OREF grant recipient seeks to understand role of matrix-degrading enzymes
Skeletal muscle atrophy, the consequence of depriving muscles of normal activity, has a profound impact on orthopaedic patients after injury, immobilization, and surgery. Recovery of muscle mass and function greatly extends the rehabilitation period, and some patients never fully recover the full use of their muscles.
The pathologic changes to muscle mass, function, and cellular properties associated with atrophy are generally well understood. But researchers know much less about what changes atrophy causes in the extracellular matrix and the enzymes that contribute to matrix degradation.
“Research has focused primarily on the effect of atrophy on muscle fibers, but the matrix is also important,” explained Xuhui Liu, MD, winner of a 2008 Research Grant from the Orthopaedic Research and Education Foundation (OREF). “If you lose the infrastructure for the muscle, then you can’t have normal muscle function.”
Dr. Liu, assistant adjunct professor in the department of orthopaedic surgery at the University of California, San Francisco (UCSF), is hoping to identify the role of an extracellular matrix-degrading enzyme in disuse-induced muscle atrophy.
OREF Research Grants encourage new investigators by providing start-up funding for up to 2 years. Dr. Liu, who earned his medical degree in China, does not currently practice medicine, but his work with clinical coprincipal investigator Hubert T. Kim, MD, associate professor of orthopaedic surgery at UCSF, made him eligible for the grant.
The science of atrophy
Muscle atrophy can begin after only a few days of disuse, and the extent of atrophy depends on the duration of disuse. At the cellular level, pathologic changes include the shrinkage of muscle fibers, the loss of nuclei from multinucleated muscle cells, and apoptosis.
The extracellular matrix forms the architecture of skeletal muscle and provides a support system for nerves and blood vessels. Pathologic changes to the extracellular matrix associated with muscle atrophy include accelerated matrix degradation with upregulation of degenerative enzymes.
Dr. Liu and his research team investigated the role of matrix metalloproteinase-2 (MMP-2), which had been identified in other research as a likely contributor to muscle atrophy. One aim of their work was to analyze the contribution of MMP-2 by correlating the upregulation of the enzyme with the timing and severity of muscle atrophy. They also investigated transcription factors believed to “switch on” the MMP-2 gene and the binding sites for those factors.
“If we can discover the mechanism that turns on MMP-2 in muscle atrophy,” said Dr. Liu, “then perhaps we can eventually find an intervention to turn it off.”
Model of atrophy
Dr. Liu conducted experiments with a mouse model of disuse-induced skeletal muscle atrophy. In one hind limb, researchers transected the Achilles tendon, which resulted in immediate retraction and acute disuse of the gastrocnemius, a major calf muscle. The other hind limb served as the control. When researchers compared the results in wild-type mice and in MMP-2 null mice, they found that the mice without MMP-2 exhibited much less atrophy. These data suggest that MMP-2 plays a critical role in the development of muscle atrophy.
To establish timelines for both MMP-2 upregulation and muscle mass loss, researchers conducted protein and tissue analyses during the 28 days after transecting the tendon. The team also correlated MMP-2 expression with matrix degradation and with the severity of atrophy as indicated by muscle fiber shrinkage, nuclei loss, and apoptosis. The MMP-2 null mice provided important data on the functional role of the enzyme in disuse-induced atrophy.
In a second series of experiments, the research team assessed the role of binding sites for a set of transcription factors that were likely candidates in regulating MMP-2 gene expression. These experiments replicated the prior series, including the same analyses over the course of 28 days, but the models carried a reporter gene—a gene with an easily measured product that served as a proxy for the expression of the MMP-2 gene.
One set of models carried a reporter attached to the complete upstream regulatory sequence for the MMP-2 gene. Another set carried a reporter with a modified MMP-2 regulatory sequence missing the two binding sites of interest. With results from two types of reporter genes, Dr. Liu was able to judge the relative contribution of the binding sites in MMP-2 regulation.
The team also employed chromatin immunoprecipitation, a state-of-the-art technique to identify transcription factors that bind to the regulatory sites at different intervals during the period of muscle disuse.
Switching off atrophy
The outcome of this OREF-funded research has led to additional funding. Drs. Liu and Kim recently received $1.1 million from the Veterans Affairs Rehabilitation Research and Development program to continue and increase the scope of this project. They will investigate the role of MMP-2 in various forms of muscle atrophy and the effect of aging in MMP-2 expression during muscle atrophy.
“So far, we’ve found that globally inhibiting MMP-2 can reduce the severity of muscle atrophy,” said Dr. Liu. “However, this treatment is not perfect because MMP-2 also plays an important role in other physiological processes, such as wound healing. We are seeking a way to block only the ‘bad’ MMP-2 in muscles, which we think can be achieved by targeting specific transcription factors that turn on the MMP-2 expression during muscle atrophy.” The goal is to identify an inhibitor that turns off MMP-2 expression associated with muscle atrophy without disrupting other normal physiologic functions of the enzyme.
“The pilot data from our animal model really encouraged me,” said Dr. Liu. “I think that in the relatively near future, we may be able to move this project to clinical trials to study the potential of inhibitors in treating muscle atrophy.”
Jay D. Lenn is a contributing writer for OREF and can be reached at firstname.lastname@example.org