AUF1 Gene Important Inducer of Muscle Repair


A new study in the laboratory of Robert J. Schneider at NYU Langone and his collaborators has uncovered a gene that plays integral roles in the repair of injured muscle throughout life. This investigation shows that this previously “overlooked” gene might play a pivotal role in “sarcopenia,” which refers to the loss of muscle tissues with age.

This collaboration between scientists at NYU Langone Medical Center and the University of Colorado at Boulder showed that the levels of a protein called AUF1 determine if stem cell populations retain the ability to regenerate muscle after injury and as mice age.

Changes in the activity of AUF1 have also been linked by past studies to human muscle diseases. More than 30 genetic diseases, known collectively as myopathies, show defective muscle regeneration and these anomalies cause muscles to weaken or waste away.

For example, muscular dystrophy is a disease in which abnormal muscles fail to function properly and undergo normal repair. Although the signs and symptoms of Duchenne Muscular Dystrophy vary, in some cases wildly, this disease develops in infants and affects and weakens the torso and limb muscles beginning in young adulthood. Sarcopenia, in healthy individuals occurs in older patients.

Skeletal muscles have a stem cell population set aside for muscle repair known as satellite cells. These cells divide and differentiate into skeletal muscle when skeletal muscle is damaged, and as we age, the capacity of muscle satellite cells to repair muscle decreases.

AUF1 is a protein that regulates muscle stem cell function by inducing the degradation of specific, targeted messenger RNAs (mRNAs). According to Robert Schneider, “This work places the origin of certain muscle diseases squarely within muscle stem cells, and shows that AUF1 is a vital controller of adult muscle stem cell fate.” He continued: “The stem cell supply is remarkably depleted when the AUF1 signal is defective, leaving muscles to deteriorate a little more each time repair fails after injury.”

The experiments in this study demonstrated that mice that lack AUF1 display accelerated skeletal muscle wasting as they age. These AUF1-depleted mice also showed impaired skeletal muscle repair following injury. When the molecular characteristics of these AUF1-depleted muscle satellite cells were examined, Schneider and his collaborators showed that auf1−/− satellite cells had increased stability and overexpression of so-called “ARE-mRNAs.” ARE mRNAs contain AU-rich elements at their tail-ends. AUF1 proteins bind to these ARE mRNAs and induce their degradation. In the absence of AUF1, muscle satellite cells accumulate ARE mRNAs. One of these ARE mRNAs includes that which encodes matrix metalloprotease, MMP9. Overexpression of MMP9 by aging muscle satellite cells causes degradation of the skeletal muscle matrix, which prevents satellite-cell-mediated regeneration of muscles. Consequently, the muscle satellite cells return to their quiescent state and fail to divide and repair skeletal muscle.

When Schneider and his coworkers and collaborators blocked MMP9 activity in auf1−/− mice, they found that they had restored skeletal muscle repair and maintenance of the satellite cell population.

These experiments suggest that repurposing drugs originally developed for cancer treatment that blocks MMP9 activity might be a way to dial down age-related sarcopenia.

“This provides a potential path to clinical treatments that accelerate muscle regeneration following traumatic injury, or in patients with certain types of adult onset muscular dystrophy,” said Schneider.

This work was published here: Devon M. Chenette et al., “Targeted mRNA Decay by RNA Binding Protein AUF1 Regulates Adult Muscle Stem Cell Fate, Promoting Skeletal Muscle Integrity,” Cell Reports, 2016; DOI: 10.1016/j.celrep.2016.06.095.

Beta-Integrin Implicated In Slow Healing Of Aged Muscles


With age, the function and regenerative abilities of skeletal muscles decrease. Therefore, the elderly can find it difficult to recover from injury or surgery.

A new study from the laboratory of Chen-Ming Fan from Johns Hopkins University has shown that a protein called β1-integrin is crucial for muscle regeneration. β1-integrin seems to provide a promising target for therapeutic intervention to combat muscle aging or disease.

