Molecular Insights into the Regulatory Mechanisms Mediated by Hypoxia-Conditioned Skeletal Muscle Exosomal miRNAs

Hypoxia alters skeletal muscle metabolism and function through complex regulatory mechanisms, including exosome-mediated microRNA (miRNA) signaling.

This study profiled exosomal miRNAs from hypoxic human skeletal muscle cells (HSMCs) to explore their roles in hypoxic adaptation.

Human skeletal muscle cells were cultured under normoxic or hypoxic conditions, and secreted exosomes were isolated for comprehensive molecular profiling. High-throughput miRNA sequencing combined with integrative bioinformatic analyses was used to uncover hypoxia-responsive regulatory networks and key miRNA hubs involved in skeletal muscle adaptation.

HSMCs were cultured under normoxic or hypoxic conditions for 24 hours. Exosomes were isolated and characterized by transmission electron microscopy, nanoparticle tracking analysis, and immunoblotting. Exosomal miRNAs (n = 3 per group) were profiled using high-throughput sequencing, followed by differential expression, target prediction, enrichment, and network analyses.

Isolated exosomes displayed typical morphology (mean size: 82.4 ± 3.2 nm) and expressed markers CD9, CD63, and TSG101. Seventy-four miRNAs were significantly dysregulated under hypoxia (23 upregulated, 51 downregulated; FDR < 0.05, |log2FC| ⩾ 1), including upregulated hsa-miR-210-3p and downregulated hsa-miR-486-5p, hsa-miR-127-3p, and hsa-miR-126-3p. Predicted targets (~2000 genes) included 451 genes differentially expressed in hypoxic versus normoxic skeletal muscle cells. Functional enrichment highlighted cancer-related, MAPK, PI3K-Akt, and HIF-1 signaling pathways, along with muscle differentiation processes. Network analysis identified hsa-miR-20a-5p as a central regulatory hub (46 targets), followed by hsa-miR-24-3p and hsa-miR-152-3p. hsa-miR-24-3p showed the strongest disease associations in the miRNA-disease network.

Hypoxia induces distinct exosomal miRNA signatures in skeletal muscle, regulating genes involved in differentiation, migration, and stress response. These findings suggest that exosome-mediated miRNA signaling contributes to hypoxia-driven muscle adaptation and intercellular communication.