Supplementary Materialsaging-08-2871-s001. which stem cell function can be linked to lipid signaling and homeostasis, and provide important new targets to stimulate muscle repair in aged individuals. [11,12]. In contrast, the Mouse Monoclonal to E2 tag alternative pathway is activated during myoblast fusion to form multinucleated myotubes, where it may regulate mitochondrial biogenesis . Tonic activation of canonical NF-B signaling in muscle fibers drives progressive muscle atrophy, in part by upregulation of the E3 ubiquitin ligases MURF and MAFbx [14,15]. Conversely, inhibition of NF-B activity in a variety of cell types, including macrophages and myofibers, can reduce inflammation and fibrosis and accelerate repair after muscle injury [16,17]. Here, we investigate the particular role of canonical NF-B signaling in the loss of muscle regenerative potential that typically occurs during normal aging. These studies reveal that selective activation of NF-B activity in muscle fibers drives dysfunction of regenerative muscle satellite cells and that life-long inhibition of NF-B activity in myofibers preserves muscle repair potential with aging via cell-non-autonomous effects on satellite cell function. Further analysis of differential gene expression in muscles with varying NF-B activity identified a secreted phospholipase (PLA2G5) as a myofiber-expressed, Licofelone NF-B-regulated gene that governs muscle regenerative capacity with age. These data suggest a model in which NF-B activation in muscle fibers increases PLA2G5 expression and drives the impairment in regenerative function characteristic of aged muscle. Importantly, inhibition of NF-B function reverses this impairment, suggesting that FDA-approved drugs like salsalate, which diminish NF-B activity, may provide new therapeutic avenues for elderly patients with reduced capacity to recover effectively from muscle injury. RESULTS Increased NF-B activity in myofibers and myotubes, but not in satellite cells alone, impairs satellite cell function Age-associated deficiencies in muscle repair slow recovery of muscle function and promote replacement of damaged myofibers with excess fat and fibrous tissue rather than newly formed muscle [2,3]. Based in part on studies in mice and humans suggesting that a pro-inflammatory microenvironment impairs physiological function [14,18,19] and limits repair potential in aged muscle , we hypothesized that alterations in canonical NF-B signaling may underwrite some of the functional changes induced in muscle during aging. Consistent with this hypothesis, muscle satellite cells isolated by fluorescence activated cell sorting (FACS, Fig. S1) from aged (24 months aged) mice showed substantially increased expression of many genes that are either direct targets or activators of the NF-B pathway, including (expression in aged WT muscle and reduced expression in aged MISR muscle (Fig. ?(Fig.3A).3A). Although present at substantially lower levels than in whole muscle tissue, was also expressed in muscle satellite cells, with higher levels in aged WT and young SCIKK mice and lower levels in young WT and aged WT mice treated with salicylate (Fig. ?(Fig.3B).3B). We therefore tested whether inhibition of expression in muscle might be sufficient to restore muscle regeneration in aged mice. Open in a separate window Physique 3 Inhibition of expression improves muscle regeneration in aged Licofelone mice(A, B) Expression of electroporation. Muscles were damaged by cryoinjury 1 day after electroporation, and regenerating myofiber size was measured 7 days after cryoinjury. Electroporation efficiency in each sample was evaluated by evaluation of mCherry-expressing myofibers. Size pubs = 500 m. (D) Performance of gene knockdown by siRNA assessed by qRT-PCR at muscle tissue harvest and in comparison to degrees of pla2g5 mRNA in muscle groups electroporated with control, scrambled siRNA (n=19 mice each group). Data stand for suggest s.e.m.; p-value computed by Student’s t check. (E) Consultant H&E staining of Licofelone muscle tissue sections at time 7 after cryoinjury from aged mice getting pla2g5 or control, scrambled siRNA. Size pubs = 100 m. (F, G) Distribution and typical of size of regenerating (centrally-nucleated) myofibers in aged mice getting control, scrambled or pla2g5 siRNA (n=6 mice per experimental group). Contralateral TA muscle groups were utilized as handles with electroporation of scrambled siRNA. Data symbolized as histograms of fibers size (E) or as mean s.e.m. (F). P-values computed by Kruskal-Wallis check for (E) and (F). Using electroporation , siRNA was co-delivered with mCherry fluorescent protein-expressing plasmid into.