Depletion of Pax7+ satellite cells does not affect diaphragm adaptations to running in young or aged mice

Abstract

Key points: Satellite cell depletion does not affect diaphragm adaptations to voluntary wheel running in young or aged mice. Satellite cell depletion early in life (4 months of age) has minimal effect on diaphragm phenotype by old age (24 months). Prolonged satellite cell depletion in the diaphragm does not result in excessive extracellular matrix accumulation, in contrast to what has been reported in hind limb muscles. Up-regulation of Pax3 mRNA+ cells after satellite cell depletion in young and aged mice suggests that Pax3+ cells may compensate for a loss of Pax7+ satellite cells in the diaphragm. Future investigations should focus on the role of Pax3+ cells in the diaphragm during adaptation to exercise and ageing.

Abstract: Satellite cell contribution to unstressed diaphragm is higher compared to hind limb muscles, which is probably attributable to constant activation of this muscle to drive ventilation. Whether satellite cell depletion negatively impacts diaphragm quantitative and qualitative characteristics under stressed conditions in young and aged mice is unknown. We therefore challenged the diaphragm with prolonged running activity in the presence and absence of Pax7+ satellite cells in young and aged mice using an inducible Pax7^CreER -R26R^DTA model. Mice were vehicle (Veh, satellite cell-replete) or tamoxifen (Tam, satellite cell-depleted) treated at 4 months of age and were then allowed to run voluntarily at 6 months (young) and 22 months (aged). Age-matched, cage-dwelling, Veh- and Tam-treated mice without wheel access served as activity controls. Diaphragm muscles were analysed from young (8 months) and aged (24 months) mice. Satellite cell depletion did not alter diaphragm mean fibre cross-sectional area, fibre type distribution or extracellular matrix content in young or aged mice, regardless of running activity. Resting in vivo diaphragm function was also unaffected by satellite cell depletion. Myonuclear density was maintained in young satellite cell-depleted mice regardless of running, although it was modestly reduced in aged sedentary (-7%) and running (-19%) mice without satellite cells (P < 0.05). Using fluorescence in situ hybridization, we detected higher Pax3 mRNA+ cell density in both young and aged satellite cell-depleted diaphragm muscle (P < 0.05), which may compensate for the loss of Pax7+ satellite cells.

Keywords: Pax3; Pax7; fluorescent in-situ hybridization.

Figure 1. Conditional depletion of satellite cells in young and aged mice

A, study design schematic demonstrating the duration of Veh and Tam treatment, washout and running in young and aged mice. B–E, representative immunohistochemistry images of Pax7 staining in young (B) Veh‐ and (C) Tam‐ treated sedentary mice, and aged (D) Veh‐ and (E) Tam‐treated sedentary mice. F, satellite cell density in young sedentary (n = 7 Veh, n = 7 Tam) and running (n = 7 Veh, n = 10 Tam) mice. G, satellite cell density in aged sedentary (n = 10 Veh, n = 12 Tam) and running (n = 9 Veh, n = 11 Tam) mice. Values are minimum, first quartile, median, third quartile and maximum; ^#main effect for age; ^*main effect for satellite cell depletion (P < 0.05).

Figure 2. Diaphragm myonuclear density in young and aged sedentary and running mice in the presence (Veh) and and absence (Tam) of satellite cells

A, single muscle fibre myonuclear density in young sedentary (n = 7 Veh, n = 6 Tam) and running (n = 8 Veh, n = 7 Tam) mice. B, single muscle fibre myonuclear density in aged sedentary (n = 7 Veh, n = 7 Tam) and running (n = 8 Veh, n = 9 Tam) mice. C, myonuclei per area from histological cross‐sections in young sedentary (n = 4 Veh, n = 5 Tam) and running (n = 4 Veh, n = 4 Tam) mice. D, myonuclei per area from histological cross‐sections in aged sedentary (n = 4 Veh, n = 5 Tam) and running (n = 4 Veh, n = 5 Tam) mice. Values are the mean ± SE; ^*main effect for satellite cell depletion (P < 0.05); †main effect for running (P < 0.05).

Figure 3. Diaphragm muscle fibre CSA in young and aged sedentary and running mice in the presence (Veh) and absence (Tam) of satellite cells

A, muscle fibre CSA in young sedentary (n = 7 Veh, n = 8 Tam) and running (n = 7, n = 10) mice. B, muscle fibre CSA in aged sedentary (n = 7 Veh, n = 9 Tam) and running (n = 9 Veh, n = 10 Tam) mice. Values are the mean ± SE; †main effect for running (P < 0.05).

Figure 4. Diaphragm muscle fibre type distribution in young and aged sedentary and running mice in the presence (Veh) and absence (Tam) of satellite cells

A, muscle fibre type distribution in young sedentary (n = 6 Veh, n = 5 Tam) and running (n = 6 Veh, n = 9 Tam) mice. B, muscle fibre type distribution in aged sedentary (n = 6 Veh, n = 7 Tam) and running (n = 5 Veh, n = 6 Tam) mice. C, representative diaphragm immunohistochemistry image visualizing MyHC type I (pink), IIa (green) and IIx (orange); scale bar = 50 μm. Values are the mean ± SE; ^*different from Tam within aged runners; †main effect for running (P < 0.05).

Figure 5. Diaphragm extracellular matrix content (ECM) in young and aged sedentary and running mice in the presence (Veh) and absence (Tam) of satellite cells

Representative muscle cross‐section visualizing WGA from (A) young and (B) aged diaphragm muscle; scale bar = 50 μm. C, ECM content in young sedentary (n = 7 Veh, n = 8 Tam) and running (n = 7 Veh, n = 9 Tam) mice. D, ECM content in young sedentary (n = 7 Veh, n = 10 Tam) and running (n = 6 Veh, n = 8 Tam) mice.

Figure 6. In vivo diaphragm breath depth at rest in young and aged sedentary and running mice in the presence (Veh) and absence (Tam) of satellite cells

A, representative ultrasound tracing of diaphragm excursion. Arrows show the magnitude of inspiration and expiration. B, breath depth in young sedentary (n = 5 Veh, n = 4 Tam) and running (n = 3 Veh, n = 4 Tam) mice. C, breath depth in aged sedentary (n = 7 Veh, n = 4 Tam) and running (n = 6 Veh, n = 6 Tam) mice. Values are the mean ± SE; †main effect for running (P < 0.05). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 7. Diaphragm Pax3 mRNA+ cells in young and aged sedentary and running mice in the presence (Veh) and absence (Tam) of satellite cells

A and A′, representative images of Pax3 mRNA+ cells (orange) overlapping with DAPI+ myonuclei (blue) in the diaphragm. B and B′, lack of Pax3 mRNA+ cells in the diaphragm using a scrambled probe (NC). C and C′, representative images of Pax3 mRNA+ cells overlaping with DAPI+ myonuclei in liver tissue (probe 1728, positive control). D and D′, only DAPI+ myonuclei in spleen tissue, indicating a lack of Pax3 mRNA+ cells (probe 1728. negative control); scale bars = 20 μm. E, Pax3+ cell density in young sedentary (n = 6 Veh, n = 6 Tam) and running (n = 5 Veh, n = 8 Tam) mice. F, Pax3+ cell density in mature sedentary (n = 6 Veh, n = 7 Tam) and running (n = 6 Veh, n = 7 Tam) mice. Values are the mean ± SE; ^*main effect for satellite cell depletion (P < 0.05).