Mammalian skeletal muscles exhibit age-related adaptive and pathological remodeling. Several muscles in particular undergo progressive atrophy and degeneration beyond median lifespan. To better understand myocellular responses to aging, we used semi-quantitative global metabolomic profiling to characterize trends in metabolic changes between 15-month-old adult and 32-month-old aged Fischer 344 × Brown Norway (FBN) male rats. The FBN rat gastrocnemius muscle exhibits age-dependent atrophy, whereas the soleus muscle, up until 32 months, exhibits markedly fewer signs of atrophy. Both gastrocnemius and soleus muscles were analyzed, as well as plasma and urine. Compared to adult gastrocnemius, aged gastrocnemius showed evidence of reduced glycolytic metabolism, including accumulation of glycolytic, glycogenolytic, and pentose phosphate pathway intermediates. Pyruvate was elevated with age, yet levels of citrate and nicotinamide adenine dinucleotide were reduced, consistent with mitochondrial abnormalities. Indicative of muscle atrophy, 3-methylhistidine and free amino acids were elevated in aged gastrocnemius. The monounsaturated fatty acids oleate, cis-vaccenate, and palmitoleate also increased in aged gastrocnemius, suggesting altered lipid metabolism. Compared to gastrocnemius, aged soleus exhibited far fewer changes in carbohydrate metabolism, but did show reductions in several glycolytic intermediates, fumarate, malate, and flavin adenine dinucleotide. Plasma biochemicals showing the largest age-related increases included glycocholate, heme, 1,5-anhydroglucitol, 1-palmitoleoyl-glycerophosphocholine, palmitoleate, and creatine. These changes suggest reduced insulin sensitivity in aged FBN rats. Altogether, these data highlight skeletal muscle group-specific perturbations of glucose and lipid metabolism consistent with mitochondrial dysfunction in aged FBN rats.

Deterioration of diaphragm function is one of the prominent factors that contributes to the susceptibility of serious respiratory infections and development of respiratory failure in patients with Duchenne Muscular Dystrophy (DMD). The NF-κB signaling pathway has been implicated as a contributing factor of dystrophic pathology, making it a potential therapeutic target. Previously, we demonstrated that pharmacological inhibition of NF-κB via a small NEMO Binding Domain (NBD) peptide was beneficial for reducing pathological features of mdx mice. Now, we stringently test the effectiveness and clinical potential of NBD by treating mdx mice with various formulations of NBD and use diaphragm function as our primary outcome criteria. We found that administering DMSO-soluble NBD rescued 78% of the contractile deficit between mdx and wild-type (WT) diaphragm. Interestingly, synthesis of a GLP NBD peptide as an acetate salt permitted its solubility in water, but as a negative consequence, also greatly attenuated functional efficacy. However, replacing the acetic acid counterion of the NBD peptide with trifluoroacetic acid retained the peptide’s water solubility and significantly restored mdx diaphragm contractile function and improved histopathological indices of disease in both diaphragm and limb muscle. Together, these results support the feasibility of using a mass-produced, water-soluble NBD peptide for clinical use.

Background: Classical NF-kappa B signaling functions as a negative regulator of skeletal myogenesis through potentially multiple mechanisms. The inhibitory actions of TNF alpha on skeletal muscle differentiation are mediated in part through sustained NF-kappa B activity. In dystrophic muscles, NF-kappa B activity is compartmentalized to myofibers to inhibit regeneration by limiting the number of myogenic progenitor cells. This regulation coincides with elevated levels of muscle derived TNF alpha that is also under IKK beta and NF-kappa B control.Methodology/Principal Findings: Based on these findings we speculated that in DMD, TNF alpha secreted from myotubes inhibits regeneration by directly acting on satellite cells. Analysis of several satellite cell regulators revealed that TNF alpha is capable of inhibiting Notch-1 in satellite cells and C2C12 myoblasts, which was also found to be dependent on NF-kappa B. Notch-1 inhibition occurred at the mRNA level suggesting a transcriptional repression mechanism. Unlike its classical mode of action, TNF alpha stimulated the recruitment of Ezh2 and Dnmt-3b to coordinate histone and DNA methylation, respectively. Dnmt-3b recruitment was dependent on Ezh2.Conclusions/Significance: We propose that in dystrophic muscles, elevated levels of TNF alpha and NF-kappa B inhibit the regenerative potential of satellite cells via epigenetic silencing of the Notch-1 gene.

