Abstract

The phytoecdysteroid 20-Hydroxyecdysone (20E) is widely recognized for its anabolic properties in skeletal muscle, yet its direct impact on mitochondrial function, the central hub of cellular energy metabolism, remains incompletely understood. This study investigated the effect of 20E on the functioning of isolated mouse skeletal muscle mitochondria to elucidate its mechanism of action at the cellular and bioenergetic level. Mitochondria were isolated from mouse skeletal muscle, and their respiratory capacity, membrane potential, and calcium retention capacity were assessed using high-resolution respirometry and fluorometric techniques in the presence of varying 20E concentrations. Our findings indicate that 20E, particularly at concentrations exceeding 100 µM, significantly suppresses mitochondrial respiration fueled by complex I substrates (glutamate and malate), suggesting a direct inhibitory effect on the electron transport chain or substrate transport. Furthermore, 20E was observed to induce a prooxidant effect, manifesting as an increase in hydrogen peroxide production, and reduced the mitochondria's ability to accumulate calcium ions, pointing towards a potential disruption of membrane integrity and calcium homeostasis. These results suggest that while 20E may have beneficial anabolic effects, its direct interaction with the mitochondrial membrane and respiratory chain at higher concentrations may present a previously unrecognized toxic mechanism, warranting caution and further investigation into its therapeutic window and cellular side effects.