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QUEST Volume 10, Number 5, SEPTEMBER/OCTOBER 2003

New Stem Cells Move From Blood to Muscle, Slow MD in Mice

MDA-funded scientists have shown for the first time that stem cell injections can prevent muscle wasting and improve strength in mice with muscular dystrophy (MD).

Kevin Campbell  
Kevin Campbell

Previous attempts at stem cell therapy for MD have met with little success. Scientists have tried injecting the cells directly into muscle or transplanting them into bone marrow, but ó whether tried in animals or in people ó neither procedure has led to enough muscle regeneration to slow the course of MD. Ideally, giving stem cells through a bone marrow transplant would send them through the bloodstream to muscles all over the body, but in reality, very few of the cells get to muscles this way.

An Italian team led by Giulio Cossu, at the University of Rome and the Stem Cell Research Institute in Milan, has isolated stem cells that normally live in blood vessels and thus can rapidly find their way to muscles when injected into an artery. Kevin Campbell, a longtime MDA grantee at the University of Iowa in Iowa City, was a member of the study team.

First, the scientists isolated the vessel-dwelling stem cells — called mesoangioblasts — from normal mice and tested them in mice lacking alpha-sarcoglycan, the muscle protein flawed in people with limb-girdle MD type 2D (LGMD2D). After a single injection into an artery in the hind leg, the cells had migrated to several leg muscles, and alpha-sarcoglycan could be detected there for at least three months afterward.

Johnny HUard  
Johnny Huard

When the injections were given three times over four months, the protein could be found in more than 50 percent of fibers in some leg muscles. Muscles in the injected leg showed less degeneration and less fibrosis (a buildup of fatty tissue) and the individual fibers were stronger than those of untreated mice. The treated mice were also better at staying on a spinning rod called a rotarod.

The scientists also isolated mesoangioblasts from mice with alpha-sarcoglycan deficiency and used a virus to give the cells an intact alpha-sarcoglycan gene. Those cells were as effective as normal cells when injected into the mice.

These results, published online by Science on July 10, suggest that people with MD could be treated with intravascular injections of their own mesoangioblasts, Campbell and Cossu say. The cells havenít yet been isolated from adult human tissue, however.

Meanwhile, a study in the July issue of Nature Cell Biology might explain why the bone marrow transplant method of delivering stem cells hasnít been very successful against MD.

Stem Cell Mouse  

MDA grantee Johnny Huard of Childrenís Hospital of Pittsburgh isolated stem cells from the muscles of mice with Duchenne MD, fixed the cells with a miniaturized version of dystrophin (the protein missing in DMD), and injected them into mice with DMD that had their bone marrow destroyed.

As reported previously, the injected cells restored the bone marrow, but only a tiny fraction of them made new muscle. In other experiments, Huard found that once muscle stem cells become bone marrow, they lose some of their ability to make muscle.

Could Proteins Activate Muscle Stem Cells?

Michael Rudnicki  
Michael Rudnicki

Two new studies hint at the possibility of using drugs to activate stem cells in people with muscular dystrophy, perhaps eliminating the need for stem cell transplants.

In the June 27 issue of Cell, MDA grantee Michael Rudnicki of the Ottawa Health Research Institute in Canada reported that Wnts — a class of hormonelike proteins — encourage the stem cells in adult muscle to become muscle. Rudnicki found that injured mouse muscles produce large amounts of Wnt protein, along with a tenfold increase in one type of muscle stem cell.

In a laboratory dish by themselves, those cells normally become blood or skin cells, but when exposed to Wnt or chemicals that mimic its effects, many of them turned into muscle cells. And when injured mouse muscles were injected with proteins that block Wnt signaling, the stem cells stopped dividing.

The findings build on previous work by Rudnicki showing that a gene called Pax7 is essential for converting muscle stem cells into adult muscle (see "Research Updates," 2000, no. 5). It now appears that Wnts might be the signal for turning on Pax7.

A study in the May 16 issue of Cell shows that interleukin-4, a protein previously thought to act only on immune cells, also has an important effect on muscle cells.

Grace Pavlath of Emory University in Atlanta showed that mice normally produce interleukin-4 after a muscle injury, and that mice with a genetic deficiency of the protein have small muscle fibers. Further experiments showed that the protein stimulates immature muscle cells to fuse with muscle fibers.

In the future, Wnts or interleukin-4 might be used to awaken stem cells that lie dormant in the muscles of people with muscular dystrophy, or to enhance the effects of a stem cell transplant.

