Chronological Age, Sex and Tissue Type as Determinants of Skeletal Muscle Extracellular Vesicle Secretion and Function

Background: Extracellular vesicles (EVs) enable complex intercellular and interorgan communication that is influenced by physiological differences. For example, aging has been associated with changes in the circulating and cell-derived abundance and composition of extracellular vesicles (EVs), but the impact of age on tissue EV secretion is poorly understood. Skeletal muscle (SkM) is one of the largest tissues in the body and is a major source of EVs. We have shown that aging decreases the expression of genes involved in EV biogenesis, secretion and cargo which correspond with changes in SkM mass, function and fiber type that occur with aging. However, it is not clear how these changes in gene expression alter SkM tissue EV secretion, diameter and composition. Methods: This study analyzed 28 total mice using an age (8 vs 19 months of age) x sex (male vs female) x SkM type (rectus femoris, plantaris, soleus, red gastrocnemius, and white gastrocnemius) study design yielding n=8 young, and n=6 old, mice for each sex and SkM type. Mice were anesthetized with isoflurane, euthanized by exsanguination and decapitation and then SkMs were rapidly collected. SkMs were rinsed in 0.1 μm-filtered PBS, cut into 5 mg pieces then incubated in serum-free cell culture media (DMEM, 25 mM glucose, 3.7 g/L sodium bicarbonate and 0.5% penicillin/streptomycin) for 24 hours @ 37°C and 5% CO 2 . EVs were isolated from conditioned media using mixed mode chromatography (Captocore 700) and quantified using nanoparticle tracking analysis (Horiba ViewSizer) with optimized, and standardized, settings. To rigorously analyze the resulting data, a 3-way ANOVA was performed with Tukey multiple comparisons testing. Results: There was no main effect observed for age, sex, or SkM type for EV abundance but there was a trend (p = 0.10) for a sex x age interaction. There was a main effect of tissue, sex and age on mean EV diameter but no significant interactions between variables. Multiple comparisons analysis did not clearly explain differences across variables. There was a main effect of sex and a significant tissue x sex x age interaction on modal EV diameter. No individual differences were observed in multiple comparisons testing for modal EV diameter. Across all samples, EV abundance (mean ± SD) = 1.45 x10 9 ± 1.77 x 10 9 EVs/mg/ml, mean EV diameter = 136 ± 36 nm, modal EV diameter = 63 ± 13 nm. Conclusions: Our data suggests that SkM tissue may be largely resilient to the influence of aging, but many questions remain. For example, our data suggests that sex may be a relevant variable in age-dependent changes in EV secretion, but more studies are required to effectively test this hypothesis. The lack of clear effect of aging could be due to SkM tissue being comprised of multiple cell types, so age-related changes in SkM tissue cell composition could mask changes in EV secretion by individual cell types (i.e., SkM myofibers). Moreover, it is not yet clear how age, sex or tissue type change the diameter of SkM tissue-derived EVs, or its composition, which could alter their function in recipient cells. Our ongoing work is focused on analyzing EV composition and resulting function in recipient cells to explore these possibilities. National Institutes of Aging: AG078859 and AG085586 and the CSU HES Alonso Quijano Undergraduate Research Scholars Program. This abstract was presented at the American Physiology Summit 2025 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.