Abstract
Skeletal muscle regeneration following injury results from the proliferation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibers. Infiltrating macrophages play an essential role in the process partly by clearing the necrotic cell debris, partly by producing cytokines that guide myogenesis. Infiltrating macrophages are at the beginning pro-inflammatory, but phagocytosis of dead cells induces a phenotypic change to become healing macrophages that regulate inflammation, myoblast fusion and growth, fibrosis, vascularization and return to homeostasis. The TAM receptor kinases Mer and Axl are known efferocytosis receptors in macrophages functioning in tolerogenic or inflammatory conditions, respectively. Here we investigated their involvement in the muscle regeneration process by studying the muscle repair following cardiotoxin-induced injury in Mer−/− mice. We found that Axl was the only TAM kinase receptor expressed on the protein level by skeletal muscle and C2C12 myoblast cells, while Mer was the dominant TAM kinase receptor in the CD45+ cells, and its expression significantly increased during repair. Mer ablation did not affect the skeletal muscle weight or structure, but following injury it resulted in a delay in the clearance of necrotic muscle cell debris, in the healing phenotype conversion of macrophages and consequently in a significant delay in the full muscle regeneration. Administration of the TAM kinase inhibitor BMS-777607 to wild type mice mimicked the effect of Mer ablation on the muscle regeneration process, but in addition, it resulted in a long-persisting necrotic area. Finally, in vitro inhibition of TAM kinase signaling in C2C12 myoblasts resulted in decreased viability and in impaired myotube growth. Our work identifies Axl as a survival and growth receptor in the mouse myoblasts, and reveals the contribution of TAM kinase-mediated signaling to the skeletal muscle regeneration both in macrophages and in myoblasts.
Highlights
Regeneration of the skeletal muscle following injury is an adaptive response of the organ
Loss of Mer did not affect the number of infiltrating CD45+ cells (Fig. 3a), the neutrophil/MΦ ratios (Fig. 3b), or the level of monocyte chemoattractant protein-1 (MCP-1), the main chemoattractant signal for MΦ recruitment [35] (Fig. 3c) in the regenerating muscle
In the present study, the involvement of Mer in the skeletal muscle regeneration process was studied by using the cardiotoxin injury model
Summary
Regeneration of the skeletal muscle following injury is an adaptive response of the organ. During the initial inflammatory phase, neutrophils arrive with the first wave of cells followed by Ly6Chigh monocytes that differentiate into inflammatory Ly6Chigh macrophages (MΦs) These cells play a critical role in orchestrating the muscle regeneration partly by phagocytosing the necrotic myofibers and dying neutrophils, partly by releasing various cytokines and growth factors. The timed switch between the two main subsets of MΦs, the Ly6Chigh ones producing pro-inflammatory cytokines (e.g., tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6)) and the healing Ly6Clow ones producing anti-inflammatory cytokines and growth factors (e.g., interleukin-10 (IL-10), transforming growth factor-β (TGF-β), and growth differentiation factor-3 (GDF3)) [6,7,8,9], is a key to the proper regeneration process, and it is thought to be induced by the efferocytosis process [10].
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