Sex-Specific Differences in Endothelial Function Are Driven by Divergent Mitochondrial Ca²⁺ Handling

Background Sex-specific differences in vasodilation are mediated in part by differences in cytosolic Ca²⁺ handling, but how variations in mitochondrial Ca²⁺ contributes to this effect remains unknown. Here, we investigated the extent to which mitochondrial Ca²⁺ entry via the MCU (mitochondrial Ca²⁺ uniporter) drives sex differences in vasoreactivity in resistance arteries. Methods and Results Enhanced vasodilation of mesenteric resistance arteries to acetylcholine (ACh) was reduced to larger extent in female compared with male mice in 2 genetic models of endothelial MCU ablation. Ex vivo Ca²⁺ imaging of mesenteric arteries with Fura-2AM confirmed higher cytosolic Ca²⁺ transients triggered by ACh in arteries from female mice versus male mice. MCU inhibition both strongly reduced cytosolic Ca²⁺ transients and blocked mitochondrial Ca²⁺ entry. In cultured human aortic endothelial cells, treatment with physiological concentrations of estradiol enhanced cytosolic Ca²⁺ transients, Ca²⁺ buffering capacity, and mitochondrial Ca²⁺ entry in response to ATP or repeat Ca²⁺ boluses. Further experiments to establish the mechanisms underlying these effects did not reveal significant differences in the expression of MCU subunits, at either the mRNA or protein level. However, estradiol treatment was associated with an increase in mitochondrial mass, mitochondrial fusion, and the mitochondrial membrane potential and reduced mitochondrial superoxide production. Conclusions Our data confirm that mitochondrial function in endothelial cells differs by sex, with female mice having enhanced Ca²⁺ uptake capacity, and that these differences are attributable to the presence of more mitochondria and a higher mitochondrial membrane potential in female mice rather than differences in composition of the MCU complex.