Progressive and Coordinated Mobilization of the Skeletal Muscle Niche throughout Tissue Repair Revealed by Single-Cell Proteomic Analysis

Abstract

Background: Skeletal muscle is one of the only mammalian tissues capable of rapid and efficient regeneration after trauma or in pathological conditions. Skeletal muscle regeneration is driven by the muscle satellite cells, the stem cell population in interaction with their niche. Upon injury, muscle fibers undergo necrosis and muscle stem cells activate, proliferate and fuse to form new myofibers. In addition to myogenic cell populations, interaction with other cell types such as inflammatory cells, mesenchymal (fibroadipogenic progenitors—FAPs, pericytes) and vascular (endothelial) lineages are important for efficient muscle repair. While the role of the distinct populations involved in skeletal muscle regeneration is well characterized, the quantitative changes in the muscle stem cell and niche during the regeneration process remain poorly characterized. Methods: We have used mass cytometry to follow the main muscle cell types (muscle stem cells, vascular, mesenchymal and immune cell lineages) during early activation and over the course of muscle regeneration at D0, D2, D5 and D7 compared with uninjured muscles. Results: Early activation induces a number of rapid changes in the proteome of multiple cell types. Following the induction of damage, we observe a drastic loss of myogenic, vascular and mesenchymal cell lineages while immune cells invade the damaged tissue to clear debris and promote muscle repair. Immune cells constitute up to 80% of the mononuclear cells 5 days post-injury. We show that muscle stem cells are quickly activated in order to form new myofibers and reconstitute the quiescent muscle stem cell pool. In addition, our study provides a quantitative analysis of the various myogenic populations during muscle repair. Conclusions: We have developed a mass cytometry panel to investigate the dynamic nature of muscle regeneration at a single-cell level. Using our panel, we have identified early changes in the proteome of stressed satellite and niche cells. We have also quantified changes in the major cell types of skeletal muscle during regeneration and analyzed myogenic transcription factor expression in satellite cells throughout this process. Our results highlight the progressive dynamic shifts in cell populations and the distinct states of muscle stem cells adopted during skeletal muscle regeneration. Our findings give a deeper understanding of the cellular and molecular aspects of muscle regeneration.

The double homeodomain protein DUX4c is associated with regenerating muscle fibers and RNA-binding proteins

“…To complete our knowledge on niche restructuration in FSHD muscles, additional components including soluble factors (laminins, collagens, fibronectin, prostaglandin E2, oncostatin M, etc.) [ 54 , 55 ] need to be investigated. Our preliminary data indicate that most of the macrophages present in affected muscles were of the M2-type (Fig.…”

Fiber-Type Shifting in Sarcopenia of Old Age: Proteomic Profiling of the Contractile Apparatus of Skeletal Muscles

“…CyTOF does suffer from several limitations, including the reduced sensitivity of metal-isotope-tagged antibodies and the longer acquisition times needed when using TOF-MS instruments. However, with these limitations being identified, there is massive scope for advances in these areas that will contribute to increasing the research possibilities of CyTOF in the future of skeletal muscle research [158][159][160]. Recently, Porpiglia et al [161] used CyTOF to study muscle stem cells (MuSCs) in the aged phenotype and showed high CD47 expression levels, which might be associated with dysfunctional MuSCs, and an impaired regenerative capacity.…”

Mass Spectrometry-Based Proteomic Technology and Its Application to Study Skeletal Muscle Cell Biology

“…Of crucial importance for the swift regeneration of damaged muscle fibers are myogenic stem cells, which are represented by the satellite cell population that is located between the sarcolemma and basal lamina in adult skeletal muscles. Satellite cell and activated muscle precursor cell proteomics has recently been carried out to identify biomarkers of fiber damage [298] and muscle aging [299].…”