day 40 (4862 interactions). Subclustering of the macrophage compartment identified five subpopulations: Mac-TLF, Mac-MHCII, Mac-rMHCII, Mac-ResL, and Classical Monocytes. Tissue-resident macrophages (Mac-TLF, CCR2-) dominated at healthy state (~55%) but were rapidly depleted at day 14, coinciding with a dramatic influx of recruited CCR2⁺ macrophages (Mac-rMHCII), which expanded to over 70% of the compartment and maintained dominance through day 40. At inflammation (day 14), the expanded Mac-rMHCII subpopulation displayed a strongly pro-inflammatory signature (Il1b, Stat2, Parp14, Apoe), and the overall macrophage compartment was enriched for cytokine response, Fc-gamma receptor, and Notch signalling pathways, while downregulating homeostatic and mitochondrial metabolic programmes, potentially contributing to impaired efferocytosis and cardiomyocyte dysfunction. Macrophage-centred communication networks expanded markedly at day 14 (1047 interactions), with resting fibroblasts (FB-R) as the primary signalling partner, driving pro-inflammatory stromal activation marked by upregulation of Ccl2, Ccl7, and Csf2. Intra-macrophage subcluster communication also intensified at this timepoint (447 interactions). These findings delineate the temporal and functional heterogeneity of cardiac macrophages during EAM progression and identify key immune-stromal interactions driving pathological cardiac remodelling. The coexistence of pro-inflammatory and transitional reparative macrophage subsets highlights the limitations of broad immunosuppression and supports precision strategies targeting CCR2-mediated recruitment, the SPP1 signalling axis, and macrophage-fibroblast crosstalk as therapeutic avenues in myocarditis and its progression.