Research Updates November-December 2007

Article Highlights:

Updates on research news as of October 2007

by Margaret Wahl on November 1, 2007 - 9:22am

QUEST Vol. 14, No. 6

Items im this article refer to research in:  Duchenne muscular dystrophy, myotonic muscular dystrophy, facioscapulohumeral muscular dystrophy and spinal muscular atrophy.

Utrophin gene therapy benefits mice with DMD

Injecting genes for the muscle protein utrophin may be a viable strategy to pursue for treating Duchenne muscular dystrophy (DMD), say researchers at McGill University in Montreal.

MDA grantees George Karpati, Basil Petrof and Josephine Nalbantoglu were part of a team that injected genes for utrophin into the leg muscles of mice missing the closely related dystrophin protein. These dystrophin-deficient mice have a disease resembling human DMD.

The advantage to injecting utrophin instead of dystrophin genes is that the immune systems of at least some children and adolescents may reject the new dystrophin as a foreign protein, while they will almost certainly accept extra utrophin, since people with DMD already make utrophin, and it won’t be foreign to them.

Newborn and adult mice showed evidence of utrophin production in the injected muscles, as well as better resistance to contraction-related damage and in some cases better force generation than on the uninjected side of the body. However, in both groups, the beneficial effects diminished over the course of a few months to a year after injection.

“A critical issue is to determine the minimum amount of utrophin that is sufficient to successfully ‘pinch-hit’ for dystrophin,” Karpati said, noting that utrophin is normally found in muscle fibers only at the places where they intersect with nerve fibers (the synapse) and that utrophin throughout the fiber membrane will be necessary to successfully treat DMD.

The other problem that must be solved is “the substantial decline of the amount of extrasynaptic [outside the synapse] utrophin over time,” Karpati added. He said it does not appear to be a problem of immune system rejection.

The authors, who published their results online July 31 in Molecular Therapy, say that utrophin therapy might be optimized by combining utrophin gene transfer with a compound that increases protein production from the patient’s own utrophin genes.

MDA is supporting research on the latter strategy (see ID of Utrophin Brake, Research Updates, September-October 2007), as well as a clinical trial to test the effects of a muscle injection of dystrophin genes into boys with DMD.

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A new “consensus statement” on clinical care in spinal muscular atrophy (SMA), published in the August issue of the Journal of Child Neurology, aims to improve and standardize the management of this disease with respect to diagnosis, pulmonary care, gastrointestinal and nutritional issues, orthopedics and rehabilitation, and end-of-life decisions.

Establishing standards of SMA care was identified as a priority by a patient advocacy group of the International Coordinating Committee (ICC) for Spinal Muscular Atrophy Clinical Trials. This committee was formed in January 2005 under the auspices of the National Institutes of Health (NIH).

Discussions at an NIH-sponsored conference in 2004, in which the goal was to formulate strategies for future clinical trials in SMA, revealed wide variations in medical practices in this disease and the need to unify these for the benefit of patients and for the conduct of future therapeutic trials. MDA supported the forming of a new SMA Standard of Care Committee through the ICC’s patient advocacy group.

The committee divided SMA patients into “nonsitters,” “sitters” and “walkers” and addressed the care needs of the three groups separately and in detail.

Some of the group’s general conclusions are that even young children with SMA should be offered independent mobility and activities of daily living, including play; that walking should be encouraged, with the use of appropriate assistive devices and orthotics; that spinal orthoses (back braces) may provide support but don’t prevent progression of spinal curvatures and may impair breathing; that surgery for scoliosis (spinal curvature) appears to benefit patients who survive beyond age 2 when curves are severe and progressive; that scoliosis surgery should be performed while pulmonary function is adequate; and that special preoperative respiratory care and neurological monitoring during surgery are desirable.

For more about the care guidelines, see SMA: Committee Presents Clinical Care Guidelines, or read the entire consensus statement at

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Excess of mu-crystallin protein added to hypotheses about FSHD

An excess of the protein known as mu-crystallin has been identified as a new addition to the array of hypotheses about the molecular basis of facioscapulohumeral muscular dystrophy (FSHD). (See Through the Looking Glass with FSH Dystrophy Researchers, Quest, March-April 2007.) The mu-crystallin findings add to and don’t necessarily contradict any of the existing FSHD hypotheses.

MDA grantee Robert Bloch at the University of Maryland School of Medicine, and colleagues, found much higher levels of the mu-crystallin protein in muscle samples from people with FSHD than from people without a neuromuscular disease or with other forms of muscular dystrophy or inflammatory muscle diseases.

The investigators, who published their results in the June issue of Experimental Neurology, suggest that changes in mu-crystallin may also explain the changes that occur in tissues other than muscle that are affected in FSHD.

Mu-crystallin is found in the retina, and retinal abnormalities affect some FSHD patients. Abnormalities in this protein have also been associated with deafness, and hearing loss sometimes occurs in FSHD.

In addition, mu-crystallin interacts with thyroid hormone, a potent signaling molecule that acts early in muscle-cell maturation.

The gene for mu-crystallin is located on chromosome 16, while the only defect known to be associated with the disease is a missing section of DNA on chromosome 4. However, investigators have long suspected that the chromosome 4 deletion may affect the activity of genes far from its location, including genes on other chromosomes.

Since the publication of this paper, lead author Patrick Reed, also at the University of Maryland, has been awarded an MDA grant to probe this and other protein abnormalities in FSHD.

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New MMD1 studies connect CUG binding protein overload to errors in molecular splicing

Two new studies from the laboratory of MDA grantee Thomas Cooper at Baylor College of Medicine in Houston have shed further light on the molecular complexities of type 1 myotonic dystrophy (MMD1) and hinted at a possible reason for some of the differences between this disease and type 2 MMD (MMD2).

