Failed reinnervation in aging skeletal muscle

Abstract

Background: Skeletal muscle displays a marked accumulation of denervated myofibers at advanced age, which coincides with an acceleration of muscle atrophy.

Methods: In this study, we evaluated the hypothesis that the accumulation of denervated myofibers in advanced age is due to failed reinnervation by examining muscle from young adult (YA) and very old (VO) rats and from a murine model of sporadic denervation secondary to neurotrypsin over-expression (Sarco mouse).

Results: Both aging rat muscle and Sarco mouse muscle exhibited marked fiber-type grouping, consistent with repeating cycles of denervation and reinnervation, yet in VO muscle, rapsyn at the endplate increased and was associated with only a 10 % decline in acetylcholine receptor (AChR) intensity, whereas in Sarco mice, there was a decline in rapsyn and a 25 % decrease in AChR intensity. Transcripts of muscle-specific kinase (21-fold), acetylcholine receptor subunits α (68-fold), ε (threefold) and γ (47-fold), neural cell adhesion molecule (66-fold), and runt-related transcription factor 1 (33-fold) were upregulated in VO muscle of the rat, consistent with the marked persistent denervation evidenced by a large proportion of very small fibers (>20 %). In the Sarco mice, there were much smaller increases in denervation transcripts (0-3.5-fold) and accumulation of very small fibers (2-6 %) compared to the VO rat, suggesting a reduced capacity for reinnervation in aging muscle. Despite the marked persistent denervation in the VO rat muscle, transcripts of neurotrophins involved in promoting axonal sprouting following denervation exhibited no increase, and several miRNAs predicted to suppress neurotrophins were elevated in VO rat.

Conclusions: Our results support the hypothesis that the accumulation of denervated fibers with aging is due to failed reinnervation and that this may be affected by a limited neurotrophin response that mediates axonal sprouting following denervation.

Keywords: Aging; Denervation; MicroRNA; Muscle atrophy; Neurotrophins; Reinnervation; Sarcopenia; Skeletal muscle.

Fig. 1

Muscle fiber type and type grouping. a Transverse muscle sections from rat vastus lateralis (VL) and mouse soleus muscle. Young adult (YA) vs very old (VO) rat VL muscle and wild-type (WT) vs Sarco mouse soleus were labeled with laminin to visualize the basal lamina and antibodies against the myosin heavy chains to visualize fiber types. Scale bars = 100 μm. Type I (blue), type IIa (red), type IIx (green), type IIb (black [no staining]) were labeled. b Fiber type proportions in YA vs VO rat and WT vs Sarco mouse. c Fiber type grouping in YA vs VO rat and WT vs Sarco mouse. In b and c, the data shown are means with standard error

Fig. 2

Protein analyses at the muscle endplate region. a Representative images of muscle endplates from YA vs VO (VL muscle) and WT vs Sarco (gastrocnemius muscle) labeled with primary antibodies against phospho-MuSK (scale bars = 10 μm). b Semi-quantitative analysis of protein intensities at the muscle endplate for rapsyn and AChRs in YA (n = 154–158 endplates) vs VO (n = 304–311 endplates) (VL muscle) and WT (n = 185 endplates) vs Sarco (n = 287–296 endplates) (gastrocnemius muscle). Values are normalized as a fold change of YA or WT, respectively. *P < 0.05 vs corresponding group within the same species

Fig. 3

Morphological and transcriptional markers of denervation. a Abundance of very small fibers in YA vs VO rat vastus lateralis muscle, WT vs Sarco mouse gastrocnemius muscle, and WT vs Sarco mouse soleus muscle. b qPCR analysis of denervation markers: MuSK, AChRa, AChRe, AChRg, cPLA2, NCAM, and RUNX1 in YA (n = 8) vs VO (n = 10) rat vastus lateralis muscle and WT (n = 8) vs Sarco mouse (n = 6) gastrocnemius and soleus muscle. TBP was used as endogenous control. Data are shown as means with standard error. T tests were performed. *P < 0.05 was considered as statistically significant

Fig. 4

Neurotrophin and receptor transcript analysis. qPCR analysis of the neurotrophins: BDNF, NGF, NT3, NT4/5, GDNF, CNTF and neurotrophin receptors: p75, NTRK1, NTRK2, NTRK3, GFRA1, and CNTFR in YA (n = 8) vs VO (n = 10) rat VL (a), Sarco gastroc (b), and Sarco soleus (c) muscles. TBP was used as endogenous control. Data are shown as means with standard error. T tests were performed. *P < 0.05 was considered as statistically significant

Fig. 5

MicroRNA analysis. MicroRNAs predicted to influence neurotrophins: a BDNF (miR-206-3p, miR-10a-5p, miR-1b, miR-195-5p and miR-497-5p), b NT3 (miR-21-5p, miR-222-3p and miR-221-3p), and c NGF (let-7b-5p and miR-98-5p) were quantified by qPCR analysis in YA (n = 8) vs VO (n = 10) rat vastus lateralis muscle and WT (n = 8) vs Sarco (n = 7) gastrocnemius muscle. miR-191a-5p was used as endogenous control. Data are shown as means with standard error. T tests were performed. *P < 0.05 was considered as statistically significant