Stem cell therapy can regenerate heart muscle in primates, according to a UW-led study. The scientists on this and related projects are seeking way to repair hearts weakened by myocardial infarctions. This all-too-common type of heart attack blocks a major artery and deprives heart muscle of oxygen.

People who survive a severe episode often continue their lives in poor health because their hearts no longer work properly. The researchers hope eventually to restore such failing hearts to normal function. Their approach uses heart cells created from human embryonic stem cells

The researchers tested the possibility of producing enough of these cardiac muscle cells to remuscularize damaged hearts in a large animal whose heart size and physiology are human-like.

Their results are reported today, April 30, in the advanced online edition of Nature. See .

Dr. Charles Murray, professor of pathology and bioengineering, and Dr. Michael LaFlamme, assistant professor of pathology, are the senior authors of the paper.

Read the details of their project and its outcomes in, and watch a video describing the research.

Preclinical studies show that gene therapy can improve muscle strength in small- and large-animal models of a fatal congenital childhood disease know as X-linked myotubular myopathy.

The findings, appearing  as the in the January 22, 2014 issue of Science Translational Medicine, also demonstrate the feasibility of future clinical trials of gene therapy for this devastating disease.

Watch a by Brian Donohue on this study.

Researchers at the  91̽��,   in France, , and in Blacksburg, Va., conducted the study.

The study was based on seminal work on local and systemic administration in a mouse model of the disease performed by Anna Buj-Bello, at ��é��é�ٳ�Dz� since 2009. The UW’s Martin K. Childers, working with Buj-Bello and Beggs groups, tested gene therapy using an engineered adenovirus vector, created by ��é��é�ٳ�Dz�. The vector carries a replacement MTM1 gene.

They used two animal models: mice with an engineered MTM1 mutation and dogs carrying a naturally occurring MTM1 gene mutation. These mutant animals appear very weak with shortened lifespans, similar to patients with myotubular myopathy.

The scientists found that both mice and dogs responded to a single intravascular injection of an adenovirus vector engineered for gene replacement therapy, produced at ��é��é�ٳ�Dz�. The treated animals had robust improvement in muscle strength, corrected muscle structure at the microscopic level, and prolonged life. No toxic or immune response was observed in the dogs.

These results demonstrate the efficacy of gene replacement therapy for myotubular myopathy in animal models and pave the way to a clinical trial in patients.

Children born with X-linked myotubular myopathy, which affects about 1 in 50,000 male births, have very weak skeletal muscles, causing them to appear floppy. They also have severe respiratory difficulties. Survival beyond birth requires intensive support, often including tube feeding and mechanical ventilation, but effective therapy is not available for patients, and most die in childhood.

Alan H. Beggs of Boston Children’s Hospital, co-senior author on the paper, has studied the mutated gene, known as MTM1, for many years and previously showed that replacing missing myotubularin protein effectively improved MTM muscles’ ability to contract.

“The implications of the pre-clinical findings are extraordinary for inherited muscular diseases,” said Childers, co-senior author on the paper, and co-principal investigator of the study with  Buj-Bello and  Beggs. “Two of our dogs treated with AAV gene therapy appear almost normal with little, if any, evidence, even microscopically, of disease caused by XLMTM.” Childers is a 91̽��professor of rehabilitation medicine and a regenerative medicine researcher.

“These results are the culmination of four years of research and show how gene therapy is effective for this genetic muscle disease,” said Buj-Bello. “We finally can envision a clinical trial in patients. These are very promising results for future trials in humans. ”

Robert W. Grange, Virginia Tech associate professor of human nutrition, foods and exercise, and Virginia Tech graduate student Jon Doering provided expertise to demonstrate the dramatic rescue of muscle function in the treated dogs. “The functional improvement was truly remarkable,” said Grange. “It is both incredibly exciting and humbling to contribute to such a meaningful project – a true highlight of our careers.”

The study was funded by the Association Francaise contre les Myopathies, the Muscular Dystrophy Association, Myotubular Trust, Genopole d’Evry, INSERM, Region d’Alsace, the Anderson Family Foundation,  the Joshua Frase Foundation,  Where There’s a Will There’s a Cure Foundation, and the  Peter Khuri Fund for Myopathy Research. National Institute of Health grants P50 N5040828, R01 AR044345, R21 AR 064503, AR 0659750 and Ro1 HL115001 also funded the work.

