Fig. 5.

Neutrophil secretome impairs myoblast fusion in accordance with reduced muscle stem cell regenerative capacity following critical-sized defects. (A) Representative image showing interspersion of neutrophils (cyan) within GFP+-regenerating myofibers (yellow) at 14 dpi. (B) Distributions of minimum Euclidean distances between neutrophils and GFP+-regenerating fibers. More than 93% of neutrophil cells are within 250 μm of the nearest regenerating fiber. n = 382 neutrophils and 410 GFP+ fibers from 20× images of four defects at 14 dpi. (C) Schematic of experiment design, whereby C2C12s are cultured in neutrophil-conditioned myoblast media for 8 (long) or 1 (short) d before addition of differentiation medium without conditioning. Fusion was assessed 72 h later. (D) Representative immunofluorescent images of C2C12s cultured for long or short durations in neutrophil-conditioned medium, then induced to differentiate for 72 h in low-serum media. Blue indicates nuclei (DAPI) and red indicates embryonic myosin heavy chain (MYH3). (Scale bar, 50 mm.) (E) Quantification of number of myotubes as percentage of total cells after short or long exposure to neutrophil-conditioned medium. *P < 0.05 by two-sided, two-sample t test after removal of one outlier. n = 29 10× images taken from three culture wells. (F) Representative immunofluorescent images of primary myoblasts harvested 28 dpi following regenerative (2-mm) and degenerative (3-mm) defects and cultured in differentiation medium for 72 h. Blue indicates nuclei (DAPI) and red indicates embryonic myosin heavy chain (MYH3). (Scale bar, 100 mm.) (G) Quantification of fusion index according to the fraction of nuclei located in myofibers containing at least two nuclei, calculated manually. *P value < 0.05 by two-sided, two-sample t test. n = 20 10× images per defect from three mice per defect size, repeated twice. Cohen’s d = 0.86. (H) Fold change in Myog expression measured by qRT-PCR following in vitro differentiation of MuSCs isolated from 2-mm and 3-mm defects at 28 dpi. Bars show mean ± SEM. **P < 0.01 by two-sided, two-sample t test. n = 3 to 4. Replicates with Cq values > 30 were excluded from further analysis.