Unveiling the neuro-vascular interplay in the skeletal muscle in health, injury and disease

Neuromuscular junctions (NMJs) are complex multicellular structures that convey motor neuron-induced responses in the skeletal muscle. Their cellular composition is well characterized, but interactions between the different cell types and with the surrounding microenvironment remain underexplored. Here, by using a panel of newly discovered mouse cell lineage markers, single-cell RNA sequencing analyses and iDisco tissue clarification, we demonstrate the existence of contacts between adjacent NMJs through their kranocytes, assembling kranocyte network-like structures within the muscle interstitium, which is essential for regulating blood flow and supporting muscle regeneration. Indeed, kranocytes do contact with the surrounding microvasculature as well, both of them uncovering a previously unexplored, expansive interactive system. Moreover, unlike the robustness shown by their counterparts, the terminal Schwann cells, we strikingly observed that kranocytes rapidly detected stressful conditions, leading to structural changes and even the loss of their connections in response to acute damage, such as muscle injury or neuromuscular disease-induced denervation. Therefore, we propose kranocytes as the interactive sensory platforms of the NMJs, working in a perpendicular axis of nerve-transmission, as key NMJ-connectors by, interconnecting adjacent NMJs and interacting with the microvasculature and the microenvironment, and also as NMJ-sensors by detecting microenvironmental inputs. Together, our data postulate kranocytes as triple connectors of the nervous, musculoskeletal and vascular systems.