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Updated: May 28, 2026

Enzymatic Isolation of Skeletal Muscle Interstitial Extracellular Vesicles
Published on: February 7, 2025
RYR1+ skeletal muscle-derived extracellular vesicles are exercise responsive and associated with insulin action
Xin Zhang1,2,3, Christopher G Vann4, David B Bartlett4,5
1Duke Molecular Physiology Institute, Duke University School of Medicine, Durham, North Carolina, 27701, USA. xin.zhang193@duke.edu.
Background:
Skeletal muscle is a central regulator of insulin sensitivity and glucose homeostasis. The ryanodine receptor 1 (RYR1) is highly expressed in skeletal muscle and plays a key role in myogenic differentiation. We hypothesize that RYR1+ extracellular vesicles (EVs) represent a skeletal muscle-derived EV subpopulation whose abundance and small RNA (smRNA) cargo are associated with aging, insulin action, and exercise responsiveness.
Methods:
We tested this hypothesis through in vitro analyses of smRNAs of skeletal muscle-derived EVs and in vivo evaluation of their exercise responsiveness and association with insulin action in older adults. Using an integrated workflow combining centrifugation, polymer-based precipitation, and single-EV sorting, we isolated RYR1+ and RYR- EVs secreted from myobundles-3D contractile skeletal muscle tissues engineered from primary muscle progenitor cells obtained from healthy donors (n = 6). We also isolated plasma EVs from 48 human participants and performed targeted high-resolution flow cytometry to evaluate EV biomarkers associated with aging, insulin action, and exercise responsiveness in older adults.
Results:
By smRNA sequencing, compared to myobundle RYR- EVs, RYR1+ EVs contained three unique microRNAs (miRNAs) and an additional 21 miRNAs with significantly greater abundance (including canonical myoMiRs miR-206, miR-1-3p, and miR-208a-3p). Of the 24 RYR1+ EV-enriched miRNAs, experimentally-supported mRNA targets (n = 422) are involved in pathways governing cell proliferation, apoptosis, senescence, insulin and glucose signaling. In two independent cohorts, including older adults with prediabetes or unknown prediabetes status, frequencies of RYR1+ EV subsets were significantly upregulated by chronic exercise with greater RYR1+ EV frequencies associated with better insulin action.
Conclusions:
These complementary in vitro and in vivo data identify skeletal muscle-derived RYR1+ EVs as upregulated by exercise and as carriers of miRNAs linked to insulin action in older adults, including those with prediabetes. These results highlight skeletal muscle-derived EVs as novel biomarkers and potential mediators of systemic metabolic regulation and healthy aging.
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