Brody School of Medicine at East Carolina University
Introduction: Bladder dysfunction varies in symptoms and etiology among males and females; yet both sexes are treated similarly. Research assessing the bladder’s physiological sex differences is lacking. Mitochondrial function is a clear indicator of cell health, is responsible for providing the cell energy and maintaining redox homeostasis. Our study aims to identify differences in mitochondrial physiology in the mucosal and detrusor smooth muscle layers in male and female mice by measuring mitochondrial respiration and protein expression.
Methods: Bladders were collected from male and female mice (C57BL/6NJ, 20-24 weeks, n=7/group) and separated into mucosal and detrusor smooth muscle layers. Tissues were permeabilized via saponin and placed in an oxygraphy-2K (O2K) for respiration analysis. Samples underwent a substrate uncoupler inhibition titration (SUIT) via substrates (pyruvate/malate, glutamate, octanoylcarnitine/malate, succinate/rotenone) added followed by a phosphocreatine clamp, cytochrome C, and FCCP. Following O2K experiments, mitochondria were isolated and prepared for nLC-MS/MS analysis. Respiration data were normalized to mass spectrometry-based sample-specific percent mitochondrial enrichment to account for individual tissue mitochondrial content.
Results: In the presence of pyruvate/malate (Complex I), female mucosal layer had decreased mitochondrial respiration whereas female detrusor had increased mitochondrial respiration compared to males. In the presence of glutamate and octanoylcarnitine/malate, male detrusor had decreased mitochondrial respiration; there was no change in the mucosal layer compared to females. In the presence of succinate/rotenone (Complex II), female detrusor mitochondrial respiration was higher compared to males. Increased Complex I activated detrusor mitochondrial respiration in females was reflected in elevated mitochondrial complex I proteins (Ndufs1, Ndufs7, Ndufv2, Ndufa12, Ndufa6, Ndufa9). Additionally, Complex III mitochondrial proteins (Uqcrh, Cyc1, Uqcrfs1, Uqcr10, Uqcrq, Uqcrc1) were also elevated in female detrusor.
Conclusions: Overall, female detrusor had increased mitochondrial respiration compared to males which was reflected in mitochondrial protein content. This novel technique of characterizing the mitochondrial proteins provides insight on upregulated/downregulated proteins in males vs females yet more research is necessary to understand the significance of these differences.
Source of Funding: DiaComp Pilot and Feasibility Grant