At least three proteolytic systems are known to be involved in muscle mass protein degradation

At least three proteolytic systems are known to be involved in muscle mass protein degradation. ubiquitin-proteasome system is Fmoc-Lys(Me)2-OH HCl not capable of initiating myofibrillar disassembly (Jagoe & Goldberg, 2001) and that additional proteolytic systems may be necessary (Tischler 1990). Therefore, questions remain as to what proteolytic systems are at work and how they coordinate protein degradation. Furthermore, the regulatory triggers mediating accelerated proteolysis and decreased protein synthesis are not well understood. Several signalling pathways including either Akt (Bodine 20012002) have been implicated in the regulation of disuse muscle mass atrophy but no direct linkages have been made between these pathways and gene targets, or even whether these components are necessary for disuse atrophy. It has also been shown that myogenic E-box-dependent mechanisms are responsible for the transactivation of several fast genes in response to inactivity (Swoap, 1998; Mitchell-Felton 2000), but the upstream pathways regulating changes in muscle mass phenotype remain elusive. Defining signalling pathways and their protein targets remains one of the biggest difficulties in the study of atrophy. This is complicated by the likelihood that several different pathways are working in parallel to mediate the atrophy process. Such complexity illustrates the need to apply a more global approach to analysing the molecular changes that occur during atrophy. In recent years, several groups have used various approaches to studying expression of multiple genes during inactivity at one time point. These include serial analysis of Fmoc-Lys(Me)2-OH HCl gene expression after 12 days of immobilization (St-Amand 2001), Affymetrix GeneChip analysis after 12 h (Bey 2003), or 21 days (Stein 2002) of hindlimb unloading, subtractive hybridization after 14 days of unloading (Cros 2001), and cDNA array analysis after 35 days of unloading (Wittwer 2002). These studies have uncovered the possibility that atrophy is usually regulated by changes in mRNA levels of genes involved in protein synthesis, proteolysis, oxidative stress, growth and cell cycle regulation and structural genes of the extracellular matrix and cytoskeleton. The current study reconfirms and significantly expands upon these findings by using Affymetrix GeneChips to monitor differential gene expression after 1, 4, 7 and 14 days of hindlimb unloading in rat soleus muscle mass. This approach not only identifies genes that are differentially expressed after unloading, but also provides a unique view of their expression patterns in relation to each other during the course of disuse. The temporal aspect of this analysis therefore provides a window into the dynamic molecular alterations that occur during disuse muscle mass atrophy. The goals of this paper are to: (1) develop Mouse monoclonal to RFP Tag a general timeline of atrophy based on the temporal expression patterns of genes involved in contraction, metabolism, cytoskeleton, extracellular matrix (ECM), protein synthesis, oxidative stress, protein processing and degradation and regulatory genes (growth, proliferation, signalling and transcription); (2) determine the extent of coordinated expression of genes that share comparable function in skeletal Fmoc-Lys(Me)2-OH HCl muscle mass (contraction, metabolism, oxidative stress and protein turnover); (3) identify the variety of distinct expression patterns among genes involved in regulating growth, proliferation, signalling and transcription; and (4) identify the expression patterns of genes not previously shown as being differentially regulated during disuse-related atrophy. Fmoc-Lys(Me)2-OH HCl METHODS Hindlimb unloading protocol and experimental design Female Wistar rats (6 weeks aged) were hindlimb unloaded (HU) for 1, 4, 7 and 14 days using a standard Fmoc-Lys(Me)2-OH HCl elastic tail cast method (Mitchell-Felton 2000). At.