Background: Extracellular vesicles (EVs) released from skeletal muscle mediate metabolic communication via microRNAs (miRNAs). While both circadian rhythms and exercise influence metabolism, the joint modulation of the muscle-derived EV miRNA landscape by circadian rhythms and chronic exercise remains undefined, particularly under the metabolic stress of obesity. Methods: Employing a 2×2 factorial design (Phase: ZT3 vs. ZT15; Condition: sedentary vs. exercise; ZT, Zeitgeber Time), EV-enriched fractions were isolated from ex vivo quadriceps muscle (QUA) cultures of high-fat diet-fed mice following an 8-week treadmill training regimen using polymer-based precipitation, and comprehensive miRNA profiling was performed by small RNA sequencing. Results: Principal component analysis (PCA) revealed that circadian phase accounted for a greater proportion of global variance in EV miRNA profiles than exercise. Differential expression analysis identified miR-1a-3p and miR-1b-5p as upregulated across both composite phase and exercise contrasts; however, condition-specific analyses indicated that this signal was primarily driven by the sedentary-phase comparison (ZT15-sed vs. ZT3-sed), in which the miR-29 family was also prominently co-upregulated, rather than constituting independent phase and exercise effects; this phase-associated signature was absent in the corresponding exercise-condition comparison. Exploratory functional enrichment of experimentally validated targets revealed phase-preferential association with metabolic and iron–heme pathways, whereas exercise-associated miRNAs mapped to signaling, inflammatory, and transcription-related networks. Conclusions: Circadian phase was the dominant contributor to global variance in muscle-derived EV-enriched miRNA profiles in obesity, as reflected by the phase-associated separation along principal component 1 (PC1, 33.47% of total variance), with exercise introducing context-dependent adaptive modulation. This study provides a foundational basis for investigating the temporal regulation of muscle secretome dynamics under high-fat diet conditions, highlighting temporal specificity as a key dimension in EV-mediated exercise physiology research.
Wang et al. (Fri,) studied this question.