Skeletal muscle cells release both small and large that have distinct effects on muscle regeneration, influenced by their sphingolipid content.
Small extracellular vesicles (sEVs) promote the migration and differentiation of muscle stem cells, aiding in muscle repair.
Large extracellular vesicles (lEVs) inhibit muscle stem cell proliferation and disrupt the transition from pro-inflammatory to anti-inflammatory responses.
The lipid composition of sEVs and lEVs, particularly the ratio of different forms of , is linked to their varying effects on muscle cells and macrophages.
Exposure to TNF-α alters the sphingolipid ratios in sEVs, impairing their regenerative functions.
These findings highlight the role of extracellular vesicle lipid content in muscle tissue regeneration and suggest potential therapeutic strategies to enhance muscle repair.
Simplified
(EVs) represent a cytokine-independent pathway though which skeletal muscle (SkM) cells influence the fate of neighbouring cells, thereby regulating SkM metabolic homeostasis and regeneration. Although SkM-EVs are increasingly being explored as a therapeutic strategy to enhance muscle regeneration or to induce the myogenic differentiation of induced pluripotent stem cells (iPSCs), the mechanisms governing their release from muscle cells remain poorly described. Moreover, because muscle regeneration involves a tightly regulated inflammatory response it also important to determine how inflammation alters SkM-EV cargo and function in order to design more effective EV-based therapies. To address this knowledge gap, we isolated and characterized the large and small EVs (lEVs, sEVs) released from SkM cells under basal conditions and in response to TNF-α, a well-established inflammatory mediator elevated in both acute muscle injury and chronic inflammatory conditions such as type 2 diabetes. We then evaluated the regenerative roles of these EV subtypes in vivo using a mouse model of cardiotoxin-induced muscle injury, with a specific focus on their bioactive sphingolipid content. Using transmission, scanning or cryo-electron microscopy, lipidomic profiling and an adenoviral construct to express labelled CD63 in myotubes, we demonstrated that SkM cells release both sEVs and lEVs primarily from the plasma membrane. Notably, sEVs were generated from specialized membrane folds enriched in the EV markers ALIX (ALG-2 interacting protein X) and TSG101, as well as lipid raft-associated lipids. During regeneration, sEVs promoted M1 macrophage polarization and migration and muscle stem cell (MuSC) differentiation, thereby accelerating muscle repair. In contrast, lEVs inhibited and promoted MuSC proliferation and impaired the transition from the pro-inflammatory to the anti-inflammatory response, an essential step for promoting MuSC differentiation. Treatment of isolated muscle fibres with SkM-EVs revealed that the distinct effects of sEVs and lEVs on MuSC behaviour and macrophage phenotype could be largely explained by differences in their lipid composition, particularly the ratio of (S1P) subspecies. However, TNF-α exposure altered these ratios in sEVs and impaired their regenerative functions on MuSC and their effect on macrophage migration and polarization. These results demonstrate for the first time the importance of the sphingolipid content of EVs released by skeletal muscle in their regenerative function within muscle tissue, largely explained by their role as carriers of different subspecies of sphingosine-1-phosphate. This suggests that modulating the sphingolipid composition of EVs could be a viable strategy to enhance the regenerative potential of muscle tissue in addition to therapeutic interventions.
Key numbers
5
Increase in Muscle Fibres >30 µm
Number of regenerated muscle fibres in mice treated with CTR-.
30%
Decrease in MuSC Activation
Percentage reduction in activated MuSCs after treatment with .
d18:1/d16:1
Altered Ratio in
Ratio of subspecies in after TNF-α treatment.
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