CD47 Promotes, While Exposure to Apoptotic Cells Destroys the Fusion Program of Differentiating Myoblasts

Cell fusion requires the activity of several phagocytic receptors and the temporary exposure of phosphatidylserine (PS) on the surface of viable myoblasts. Recently, we reported that these receptors turn myoblasts into potent phagocytic cells. Since cell fusion and phagocytosis share many molecules and mechanisms in myoblasts, we aimed to investigate how myoblasts choose between the two pathways during fusion. To prevent accidental uptake, viable cells express "don't eat-me" signals. By analyzing RNA sequencing data, we found that differentiation affected the expression of multiple "don't eat-me" genes in the C2C12 mouse myoblast cells, including upregulation of Sirpα, a receptor for CD47. The same was observed in differentiating myoblasts in vivo following cardiotoxin-induced injury in mouse skeletal muscle. Treatment of differentiating C2C12 cells with anti-CD47 antibody significantly reduced cell fusion but did not affect cell survival or differentiation. Both CD47 and SIRPα appeared at contact points of fusing myoblasts. Blocking CD47 signaling increased the uptake of viable red blood cells but only slightly increased the uptake of viable myoblasts. Blocking thrombospondin-1, another CD47 ligand, also inhibited fusion. Inhibiting CD47 signaling did not impact the engulfment of apoptotic cells. However, long-term exposure to continuously PS-expressing apoptotic cells disrupted myotube formation by inhibiting PIEZO1 activation, leading to syncytia formation. Overall, our data show that differentiating myoblasts upregulate CD47 to avoid accidental phagocytosis of live cells but mainly to promote myoblast fusion. Therefore, the activity of this signaling pathway contributes to the decision-making between the two processes that would compete with each other during myoblast differentiation.