Regulation of the muscle fiber microenvironment by activated satellite cells during hypertrophy

Abstract

Our aim in the current study was to determine the necessity of satellite cells for long-term muscle growth and maintenance. We utilized a transgenic Pax7-DTA mouse model, allowing for the conditional depletion of > 90% of satellite cells with tamoxifen treatment. Synergist ablation surgery, where removal of synergist muscles places functional overload on the plantaris, was used to stimulate robust hypertrophy. Following 8 wk of overload, satellite cell-depleted muscle demonstrated an accumulation of extracellular matrix (ECM) and fibroblast expansion that resulted in reduced specific force of the plantaris. Although the early growth response was normal, an attenuation of hypertrophy measured by both muscle wet weight and fiber cross-sectional area occurred in satellite cell-depleted muscle. Isolated primary myogenic progenitor cells (MPCs) negatively regulated fibroblast ECM mRNA expression in vitro, suggesting a novel role for activated satellite cells/MPCs in muscle adaptation. These results provide evidence that satellite cells regulate the muscle environment during growth.

Keywords: Pax7; Tcf4; extracellular matrix; fibroblast; myogenic progenitor cells.

Figure 1.

Conditional depletion of satellite cells results in attenuation of myonuclear accretion 8 wk following SA surgery (SA-8). A) Study design diagram demonstrating time of tamoxifen treatment, SA surgery, and harvest of tissue. B) Pax7 immunohistochemistry on plantaris muscle cross-section from vehicle-treated Pax7-DTA mice to identify satellite cell nuclei (green), counterstained with DAPI (blue). Pax7⁺/DAPI⁺ nuclei (arrowheads) were counted. Scale bar = 100 μm. C) Pax7 immunohistochemistry on plantaris muscle cross section from tamoxifen-treated Pax7-DTA mice. D) Quantification of Pax7⁺/DAPI⁺ nuclei, presented as mean ± se Pax7⁺ nuclei per fiber; n = 10–12 mice/group. E) Dystrophin (red) immunohistochemistry, costained with DAPI (blue). DAPI⁺ nuclei residing within the dystrophin border were classified as myonuclei (arrows). Scale bar = 50 μm. F) Quantification of myonuclei, presented as mean ± se myonuclei per fiber; n = 6. *P < 0.05 for surgery between condition-matched groups; ^†P < 0.05 for tamoxifen between condition-matched groups.

Figure 2.

Conditional depletion of satellite cells results in attenuation of muscle hypertrophy 8 wk following SA surgery (SA-8). A) Time course of plantaris growth. Muscle wet weight normalized to body weight (mg/g) 1, 2, 4, and 8 wk following surgery, presented as means ± se; n = 4–12. B) Quantification of plantaris muscle fiber CSA following dystrophin immunohistochemistry (see Fig. 1E), presented as mean ± se CSA (μm²); n = 6. C) Histogram of binned distribution of fiber CSA following dystrophin immunohistochemistry. *P < 0.05 for surgery between condition-matched groups; ^†P < 0.05 for tamoxifen between condition-matched groups.

Figure 3.

Plantaris fiber type shift 8 wk following SA surgery (SA-8) is independent of satellite cell content. A) Immunohistochemical analysis of plantaris muscle cross sections for myosin heavy chain (MHC) type I (pink), type IIa (green) and type IIb (blue). Unstained fibers are MHC type IIx. Scale bar = 100 μm. B) Quantification of MHC-specific fiber CSA, presented as mean ± se CSA (μm²); n = 6. C) Quantification of the relative frequency of the different fiber types, presented as mean ± se relative frequency; n = 6. *P < 0.05 for surgery between condition-matched groups; ^‡P = 0.1 for tamoxifen between condition-matched groups (trend toward significant effect).

Figure 4.

Decreased whole-muscle function, but not single-fiber function, in satellite cell-depleted muscle 8 wk following SA surgery (SA-8). A) Specific force (kN/m²) in permeabilized single fibers isolated from the plantaris of vehicle-treated and satellite cell-depleted animals, presented as mean ± se specific force; n = 19–29 fibers/group. B) Specific force in the plantaris of vehicle-treated and satellite cell-depleted animals, presented as mean ± se specific force; n = 8 mice/group. ^†P < 0.05 for tamoxifen between condition-matched groups.

Figure 5.

Depletion of satellite cells is associated with increased ECM deposition and fibroblast content 8 wk following SA surgery (SA-8). A) Plantaris muscle cross sections incubated with fluorescently labeled wheat germ agglutinin quantifies N-acetyl-d-glucosamine (red) a major component of the ECM. Scale bar = 100 μm. B) Quantification of area occupied by wheat germ agglutinin staining, presented as mean ± se area of wheat germ agglutinin per fiber; n = 4–6. C) Tcf4 immunohistochemistry (red) on plantaris muscle cross sections counterstained with DAPI (blue). Scale bar = 100 μm. D) Quantification of Tcf4⁺/DAPI⁺ fibroblasts, presented as mean ± se Tcf4⁺ fibroblasts per area of muscle (mm²); n = 6. *P < 0.05 for surgery between condition-matched groups; ^†P < 0.05 for tamoxifen between condition-matched groups.

Figure 6.

MPCs and MPC-CM decrease ECM gene expression in mouse primary muscle fibroblasts. A) Relative mRNA expression of various ECM components in fibroblasts that were cocultured with MPCs or fibroblasts; n = 5 isolates/group. B) Relative mRNA expression of various ECM components in fibroblasts that were cocultured in MPC- or fibroblast-CM; n = 3–5 isolates. *P < 0.05 for coculture with MPCs/MPC-CM: ^‡P = 0.1 for coculture with MPCs/MPC-CM (trend toward significant effect).