Fig 2: Biomarkers of neuromuscular junctions remodelling in response to 10‐day bed restNeural cell adhesion molecule (NCAM)‐positive fibres expressed as a percentage of the total numbers of fibres (A). NCAM‐positive staining at baseline (BR0) (B), 5 days of bed rest (BR5) (C, D) and 10 days of bed rest (BR10) (E–G). Serum C‐terminal agrin fragment (CAF) levels measured at BR0, BR5 and BR10 (H). AGRN gene expression (encoding for agrin) (I); CHRNA1 gene expression (encoding for acetylcholine receptor α1 subunit) (L) and HOMER2 gene expression (encoding for homer2 protein) (M). All RNA transcripts are reported as normalized read count at BR0, BR5 and BR10. Results shown as means ± SD, individual data represented as scatter plots. * P < 0.05 BR10 vs BR0.

Fig 3: Association between carrier status of rs2710873 (AGRN) and; appendicular lean mass (A total, B males, C females), whole body lean mass (D total, E males, F females) and plasma C-terminal agrin fragment (CAF) (G total, H males, I females) in the GenoFit cohort (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig 4: Growth factor expression pattern in myoneurovascular triculture. A Cell culture supernatant from the various groups was taken after 21 DIV and monitored for the secretion of growth factors like amphiregulin, insulin growth factor-binding proteins (IGFBP) 3, 4, and 6 subtypes. Motor neuron-myocyte coculture significantly increased production of amphiregulin, which is a potent mitogen of neural stem cells and has been shown to promote neurite outgrowth. IGFBPs regulate the activity of insulin-like growth factors (IGFs), wherein IGBP-4 is specifically inhibitory to IGF-1 and IGF-2. We observed high levels of extracellular IGFBP-4 when endothelial cells were cocultured with myocytes (MYO + EC) compared to other groups indicating that such a system can result in inhibiting IGF-1 and IGF-2-mediated effects on cells. Although not significant, the presence of motor neurons was found to increase levels of IGFBP-6, which is a regulator of IGF-2 signaling pathway. IGFBP-6 is significantly upregulated in spinal motor neurons post injury and has been found to be neuroprotective. We did not observe any significant difference in IGFBP-3 expression among the different experimental groups. Kruskal–Wallis test followed by Dunn's multiple comparison test was performed, n = 3/group was analyzed, and p < 0.05 was considered for significance. B Agrin expression by different cell combinations after 21 DIV was quantified from cell culture supernatants using an Agrin-specific ELISA assay. Endothelial cells alone (MYO + EC) as well as in combination with motor neurons (MYO + MN + EC) lead to elevated levels of Agrin expression as compared to myocyte monocultures. Ordinary one-way ANOVA with Tukey’s multiple comparison test was performed. n = 6/group was analyzed, and p < 0.05 was considered for significance

Fig 5: Sedentary aging is associated with reduced muscle performance and neuromuscular disturbance in vivo. (a) In vivo characterization of male participants. (b) Leg lean mass, measured by DEXA. (c) Isometric unilateral knee extension MVC, measured in a dynamometer. (d) Specific force, calculated as MVC per leg lean mass. (e) Muscle performance, measured as force exerted during repeated maximal knee extension concentric contractions, expressed relative to MVC. (f) CAF, measured in plasma by ELISA. Statistical analysis was conducted on log transformed values. (g) Muscle fiber cross sectional area measured using immunohistochemical analyses of muscle biopsy cross‐sections. All data are means ± SEM except CAF which is shown as geometric means with 95% CI. N: LLEX: 7, SED: 6, Young: 9. Statistics: Data were analyzed by unpaired two‐tailed t‐tests, with significance indicated by *p < 0.05, **p < 0.01, ***p < 0.001. CAF, C‐terminal Agrin Fragment; DEXA, dual‐energy X‐ray absorptiometry; MVC, maximal voluntary contraction.