Although cytokine-induced nuclear factor kappaB (NF-kappaB) pathways are involved in muscle wasting subsequent to disease, their potential role in disuse muscle atrophy has not been characterized. Seven days of hind limb unloading led to a 10-fold activation of an NF-kappaB-dependent reporter in rat soleus muscle but not the atrophy-resistant extensor digitorum longus muscle. Nuclear levels of p50 were markedly up-regulated, c-Rel was moderately up-regulated, Rel B was down-regulated, and p52 and p65 were unchanged in unloaded solei. The nuclear IkappaB protein Bcl-3 was increased. There was increased binding to an NF-kappaB consensus oligonucleotide, and this complex bound antibodies to p50, c-Rel, and Bcl-3 but not other NF-kappaB family members. Tumor necrosis factor alpha (TNF-alpha) and TNF receptor-associated factor 2 protein were moderately down-regulated. There was no difference in p38, c-Jun NH(2)-terminal kinase or Akt activity, nor were activator protein 1 or nuclear factor of activated T cell-dependent reporters activated. Thus, whereas several NF-kappaB family members are up-regulated, the prototypical markers of cytokine-induced activation of NF-kappaB seen with disease-related wasting are not evident during disuse atrophy. Levels of an anti-apoptotic NF-kappaB target, Bcl-2, were increased fourfold whereas proapoptotic proteins Bax and Bak decreased. The evidence presented here suggests that disuse muscle atrophy is associated with activation of an alternative NF-kappaB pathway that involves the activation of p50 but not p65.

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Investigating the molecular mechanisms underlying sarcopenia in humans with the use of microarrays has been complicated by low sample size and the variability inherent in human gene expression profiles. We have conducted a study using Affymetrix GeneChips to identify a molecular signature of aged skeletal muscle. The molecular signature was defined as the set of expressed genes that best distinguished the vastus lateralis muscle of young ( n = 10) and older ( n = 12) male subjects, when a k-nearest neighbor supervised classification method was used in conjunction with a signal-to-noise ratio gene selection method and a holdout cross-validation procedure. The age-specific expression signature was comprised of 45 genes; 27 were upregulated and 18 were downregulated. This signature also correctly classified 75% of the muscle samples from young and older subjects published by an independent laboratory, based on their expression profiles. The signature revealed increased expression of several genes involved in mediating cellular responses to inflammation and apoptosis, including complement component C1QA, Galectin-1, C/EBP-β, and FOXO3A, among others. The increased expressions of genes that regulate pre-mRNA splicing, localization, and modification of RNA comprise markers of the aging signature. Downregulated genes in the signature were the glutamine transporter SLC38A1, a TRAF-6 inhibitory zinc finger protein, and membrane-bound transcription factor protease S2P, among others. The sarcopenia signature developed here will be useful as a molecular model to judge the effectiveness of exercise and other therapeutic treatments aimed at ameliorating the effects of muscle loss associated with aging.

Skeletal muscle atrophy is associated with a marked and sustained activation of nuclear factor-kappaB (NF-kappaB) activity. Previous work showed that p50 is one of the NF-kappaB family members required for this activation and for muscle atrophy. In this work, we tested whether another NF-kappaB family member, c-Rel, is required for atrophy. Because endogenous inhibitory factor kappaBalpha (IkappaBalpha) was activated (i.e., decreased) at 3 and 7 days of muscle disuse (i.e., hindlimb unloading), we also tested if IkappaBalpha, which binds and retains Rel proteins in the cytosol, is required for atrophy and intermediates of the atrophy process. To do this, we electrotransferred a dominant negative IkappaBalpha (IkappaBalphaDeltaN) in soleus muscles, which were either unloaded or weight bearing. IkappaBalphaDeltaN expression abolished the unloading-induced increase in both NF-kappaB activation and total ubiquitinated protein. IkappaBalphaDeltaN inhibited unloading-induced fiber atrophy by 40%. The expression of certain genes known to be upregulated with atrophy were significantly inhibited by IkappaBalphaDeltaN expression during unloading, including MAFbx/atrogin-1, Nedd4, IEX, 4E-BP1, FOXO3a, and cathepsin L, suggesting these genes may be targets of NF-kappaB transcription factors. In contrast, c-Rel was not required for atrophy because the unloading-induced markers of atrophy were the same in c-rel(-/-) and wild-type mice. Thus IkappaBalpha degradation is required for the unloading-induced decrease in fiber size, the increase in protein ubiquitination, activation of NF-kappaB signaling, and the expression of specific atrophy genes, but c-Rel is not. These data represent a significant advance in our understanding of the role of NF-kappaB/IkappaB family members in skeletal muscle atrophy, and they provide new candidate NF-kappaB target genes for further study.

Muscular inactivity leads to atrophy, weakness, and decreased fatigue resistance. In order to provide a window into the dynamic processes that underlie muscle atrophy, we performed global gene expression analysis of rat soleus muscles using Affymetrix GeneChips at 1, 4, 7 and 14 days of hindlimb unloading. Expression of 309 known genes was significantly changed by at least 2-fold (212 upregulated, 97 downregulated). K-means clustering was used to divide these genes into co-regulated clusters based on the similarity of temporal expression patterns. This allowed the development of a timeline of the atrophy process with respect to the behaviour of genes in a broad array of functional categories. Regulatory genes were often upregulated early, in either a transient or sustained manner, but they also populated clusters with later patterns of activation, suggesting different phases of molecular adaptations. Other early events were the activation of ubiquitination genes and downregulation of protein chaperones. In comparison, clusters representing slightly delayed activation patterns included genes involved in fast contraction, glycolysis, translational inhibition, oxidative stress, protein degradation, and amino acid catabolism. Downregulated genes exhibited fewer unique expression patterns and included structural and regulatory genes of the extracellular matrix and cytoskeleton, and genes that define a slow-oxidative phenotype. Other novel findings include the tight co-activation of proteasome subunit and ubiquitination genes, differential regulation of serine proteases and serine protease inhibitors, and the identification of transcriptional, signalling, growth and cell cycle genes that probably play a role in the atrophy process. The present work has uncovered temporal patterns of gene expression that highlight the molecular processes that underlie muscle atrophy and provide new avenues for study.