Single cell deconstruction of murine volumetric muscle loss reveals inflammatory imbalances preventing muscle stem cell mediated regeneration

Volumetric muscle loss (VML) overwhelms the innate regenerative capacity of mammalian skeletal muscle (SkM), leading to numerous disabilities and reduced quality of life. Immune cells are critical responders to muscle injury and guide tissue resident stem cell and progenitor mediated myogenic repair. However, how immune cell infiltration and inter-cellular communication networks are altered following VML and drive pathological outcomes remains under-explored. Herein, we characterize the cellular and molecular mechanisms of VML injuries that result in fibrotic degeneration or regeneration of SkM. Following degenerative VML injuries, we observe heightened infiltration of a previously uncharacterized population of Natural Killer (NK) cells as well as persistence of neutrophils beyond two weeks post injury. Functional validation of NK cells revealed an antagonistic role on neutrophil accumulation in part via CCR1 mediated chemotaxis. The persistent infiltration of neutrophils in degenerative VML injuries was found to contribute to impairments in muscle stem cell regenerative function, which was also attenuated by transforming growth factor beta 1 (TGFb1). Blocking TGFb signaling antagonized neutrophil accumulation, reduced fibrosis, and improved muscle specific force. Collectively, these results enhance our understanding of immune cell-stem cell crosstalk that drives regenerative dysfunction and provide further insight into possible avenues for fibrotic therapy exploration. Overall design: 8-10 week old C57B6J mice were uninjured or received bilateral 2mm or 3mm muscle loss defects to the rectus femoris and were humanely euthalized 7, 14, 28, or 42 days post injury. Quadriceps were dissected using sterile surgical instruments and enzymatically digested. Live cells were FACS-enriched using propidium iodide negative selection, then loaded into the 10X Genomics chromium single cell controler and single cells were captured into nanoliter-scale gel bead-in-emulsions. cDNAs were prepared using the second generation single cell 3? Protocol as per manufacturer?s instructions and sequenced on a Novaseq. Each sample is a pool of two mice (4 defects).