Fusion-Independent Satellite Cell Communication to Muscle Fibers During Load-Induced Hypertrophy

Abstract

The "canonical" function of Pax7+ muscle stem cells (satellite cells) during hypertrophic growth of adult muscle fibers is myonuclear donation via fusion to support increased transcriptional output. In recent years, however, emerging evidence suggests that satellite cells play an important secretory role in promoting load-mediated growth. Utilizing genetically modified mouse models of delayed satellite cell fusion and in vivo extracellular vesicle (EV) tracking, we provide evidence for satellite cell communication to muscle fibers during hypertrophy. Myogenic progenitor cell-EV-mediated communication to myotubes in vitro influences extracellular matrix (ECM)-related gene expression, which is congruent with in vivo overload experiments involving satellite cell depletion, as well as in silico analyses. Satellite cell-derived EVs can transfer a Cre-induced, cytoplasmic-localized fluorescent reporter to muscle cells as well as microRNAs that regulate ECM genes such as matrix metalloproteinase 9 (Mmp9), which may facilitate growth. Delayed satellite cell fusion did not limit long-term load-induced muscle hypertrophy indicating that early fusion-independent communication from satellite cells to muscle fibers is an underappreciated aspect of satellite cell biology. We cannot exclude the possibility that satellite cell-mediated myonuclear accretion is necessary to maintain prolonged growth, specifically in the later phases of adaptation, but these data collectively highlight how EV delivery from satellite cells can directly contribute to mechanical load-induced muscle fiber hypertrophy, independent of cell fusion to the fiber.

Keywords: Mmp9; Nr4a1; Pax7-DTA; extracellular vesicles; miRNA; tdTomato.

Graphical Abstract

Figure 1.

Delayed satellite cell fusion and EV-dependent communication to muscle fibers during 7 days of MOV. (A) Confocal image of a tdT-expressing satellite cell (red) overlaid with DAPI (blue) on a muscle fiber (stained for F-actin, gray), illustrating Pax7Cre-mediated recombination of the floxed tdT transgene in the presence of tamoxifen. (B) Histochemical image depicting Pax7 immunostaining (green) that coincided with tdT fluorescence (red) in the correct anatomical location (beneath laminin, gray, and overlaid with DAPI, blue) after tamoxifen treatment. (C) Flow cytometry plot illustrating tdT+ cells coexpressing Vcam, which was used to identify and purify satellite cells via FACS in tamoxifen-treated mice; Vcam+/Cd31−/Cd45−/Sca1− cells were purified in vehicle-treated mice, as described previously, and tdT recombination was minimal (not shown). (D) Gene expression to confirm N-WASp recombination from in vivo-treated vehicle (N-WASp+/tdT−) versus tamoxifen-treated (N-WASp−/tdT+) primary MPCs purified via FACS, n = 2 biological replicates per condition. (E) Nanoparticle tracking analysis showing EV particle abundance normalized to cell count from N-WASp+/tdT− and N-WASp−/tdT+ MPCs, n = 2 biological replicates per condition. (F) Relative tdT mRNA abundance in EVs isolated from N-WASp+/tdT− (n = 2 biological replicates) and N-WASp−/tdT+ MPCs (n = 3 biological replicates). (G) Proliferation in N-WASp−/tdT+ and N-WASp+/tdT− MPCs (n = 3 biological replicates per condition). (H) MyHC area after 30 h of differentiation (n = 3 biological replicates per condition). (I) Fusion index (n = 3 biological replicates per condition). (J) Representative images of MPC fusion in N-WASp+/tdT− versus N-WASp−/tdT+ conditions; green is MyHC and blue is DAPI. (K) Study design schematic illustrating vehicle (Veh) and tamoxifen (Tam) treatment followed by sham or 7 days of MOV in N-WASp/tdT mice. (L) Satellite cells normalized to muscle fiber count in N-WASp+/tdT− (n = 5 sham and 5 MOV) versus N-WASp−/tdT+ (n = 6 sham and 7 MOV) mice. (M) Representative images of myonuclei in isolated single muscle fibers from MOV mice. (N) Myonuclear content analysis on isolated single muscle fibers. (O) Representative image of tdT fluorescence in MOV N-WASp−/tdT+ muscle fibers despite the prevention of satellite cell-mediated myonuclear accretion. (P) Study design schematic illustrating the treatment of wild type C57BL/6J myotubes with EVs from N-WASp−/tdT+ MPCs. (Q) Representative image showing tdT puncta (red) in C57BL/6J myotubes (stained for F-actin, green, and DAPI, blue) incubated with N-WASp−/tdT+ MPC EVs. *P < 0.05, ^#P < 0.05 effect of MOV, scale bars = 100 µm, all data are presented as mean ± SE.

Figure 2.

Evidence for the impact of EV-mediated communication to muscle fibers in vitro and in vivo. (A) Study design schematic illustrating vehicle (Veh, SC+) and tamoxifen (Tam, SC−) treatment followed by sham or 7 days of MOV in Pax7-DTA mice. (B) Mmp9 mRNA levels in sham versus MOV in the presence and absence of satellite cells; n = 6 mice per group, two separate pools of three plantaris muscles for each group resulting in n = 2 pools per group. (C) miRNAs enriched in MPC EVs that influence Mmp9 levels in different experimental models; miR-206 was the most abundant miRNA measured. (D) Summary of evidence for miRNAs that are enriched in MPC EVs that affect Mmp9 via direct 3′-UTR targeting or indirectly via experimental manipulation using miRNA mimics and/or antagomirs (see “Results” section for specific studies). (E) DIANA miRPath analysis of miRNAs enriched in MPC EVs using the top 100 miRNAs. (F) Mmp9 mRNA levels in C57BL/6J myotubes incubated with MPC EVs for 12 or 24 h; one primary cell line was used to generate myotubes and was incubated with MPC EVs from two separate cell lines at each time point (n = 2 per time point: 12 and 24 h control, 12 and 24 h EV treatment). *Adjusted P < 0.05, all data are presented as mean ± SE.

Figure 3.

Delayed satellite cell fusion does not inhibit long-term muscle hypertrophy. (A) Study design schematic illustrating vehicle (Veh) and tamoxifen (Tam) treatment followed by sham, 2 weeks, or 8 weeks of MOV in N-WASp (2 weeks) and N-WASp/tdT (8 weeks) mice. (B) Myonuclear content analysis on isolated single muscle fibers at 2 weeks (n = 5 sham and 8 MOV N-WASp+, n = 7 sham and 11 MOV N-WASp−) and 8 weeks (n = 4 sham and 4 MOV N-WASp+/tdT−, n = 4 sham and 5 MOV N-WASp−/tdT+). (C) Satellite cells normalized to muscle fiber count after 8 weeks of MOV. (D) Plantaris muscle wet weight normalized to body weight. (E) Representative plantaris muscle cross-sections labeled with dystrophin (pink), scale bar = 500 µm. (F) Magnified representative images of dystrophin, scale bar = 50 µm. (G) Average muscle fiber cross-sectional area (CSA). (H) Representative images of ECM glycosaminoglycan area evaluated by WGA staining, scale bar = 50 µm. (I) WGA area normalized to muscle fiber count. *P<0.05, ^#P < 0.05 effect of MOV, all data are presented as mean ± SE.