Differential requirement for satellite cells during overload-induced muscle hypertrophy in growing versus mature mice

Abstract

Background: Pax7+ satellite cells are required for skeletal muscle fiber growth during post-natal development in mice. Satellite cell-mediated myonuclear accretion also appears to persist into early adulthood. Given the important role of satellite cells during muscle development, we hypothesized that the necessity of satellite cells for adaptation to an imposed hypertrophic stimulus depends on maturational age.

Methods: Pax7^CreER-R26R^DTA mice were treated for 5 days with vehicle (satellite cell-replete, SC+) or tamoxifen (satellite cell-depleted, SC-) at 2 months (young) and 4 months (mature) of age. Following a 2-week washout, mice were subjected to sham surgery or 10 day synergist ablation overload of the plantaris (n = 6-9 per group). The surgical approach minimized regeneration, de novo fiber formation, and fiber splitting while promoting muscle fiber growth. Satellite cell density (Pax7+ cells/fiber), embryonic myosin heavy chain expression (eMyHC), and muscle fiber cross sectional area (CSA) were evaluated via immunohistochemistry. Myonuclei (myonuclei/100 mm) were counted on isolated single muscle fibers.

Results: Tamoxifen treatment depleted satellite cells by ≥90% and prevented myonuclear accretion with overload in young and mature mice (p < 0.05). Satellite cells did not recover in SC- mice after overload. Average muscle fiber CSA increased ~20% in young SC+ (p = 0.07), mature SC+ (p < 0.05), and mature SC- mice (p < 0.05). In contrast, muscle fiber hypertrophy was prevented in young SC- mice. Muscle fiber number increased only in mature mice after overload (p < 0.05), and eMyHC expression was variable, specifically in mature SC+ mice.

Conclusions: Reliance on satellite cells for overload-induced hypertrophy is dependent on maturational age, and global responses to overload differ in young versus mature mice.

Keywords: Development; Fiber splitting; Muscle hypertrophy; Pax7; Regeneration; Synergist ablation.

Fig. 1

Conditional depletion of satellite cells (Pax7+) in young and mature mice before synergist ablation surgery to overload the plantaris muscle for 10 days (OV). a Study design schematic demonstrating the duration of vehicle (satellite cell-replete, SC+) and tamoxifen (satellite cell-depleted, SC-) treatment, washout, and sham or OV surgery in young and mature mice. b–e Immunohistochemistry (IHC) representative images visualizing b laminin (green), c myonuclei (blue), d Pax7 (red), and e merged fields, where satellite cells are identified as Pax7+/DAPI+. White arrows denote the same satellite cell in each image; scale bar=20 μm. f Satellite cell density in young sham (n = 7 SC+, n = 6 SC-) and OV (n = 8 SC+, n = 6 SC-) mice. g Satellite cell density in mature sham (n = 9 SC+, n = 7 SC-) and OV (n = 6 SC+, n = 7 SC-) mice. *p < 0.05 compared to analogous SC+ group

Fig. 2

Satellite cell-mediated myonuclear accretion is prevented in satellite cell-depleted (SC-) but not satellite cell-replete (SC+) mice after synergist ablation overload of the plantaris for 10 days (OV). a–d Representative image of myonuclei per fiber length after OV in a young SC+, b mature SC+, c young SC-, d and mature SC- mice; scale bar=20 μm. e Myonuclei per 100 mm in young sham (n = 6 SC+, n = 5 SC-) and OV (n = 6 SC+, n = 6 SC-) mice. f Myonuclei per 100 mm in mature sham (n = 9 SC+, n = 7 SC-) and OV (n = 6 SC+, n = 7 SC-) mice. *p < 0.05 compared to respective sham

Fig. 3

Changes in embryonic myosin heavy chain expression (eMyHC) and muscle fiber number per whole cross section in young versus mature satellite cell-replete (SC+) and -depleted (SC-) mice after synergist ablation overload of the plantaris for 10 days (OV). a eMyHC expression after OV in young (n = 8 SC+, n = 6 SC-) and mature (n = 7 SC+, n = 7 SC-) mice, *p < 0.05 SC+ versus SC- OV within each age group. b IHC representative image visualizing eMyHC fibers of differing size in a mature SC+ mouse; green arrow points to a small-caliber eMyHC+ muscle fiber <300 μm². c Muscle fiber number in young sham (n = 7 SC+, n = 6 SC-) and OV (n = 8 SC+, n = 6 SC-) mice. d Muscle fiber number in mature sham (n = 9 SC+, n = 7 SC-) and OV (n = 6 SC+, n = 7 SC-) mice. *p < 0.05 compared to respective sham. e–f Representative image showing central nuclei (white arrows) in eMyHC+ (green) and eMyHC- (unstained) muscle fibers (pink borders). Scale bar=50 μm

Fig. 4

Average muscle fiber cross-sectional area (CSA) increases in young and mature satellite cell-replete (SC+) and mature satellite cell-depleted mice (SC-), but not young SC- mice after synergist ablation overload of the plantaris for 10 days (OV). IHC representative images visualizing muscle fiber borders (pink) in a young sham (SC+, SC-) and OV (SC+, SC-) mice, and b mature sham (SC+, SC-) and OV (SC+, SC-) mice; scale bar=20 μm. c Average muscle fiber CSA in young sham (n = 7 SC+, n = 6 SC-) and OV (n = 8 SC+, n = 6 SC-) mice, excluding fibers <300 μm². d Average muscle fiber CSA in mature sham (n = 9 SC+, n = 7 SC-) and OV (n = 6 SC+, n = 7 SC-) mice, excluding fibers <300 μm², but including eMyHC+ fibers. e Average muscle fiber CSA in mature mice, excluding fibers <300 μm², as well as eMyHC+ fibers. *p < 0.05 compared to respective sham

Fig. 5

Dynamic continuum of satellite cell (SC) participation in overload-induced muscle fiber hypertrophy throughout the lifespan. Satellite cells are required for hypertrophic growth in young adult mice (2.5 months of age), are not required for hypertrophic growth in mature adult mice (>4 months of age), and are not permissive for hypertrophy in aged anabolic resistant mice (>24 months of age, refer to Lee et al. [41]). CSA cross-sectional area