Slowing muscle atrophy: putting the brakes on protein breakdown

Physiologists and neurobiologists generally view muscle atrophy as a response of specific skeletal muscles to denervation or disuse. However, fiber atrophy also occurs systemically in poor nutritional states and diverse diseases, including cancer, uncontrolled diabetes, uraemia, and sepsis (Lecker et al. 1999). Beyond its motile function, skeletal muscle in mammals serves as a protein reservoir that is mobilized in stressful states as a source of amino acids for energy metabolism. The muscle atrophy and reduction in strength are major factors contributing to the morbidity in these disease states. Recent work has demonstrated that rapid atrophy in various models of these diseases is due primarily to enhanced degradation of muscle protein, although protein synthesis may also fall (Lecker et al. 1999). In all these models, a common set of transcriptional and enzymatic adaptations develop that appear to enhance the cell's capacity for protein breakdown (Jagoe et al. 2002). A better understanding of the pathways of protein degradation activated during muscle atrophy is therefore of scientific and therapeutic interest. In muscle, the majority of intracellular proteolysis occurs through the ubiquitin- proteasome system. The concerted action of ubiquitin-conjugating enzymes link chains of the polypeptide cofactor ubiquitin to proteins to mark them for degradation. This tagging process leads to their recognition by the proteasome, a very large ATP-dependent proteolytic complex that degrades these proteins to small peptides (Glickman & Ciechanover, 2002). This system catalyses not only the rapid breakdown of regulatory and abnormal proteins, but also the slower degradation of the bulk of proteins, including myofibrillar proteins. This pathway has also been shown to be of primary importance in the enhanced proteolysis in atrophying muscles (Lecker et al. 1999). However, other cell proteases (e.g. calpains, caspases, lysosomal cathepsin) may also contribute to this accelerated degradation. Although cells contain hundreds of different ubiquitin-conjugating enzymes (E3s), three have been shown to play key roles in the activation of proteolysis during atrophy. Atrogin-1/MAFbx and MURF-1 are both newly discovered ubiquitin-protein ligases (commonly called E3s) which link ubiquitin to specific proteins (Bodine et al. 2001; Gomes et al. 2001). Both are found exclusively in striated muscle and are dramatically induced in all forms of muscle wasting studied to date in rodents. Moreover, knockout mice lacking these enzymes show reduced muscle atrophy following denervation (Bodine et al. 2001). Another ubiquitin-protein ligase, E3α (also called Ubr1/Ubr2), has also been implicated in muscle atrophy (Lecker et al. 1999). It acts specifically on proteins with hydrophobic or basic amino acids on their N-termini (called the N-end rule pathway). In extracts of atrophying muscles, inhibitors of E3α reduce ubiquitin conjugation rates towards normal levels. Because cellular proteins begin with methionine, and most are acetylated, the specific proteins ubiquitinated by E3α and how they are generated are quite unclear. One possibility is that an unidentified proteolytic enzyme (e.g. a calpain - see below) clips muscle proteins and generates proteins with amino-terminal residues that are recognized by this E3. The elegant study by Tidball & Spencer (2002) in this issue of The Journal of Physiology presents strong evidence that another type of protease is critical in muscle atrophy. Calpains are a diverse group of more than 10 Ca2+-dependent proteases, including m- and μ-calpains found in vertebrate muscles and the endogenous protein inhibitor of calpains, calpastatin. The calpains were first discovered by their capacity to break down muscle Z bands and have long been suspected to contribute to muscle wasting. However, their in vivo functions and possible roles in muscle atrophy are still unclear. To test whether a calpain is important in the development of atrophy, Tidball & Spencer genetically overproduced the specific inhibitor of these enzymes, calpastatin, in muscle. The muscles expressing high levels of this inhibitor were partially resistant to disuse atrophy induced by hindlimb suspension. These interesting experiments raise a number of questions. If the calpain pathway is important in muscle atrophy, it is surprising that potent synthetic calpain inhibitors have failed to slow protein breakdown in the same in vitro models where proteasome inhibitors are effective. Also, what are the protein substrates in muscle for the calpains, and which calpains are involved? Do the calpains catalyse general proteolysis directly, or just trigger this process? Finally, does calpastatin overproduction prevent or diminish other types of muscle atrophy? It seems likely that multiple proteolytic pathways are activated in muscle as it atrophies. This coordinated activation of multiple enzymes suggests that each functions to remove distinct cellular components. If parallel pathways degrade different muscle proteins, it might explain why the knockouts in mice of atrogin-1/MAFbx and MURF-1, and transgenic overproduction of calpastatin by Tidball & Spencer (2002) could each partially block the development of atrophy. Information on the classes of cell proteins lost in these conditions should help test this idea, as would mating these animals to test whether inactivating the individual atrophy-related E3s and calpains have additive effects in diminishing atrophy. Another attractive possibility is that the functioning of these proteolytic systems is linked. For instance, a member of the calpain family could be the elusive protease that generates proteins with the novel N-terminal amino groups recognized by E3α. In such a model, atrophy of muscle would be a multi-step proteolytic process. Conclusive evidence for this model will require biochemical identification of the critical proteins degraded or ubiquitinated by these enzymes. Until this happens, it will be difficult to predict if these proteolytic systems work in series or in parallel. Although much work lies ahead, the major players in this excessive proteolysis now appear to be coming into focus.