Rudnicki said heís testing delivery of Wnt genes in mice with Duchenne MD and plans to start injecting the mice with Wnt proteins.

Mice Lacking Dystrophin Make It With New Construct

MDA grantee Stephen Wilton, a molecular biologist at the Centre for Neuromuscular and Neurological Disorders of the University of Western Australia in Perth, was on a research team that coaxed mice with severe dystrophin deficiency and symptoms of Duchenne muscular dystrophy (DMD) to begin making dystrophin, the protein needed but missing in the disease.

Mice Lacking Dystrophin  

The researchers, who published their results in the August issue of Nature Medicine, gave the mice a combination of an antisense oligo molecule, which they designed to snip out the flaw (mutation) in the genetic instructions for dystrophin in these mice, and a substance called F127, which helped with delivery of the antisense to muscle cells.

No viruses, which have led to unwanted immunologic reactions in other studies, were used.

After an injection of this construct into a leg muscle, the mice made near-normal dystrophin in about 20 percent of the injected areaís muscle fibers. The antisense apparently allowed the cells to skip over the flaw, which acts as a "stop sign" to interrupt the normal gene-to-protein manufacturing process. The treated cells were able to run through the stop sign and follow the remainder of the dystrophin "recipe," making a functional protein.

The mouse muscles showed improved force generation, and the mice accepted the newly made protein without mounting an immunologic attack against it.

Wilton noted that the researchers need to find ways to make the drug more stable and more biologically active, as well as ensuring its long-term safety, since human treatments would likely have to be given over a long period of time. He also cautioned that not all human dystrophin mutations can be addressed by this strategy.

Transport Gene May Be Part of Inclusion-Body Myositis

Variations in a gene for the transthyretin protein, which transports one of the hormones secreted by the thyroid gland, may be a factor in at least some cases of inclusion-body myositis (IBM), the most common progressive muscle disease of older people, according to MDA grantee Valerie Askanas and colleagues. (For more on IBM, see "Advances in Inclusion-Body Myositis," Quest, April 2001.)

Valerie Askanas  
Valerie Askanas

Askanas, a physician-scientist at the University of Southern California in Los Angeles, recently led a team that made this connection and published the finding in the July 22 issue of Neurology.

Most cases of IBM donít appear to be genetic, but researchers have theorized that genetic factors may predispose some people to developing the condition after about age 50.

Previous investigations have shown that flaws in the transthyretin gene can lead to deposits of a starchlike material called amyloid in the heart and other organs, and that such amyloid deposits in muscle tissue are a hallmark of IBM.

The researchers identified a patient who had cardiac amyloid deposits and IBM, a combination they say is related to a mutation in his transthyretin gene. Askanas said identifying this and other predisposing genes could provide clues for understanding IBM and might be helpful in developing treatments.

ĎDesignerí CoQ10 and Vitamin E Under Study

MDA grantee Michael Murphy is developing new, possibly more potent versions of coenzyme Q10 and vitamin E, antioxidants that have shown mixed results against amyotrophic lateral sclerosis (ALS), Friedreichís ataxia (FA), and mitochondrial diseases.

Designer CoQ10  

All three diseases involve a breakdown of mitochondria, the factories within cells that use oxygen to produce energy. The backlog of oxygen and "fuel" inside the mitochondria leads to oxidative stress — an accumulation of harmful oxygen-based free radicals.

CoQ10 is a component of mitochondria, and vitamin E is found in green, leafy vegetables. Both are available as over-the-counter supplements.

CoQ10 appears to work wonders against mitochondrial disease caused by coQ10 deficiency. But the two antioxidants have produced only slight benefits in ALS, FA and other mitochondrial diseases — not the increases in strength that had been hoped for.

Murphy, a scientist with the Medical Research Council (the United Kingdomís equivalent of the U.S. National Institutes of Health), hopes to improve the effects of coQ10 and vitamin E by making it easier for them to get into mitochondria.

To that end, heís attached both substances to a chemical (TPMP) that penetrates mitochondria, making what he calls MitoQ and MitoVit E.

In the April 29 issue of Proceedings of the National Academy of Sciences, he reported that normal mice given MitoQ or MitoVit E through their drinking water accumulated the compounds in mitochondria in their muscles, brains, hearts and other organs.

When tested on cells and isolated mitochondria in a laboratory dish, the compounds accumulate several hundredfold more than currently available forms of vitamin E and coQ10, he said. Next, he plans to test the compounds in mice with mitochondrial defects.