Muscle stem cells are the primary source of muscle regeneration after muscle injury from exercise, accidents, or surgery. These specialized adult stem cells lie dormant in the muscle tissue, and muscles even have them stored off to the side of the individual muscle fibers. Because of their location, these muscle stem cells are known as muscle “satellite cells.” After damage, these satellite cells awaken and proliferate, and go on to make new muscle fibers and restore muscle function. Some satellite cells return to dormancy, which allows the muscle to keep a reservoir of healing cells that can repair the muscle over and over again. Fan and her colleagues determined that proteins called integrins, and in particular, β1-integrin, are integral for maintaining the cycle of hibernation, activation, proliferation, and then return to hibernation, in muscle stem cells.

Integrins are cell surface proteins that provide tight connections between cells and the immediate external environment.

Integrin Dimer Structure: Globular domain structures of α and β subunits in a stable dimer. Ligand binding happens at the interface of the αI (left panel) or β-propeller (right panel) and the βI domain.
Integrin Dimer Structure: Globular domain structures of α and β subunits in a stable dimer. Ligand binding happens at the interface of the αI (left panel) or β-propeller (right panel) and the βI domain.

Without integrins, almost every stage of the regeneration is disrupted. Fan and her group predicted that defects in β1-integrin likely contribute to aging, which is associated with reduced muscle stem cell function and decreased quantities of muscle stem cells. This means that healing after injury or surgery is very slow, which can cause a long period of immobility and an accompanying loss of muscle mass. Inefficient muscular healing in the elderly is a significant clinical problem. Therapeutic approaches would be quite welcome by the aging population and their physicians. One way to improve muscle regeneration would be to stimulate muscle satellite cells in older individuals.

Fan and others determined that β1-integrin function is diminished in aged muscle stem cells. When they artificially activated integrins in aged mice, their regenerative abilities were restored to youthful levels. Improvement in regeneration, strength, and function were also seen when this treatment was applied to animals with muscular dystrophy, which underscores the potential importance of such an approach for the treatment of muscle disorders.

Muscle stem cells use β1-integrin to interact with many other proteins in the external environment of the muscle. Among this forest of proteins in the external environment of the muscle, Fan and her coworkers found one called fibronectin that might be the most relevant. They discovered that aged muscles contain substantially less fibronectin compared to young muscles. Like β1-integrin, eliminating fibronectin from young muscles makes them function as though they were old. However, restoring fibronectin to aged muscle tissue restores muscle regeneration to youthful levels. Fan’s group demonstrated a strong link between β1-integrin, fibronectin and muscle stem cell regeneration.

Taken together, the results show that aged muscle stem cells with compromised β1-integrin activity and aged muscles with insufficient amount of fibronectin both root causes of muscle aging. This makes β1-integrin and fibronectin very promising therapeutic targets.

This work appeared in the following journal: Michelle Rozo et al., “Targeting β1-integrin signaling enhances regeneration in aged and dystrophic muscle in mice,” Nature Medicine, 2016; DOI: 10.1038/nm.4116.

LIF Increases Muscle Satellite Expansion in Culture and Transplantation Efficiency


Transplantation of satellite stem cells, which are found in skeletal muscles, might potentially treat degenerative muscle diseases such as Duchenne muscular dystrophy. However, muscle satellite cells have an unfortunate tendency to lose their ability to be transplanted then they are grown in culture.

In order to generate enough cells for transplantation, the cells are isolated from the body and then they must be grown in culture. However, in order to properly grow in culture, the cells must be prevented from differentiating because fully differentiated cells stop growing and die soon after transplantation. Several growth factors, cytokines, and chemicals have been used in muscle satellite cell culture systems. Unfortunately, the optimal culture conditions required to maintain the undifferentiated state, inhibit differentiation, and enhance eventual transplantation efficiency have not yet been established satisfactorily.

Because it is impossible to extract enough satellite cells for therapeutic purposed from biopsies, these cells must be expanded in culture. However this very act of culturing satellite cells renders them inefficient for clinical purposes. How can we break away from this clinical catch-22?

