Cardiac Macrophages and Fibroblasts Modulate Atrial Fibrillation Maintenance

Background: Nonmyocytes may contribute to regional adaptive changes during persistent atrial fibrillation (PsAF), favoring its perpetuation. We aimed to investigate the differential features of fibroblast and macrophage populations within individual-specific atrial regions associated with PsAF maintenance. Methods: The study was conducted in 2 pig models of PsAF with and without infarct-related substrate (N=27 and N=27, respectively) and further validated in humans with PsAF (N=20). Sham-operated pigs (N=9), healthy animals (N=4), and patients in sinus rhythm (N=7) were used as comparative controls. In pigs, in vivo high-density instantaneous frequency modulation maps were used to identify atrial regions associated with PsAF maintenance (drivers). Regional cellular composition and phenotypic states of fibroblast and myeloid lineages were determined using flow cytometry, single-cell RNA sequencing, immunohistochemistry, and proteomic analyses. The functional relevance of driver regions was further studied in patients with symptomatic PsAF undergoing ablation. Flow cytometry and single-cell RNA sequencing analyses were performed in tissue samples of the left atrial appendage in a complementary cohort of patients with PsAF undergoing thoracoscopic-guided ablation. Results: PsAF terminated acutely in 12 of 14 pigs undergoing mapping and ablation of driver regions. In humans, driver ablation was associated with 90% AF-freedom (on/off antiarrhythmic drugs) after 2 years of follow-up. Samples from nonablated pigs revealed a phenotypic shift towards ACTA2 (actin alpha 2)-fibroblasts and PTX3-fibroblasts during PsAF. Although ACTA2-fibroblasts were highly preserved in human samples, paired comparisons in pig samples showed that PTX3 (pentraxin 3)-fibroblasts were enriched only in driver regions. PsAF also showed changes in myeloid cells towards inflammatory profiles. However, regional analysis revealed that, in both humans and pigs with PsAF, driver regions were enriched in cardiac resident macrophages with transcriptomic and proteomic profiles favoring cardiomyocyte homeostasis and cell survival. Conclusions: PsAF shows differential regional changes in fibroblast and myeloid populations with distinctive gene signatures in areas that drive the overall arrhythmia.