title: Transcriptional profiling to identify therapeutic targets influencing skeletal muscle atrophy
Creators: name: Fisher, Andrew
Publisher: name: University of Liverpool Repository
Year: 2013

description: Skeletal muscle atrophy is characterised by a loss of muscle weight and volume involving a reduction in muscle fibre diameter in the absence of degenerative changes and/or a reduction in the actual number of fibres. In humans there are a wide range of stimuli for skeletal muscle atrophy including bed rest, extreme training, malnutrition and cancer. The underlying cellular and molecular mechanisms governing the process of atrophy are beginning to be elucidated. However, there remains much to be learnt to allow the development of rational therapeutic interventions. A miniature neuromuscular stimulator was used to impose artificial levels of activity on the rat Tibialis anterior muscle in vivo. Continuous electrical stimulation at a frequency of 20 Hz for 7 days resulted in a 12% (+/- 2%) decrease in muscle weight. Foxo1 is known to play a central role in skeletal muscle atrophy. Therefore transcriptional changes in 12 Foxo1 target genes were measured following artificial in vivo stimulation and compared with changes in the same genes in published in vitro models of muscle atrophy. The standard in-vitro models used were treatment of C2C12 myotubes for 24 hours with glucose free media, giving a 56% (+/- 2%) decrease in myotube diameter, or 24 hours treatment with 1μM dexamethasone (Dex), which we found produced no significant change in myotube diameter. Marked transcriptional differences were observed between in vivo and in vitro atrophy models. These differences highlighted the need to develop a model based on muscle inactivity that more clearly reflected muscle behaviour in vivo. A tetrodotoxin (TTX) nerve cuff was used to block all efferent impulse activity in the common peroneal nerve, and therefore to induce progressive disuse atrophy in the dorsiflexors over a period of 14 days. This resulted in a maximal 51% (+/-1%) loss in mass of the treated Tibialis anterior muscle compared to that in the untreated control limb. This atrophy was achieved in the absence of any histological signs of damage or degeneration of the muscle fibres. It was therefore possible to block muscle activity for 14 days, and then reverse the blockade, allowing the same muscle fibres to recover without detriment over the subsequent 7 days. Microarray analysis was used to compare genome-wide transcript changes following 3, 7 and 14 days of nerve blockade, 14 days of nerve block with 7 days of recovery, or 7 days electrical stimulation at 20Hz. Following quality control and normalisation of data(n=4), systematic bioinformatics analysis including GO-term enrichment revealed key signalling pathways involved in the process of disuse atrophy. A working hypothesis was developed which centres on the transcriptional control of myogenin (Myog) through a sensory role of the neuromuscular junction. This involves both class II histone deacetylases (Hdac4/5) and the janus kinase and signal transducer and activator of transcription (Jak/Stat) pathway, and influences potential downstream Myog targets including the ubiquitin E3 ligases Trim63 and Fbxo32, as well as genes involved in neuromuscular junction formation. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to confirm the temporal profiles for expression of key genes central to this model. These included a 33-fold increase in Myog mRNA after 3 days nerve block, and a 180-fold increase in transcript for the alpha subunit of the nicotinic acetylcholine receptor after 14 days of nerve blockade. To establish whether our findings were transferable to atrophy induced by other mechanisms, and to examine potential chemical interventions, we returned to the C2C12 in vitro model. The transcript levels of the same key genes were measured in myotubes following treatment with glucose-free media as well as media containing chemical inhibitors of the signalling pathways central to our hypothesis: the class II Hdac inhibitor MC1568, the Stat3 inhibitor S3I-201, and in addition to the Class I Hdac inhibitor MGCD0103. Differences were again observed in the patterns of change in transcript levels between in vivo and in vitro treatments as well as between in vitro treatment groups. The atrophy associated with glucose starvation was a 56% (+/- 2.%) reduction in myotube diameter relative to cells grown in normal media. While MC1568 and S3I-201 treatment reduced this in vitro atrophy by a small amount (to 42% +/- 4% and 21% +/- 6%, respectively, relative to cells grown in normal media), surprisingly the class I Hdac inhibitor MGCD0103 markedly blunted starvation atrophy with a decrease in myotube diameter of only 3% (+/- 7%). The discovery of therapeutic targets that could alleviate skeletal muscle atrophy is an exciting and potentially productive avenue of research. Skeletal muscle atrophy has wide ranging causes and debilitating consequences for patients. This project represents a starting point in the systematic development of drugs that could benefit these patients by improving quality of life and decreasing morbidity.