Shin’ichi Takeda from the National Center of Neurology and Psychiatry and his colleagues have used growth factors to maintain muscle satellite cell efficiency during cell culture. In particular, Takeda and others used a growth factor called leukemia inhibitory factor (LIF). LIF effectively maintains the undifferentiated state of the satellite cells and enhances their expansion and transplantation efficiency. LIF is also thought to be involved in muscle regeneration.

This is the first study on the effect of LIF on the transplantation efficiency of primary satellite cells,” said Shin’ichi Takeda of the National Center of Neurology and Psychiatry. “This research enables us to get one step closer to the optimal culture conditions for muscle stem cells.”

The precise mechanisms by which LIF enhances transplantation efficiency remain unknown. Present work is trying to determine the downstream targets of LIF. Identifying the precise mechanisms by which LIF enhances satellite cell transplantation efficiency would help to clarify the functional importance of LIF in muscle regeneration, and, even more importantly, further its potential application in cell transplantation therapy.

The reference for this paper is: N. Ito et al., “Enhancement of Satellite Cell Transplantation Efficiency by Leukemia Inhibitory Factor,” Journal of Neuromuscular Diseases, 2016; 3 (2): 201. DOI: 10.3233/JND-160156.

Human Muscle Satellite Cells Isolated and Characterized


A research group from the University of California, San Francisco have isolated and characterized human muscle stem cells. In addition, they have established that these stem cells can robustly replicate and repair damaged muscles when they are grafted onto an injured site. These remarkable findings might open the door to potential treatments for patients with severe muscle injuries, paralysis or genetic diseases that adversely affect skeletal muscles (e.g., muscular dystrophy).

Jason Pomerantz, MD is an assistant professor of plastic and reconstructive surgery at UCSF, and served as the managing author of this work. “We’ve shown definitively that these are bona-fide stem cells that can self-renew, proliferate and respond to injury,” said Pomerantz.

Badly damaged muscles can suffer terrible depletion of their native populations of stem cells or even obliteration of the stem cell niches and populations. Since such muscles have lost the very things that can heal them, these muscles will not be able to heal the damage they have sustained. This very fact represents a terrible hurdle for physicians who specialize in patients who have been crippled by muscle injury and paralysis. One of the worse cases is those conditions that cause damage or paralysis in the critical small muscles of the face, hand and eye, according to Pomerantz.

When muscles are badly damaged, they can lose the native populations of stem cells that are needed to heal. This has posed a major roadblock for treating patients crippled by muscle injury and paralysis, particularly in the critical small muscles of the face, hand and eye, Pomerantz said.

Fortunately, there have been remarkable surgical advances in restoring nerves in damaged muscles. Unfortunately, if the healing process takes too long, the stem cell pool is exhausted and the regenerative capacity is attenuated and eventually. Such injured muscles fail to connect to the nerve tissue and without accompanying motor and sensory nerves, skeletal muscles then to degenerate.

“This is partly why we haven’t had major progress in treating these patients in 30 years,” Pomerantz said. “We know we can get the axons there, but we need the stem cells for there to be recovery.”

A group of stem cells called “satellite cells” line the borders of muscle fibers and, in mice, can function as stem cells and contribute to muscle growth and repair. Until now, however, it wasn’t clear whether human satellite cells worked the same way. It was also terribly unclear how to isolate muscle satellite cells from human tissue samples or even adapt them to help treat patients with muscle damage.

Muscle satellite cells in section

Pomerantz and colleagues tackled this problem used muscle tissue from surgical biopsies of muscles of the head, trunk and leg. Then they used antibody staining to show that human satellite cells can be identified by the expression of the transcription factor PAX7 in combination with the cell-surface proteins CD56 and CD29. Pomerantz and his colleagues use this molecular signature to isolate populations of human satellite cells from these patient biopsies. Then they grafted these satellite cells into mice with damaged muscles whose own muscle stem-cell populations had been depleted. Five weeks after the transplantation, these human cells had successfully integrated into the mouse muscles and divided to produce families of daughter stem cells; effectively replenishing the stem cell niche and repairing the damaged muscle tissue.

