hiPSC-derived new approach methodology (NAM) recapitulates hallmarks of atrial fibrillation

Background and aim Human in vitro models for atrial fibrillation (AF) remain limited in reproducing disease-specific remodeling. We leveraged a human-induced pluripotent stem cell-derived atrial cardiomyocyte (hiPSC-aCM) model, which was co-cultured with human atrial cardiac fibroblasts (haCFs) and subjected to an intermittent tachypacing protocol (ITPP) to mimic AF-associated phenotypes. Materials and methods Human iPSC-derived atrial cardiomyocytes (hiPSC-aCMs) were differentiated, purified, matured, and cultured alone or co-cultured with human atrial cardiac fibroblasts at defined seeding ratios (e.g., 90/10 or 70/30 hiPSC-aCM/haCF). Cultures were subjected to 7 days of intermittent tachypacing using electrical field stimulation. Functional electrophysiology and calcium handling were assessed by high-throughput optical mapping, arrhythmia inducibility testing, and genetically encoded or chemical calcium indicators. Structural and molecular remodeling were evaluated by immunocytochemistry, qPCR, and Western blot analysis. Results ITPP increased spontaneous action potential (AP) frequency and shortened AP duration at 80% repolarization (APD 80% ), while reducing calcium transient (CaT) upstroke slope and amplitude. Co-cultures at a 70/30 hiPSC-aCM/haCF ratio exhibited marked structural remodeling, with a >100-fold increase in Collagen III expression ( P < 0.0001), decreased excitability (ΔHz = –61 ± 6%, P < 0.0001), prolonged APD 80% (Δ = +130 ± 10%, P < 0.0001), increased triangulation (ΔAPD Tri = +143 ± 13%, P < 0.0001), reduced upstroke slope (Δ = –66 ± 6%, P < 0.0001), conduction block (Δ = –52 ± 18%, P = 0.026), and impaired Ca 2 ⁺ handling (upstroke slope Δ = –50 ± 8%, P < 0.0001; ΔdF/F0 = –34 ± 9%, P = 0.0003). Applying ITPP to the 70/30 co-culture induced AF-like remodeling, with increased spontaneous activity (ΔHz = +25 ± 8%, P = 0.02), shortened APD 80% (Δ = –16 ± 6%, P = 0.01), and reduced conduction velocity (ΔCV 100/0 vs. 70/30 = –27 ± 15%, P = 0.0005). Mechanistically, ITPP led to downregulation of phospholamban (PLN) and altered PLN-SERCA2a balance, along with upregulation of TGFB1 and natriuretic peptides, reflecting calcium-handling dysfunction and pro-fibrotic signaling. Conclusion Together, these findings establish a human-relevant platform in 2D culture that recapitulates key AF-associated structural and electrophysiological remodeling for mechanistic studies and therapeutic screening.