In one set of experiments, Cooper, with colleagues N. Muge Kuyumcu-Martinez and Guey-Shin Wang, uncovered the probable mechanism by which a protein known as CUGBP1 (CUG binding protein 1) becomes abnormally elevated in the heart and skeletal muscle cells in MMD1 and the effects of CUGBP1 excess.

The investigators, who published their findings online in October in Molecular Cell, describe how the presence of extra genetic material on chromosome 19 in type 1 MMD leads to a toxic increase of CUGBP1.

Since the extra genetic material (RNA) consists of a string of chemical triplets (cytosine, uracil and guanine, or CUG), it had been proposed that CUGBP1 would stick to these and that its levels elsewhere in the cell would decrease, a phenomenon that occurs in MMD1 with at least one other protein. However, data from many studies have shown just the opposite: CUGBP1 is unexpectedly elevated in MMD1-affected muscle cells.

Cooper and colleagues say that production of the extra repeated CUG (CUG “repeat”) RNA triggers the activation of an enzyme called protein kinase C, which in turn causes extra phosphate groups to be attached to the CUGBP1 molecules. These phosphate groups (a phosphorus atom surrounded by four oxygen atoms) make CUGBP1 more stable than it otherwise would be, so it lasts longer and builds up in the cell nucleus.

Then, they say, the extra CUGBP1 affects the way other muscle proteins are constructed by changing a molecular process called splicing. For example, abnormalities in the way a chloride channel protein and an insulin receptor protein are constructed probably underlie myotonia (prolonged muscle contraction) and insulin resistance, respectively. Both of these are features of MMD1.

The researchers note that reducing CUGBP1 levels could be explored as a potential treatment for MMD1.

In a separate set of experiments, Cooper and several other Baylor scientists describe how mice with 960 CUG repeats (normal in humans is 3-37) in the MMD1-associated gene on chromosome 19 have elevated CUGBP1 and the same type of heart defects seen in humans with type 1 MMD.

In a paper published online in September in the Journal of Clinical Investigation, they say mice with these extra CUG repeats develop heart rhythm abnormalities and cardiac muscle deterioration that mirror those seen in the human disease, and that the cardiac muscle protein troponin T is incorrectly constructed because of errors in splicing.

They suggest that the elevated levels of CUGBP1 contribute to the incorrect splicing during synthesis of troponin T and therefore to the heart abnormalities, although they say their results make it likely that there are additional errors in the synthesis of other cardiac proteins.

MMD2, a disease that has many similarities to MMD1, results from extra CCUG repeats in a chromosome 3 gene. Last year, MDA grantees at the University of Rochester (N.Y.) and the University of Florida found that CUGBP1 is not elevated in MMD2, which, Cooper says, may account for some of the differences in the two diseases.

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Decorin protein pushes damaged muscle toward repair

Researchers at the University of Pittsburgh and Children’s Hospital of Pittsburgh have found that treating distressed muscles with a protein called decorin prevents scar tissue formation and improves regeneration and repair.

The team included Xiao Xiao and Johnny Huard, both of whom had MDA funding for related work from 2002 to 2005.

In a paper published online July 3 in Molecular Therapy, Yong Li and colleagues describe test-tube experiments and mouse experiments showing that decorin increases production of proteins related to muscle regeneration and decreases production of myostatin, a protein known to limit muscle fiber growth. They also found that decorin neutralizes the effects of a protein that stimulates scar tissue formation.

Mice with a disease resembling Duchenne muscular dystrophy (DMD) showed better muscle regeneration and less scar tissue formation in muscles that had been treated with decorin genes than they showed in their untreated muscles.

“It is possible that decorin increases muscle fiber growth and limits the overgrowth of connective tissues,” the researchers write. “These findings indicate that decorin could be very useful in promoting the healing of muscles damaged by injury or disease.”

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Saving muscle may require moving beyond myostatin blocking

The myostatin protein, known to limit muscle growth, probably works in at least two distinct pathways, and it doesn’t work alone, says Se-Jin Lee at Johns Hopkins University in Baltimore online Aug. 29 in PLoS One.

Myostatin has been a target of molecular biologists seeking to increase or preserve muscle in the face of degenerative diseases like muscular dystrophy.

Lee, who had MDA support for this study, says myostatin appears to regulate local growth of muscle in response to specific events, such as injury, and he speculates that it may also regulate the overall balance between fat and muscle in the body in response to general conditions, such as nutritional status.

His findings suggest that local control of muscle growth may be achieved through regulating how much myostatin is activated in a particular location at any given time and that global control may depend on how much myostatin is circulating in the bloodstream.

In addition, Lee says, his studies demonstrate that at least three proteins contribute to regulation of muscle mass in mice: myostatin, which limits muscle fiber growth, in addition to follistatin and follistatin-related protein, which work against myostatin to promote muscle fiber growth.

Mice with extra follistatin genes and no myostatin genes had four times the normal amount of muscle mass. (Mice lacking myostatin genes with the normal amount of follistatin have twice the normal amount of muscle.)

Understanding all the factors involved in the number and size of muscle fibers is essential to developing optimum treatments, the investigators say.

“The studies presented here demonstrate that the capacity for promoting muscle growth by targeting this general signaling pathway is far greater than previously appreciated,” they write, noting that most efforts in this regard have focused on inhibiting myostatin’s activity alone.

“The finding that myostatin is not the sole regulator of muscle mass in mice raises the question as to whether targeting myostatin alone will be the most effective strategy for manipulating this signaling pathway in humans.”

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