This characterization of human muscle stem cells and the ability to transplant them into injured muscles has varied and wide-ranging implications for patients who are presently suffering from muscle paralysis, whose damaged muscles have lost the ability to regenerate. Additionally, protocols that allow us to isolate and manipulate human stem cells also may have applications for understanding why our muscles lose their regenerative capacity during normal aging or in the case of genetic diseases such as muscular dystrophy.

“This gives us hope that we will be able to extract healthy stem cells from other muscles in the patient’s body and transplant them at the site of injury,” Pomerantz said. “If replenishing a healthy muscle stem cell pool facilitates reinnervation and recovery, it would be a significant leap forward.”

These findings appeared the Sept. 8 edition in the open access Cell Press journal, Stem Cell Reports.

Lab-Grown Muscle FIbers Aid in Studying Muscular Dystrophy


Skeletal muscle is the most abundant tissue in the human body, but, strangely, growing large quantities of it in the laboratory have proven rather challenging. While it is possible to reprogram other mature cells into heart muscle cells, or neurons, differentiating cells into skeletal muscle cells has simply not worked. So where do we go from here?

A new study from Brigham and Women’s Hospital (BWH) published in Nature Biotechnology has identified and even mimicked integral cues in the development of skeletal muscle. They used these cues to grow millimeter-long muscle fibers that are capable of contracting in the laboratory. This new method for growing functional muscle fibers in the laboratory potentially offer a better model for studying muscle diseases such as muscular dystrophy and for testing new treatments for these diseases.

Previous studies have used genetic modification techniques to grow small numbers of skeletal muscle cells in the laboratory. However, this new technique, which is the result of a collaboration between BWH and Harvard Stem Cell Institute, has produced a way to grow large numbers of skeletal muscle cells for use in clinical applications.

Olivier Pourquié of Harvard Medical School said, “We took the hard route: we wanted to recapitulate all of the early stages of muscle cell development that happen in the body and recreate that in a dish in the lab. We analyzed each stage of early development, and generated cell lines that glowed green when they reached a each stage. Going step by step, we managed to mimic each stage of development and coax cells toward muscle cell fate.”

The team found that a combination of secreted factors are important at the very early stages of embryonic development to stimulate muscle differentiation. By recapitulation this cocktail in the laboratory, Pourquié and his colleagues were able to mature muscle fibers in the laboratory from mouse or human pluripotent stem cells. Additionally, they produced muscle fibers in mice afflicted with muscular dystrophy by using muscle satellite cells. It is unknown if this method could help humans who suffer from muscular dystrophy, as more research is needed.

“This has been the missing piece: the ability to produce muscle cells in the lab could give us the ability to test out new treatments and tackle a spectrum of muscle diseases,” Pourquié said.

This new method also has the potential to help researchers study other muscle diseases, such as sarcopenia, or degenerative muscle loss and cachexia, the wasting away of muscle that typically occurs during severe illness.

Gene Controls Proliferation of Muscle Stem Cells


Fortunately, skeletal muscles have a high potential for regeneration, unlike other organs. When injured, muscle stem cells, known as satellite cells and located between the individual muscle fibers, rapidly begin to proliferate and subsequently replace the damaged muscles cells. New research from researchers from the Max Planck Institute for Heart and Lung Research in Bad Nauheim, Germany, have shown that a protein called Prmt5 plays a key role in regulating the activity of muscle satellite cells. These data gave rise to new studies that would like to examine the impact of Prmt5 in muscle disorders.

Satellite cells in skeletal muscles are small, spherical stem cells in between the individual muscles fibers. Normally, these cells remain almost completely inactive, but when a muscle is immediately begin to proliferate and heal the injury by replacing damaged muscles fibers.

satellite_cells

When satellite cells react to an injury, they undergo a transition from their inactive state to one of increased activity. This transition must be finely balanced because uncontrolled proliferation of satellite cells in healthy muscle tissue increases the risk of tumor formation. Conversely, muscle regeneration is impeded if the satellite cells are not activated fast enough when muscles are injured.

satellite cells
satellite cells

Now a research team headed by Thomas Braun from the Max Planck Institute for Heart and Lung Research in Bad Nauheim has now identified a gene that plays a decisive role in regulating the activity of satellite cells. Braun and his colleagues isolated muscle satellite cells from laboratory mice and identified 120 genes that are instrumental for the function of these cells.

