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

This dataset contains the primary and processed data supporting the manuscript “Sex Differences in Mitochondrial Ca²⁺ during Ischemia/Reperfusion Injury: A Role for S-Nitrosylation.” The files are organized according to the figures presented in the publication to facilitate transparency, reproducibility, and data reuse. ZIP File Structure and Data Organization The ZIP archive is organized into individual folders corresponding to each figure included in the manuscript (e.g., Figure 1, Figure 2, Supplementary Figures). Each figure folder contains the experimental data used to generate the final panels shown in the publication. Within each figure folder, users will find: · GraphPad Prism files (.pzfx) containing statistical analyses and graph generation. · Microsoft Excel files (.xlsx) containing raw and processed numerical datasets. · PowerPoint files (.pptx) including assembled figure panels and annotated experimental outputs. · Western blot datasets, including uncropped images and quantification files when applicable. Western blot data include representative blots, source images, and associated quantification used for figure preparation. All datasets are labeled to correspond directly with the figure panels presented in the manuscript. No specialized software beyond standard laboratory analysis tools (GraphPad Prism, Microsoft Excel, and PowerPoint) is required to access the files. Associated Publication Abstract 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 Langendorff 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.