Mitochondrial NOX4 drives atrial fibrillation via redox-dependent structural remodeling and fibrosis

Mitochondrial oxidative stress is a critical contributor to age-related cardiovascular disease, including cardiac arrhythmia. While our previous work demonstrated that mitochondrial NOX4 overexpression induces ventricular arrhythmias via altered membrane repolarization and structural remodeling in the ventricular myocardium, its role in atrial remodeling and AF remains unclear. In this study, we investigated the role of mitochondrial NOX4 in the initiation and maintenance of atrial fibrillation (AF) and investigated the underlying arrhythmogenic mechanisms using a transgenic mouse model (Nox4TG) with mitochondria-targeted NOX4 overexpression. We performed telemetry, intracardiac recordings, patch-clamp electrophysiology, calcium imaging, and histological analyses, and high-resolution optical mapping to assess structural and functional changes. Nox4TG mice exhibited significantly higher incidence of spontaneous and pacing-induced AF episodes compared to wild-type controls, despite preserved action potential duration, sodium current density, and conduction velocity. RyR2-mediated calcium spark mass was reduced, suggesting localized redox modifications; however, sarcoplasmic reticulum (SR) calcium content and fractional release remained intact due to upregulation of SERCA2 and phospholamban phosphorylation. Nox4TG atria showed significant structural remodeling, with increased expression of periostin, α-SMA, calpain2, and interstitial fibrosis. Optical mapping indicated preserved conduction velocity, suggesting that fibrosis, rather than electrical remodeling, caused arrhythmia without affecting global conduction. Inhibiting NOX4 with Setanaxib reduced AF duration. These findings demonstrate that mitochondrial NOX4 promotes AF not by altering ionic currents or promoting RyR2 leak, but through redox-sensitive fibrotic remodeling. Our results underscore the chamber-specific consequences of oxidative stress and support therapeutic targeting of mitochondrial NOX4 to mitigate atrial remodeling and AF in aging hearts.