Sex Differences in Mitochondrial Ca2+ during Ischemia/Reperfusion Injury: A role for S-Nitrosylation

Sex differences in cardiac ischemia/reperfusion (I/R) injury have been reported, but the mechanisms underlying these differences remain poorly understood. As mitochondrial Ca2+ accumulation plays an important role in I/R injury, we examined whether sex specific differences occur.To monitor mitochondrial Ca2+ in Langendorf perfused hearts, we used a genetically encoded, mitochondrially targeted Ca2+ indicator (R-GECO1) delivered via an adeno-associated viral vector (AAV9). Male hearts accumulated significantly more mitochondrial Ca2+ during 20 minutes of ischemia than female hearts. Interestingly, sex differences in Ca2+ accumulation during ischemia were not observed in hearts from mice lacking the mitochondrial Ca2+ uniporter (MCU), suggesting an important role for MCU. As nitric oxide (NO) and its posttranslational modification S-nitrosylation have been suggested to modulate sex differences in Ca2+ homeostasis, we inhibited NO signaling in female hearts, which increased mitochondrial Ca2+ accumulation, while treatment of male hearts with an NO donor reduced mitochondrial Ca2+ levels, indicating that S-nitrosylation modulates Ca2+ uptake during ischemia in a sex-dependent manner.Using a biotin-switch assay in isolated mitochondria, we found increased S-nitrosylation of MCU in females compared to males. Finally, isolated male mitochondria exposed to an NO donor exhibited reduced Ca2+ uptake, comparable to untreated female mitochondria. Taken together, these findings suggest that S-nitrosylation of MCU reduces mitochondrial Ca2+ uptake during ischemia, uncovering a new layer of redox regulated mitochondrial function, with sex as a critical determinant.