Evidence supporting a morpho-functional interaction between telocytes and satellite cells in damaged skeletal muscle

Telocytes (TCs) represent unique stromal cells characterized by a small cell body and distinctive extremely long, thin and moniliform cytoplasmic extensions called telopodes alternating slender segments (podomers) with dilatations (podoms). TCs have been identified in the skeletal muscle interstitium with their telopodes being strategically positioned in the close vicinity of striated myofibers with regenerative features, nerve endings, small blood vessels, and often of resident muscle stem cells, namely satellite cells (SCs). A “nursing” role for TCs in SC-mediated skeletal muscle regeneration has been supposed; however, to date there is no experimental evidence demonstrating a morpho-functional interaction between these two cell types in an injured skeletal muscle. Hence, the aim of present morphological study was to explore the presence and the behavior of TCs in an ex vivo murine model of skeletal muscle (extensor digitorum longus) damage induced by forced eccentric contraction (EC) in isometric condition, focusing on their interaction with SCs. EC-damaged skeletal muscles showed evidence of structural and ultrastructural injury as judged by light microscopic and transmission electron microscopy (TEM) examination and along with significant electrophysiological changes of sarcolemnic properties. Specifically, we observed a resting membrane potential depolarization, an increase of membrane capacitance, a reduction of membrane resistance and a reduction of the outward K+ current amplitude, leading to an overall alteration of myofiber excitability. TCs were identified in both control and EC-injured muscles within the interstitium, alongside the myofibers and in close vicinity of vascular structures either by CD34/CD31 double confocal immunofluorescence staining (i.e. TCs: CD34+/CD31–; endothelial cells: CD34+/CD31+) or by TEM. Of note, in EC-damaged muscles, the telopode network was more extended and arranged around activated SCs displaying nuclear positivity for Pax7, the most reliable marker of SCs, and for MyoD, the SC activation marker. TEM analysis clearly demonstrated the presence of TCs invading the SC niche passing with their telopodes a broken basal lamina to contact the underlying activated SC that exhibited a swollen appearance and an euchromatic nucleus. The interaction between TCs and SCs was confirmed by in vitro experiments performed by culturing single living endomysial sheath-covered myofibers isolated from EC-damaged muscle and the derived stromal cells/TCs and SCs. Interestingly, TCs from EC-damaged myofibers showed an increased expression of vascular endothelial growth factor (VEGF)-A, whose role in promoting myoblast proliferation and differentiation is documented. SCs isolated from the same samples exhibited an increased MyoD expression as well as a major tendency to fuse into myotubes. These findings establish for the first time a morphological interaction between TCs and SCs in a damaged muscle and suggest a juxtacrine–paracrine cell-cell interaction involving VEGF-A, which worth investigating further.