Stroke Risk May Rise With IVIg Use, Caution Urged

IVIg (intravenous immunoglobulin) is used to redirect the immune system when it has mistakenly attacked the bodyís own tissues.

The "IV" part of IVIg means intravenous, which is the method of administration. The "Ig" refers to immunoglobulins, immune-system proteins, which are taken from a number of donors. These proteins have complex activities that seem to "confuse" the immune system and thus redirect it.

IVIg is used in various autoimmune neuromuscular conditions, such as myasthenia gravis, Lambert-Eaton myasthenic syndrome and dermatomyositis, usually when other treatments have failed.

In general, it has an excellent safety record, experts say, with side effects usually limited to temporary headaches, chills, muscle pain and similar discomforts. But one potential effect causes concern ó the possibility of an increased risk of stroke.

In the June 9 issue of Neurology, researchers James Caress at Wake Forest University in Winston-Salem, N.C., and colleagues, report on 16 IVIg-associated strokes that occurred over a four-year period at Wake Forest University Baptist Medical Center and other centers in the region. However, the report notes that stroke occurred in less than 1 percent of patients who received IVIg treatment during the study period.

Ten of the 16 people who had strokes that were apparently IVIg-associated had neuromuscular diseases, and six had blood disorders. Fifteen had one or more known stroke risk factors, such as high blood pressure, diabetes or previous vascular conditions.

The researchers believe the factors that could lead to stroke after IVIg treatment include an elevation in blood viscosity (resistance to flow), a rapid change in viscosity, sudden expansion of volume inside the blood vessels, spasm of a cerebral artery, or the introduction of substances that cause clotting or dilate or constrict blood vessels along with the immunoglobulins.

In an accompanying editorial, Marinos Dalakas of the National Institutes of Health and Wayne Clark of Oregon Health Sciences University in Portland recommend that patients who need IVIg but have stroke risk factors should receive measurements of blood viscosity before the infusion, with lowering of this value if necessary. They also recommend a slow rate of infusion, in a hospital setting, and possibly an ultrasound examination of the arms and legs to search for hidden clots, especially if the person has been immobile for a long time.

The authors say that IVIg can have dramatic benefits that justify its use, but that such use should be with great care, with precautions taken to identify and, when possible, correct any risk factors for complications.


Pompeís Study Now Includes Younger Babies

In March, the biotech company Genzyme of Cambridge, Mass., in conjunction with MDA, announced the opening of a new clinical trial to test a laboratory-engineered acid maltase enzyme to replace the one thatís missing in Pompeís disease, also known as acid maltase deficiency. (An enzyme is a protein that speeds chemical reactions; the acid maltase enzyme breaks down stored sugar.)

That trial is still seeking participants ages 6 months to 3 years. For details, see "Research Updates," May-June.

The laboratory-engineered enzyme is designed to break down stored sugar in muscle fibers.

Now, the company is seeking babies 6 months old or younger to participate in a separate enzyme replacement trial for Pompeís.

The babies must have a clear diagnosis of infantile-onset Pompeís disease with heart enlargement and very low acid maltase enzyme activity. Candidates must be free of major medical conditions not associated with Pompeís disease, have adequate respiratory function and meet many other study criteria.

The U.S. part of the trial is being conducted at these centers:

  • University of Florida, Gainesville
    Contact Julie Berthy at (352) 392-6431 or jberthy@ufl.edu.

  • Duke University, Durham, N.C.
    Contact Stephanie DeArmey at (919) 681-1946 or dearm001@mc.duke.edu.

  • Childrenís Hospital Medical Center,
    Cincinnati, Ohio
    Contact Michelle Wojtasiak at
    (513) 636-1116 or michelle.wojtasiak@cchmc.org.


There are also centers in France and the United Kingdom.

High Lactic Acid in Mitochondrial Disease

A high level of lactic acid in the blood — which can damage the nervous system and muscles — is a common effect of many mitochondrial disorders.

The drug dichloroacetate (DCA) has shown some promise in lowering lactic acid levels and improving symptoms in some people, but it can have undesirable side effects, such as liver and nerve damage.

Now, investigators at the University of Florida in Gainesville, working under a grant from the U.S. Food and Drug Administration, want to find out how effective DCA is, and whether they can reduce its side effects by adding another drug, nitisinone (Orfadin).

