Mechanical stimulation induces electrical coupling via TNTs through calcium-dependent mechanisms in C2C12 cells

Intercellular communication and cellular material exchange are essential for the development, tissue repair, and survival of multicellular organisms. Tunneling nanotubes (TNTs) serve as long-distance intercellular connections that facilitate the transmission of biochemical and electrical signals between cells. However, the mechanisms underlying this process remain largely unknown. This study uses mechanical stimulation to explore the transmission and mechanisms of mechano-electrical signals mediated by TNTs. We assessed both gap junction (GJ)-mediated and TNT-mediated intercellular electrical coupling in HEK cells, A549 cells, and C2C12 cells. Our results show that the coupling ratio was lower in HEK cells compared to A549 cells, with the highest ratio observed in C2C12 cells. Additionally, the strength of TNT-mediated electrical coupling decreased as TNT length increased. Using C2C12 cells as a model, we found that Ca²⁺ influx and the gap junction protein Connexin 43 (Cx43) play roles in TNT-mediated electrical coupling induced by mechanical stimulation. Mechanical stimulation triggers extracellular Ca²⁺ entry into C2C12 cells via mechanosensitive and T-type calcium channels. Our experiments also demonstrate that TNT-mediated electrical coupling between C2C12 cells is predominantly unidirectional. These findings provide new insights into the mechanisms of intercellular electrical signal transmission.