Heterogeneous Macrophage Activation in Acute Skeletal Muscle Sterile Injury and mdx^5cv Model of Muscular Dystrophy

Monocytes/macrophages promote the repair of acutely injured muscle while contributing to dystrophic changes in chronically injured muscle in Duchenne muscular dystrophy (DMD) patients and animal models including mdx and mdx^5cv mice. To elucidate the molecular mechanisms underlying this functional difference, we compared the transcriptomes of intramuscular monocytes/macrophages from wild-typed (WT) uninjured muscles, WT acutely injured muscles, and mdx^5cv dystrophic muscles, using single cell-based RNA sequencing (scRNA-seq) analysis. Our study identified multiple transcriptomically diverse monocyte/macrophage subclusters, which appear to be induced by the intramuscular microenvironment. They expressed feature genes differentially involved in muscle inflammation, regeneration, and extracellular matrix (ECM) remodeling, but none of them conform to strict M1 or M2 activation. The Gpnmb⁺Spp1⁺ macrophage subcluster, an injury-associated subcluster that features the signature genes of reported scar-associated macrophages (SAMs) involved in ECM remodeling and fibrosis, is present transiently in acutely injured muscle and persistently in chronically injured dystrophic muscle, along with the persistence of monocytes. Our findings suggest that the persistent monocyte/macrophage infiltration and activation induced by continuous injury may underlie the pathogenic roles of macrophages in mdx^5cv muscles. Controlling muscle injury and subsequent macrophage infiltration and activation may be important to the treatment of DMD.