Mitochondrial calcium overload is sexually dimorphic and leads to ultrastructural changes in isolated cardiac mitochondria

Heart failure is a leading contributor to death across the globe, and sex hormones are postulated to be responsible for the disparities of heart failure rates between men and premenopausal women. Differences in calcium handling has been postulated as an explanation, so a sex as a biological variable study was conducted using isolated cardiac mitochondria from Sprague Dawley rats to determine if there are different calcium effects. In this study, we assessed the rate and capacity of calcium uptake using spectrofluorimetry, effect of calcium on ADP-stimulated respiration using a respirometer, calcium handling mRNA abundance using qPCR, and mitochondrial ultrastructure using cryo-electron tomography (cryo-ET). Calcium tolerance was established to be between 0 and 20 µM CaCl 2 and confirmed by absorbance and fluorescence-based assays to rule out the well-known permeability transition phenomenon. Oxidative capacities between the sexes under calcium challenges were compared to reveal novel findings that female isolated cardiac mitochondria had 12.5% higher rates of ADP-stimulated respiration at every added concentration of calcium (5 – 20 µM; p-value = 0.0075). The leading hypothesis is the reduction in oxidative phosphorylation experienced in the calcium overloaded state strongly correlates with how much calcium is retained in mitochondria. To interrogate this, differences in initial calcium uptake rates and total activity of calcium handling proteins were quantified. Male mitochondria were shown to have both higher mitochondrial calcium uniporter (MCU) uptake and activity (p-values =0.01749 and 0.00019, respectively) at concentrations of added calcium ≥ 10 µM. Calcium handling machinery abundance was assessed using qPCR and showed that there were no differences in mRNA expression of left ventricular tissue samples. Isolated mitochondria were also imaged via cryo-ET to examine calcium-induced changes in cristae morphology in the presence of calcium-phosphate granules. Sexually dimorphic structural changes were assessed to help explain depressions in respiratory capacity which align with similar findings using guinea pig cardiac mitochondria. These data highlight key sex differences regarding the mitochondrial response to calcium and subsequent oxidative impairments that occur during the progression of heart failure to help explain a possible new etiology of this increasingly important pathology and the mechanism by which cardioprotection is afforded to premenopausal women. The work detailed herein was funded by NSF CAREER MCB 2237117. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.