Meta-analysis of expression signatures of muscle atrophy: gene interaction networks in early and late stages

Enrica Calura 1 Stefano Cagnin 0 Anna Raffaello 0 Paolo Laveder 0 Gerolamo Lanfranchi 0 Chiara Romualdi 0 0 CRIBI Biotechnology Centre and Department of Biology, University of Padova , via U. Bassi 58/B 30121 Padova , Italy 1 Department of Biology, University of Ferrara , Via L. Borsari 46 I-44100 Ferrara , Italy Background: Skeletal muscle mass can be markedly reduced through a process called atrophy, as a consequence of many diseases or critical physiological and environmental situations. Atrophy is characterised by loss of contractile proteins and reduction of fiber volume. Although in the last decade the molecular aspects underlying muscle atrophy have received increased attention, the fine mechanisms controlling muscle degeneration are still incomplete. In this study we applied metaanalysis on gene expression signatures pertaining to different types of muscle atrophy for the identification of novel key regulatory signals implicated in these degenerative processes. Results: We found a general down-regulation of genes involved in energy production and carbohydrate metabolism and up-regulation of genes for protein degradation and catabolism. Six functional pathways occupy central positions in the molecular network obtained by the integration of atrophy transcriptome and molecular interaction data. They are TGF- pathway, apoptosis, membrane trafficking/cytoskeleton organization, NFKB pathways, inflammation and reorganization of the extracellular matrix. Protein degradation pathway is evident only in the network specific for muscle short-term response to atrophy. TGF- pathway plays a central role with proteins SMAD3/ 4, MYC, MAX and CDKN1A in the general network, and JUN, MYC, GNB2L1/RACK1 in the short-term muscle response network. Conclusion: Our study offers a general overview of the molecular pathways and cellular processes regulating the establishment and maintenance of atrophic state in skeletal muscle, showing also how the different pathways are interconnected. This analysis identifies novel key factors that could be further investigated as potential targets for the development of therapeutic treatments. We suggest that the transcription factors SMAD3/4, GNB2L1/RACK1, MYC, MAX and JUN, whose functions have been extensively studied in tumours but only marginally in muscle, appear instead to play important roles in regulating muscle response to atrophy. - Background Atrophy is a complex modification occurring in skeletal muscles as a result of a variety of causes such as damages to neural connections, disuse or unloading, fasting and also as a consequence of many diseases including diabetes, sepsis, acidosis or cancer. The variety of conditions inducing atrophy implies different molecular triggers and signalling pathways for muscle wasting. However, regardless of the stirring event, skeletal muscle atrophy is generally characterized by a decrease in protein content, fiber diameter, force production, and fatigue resistance. The dynamic regulation of skeletal muscle mass depends on the balance between overall rates of protein synthesis and degradation. It is now established that these two biochemical processes appear to be coordinated by complex signalling networks. During hypertrophy, the rate of synthesis of muscle contractile proteins is much higher than the rate of degradation that results in an increase of the size of the existing muscle fibers. On the contrary, enhanced protein breakdown is the primary cause of the rapid loss of muscle proteins that occurs during atrophy [1-3]. Significant advancements have been recently made in the understanding of the signalling pathways mediating skeletal muscle atrophy and its opposite process of hypertrophy [4-9]. It has become clear that the activity or inactivity of the IGF-1/Insulin/Akt/FoxO pathway determines whether a muscle will increase protein synthesis and growth (hypertrophy), or undergo protein breakdown and atrophy. In particular, IGF-1 stimulation induces hypertrophy of skeletal muscle by stimulating the phosphatidylinositol 3-kinase (PI3K)-Akt pathway, resulting in the downstream activation of proteins required for protein synthesis [10,11]. Downstream of PI3K-Akt signal, IGF-1 activates also mTOR and p70S6K. However, mTOR can be activated directly by amino acids, causing a subsequent stimulation of p70S6K activity [12,13]. Thus, mTOR seems to have a central role in integrating a variety of growth signals, from simple nutritional stimulation to activation by protein growth factors, resulting in protein synthesis. Akt activates mTOR by phosphorylation [14], and both Akt and mTOR phosphorylation are increased during muscle hypertrophy [15]. Conversely, when the activity of the IGF-1/Akt/FoxO pathway decreases, the transcription factors FoxO1 and 3 are activated and the two muscle specific E3 ubiquitin ligases atrogin-1 (or MAFbx, muscle atrophy F-box) and MuRF-1 (muscle ring finger 1) are induced [16,17]. These proteins have been identified by genomic experiments designed to uncover new markers of the atrophy process [18,19] and their expression is increased significantly in several types of muscle atrophy, demonstrating the predominant role of the ubiquitin-proteasome pathway during the progression of muscle wasting [20]. The NFKB signalling cascade also plays an important role in the control of muscle degradation. First hints on involvement of NFKB in muscle wasting came from the up-regulation of this gene during disuse atrophy [21] and sepsis [22]. Additionally, experiments in cultured myotubes demonstrated that the block of this transcription factor by overexpression of a mutant form of I-kB, that is insensitive to degradation by the proteasome, inhibits protein loss induced by tumor necrosis factor- (TNF-) [23]. Not only the IGF-1/PI3K/Akt/FoxO and the NFKB signalling cascades are involved in the control of muscle mass upon skeletal muscle atrophy. Recent studies in cultured myotubes, mouse models and natural mutations demonstrated that also myostatin is a potent regulator of skeletal muscle mass [24-28]. The mechanism by which myostatin inactivation leads to muscle growth is still controversial. Recently, it was proposed that the myostatin signalling pathway could be linked to the IGF-1/PI3K/Akt pathway. McFarlane and co-workers showed in cultured myotubes and in mouse skeletal muscle that treatment with myostatin was associated with a reduction of fiber size, and with induction of the muscle-specific E3 ubiquitin ligases atrogin-1 and MuRF-1 [29]. This study showed that the atrophic effects observed were mediated by dephosphorylation and inhibition of Akt and the consequent activation of FoxO1. Although in the last decade, with the application of genomic technologies such as global gene expression profiling, the molecular networks underlying several types of atrophy have been studied in deeper details, the fine mechanisms that control muscle wasting and loss of functional capacity are still (...truncated)