Mitochondrial calcium homeostasis mediated by estradiol contributes to atrial fibrillation protection

Background Atrial fibrillation (AF) exhibits marked sex disparities, with premenopausal women showing lower incidence than age-matched men. However, the molecular mechanisms underlying estrogen's cardio protective effects remain unclear. Mitochondrial calcium ( Ca 2+ _m ) mishandling is a key driver of AF, but it is unknown whether estrogen regulates Ca 2+ _m homeostasis through Mitochondrial Calcium Uniporter (MCU). Methods Ovariectomized (OVX) female Sprague-Dawley rats were subjected to atrial pacing-induced AF for evaluation. Cardiac calcium dynamics, mitochondrial membrane potential (ΔΨm), and expression of calcium-regulating proteins (MICU1, NCX, LETM1) were assessed. In vitro, H9C2 cardiomyocytes under electrical stimulation (0.2 V/cm, 24h) were treated with estradiol (500 nM) or subjected to MCU knockdown (CRISPR-Cas9). Results OVX exacerbated AF susceptibility in rats, as evidenced by prolonged AF duration, reduced serum estradiol, and disrupted myocardial calcium homeostasis. OVX-AF hearts exhibited upregulated MICU1, NCX, and LETM1, alongside ΔΨm collapse (JC-1 monomer). Under electrical stimulation, cardiomyocytes displayed calcium homeostasis dysregulation, decreased ΔΨm, elevated ROS levels, along with concurrent downregulation of both MCU and ERβ protein expression, Estradiol supplementation normalized [Ca 2+ ]mt,restored ΔΨm. Strikingly, MCU knockdown abolished estradiol's protective effects, inducing irreversible [Ca 2+ ]mt overload and a surge in reactive oxygen species (ROS). Conclusions We reveal that estradiol modulates MCU-mediated mitochondrial calcium homeostasis to ameliorate AF-related cellular phenotypes in vitro, implicating the estrogen-MCU axis as a promising intervention target, though its in vivo cardioprotective effects demand additional investigation. Estrogen deficiency disrupts this axis, triggering maladaptive upregulation of MICU1/NCX/LETM1 and calcium-driven remodeling. Targeting ERβ-MCU signaling may offer novel therapeutic strategies for AF, particularly in hypoestrogenic states such as menopause.