NADPH oxidase-dependent reactive oxygen species upregulate K_Ca3.1 to promote atrial fibroblast activation

Atrial fibroblast activation is a central cellular event driving atrial fibrotic remodeling, which plays a critical role in the initiation and maintenance of atrial fibrillation (AF). Our previous studies have identified the intermediate-conductance Ca²⁺-activated potassium channel K_Ca3.1 as a key mediator of reactive atrial fibrosis. However, the upstream mechanisms regulating its expression under pro-fibrotic stimulation remain incompletely defined. The present study aimed to investigate whether NADPH oxidase (NOX)-derived reactive oxygen species (ROS) regulate K_Ca3.1 expression and function in atrial fibroblasts. Primary rat atrial fibroblasts were stimulated with angiotensin II (Ang II). Ang II markedly increased intracellular ROS generation through AT1 receptor-dependent activation of NOX. Pharmacological inhibition with Diphenyleneiodonium (DPI) or CRISPR-mediated deletion of NOX2 or NOX4 significantly suppressed Ang II-induced upregulation of K_Ca3.1 expression and channel activity. Exposure to hydrogen peroxide (H₂O₂) alone was sufficient to enhance K_Ca3.1 expression and TRAM-34-sensitive currents. Functionally, pharmacological blockade or genetic deletion of K_Ca3.1 markedly attenuated ROS-induced fibroblast proliferation, migration, and myofibroblast differentiation. Mechanistically, ROS activated c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2), and inhibition of either pathway suppressed K_Ca3.1 promoter activity and expression. Collectively, these findings identify a NOX-ROS-JNK/ERK-K_Ca3.1 signaling axis that drives atrial fibroblast activation and may represent a potential therapeutic target in oxidative stress-associated atrial remodeling.