Supplementary Material for: 3-Hydroxybutyrate Prevents Pulmonary Vein Arrhythmogenesis by Regulating Calcium/Calmodulin-Dependent Protein Kinase II Signaling and Oxidative Stress

Introduction Atrial fibrillation is the most common sustained cardiac arrhythmia, primarily caused by arrhythmogenic activity within the pulmonary veins (PVs). Enhanced ketone body utilization can prevent heart failure by reducing inflammation and oxidative stress, factors crucial for atrial fibrillation pathogenesis. However, the role of ketone bodies in PV arrhythmogenesis remains unclear. This study investigated the effect of 3-hydroxybutyrate (3-OHB), the most abundant ketone body, on PV arrhythmogenesis and explored the underlying mechanisms. Methods Using conventional microelectrodes and the patch-clamp technique, we analyzed electrophysiological characteristics and ionic currents in isolated rabbit PV tissues and single PV cardiomyocytes. Results The effects of 3-OHB (10 mM) on reactive oxygen species (ROS) production, calcium homeostasis, and phosphorylated calcium/calmodulin-dependent protein kinase II (CaMKII) expression were analyzed through confocal microscopy and Western blotting. The results indicated that 3-OHB dose-dependently reduced PV spontaneous activity, contractility and diastolic tension. However, these effects were blocked by KN93 and AIP, a CaMKII inhibitor. Moreover, 3-OHB reduced late sodium and L-type calcium currents in PV cardiomyocytes; these effects were blocked by KN93 and AIP. Furthermore, 3-OHB reduced cytosolic ROS levels and phosphorylated CaMKII expression. Conclusion 3-OHB prevents PV arrhythmogenesis by modulating ionic currents, reducing ROS production, and regulating CaMKII signaling. Thus, 3-OHB holds promise as an antiarrhythmic agent.