Clenbuterol and other beta2-adrenergic agonists are effective at inducing muscle growth and attenuating muscle atrophy through unknown mechanisms. This study tested the hypothesis that clenbuterol-induced growth and muscle sparing is mediated through the activation of Akt and mammalian target of rapamycin (mTOR) signaling pathways. Clenbuterol was administered to normal weight-bearing adult rats to examine the growth-inducing effects and to adult rats undergoing muscle atrophy as the result of hindlimb suspension or denervation to examine the muscle-sparing effects. The pharmacological inhibitor rapamycin was administered in combination with clenbuterol in vivo to determine whether activation of mTOR was involved in mediating the effects of clenbuterol. Clenbuterol administration increased the phosphorylation status of PKB/Akt, S6 kinase 1/p70(s6k), and eukaryotic initiation factor 4E binding protein 1/PHAS-1. Clenbuterol treatment induced growth by 27-41% in normal rats and attenuated muscle loss during hindlimb suspension by 10-20%. Rapamycin treatment resulted in a 37-97% suppression of clenbuterol-induced growth and a 100% reduction of the muscle-sparing effect. In contrast, rapamycin was unable to block the muscle-sparing effects of clenbuterol after denervation. Clenbuterol was also shown to suppress the expression of the MuRF1 and MAFbx transcripts in muscles from normal, denervated, and hindlimb-suspended rats. These results demonstrate that the effects of clenbuterol are mediated, in part, through the activation of Akt and mTOR signaling pathways.

Nat. Med. 11, 199–205 (2005) In Methods, the sequence of the peptide used to generate the anti-SHIP2 polyclonal antibody should read PRGPSDYGRPLSFPPPRIRESIQED.

Summary membrane phospholipid phosphatidylinositol-4,5-bis- phosphate to phosphatidylinositol-3,4,5-trisphosphate, Skeletalmusclesizedependsuponadynamicbalance creating a lipid binding site on the cell membrane forbetween anabolic (or hypertrophic) and catabolic (or a serine/threonine kinase called Akt (or PKB—protein atrophic) processes. Previously, no link between the kinase B). The subsequent translocation of Akt to themolecular mediators of atrophy and hypertrophy had membrane facilitates its phosphorylation and activation been reported. We demonstrate a hierarchy between by the kinase PDK-1 (Cantley, 2002; Datta et al., 1999;the signals which mediate hypertrophy and those Vivanco and Sawyers, 2002). Cell growth and survival which mediate atrophy: the IGF-1/PI3K/Akt pathway, inavarietyoftissuesandcelltypesinresponsetoIGF-1,which has been shown to induce hypertrophy, pre- insulin, and other growth factors is critically mediated ventsinductionofrequisiteatrophymediators,namely by Akt (Datta et al., 1999; Vivanco and Sawyers, 2002).the muscle-specific ubiquitin ligases MAFbx and

Skeletal muscles adapt to changes in their workload by regulating fibre size by unknown mechanisms1,2. The roles of two signalling pathways implicated in muscle hypertrophy on the basis of findings in vitro3,4,5,6, Akt/mTOR (mammalian target of rapamycin) and calcineurin/NFAT (nuclear factor of activated T cells), were investigated in several models of skeletal muscle hypertrophy and atrophy in vivo. The Akt/mTOR pathway was upregulated during hypertrophy and downregulated during muscle atrophy. Furthermore, rapamycin, a selective blocker of mTOR7, blocked hypertrophy in all models tested, without causing atrophy in control muscles. In contrast, the calcineurin pathway was not activated during hypertrophy in vivo, and inhibitors of calcineurin, cyclosporin A and FK506 did not blunt hypertrophy. Finally, genetic activation of the Akt/mTOR pathway was sufficient to cause hypertrophy and prevent atrophy in vivo, whereas genetic blockade of this pathway blocked hypertrophy in vivo. We conclude that the activation of the Akt/mTOR pathway and its downstream targets, p70S6K and PHAS-1/4E-BP1, is requisitely involved in regulating skeletal muscle fibre size, and that activation of the Akt/mTOR pathway can oppose muscle atrophy induced by disuse.