By David N. Leff
In the gym or locker room, victory-fixated athletes shoot up or pop down anabolic steroids, such as testosterone and its derivatives, aiming to build up their muscles. These illicit drugs have severe side effects.
¿They are terrible,¿ observed molecular biologist David Glass, who directs the muscle atrophy program at Regeneron Pharmaceuticals Inc. in Tarrytown, N.Y. But he pointed out that even legitimate therapeutic corticosteroids such as dexamethasone, an anti-inflammatory and immunosuppressant agent, often have other dire side effects ¿ i.e., muscle atrophy.
¿It¿s interesting that we don¿t think of muscle atrophy as a distinct disease entity,¿ Glass pointed out, ¿but it occurs with devastating consequences in a wide variety of diseases. When you point this out to people, they remember and say, Oh yes, I¿ve seen that.¿ In cancer we¿ve all seen patients who have this profound wasting; they get really thin and highly debilitated from losing a lot of muscle. We¿ve seen that as well in AIDS patients,¿ he continued. ¿Even during aging, your muscle mass, and ability to build muscle, go down significantly.
¿Also, in injuries where you¿re put in a plaster cast, or your leg is braced, or you¿re in for long-term bed rest,¿ Glass went on, ¿you have profound muscle atrophy. And, for example, if you cast a leg, or suffer a ligament tear and have to brace a limb for a while, it takes quite a long time ¿ on the order of nine months ¿ for the wasted muscle to recover. What¿s more, there are no good drugs or therapies for preventing muscle atrophy or accelerating muscle regeneration.¿
Glass is senior author of a paper in the current online issue of Science, dated Oct. 25, 2001. Its title: ¿Identification of ubiquitin ligases required for skeletal muscle atrophy.¿ The article¿s first author is muscle physiologist Sue Bodine at Regeneron.
¿We report discovering two genes that are required for inflicting muscle atrophy,¿ Glass told BioWorld Today. ¿If we knock out either one of them genetically, then we can spare the atrophy condition. One of these genes, muscle RING finger 1 [MuRF1], which was previously known, but not its role, resides on the short arm of human chromosome 2; the other, muscle atrophy F-box [MAFbx], on chromosome 8¿s long arm, remained until now undiscovered.
¿We figured out,¿ Glass continued, ¿that both are involved in a protein-breakdown process called ubiquination. The way that works is you can target a specific protein for breakdown by this type of gene product. The two genes are up-regulated during muscle atrophy, so presumably they¿re targeting specific muscle proteins for degradation. Therefore, when we deleted them through knockout [KO] mice, 56 percent of MAFbx-minus animals could spare muscle, compared to 36 percent of muscle-sparing in MuRF1-deleted KOs.¿
A First¿ Find And A Medical Hope
Glass added, ¿It¿s never been shown before that one could, by interdiction or removal of two genes, spare muscle from atrophy. This phenotype, to my knowledge, has not been demonstrated. It really gives hope that blockade of a single degradation pathway, in the case of these genes, could have a profound effect on muscle wasting. Our hope is to apply it therapeutically in human patients some day.
¿Ubiquitin-ligase,¿ he explained, ¿which actually breaks down muscle protein, is an enzyme or enzyme complex that attaches the ubiquitin molecule to a substrate. When ubiquitination was discovered 20 years ago,¿ Glass added, ¿people thought of it as merely a garbage disposal process; just taking misfolded or nonfunctional proteins and breaking them down. With discovery of these ligases, what¿s really being appreciated is that one can target perfectly fine, functional proteins and break them down at this point. It¿s sort of exciting. Rather than phosphorylating a protein kinase, for example, to get a signal, you can simply say, OK, there¿s an inhibitory protein that¿s blocking a reaction. I¿ll just break it down to remove it.¿¿
Glass and his co-authors set up three different in vivo experiments to analyze muscle atrophy¿s onset and course by as many contrasting physical triggers. ¿The first thing we did,¿ Glass recounted, ¿was to ask the question: Is atrophy in various conditions similar in terms of its time course?¿ That would give us clues as to whether a single pathway was involved, or perhaps many different pathways.¿
The team initially made knockout mice, deleting the two genes at the root of their muscle-breakdown findings. ¿Over-expression of MAFbx in myotubes [key skeletal-muscle fibers] produced atrophy,¿ the Science paper reported, ¿while mice deficient in either MAFbx or MuRF1 were found to be resistant to atrophy.¿
In the first experiment, Glass related, ¿Sue Bodine severed the sciatic nerve to the calf muscle of those KO mice, thus denervating the entire lower limb. When she cut the nerve to the muscle, that muscle became, of course, nonfunctional, so much less atrophy occurred than we expected. The second model,¿ he went on, ¿physically immobilized the limb of a rat, as you would with a plaster cast. Sue put a screw through the rat¿s heel, which fixed the joint and blocked flexion of the limb, thereby immobilizing the muscles.
¿The third model was unweighting ¿ physically suspending the hind limb. The rat could still move around, but with the limb hanging free, its muscles could not contract against a force. Then Sue took muscle with various time points after those perturbations, and showed that we got a very defined amount of atrophy in the same time course, which was similar in all rat models.¿
On Track Of New Medicaments
Looking to the future, Glass allowed, ¿We now need to develop new medicines. We need to figure out inhibitors of these two gene products. Not necessarily the genes themselves ¿ not at the DNA level ¿ but the proteins the genes encode. So we¿re working on doing that. We¿re also working on figuring out what the targets of these ligases are ¿ that is, which muscle proteins do they target for breakdown? Obviously, that would help us figure out what proteins are critical to blocking and maintaining atrophy.
¿The paper in Science represented significant validation of our preclinical experiments,¿ he observed. ¿So we¿re now in the process,¿ Glass concluded, ¿of beginning to screen for those therapeutic enzyme inhibitors.¿