Study participation requirements include having a clear diagnosis of mitochondrial disease with high lactic acid levels, the ability to fast for eight to 12 hours while maintaining blood sugar levels, and an agreement to follow a restricted diet during the study.

Contact Peter Stacpoole or Margaret Francis at (352) 392-2321 or francma@medicine.ufl.edu.


Arthritis Medication in Dermatomyositis

A study of etanercept (Enbrel) for dermatomyositis that isnít responding to the usual treatments is still open to participants between the ages of 13 and 65.

Enbrel, which is approved for use in adult and juvenile rheumatoid arthritis and psoriasis-associated arthritis, blocks a body chemical called tumor necrosis factor, thought to be involved in inflammation.

Prospective participants must have been on the same medication regimen for the two months prior to trial entry and be free of infection or poorly controlled diabetes.

Study-associated lab tests, examinations and medications are free to participants. The trial is being conducted by neurologist Kumaraswamy Sivakumar at St. Josephís Hospital and Medical Center in Phoenix.

Contact Valentina Apostol at (602) 406-6364 or vapostol@chw.edu.


Exercise in CMT

Graduate student Jill Pascoe, who is pursuing a doctoral degree in physical therapy at Arizona School of Health Sciences in Mesa, is conducting a study of how and why people with Charcot-Marie-Tooth disease (CMT) exercise or donít exercise and what effect their exercise habits may have on their physical, social or mental well-being.

Participants, who must be 21 or older and have a diagnosis of CMT, are asked to fill out an anonymous questionnaire on the Internet at www.ashs.edu/survey/jpascoe/index.htm.

The study will include at least 100 people and is open through November.



Uncovering Causes of Cardiac Problems in MDs


Doctors have long been aware that many types of muscular dystrophy can affect the heart, but until recently, they understood little of the details of these problems or what precisely caused them.

Todayís molecular and genetic advances have allowed investigators to study the heart defects in ways that until recently werenít possible.

In September, MDA will host a meeting of medical and scientific specialists, who will discuss research in the cardiac aspects of neuromuscular disease. Among likely topics for discussion will be the following findings, published in the May issue of Neuromuscular Disorders unless otherwise noted:

Loss of Dystrophinís Two Attachments in Heart Cells Likely Culprit in DMD

T.A. Hainsey and colleagues in the Department of Molecular and Cellular Biochemistry at Ohio State University conducted a series of experiments in mice with Duchenne muscular dystrophy (DMD) to determine more precisely what goes wrong in the heart in this dystrophin-deficiency disorder.

The researchers wanted to find out whether the heart problems often seen in these dystrophies are caused by the absence of dystrophin in the heart muscle cells or in blood vessel cells.

They also wanted to determine what part of the dystrophin molecule is essential in the heart.

To find out, they started with mice that were bred to lack both dystrophin and another protein, utrophin. These mice develop an obvious cardiac muscle problem, or cardiomyopathy.

They then bred some of these mice to have a shortened form of dystrophin that only attached to proteins in the cell membrane, a sheath surrounding each muscle fiber. This short dystrophin molecule didnít attach to the inside of the cell.

They bred other mice to have a shortened form of dystrophin that attached inside the cell and to the membrane proteins but lacked a section in its middle.

Somewhat surprisingly, the mice that got the dystrophin that attached only to the membrane proteins and not to the cellís interior showed a more severe cardiomyopathy than those that had no dystrophin at all.

In contrast, those bred with dystrophin lacking only a midsection but with both connecting ends intact had hearts as healthy as those of normal mice.

Since neither form of shortened dystrophin was detected in the blood vessels in these mice, the researchers concluded that blood vessel dystrophin probably isnít needed to prevent cardiomyopathy.

They also concluded that dystrophin has to attach to the membrane and to the cellís interior and be present in heart muscle cells for cardiomyopathy to be avoided.

Such deductions are important for designing treatments for cardiomyopathy related to dystrophin deficiency.

Direct Heart Damage Can Play Role in LGMDs

Marina Fanin and colleagues at the University of Padua in Italy studied six people with a form of limb-girdle muscular dystrophy (LGMD) that results from flaws in the gene for beta-sarcoglycan, a protein in the muscle cell membrane. There are four sarcoglycans in this membrane — alpha, beta, gamma and delta — and they rely on dystrophin and on one another for proper positioning.

Three of the six study subjects had no detectable cardiac abnormalities, while three had various degrees of cardiac involvement. One person died at age 14 of severe cardiomyopathy and heart failure.

