Simon-Chica, AnaQuintanilla, Jorge GTorroja, CarlosCouselo-Seijas, MarinelaToda, HarukaLee, PeterBenguria, AlbertoRevilla, ConcepciónRedondo-Rodríguez, AndrésAlfonso-Almazán, José ManuelGarcía-Escolano, AlbaMarina-Breysse, ManuelGalán-Arriola, CarlosVera-Pedrosa, María LinarejosLa Rosa, GiulioDopazo, AnaSánchez-Cabo, FátimaGarcía-Torrent, María JesúsOrtega-Hernández, AdrianaIbáñez, BorjaNúñez, EstefaníaGómez-Garre, DulcenombreMorillo, CarlosGreiner, JoachimKohl, PeterPérez-Villacastín, JuliánPérez-Castellano, NicasioJalife, JoséDomínguez, JavierVázquez, JesúsCarnero-Alcázar, ManuelFilgueiras-Rama, David2026-07-162026-07-162026-03-13Circ Res. 2026 Mar 13;138(6):e326291.https://hdl.handle.net/20.500.12105/27601Nonmyocytes 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. 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. 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 (pentraxin 3)-fibroblasts during PsAF. Although ACTA2-fibroblasts were highly preserved in human samples, paired comparisons in pig samples showed that PTX3-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. PsAF shows differential regional changes in fibroblast and myeloid populations with distinctive gene signatures in areas that drive the overall arrhythmia.This work was supported by the European Union Horizon 2020 research and innovation program (grant agreement no. 965286), the Ministry of Science and Innovation (MCIN; PID2023-150456OB-I00 and PGC2018-097019-B-I00) funded by MCIN/AEI (Agencia estatal de Investigacion)/10.13039/501100011033, the Instituto de Salud Carlos III (ISCIII; Fondo de Investigacion Sanitaria grant PRB3; PT17/0019/0003- ISCIII-SGEFI/FEDER, ProteoRed), the Fundacion Interhospitalaria para la Investigacion Cardiovascular, Sociedad Espanola de Cardiologia, and la Caixa Banking Foundation (HR17-00247). The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the ISCIII, MCIN, and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). Microscopy experiments were performed at the Microscopy and Dynamic Imaging Unit, CNIC, Infraestructuras Cientificas y Tecnicas Singulares (ICTS)-Red Distribuida de Imagen Biomedica (ReDib), cofunded by MCIN/AEI/10.13039/501100011033 and Fondo Europeo de Desarrollo regional, (FEDER) Una manera de hacer Europa (ICTS-2018-04-CNIC-16). A. Simon-Chica was supported by a PhD fellowship from La Caixa (LCF/BQ/DR19/11740029). A. Simon-Chica, J. Greiner, and P. Kohl are members of the German Center of Research Excellence 1425 (Deutsche Forschungsgemeinschaft, DFG): 422681845. M. Couselo-Seijas was supported by MICIU/AEI/10.13039/501100011033 and by FSE+ (grant no. JDC2023-050723-I).engVoRatrial appendageatrial fibrillationfibroblastsmacrophages41664919CIRCULATION RESEARCHopen access