A. Skeletal muscle cryosections were immunostained for Pax7, M-Cadherin and NCAM (green). Laminin immuno-detection is shown in red and Hoechst labels nuclei (blue). Sublaminar mono-nucleated cells expressing these markers were identified in association with both young and old myofibers. B. The total numbers of Pax7, NCAM and M-Cadherin⁺ satellite cell nuclei were quantified per 10mm² for both young and old muscle. *, P ≤ 0.05 (old compared to young at ‘pre’ condition). C. Total numbers of Pax7⁺ nuclei were analyzed at the pre and 2wk (resting and immobilization phase) and the 2wk+3d and 4wk time points (regeneration phase). *, P ≤ 0.05 (young compared to old at 2wk + 3d). D. Western blotting for Pax7 was performed on whole muscle protein isolates (quantified in E), using actin as a loading control. * P ≤ 0.05, old compared with young. Data are means ± s.d. n=10 for immunostained cryosections, n=6 for Western blot analysis.

Immunodetection of: (A) TGF-β (green) and laminin (red), (B) P-Smad3 (green) and laminin (red) is shown for 10μm skeletal muscle cryosections. Hoechst labels nuclei (blue). C. Western blotting for TGF-β, P-Smad3, p15, p21, p16 and p27 from whole muscle protein lysates; quantified in D. Actin was used as loading control. *, P ≤ 0.05, old compared with young. Significant age-specific elevation of TGF-β/pSmad and of CDK inhibitors, p15 and p21 was detected in old muscle as compared to young. E. Activated satellite cells were cultured for 24hr in OPTI-MEM containing age-matched human sera in the presence of 25ng/ml recombinant TGF-β1. Myogenic responses were analyzed and quantified (F), based on the co-expression of desmin/BrdU. Data are means ± s.d., n = 10-15 for immunodetection of cryosections, n = 6 for Western blotting analysis, n=6 for myogenic culture experiments.

A. Young and old satellite cells were isolated and cultured in the presence of young or old sera, with or without experimentally-induced Notch activation (immobilized Delta ligand for old cells) or Notch inhibition (treatment with gamma secretase inhibitor (GSI) for young cells). After seven days of culture, cells were fixed and analyzed for myogenic responses (generation of desmin/BrdU co-stained cells). Quantification is shown in B. *, P ≤ 0.05 (Y + Y sera compared to O + O sera and Y + Y sera/GSI, O + O sera/Delta compared to O + O sera). n = 6. Representative immunostaining is depicted in A; desmin (green) BrdU (red), Hoechst (blue) labels nuclei. Activation of Notch is required for myogenic properties of young satellite cells and rescues myogenic potential of old human satellite cells even in the presence of aged human sera. Young satellite cells did not have significantly higher myogenic potential when Notch was experimentally activated, as compared to control young cells, suggesting that young myogenic responses are at the optimal high (not shown). C. Treated cells were analyzed for the expression of p15 and p21 by Western blot; quantified in D. Actin serves as loading control. *, P ≤ 0.05, Y + GSI compared to Y+Y and O + Delta compared to O+O. Data are means ± s.d., n = 6 for immunodetection assays and Western blot analyses. Forced activation of Notch reduces levels of p15 and p21, while experimental attenuation of Notch activity increases the levels of these CDK inhibitors in satellite cells.

A. Scheme of experimental setup, as described in text. B. Muscle histology from resting state (pre), immobility-atrophy (2wk imm.) and loading-recovery (3d recovery, 4wk recovery) was analyzed by haematoxylin and eosin (H & E) of 10-μm skeletal muscle cryosections. During immobilization phase of the study areas of severe degeneration and scar tissue formation were evident in old muscle (yellow arrows), versus healthy maintenance of young immobilized muscle (white arrows). Scale bar = 100μm. n = 10.

A. Cryosections were analyzed by immunostaining for co-expression of nuclear Pax7⁺/active Notch in resident satellite cells. B. Delta (red) and dystrophin (green) immuno-detection is shown for 10μm skeletal muscle cryosections. Hoechst labels nuclei (blue). C. Western blot of Notch and Delta levels on whole muscle protein isolates for 2wk+3d; quantified in D. *P ≤ 0.05, old compared with young for both Notch and Delta. E. Quantification of Notch/Pax7 double-positive myofiber-associated cells from cryosections. Data are means ± s.d., n = 10-15 for immunodetection of cryosections. n = 6 for Western blotting analysis. As compared to young tissue, in old muscle loaded after immobility, there is significant down-regulation of Delta, active Notch and decline in numbers of myofiber-associated myogenic cells that co-express Pax7 and active Notch.

A. Western blot on whole muscle protein isolates was performed for P-Erk and Erk, quantified in B where *, P ≤ 0.05, old compared with young. Similar levels of Erk, but lower levels of pErk were detected in old muscle as compared to young. C. Western blot of Notch, Delta, P-Erk and Erk was performed (quantified in D), following 24hr exogenous addition of 10ng/ml FGF or 10μM MEK inhibitor (MEKi) to isolated human satellite cell cultures *, **P ≤ 0.05, +FGF compared to Control, and +MEKi compared to Control. Experimental attenuation of MAPK decreases levels of Delta and active Notch, while induction of MAPK up-regulates Delta and active Notch. E. Activated satellite cell, cultured as in (C), were analyzed for myogenic responses and quantified (F), based on the co-expression of desmin/BrdU Data are means ± s.d., n = 6 for all panels indicated. Myogenic responses of both young and old human satellite cells were enhanced by forced activation of MAPK and were diminished by attenuation of MAPK.