Piezo1-dependent activation of stromal cells ignites muscle inflammation in exercise and injury and is associated with inflammaging

As the actuator of movement and a key regulator of organismal metabolism, skeletal muscle is a site at which inflammatory responses must be carefully calibrated to counteract imposed stressors while preventing protracted functional impairments. Exercise, injury, and aging are common forms of stress associated with inflammation, particularly in skeletal muscle, yet the specific inducers and sensors driving such inflammation are not fully understood. Multi-pronged assessment of acute and chronic endurance-exercise models allowed us to evidence a role for MmSCs in transducing exercise-induced mechanical stress into local inflammatory responses. We identified the mechanosensitive ion channel Piezo1 as the molecular sensor of such mechanical stress. We also demonstrated that mechanosensing by stromal cells is necessary for appropriately timed inflammatory and myogenic responses to acute muscle injury and is associated with age-related muscle inflammation. Taken together, these findings highlight recognition of altered tissue stiffness by the Piezo1:MmSC pair as a fundamental mechanism of stress-induced immunomodulation in skeletal muscle. Overall design: To investigate the potential of acute exercise (AEX/exe) and exercise training (EXT) to modulate muscle mesenchymal stromal cells (MmSCs), 1000 MmSCs were cytofluorometrically sorted (PDGFRa+Sca1+CD31-CD45-DAPI-) from hindlimb muscles of 8 to 10-week old AEX and EXT mice, as well as from sedentary (SED) controls. MmSCs were sorted directly into TCL buffer for subsequent RNA isolation, library construction, and population-level RNA-seq. To determine whether mechanical stress was sufficient to induce an inflammatory reaction, MmSCs were cytofluorometrically sorted and briefly cultured in the presence of the Piezo1-specific agonist, Yoda1 (25uM), or vehicle prior to lysis in TCL buffer, library construction and population-level RNA-seq. For loss-of-function experiments, Pdgfra-Cre.Piezo1f/f (mutant) and Pdgfra-Cre.Piezo1+/+ (WT) mice were injured by intramuscular injection of cardiotoxin (CTX), and tibialis anterior (TA) muscles were dissected and homogenized in TRIzol for RNA isolation, library construction and whole-tissue RNA-seq on days 1, 3 and 7 of recovery. Hearts from a separate cohort of Piezo1 mutant and WT littermates were also dissected 3 days after CTX injury to skeletal muscles (perfused), and RNA was extracted and used for library construction as before. Whole-tissue RNA-seq was also performed on Qd muscles, hearts (perfused), and liver specimens (perfused) from separate cohorts of uninjured, SED mutant and WT littermates. MmSCs were sorted from hindlimb muscles of mutant and WT mice on day 1 after AEX (versus SED controls) or on day 1 after cardiotoxin (CTX)-induced injury. Sorting and sequencing was the same as for AEX and EXT experiments described above.