Research Summary

Our laboratory is using computational and molecular approaches to better understand the pathways that mediate muscle protein loss associated with disuse atrophy. Several studies in our lab have been geared towards identifying genes that are necessary or sufficient for the induction and progression of skeletal muscle atrophy.

Microarray Studies

Disuse Atrophy in Rodents

In order to better understand the general process of inactivity induced protein loss in muscle we have used Affymetrix GeneChips to analyze the early time course of gene expression changes that occur during muscle unloading. Soleus muscles were studied after 1, 4, 7 and 14 days of unloading and time matched controls. Approximately 300 different gene products were differentially expressed by at least 2-fold and these belonged to a variety of different functional categories. In particular, changes were seen in genes that underlie the early decrease in translational activity and increase in protein degradation. Novel findings include the activation of two ubiquitin-protein ligases not previously characterized with atrophy, coordinate upregulation of proteasome subunits, and upregulation of other genes involved in proteolysis such as the cathepsins and serine proteases (Stevenson et al, 2003). Ongoing work is testing whether overexpression of these gene products (via adenoviral infection), either individually or in combination, is necessary or sufficient for muscle atrophy in cell culture and whole muscle models.

Sarcopenia in Humans

Sarcopenia is an “age-related” loss of muscle mass and function, which causes muscle weakness, limited mobility, and a greater susceptibility to injury. We have used the Affymetrix HG-U133A GeneChip to interrogate the expression of ~14,000 genes in the vastus lateralis muscle of 10 young (19-25 years old) and 12 older (70-80 years old) ambulatory males. We have improved upon previous work by increasing sample size and using leave-one-out cross validation (LOOCV) to refine our understanding of the molecular events underlying sarcopenia. This was accomplished through the elimination of probe sets from the signature that exhibited high within-group variability, which would normally be deemed significant using more standard parametric tests (i.e.- t-test). We were also able to validate the newly defined gene expression signature of aged skeletal muscle using published data generated in an independent laboratory. Knowledge of the set of genes that define sarcopenia is important as this information can be used to assess the plasticity of skeletal muscle in response to exercise training or pharmacological interventions; allowing us to gain a better understanding of the molecular elements responsible for the recalcitrant nature of aged skeletal muscle. The present work identified 44 genes that define the molecular signature of sarcopenia, which was determined through LOOCV (internal) and validated using an independent dataset (external). The computational methods used to identify these genes and the biological description of this signature is described (In preparation).

Involvement of NFkB in Disuse Atrophy

We are also studying the role of nuclear factor-kB (NF-kB) and IkB in disuse muscle atrophy. While NF-kB is involved in muscle wasting subsequent to disease, its potential role in disuse atrophy had not until recently been characterized. In a recent project we showed that 7 days of hindlimb unloading led to a 10-fold activation of an NF-kB-dependent reporter in rat soleus muscle but not the atrophy resistant extensor digitorum longus muscle. Nuclear levels of p50 and c-Rel were upregulated but p52 and p65 were unchanged in unloaded solei. Gel-shift assays showed the involvement of p50, c-Rel, and Bcl-3 but not other NF-kB family members. This work suggests that disuse muscle atrophy is associated with activation of an alternative NF-kB pathway that involves the activation of p50 but not p65. We have also shown that atrophy during muscle unloading is inhibited in p50 and bcl-3 knock out mice indicating that NFkB transcription factors play an important role in disuse atrophy.

Development of Adenoviral Vectors

The microarray data described above has allowed us to compile a list of genes that may play regulatory roles in the process of atrophy. We are characterizing the role of selected genes from this list using genetic approaches (overexpression and inhibition of genes) in both muscle cell culture and in whole muscle. For most of the proposed work, we are employing conditional expression approaches that allow for the overexpression of genes in mature myocytes or in whole muscle. In this way we will avoid the problems encountered when genes are overexpressed (or inhibited) throughout development, thereby complicating interpretations due to the inability to distinguish pre and post differentiation effects. By identifying genes involved in atrophy we will be in a better position to develop more effective nutritional or pharmacolgical countermeasures to combat the deleterious changes in muscle function due to atrophy.