In space, the significant loss of muscle mass results primarily from an accelerated degradation of muscle proteins. Earlier research has indicated that a specific pair of enzymes plays an important role in this activation of protein breakdown in atrophying muscles, and Dr. Alfred L. Goldberg and coworkers are identifying the mechanisms that activate protein breakdown. This work has already led to the discovery of a new degradative enzyme, atrogin-1, that is specifically involved in muscle atrophy, including that seen in numerous disease states on Earth.
Overview
The Activation of Protein Breakdown in Muscle Upon Unloading and Possible Countermeasures
Principal Investigator:
Alfred L. Goldberg, Ph.D.
Organization:
Harvard Medical School
Technical Summary
Specific Aims
- To clarify the mechanisms that activate the ubiquitin (Ub)-proteasome pathway during muscle atrophy induced by hind-limb suspension and by glucocorticoids which may also contribute to the muscle wasting in astronauts. We have made the surprising finding that one set of ubiquitination enzymes, E214K and E3α, which comprise the "N-end rule" pathway, catalyze most of the ubiquitination in atrophying muscles. Therefore, we shall pursue studies to elucidate their special role and how this pathway is activated and contributes to muscle wasting.
- To determine whether pharmacological inhibitors of the ubiquitin-proteasome pathway could be useful as countermeasures to reduce muscle proteolysis and atrophy and to synthesize novel types of inhibitors of this pathway.
- We shall test whether inhibition of the "N-end rule" pathway might be an effective way to prevent atrophy through in vivo studies of transgenic or knockout mice with defects in E3α or E214K. Related biochemical studies will attempt to identify more potent inhibitors of E3α.
- Our other approach toward countermeasure development will be to develop agents that partially inhibit proteasome function in muscle. Because available proteasome inhibitors can block its active sites completely, they are potentially dangerous and can only be used against life-threatening diseases. We recently discovered inhibitory sites in the proteasome by which certain peptides can feedback and retard (but not block) protein breakdown. We hope to synthesize safer types of inhibitors that function by this novel allosteric mechanism to reduce partially muscle protein degradation.
- We shall test whether inhibition of the "N-end rule" pathway might be an effective way to prevent atrophy through in vivo studies of transgenic or knockout mice with defects in E3α or E214K. Related biochemical studies will attempt to identify more potent inhibitors of E3α.
- By using a gene microarray analysis, we hope to identify the spectrum of genes whose transcription rises or falls during muscle atrophy induced by hind-limb suspension or glucocorticoid treatments. Although several changes in transcription have been described in atrophying muscles, in order to fully understand the critical adaptations leading to the loss of mass and functional capacity, it is necessary to obtain a more complete picture of the changes occurring in muscle gene expression. Our initial experiments have already uncovered large increases in seven unidentified mRNAs. Such mRNAs could be useful markers to monitor muscle wasting and the efficacy of countermeasures. Also, identification of their functions could suggest new targets for pharmacological intervention. We shall also carry out a similar analysis of human muscle biopsies taken before and during prolonged bed rest (provided by W. Evans and coworkers) in order to test if insights gained from studies of the rodent models are applicable to atrophying human muscles.
- To identify possible nonpharmacological approaches to reduce protein breakdown, we shall investigate the biochemical adaptations that occur in certain animals to suppress muscle proteolysis and preserve muscle mass. We shall study muscles in two unusual physiological states: in black bears during winter (using biopsies provided by H. Harlow and coworkers) and in rats fed very low protein diets in which muscle protein is preserved despite disuse and decreased caloric intake. In addition, since muscle protein breakdown decreases in rats on protein-deficient diets, we shall test whether in such animals there is less atrophy upon hind-limb suspension.
This project's funding ended in 2004