Age-dependent alteration in muscle regeneration: the critical role of tissue niche

Abstract

Although adult skeletal muscle is composed of fully differentiated fibers, it retains the capacity to regenerate in response to injury and to modify its contractile and metabolic properties in response to changing demands. The major role in the growth, remodeling and regeneration is played by satellite cells, a quiescent population of myogenic precursor cells that reside between the basal lamina and plasmalemma and that are rapidly activated in response to appropriate stimuli. However, in pathologic conditions or during aging, the complete regenerative program can be precluded by fibrotic tissue formation and resulting in functional impairment of the skeletal muscle. Our study, along with other studies, demonstrated that although the regenerative program can also be impaired by the limited proliferative capacity of satellite cells, this limit is not reached during normal aging, and it is more likely that the restricted muscle repair program in aging is presumably due to missing signals that usually render the damaged muscle a permissive environment for regenerative activity.

Fig. 1

Schematic model depicting muscle regeneration. (a) Schematic representation of the four interrelated and time-dependent phases underlying muscle regeneration. The relevant biological responses, activated after cardiotoxin (CTX) injection, are indicated. (b) Schematic model outlining the relevant markers expressed by satellite cells during the different stages of regeneration. (h: hours; d: days). stages of muscle regeneration that could be impaired in muscle aging

Fig. 2

Immunofluorescence analysis showing that aged satellite cells, in culture, express specific markers of activated and proliferating satellite cells, such as Pax7, desmin, MyoD, and Ki67

Fig. 3

Proliferative capacity of human muscle precursor cells (myoblasts) isolated from three groups of subjects (young, old sedentary and old active). The proliferative lifespan was determined on five distinct cultures isolated from five different donors in each group of subjects

Fig. 4

p16 mRNA expression (a) and Telomere length (b) were measured by qRT-PCR and qPCR respectively in proliferative myoblasts at the beginning of their lifespan (P) and in senescence myoblasts (S). Values are mean ± standard deviation, n = 5 and 10 for young and old respectively

Fig. 5

Proliferation of aged satellite cells is impinged in autologous culture conditions. Immunofluorescence analysis for BrdU incorporation in satellite cells obtained from young (30.3 ± 1.8 year-old) and old (83.3 ± 6.3 year-old) subjects, cultured in either autologous serum (AS) or heterologous serum (HS). Old-derived satellite cells showed a 21.3 % of BrdU-positive cells when cultured in autologous (homochronic) serum (middle panel), whereas old-derived satellite cells showed a 32.5 % of BrdU-positive cells when cultured in heterologous (heterochronic from young donors) (right panel). Young-derived satellite cells showed a 51 % of BrdU-positive cells (left panel). No major differences in the percentage of BrdU-positive cells were observed when young-derived satellite cells were cultured in either autologous (homochronic) or heterologous (heterochronic from old donors) serum. Bottom panels show Hoechst nuclear staining

Fig. 6

p53 and p21 expression is increased in aged myoblasts. Real time PCR for the expression of p53 and p21 on myoblasts obtained from young (21.6 ± 2.23 year-old) and old (73.37 ± 2.66 year-old) subjects. Data are represented as average ± SEM. (p53: p = 0.02; p21: p = 0.03)

Fig. 7

Differentiation of aged satellite cells is compromised in autologous culture conditions. Immunofluorescence analysis for MyHC expression. Muscle differentiation was evaluated analysing the expression of MyHC, which resulted dramatically reduced in aged satellite cells cultured in autologous serum (5 % AS) and partially recovered in satellite cells cultured in heterologous serum (5 % HS). Nuclei (blue) were stained with Hoechst

Fig. 8

TNFα levels and NFkB expression increase in the muscle of old subjects. (a) Elisa assay to evaluate the concentration of TNFα in muscles of young and old females and males indicated. Data are represented as average ± SEM. n = 12 old females, n = 17 young females, n = 15 old males, n = 12 young males. (b) Left panel: western blot quantification of phosphorylated active forms of NF-κB p65 subunit (P-p65). Subjects were categorized by age (young, <40 years; and old, >70 years) and by gender. Quantification was performed using ImageJ software and normalized to β-tubulin used as loading controls. *p < 0.021, one-way ANOVA test; •p < 0.001 and Δp = 0.012, Student’ t test. Right panel shows representative western blot analysis for P-p65 and total p65 in female and male of different age