Previous research has suggested that heart disease in sarcoglycan-deficient LGMD arises from a combination of blood vessel abnormalities and direct damage to the heart muscle from sarcoglycan loss. But patients lacking alpha- and gamma-sarcoglycan, which arenít found in blood vessels, also can develop cardiomyopathy. This cardiac damage is therefore likely to be related to sarcoglycan loss in the heart itself.

Beta-sarcoglycan, in contrast, is found in blood vessels and in muscle cells, as is delta-sarcoglycan. So, in these types of LGMD, the absence of the needed sarcoglycan in both tissues may contribute to the cardiac abnormalities.

The authors of the beta-sarcoglycan LGMD study recommend that doctors be alert to the risk of cardiomyopathy in people with beta-sarcoglycan-deficient LGMD.

Another idea thatís been considered is that cardiomyopathy in sarcoglycan or dystrophin deficiency could be a secondary effect of the absence of these proteins in skeletal muscles (for instance, from changes in exercise and blood flow patterns).

But last year, a team including MDA grantee Elizabeth McNally of the University of Chicago found that when mice missing gamma-sarcoglycan in both skeletal and cardiac muscle had this protein restored to their skeletal muscles alone, they still developed cardiomyopathy.

This told the investigators, who published their study in the July 2002 issue of the FASEB Journal, that the heart damage in this type of sarcoglycan-deficient LGMD isnít caused by skeletal muscle abnormalities, since the protein was restored there; isnít secondary to blood vessel dysfunction, since the protein doesnít belong in blood vessels; and therefore must be caused by the direct effects of gamma-sarcoglycan loss on the heart muscle cells.

Cardiac Defects in MMD2 Probably Not Neurological

Peter Flachenecker and colleagues at the University of Wurzburg in Germany say that cardiac rhythm abnormalities are fairly common in type 2 myotonic dystrophy (MMD2). In this type of cardiac disease, the heart beats too slowly, too fast or irregularly.

When the team studied 16 people with MMD2, they found four (25 percent) of them had cardiac involvement, mostly rhythm disturbances. They theorized that these rhythm problems could be due to the signals the hearts were receiving from the nervous system.

But when these 16 people were compared with 16 people without any known disorder on several tests to evaluate the part of the nervous system that controls the heartbeat, the results in the two groups were similar.

The investigators concluded that autonomic nervous system abnormalities are an unlikely cause of heart rhythm disturbances in MMD2 and that direct heart involvement, even in these cases, is a more likely culprit.

Gene Transfer Corrects Cardiac Problem in DMD Mice

Dongsheng Duan  
Dongsheng Duan

It seems likely that direct protein deficiencies resulting from genetic flaws are the principal culprits in MD-associated cardiomyopathy. Some investigators are looking into replacement of these genes or the proteins that come from their instructions.

MDA grantees Dongsheng Duan at the University of Missouri in Columbia and Jeffrey Chamberlain at the University of Washington in Seattle were part of a team that recently explored the possibility of correcting dystrophin deficiency, the underlying cause of muscle degeneration in Duchenne and Becker muscular dystrophies (DMD and BMD), in the hearts of mice that lack this protein and show signs of DMD.

Jeff Chamberlain  
Jeff Chamberlain

At a June meeting of the American Society of Gene Therapy in Washington, D.C., the investigators reported that they could successfully deliver a highly miniaturized dystrophin (microdystrophin) gene to the hearts of these mice with considerable benefit to the tissue.

The investigators inserted the microdystrophin genes into adeno-associated viruses for transport into muscle cells, then injected them into the mouse hearts. AAV is considered the safest and most effective virus for gene transfer to muscle, but itís also among the smallest; hence, the need to miniaturize the large dystrophin gene.

The mice that received the genes showed dystrophin protein production in their heart muscle cells for at least 10 months. In addition, a group of cell proteins that requires dystrophin for its assembly was restored and located in its normal position in the cellsí protective membrane.

The investigators consider this study "an important first step" in understanding and potentially treating the heart disease of DMD and BMD. A paper with the complete results is slated for the Sept. 30 issue of Circulation.


For up-to-the-minute news on MDA research developments, visit MDAís Web site at www.mdausa.org. Click on "Research" for information on recent research developments and active clinical trials, and links to major medical/research sites. Look at the Web siteís "Whatís New" section for news bulletins about breaking research announcements.

For research news about amyotrophic lateral sclerosis, see The MDA/ALS Newsletter or go to www.als.mdausa.org.


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