Abstract Body (Do not enter title and authors here): Background: Atrial fibrillation (AF) is associated with aging and increased expression of senescence-associated secretory phenotype (SASP) markers. It remains unclear whether AF directly exacerbates atrial aging and whether inhibition of oxidative pathways can reverse this effect.

Hypothesis: We hypothesized that persistent AF (persAF) increases atrial SASP expression and that targeted knockdown of NADPH oxidase 2 (NOX2) – a major enzymatic source of oxidative injury - mitigates these increases and attenuates AF development.

Methods: Canines underwent rapid atrial pacing (RAP, 600 bpm) to induce persAF (93-160 days; n=6), with non-paced animals as controls (n=7). Additional animals received either plasmids expressing NOX2 shRNA (n=7) or scramble shRNA (n=5) delivered to the atrial myocardium via direct injection and electroporation. Expression of senescence markers (IL-1β, IL-6, p16, p21, p53, and PAI-1) was assessed in atrial regions left atrial appendage (LAA), left atrial free wall (LAFW), posterior left atrium (PLA), right atrial appendage (RAA), posterior left atrium (PLA), right atrial free wall (RAFW) and posterior right atrium (PRA) via quantitative RT-PCR. Ganglionated plexi (fat pads) from RAP (n=5) and control animals (n=3) were similarly analyzed.

Results: PersAF significantly elevated SASP markers in atrial tissue, notably IL-1β, IL-6, and p21 (p<0.001 in LAA). Upregulation was widespread across atrial regions but particularly pronounced in the left atrial appendage (LAA) (panel A). NOX2 knockdown significantly blunted senescence marker expression, especially p21, across multiple atrial regions compared to scramble controls (p<0.01 in LAA, PLA, RAFW; p<0.001 in LAFW) (panel B, C). In atrial fat pads, persAF significantly increased IL-1β and IL-6 (p<0.05), consistent with the atrial tissue findings (panel D). NOX2 knockdown also significantly delayed AF onset and reduced AF burden (Fig. 2).

Conclusion: Persistent AF markedly upregulates senescence markers across atrial myocardium, implicating a pro-aging phenotype induced by AF. Targeted NOX2 knockdown effectively rescues this SASP marker upregulation – along with a significant decrease in AF burden - highlighting NOX2 as a potential therapeutic target for slowing cardiac aging associated with AF.