eNOS uncoupling drives oxidative stress and arrhythmogenic vulnerability in experimental hypothyroidism

Cardiac arrhythmias are a major cause of morbidity and sudden cardiac death worldwide, and hypothyroidism has emerged as an important risk factor. Among the mechanisms involved in cardiac electrical stability, nitric oxide (NO) signaling, particularly via endothelial nitric oxide synthase (eNOS), plays a critical role in cardiac excitation-contraction coupling. Here, we investigated whether hypothyroidism increases susceptibility to stress-induced arrhythmias through eNOS uncoupling and excessive reactive oxygen species (ROS) production.

Hypothyroidism was induced in Swiss mice by methimazole administration in the drinking water for 21 days. In vivo electrocardiographic recordings revealed a marked increase in stress-induced arrhythmias following dobutamine/caffeine challenge. At the cellular level, left ventricular cardiomyocytes from hypothyroid mice exhibited elevated ROS production, electrical instability, enhanced Na⁺ and Ca²⁺ currents, and reduced repolarizing K⁺ currents, resulting in action potential prolongation. Unbalanced oxidative stress was associated with redox-dependent Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation, contributing to intracellular Ca²⁺ instability and triggered activity in cardiomyocytes. Mechanistically, hypothyroidism promotedeNOS uncoupling in cardiomyocytes, evidenced by a reduced eNOS dimer/monomer ratio, leading to increasedROSgeneration and impaired NO production. Pharmacological restoration of eNOS coupling with tetrahydrobiopterin (BH4) normalized ROS and NO at the cellular level, attenuated redox-dependent CaMKII activation, and significantly reduced arrhythmia incidence in vivo.

Together, these findings demonstrate that eNOS uncoupling is a key driver of oxidative stress and arrhythmogenic vulnerability in experimental hypothyroidism, suggesting that restoration of eNOS coupling may represent a potential therapeutic strategy to reduce arrhythmogenic risk in hypothyroidism.