Next, they switched off one of these genes, Prmt5, in the satellite cells of adult mice. “In healthy mice, switching off Prmt5 in the satellite cells had no effect on the muscles. But when the mice had a muscle injury, the results were completely different”, says Ting Zhang, the study’s lead author. No signs of regeneration were observed in Prmt5-deficient mice, but the muscles of control mice that had an active Prmt5 gene healed normally. “Instead of growing new muscle tissue, the mice without Prmt5 eventually developed clear signs of fibrosis”.

Braun and others further examined how Prmt5 regulates muscle regeneration. In mice without Prmt5, the number of satellite cells was noticeably reduced. Prmt5 seems to regulate proliferation activity of satellite cells. Furthermore, these results indicated that Prmt5 also prevents satellite cells from dying prematurely and plays a key role in transforming them into functional muscle fibers.

Braun and his colleagues hope their study will help them gain a better understanding of muscle disorders in humans. “The loss of muscle tissue in the absence of Prmt5 shows clear parallels to degenerative muscle disorders such as Duchenne muscular dystrophy”, says Johnny Kim, a member of Braun’s working group. In fact, the Bad Nauheim team now hopes that in the future, mice lacking the Prmt5 gene can serve as models for this particular disorder. “But we also want to study the etiological effects of Prmt5 regarding the genesis of muscular hypertrophies and certain tumor types,” Kim adds.

“In Body” Muscle Regeneration


Researchers at Wake Forest Baptist Medical Center’s Institute for Regenerative Medicine have hit upon a new strategy for tissue healing: mobilizing the body’s stem cells to the site of injury. Thus harnessing the body’s natural healing powers might make “in body” regeneration of muscle tissue is a possibility.

Sang Jin Lee, assistant professor of Medicine at Wake Forest, and his colleagues implanted small bits of biomaterial scaffolds into the legs of rats and mice. When they embedded these scaffolds with proteins that mobilize muscle stem cells (like insulin-like growth factor-1 or IGF-1), the stem cells migrated from the muscles to the bioscaffolds and formed muscle tissue.

“Working to leverage the body’s own regenerative properties, we designed a muscle-specific scaffolding system that can actively participate in functional tissue regeneration,” said Lee. “This is a proof-of-concept study that we hope can one day be applied to human patients.”

If patients have large sections of muscle removed because of infections, tumors or accidents, muscle grafts from other parts of the body are typically used to restore at least some of the missing muscle. Several laboratories are trying the grow muscle in the laboratory from muscle biopsies that can be then transplanted back into the patient. Growing muscle on scaffolds fashioned from biomaterials have also proven successful.

Lee’s technique overcomes some of the short-comings of these aforementioned procedures. As Lee put it, “Our aim was to bypass the challenges of both of these techniques and to demonstrate the mobilization of muscle cells to a target-specific site for muscle regeneration.”

Most tissues in our bodies contain a resident stem cell population that serves to regenerate the tissue as needed. Lee and his colleagues wanted to determine if these resident stem cells could be coaxed to move from the tissue or origin, muscle in this case, and embeds themselves in an implanted scaffold.

In their first experiments, Lee and his team implanted scaffolds into the leg muscles of rats. After retrieving them several weeks later, it was clear that the muscle stem cell population (muscle satellite cells) not only migrated into the scaffold, but other stem cell populations had also taken up residence in the scaffolds. These scaffolds were also contained an interspersed network of blood vessels only 4 weeks aster transplantation.

In their next experiments, Lee and others laced the scaffolds with different cocktails of proteins to boost the stem cell recruitment properties of the implanted scaffolds. The protein that showed the most robust stem cell recruitment ability was IGF-1. In fact, IGF-1-laced scaffolds had four times the number of cells as plain scaffolds and increased formation of muscle fibers.

“The protein [IGF-1] effectively promoted cell recruitment and accelerated muscle regeneration,” said Lee.

For their next project, Lee would like to test the ability of his scaffolds to promote muscle regeneration in larger laboratory animals.