Morayma Reyes, professor of pathology and laboratory medicine, and Hannele Ruohola-Baker, professor of biochemistry and associate director of the Institute for Stem Cell and Regenerative Medicine, headed the 91̽�� team that made the discovery. The first authors of the paper were Nicholas Ieronimakis, 91̽��Department of Pathology; and Mario Pantoja, 91̽��Department of Biochemistry.

They explained that the mice in their study, like boys with the gender-linked inherited disorder, are missing the gene that produces dystrophin, a muscle-repair protein. Neither the mice nor the affected boys can replace enough of their routinely lost muscle cells. In people, muscle weakness begins when the boys are toddlers, and progresses until, as teens, they can no longer walk unaided.  During early adulthood, their heart and respiratory muscles weaken. Even with ventilators to assist breathing, death usually ensues before age 30. No cure or satisfactory treatment is available. Prednisone drugs relieve some symptoms, but at the cost of severe side effects.

The disabling, then lethal, nature of the rare disease in young men presses scientists to search for better therapeutic agents. Reyes and Ruohola-Baker are seeking ways to suppress the disorder’s characteristic functional and structural muscle defects.

Ruohola-Baker’s lab originally identified the sphingosine 1-phosphate (S1P) pathway as a critical player in ameliorating muscular dystrophy in flies. Her lab did this through a large genetic suppressor screen using the fruit fly, Drosophila melanogaster. Sphingosine 1-phosphate is found in the cells of most living beings from yeasts to mammals. Named after the enigmatic sphinx, this cell signal is important in many activities of living cells, from migration to proliferation. The multi-talented, bioactive lipid is essential, Reyes said, in turning stem cells into specific types of cells, in regenerating damaged tissue, and in inhibiting cell death. Without cell receptors for sphingosine 1-phosphate, an embryo would fail to develop.

Other scientists had observed that levels of sphingosine 1-phosphate are lower in the muscles of mice with the muscular dystrophy mutation, and that certain cell repair pathways involving this signal are impaired. However, sphingosine 1-phosphate couldn’t be administered as a drug because it is rapidly used up.

Instead, Reyes and Ruohola-Baker sought to prevent the sphingosine 1-phosphate occurring naturally in the body from degrading. A fruit fly model of Duchenne muscular dystrophy allowed Ruohola-Baker’s lab to rapidly score small molecule therapy candidates for raising the level of sphingosine 1-phosphate. Flies with the genetic defect act normally after they hatch and fly around, but in a few weeks, due to muscle degeneration, they are flightless. By using insect activity monitors, the scientists assessed the effects of drug and gene therapy candidates on the flies’ ability to move.

This screening tool led to the discovery that a small molecule with a long name, 2-acetyl4 (5)-tetrahydroxybutyl imidazole, or THI for short, blocks an enzyme that breaks down sphingosine 1-phosphate.

“It’s interesting to note that THI is a trace component of Caramel Color III, which the U.S. Food and Drug Administration categories as ‘generally recognized as safe’,” said Reyes. The substance is also found in very tiny amounts in burnt sugar, brown sugar, beer, cola and some candies.

The researchers added a purified, concentrated form of THI to the food of young flies with the muscular dystrophy-like mutation. They confirmed that the THI alleviated muscle wasting in the flies. A few other drugs, including a THI derivative and an unrelated drug now in clinical trials for rheumatoid arthritis, also showed beneficial effects in fruit flies.

The study of THI then switched from insects to mammals. Reyes lab began by treating old dystrophic mice with direct injection of THI. Later, the researchers simply added the compound to the drinking water in the habitats of young dystrophic mice. These mice were comparable in developmental stage to human teens who have muscular dystrophy genetic variation.

“We observed that treatment with THI significantly increased muscle fiber size and muscle-specific force in our affected mice,” Reyes said.  “We also saw that other hallmarks of impaired muscle regeneration – fat deposits and fibrosis [scar tissue] accumulation – were also lower in the THI-treated mice.”

The research team linked the desired regenerative effects in the mice to the response of muscle-forming cells and the subsequent regrowth of muscle fibers. A type of sphingosine 1-phosphate, and cell receptors for it, also were observed in the cells in the regenerating muscle fibers. The researchers proposed that sphingosine 1-phosphate turned up the dial on the regulators for the biochemical pathways that mediate skeletal muscle mass and muscle function.

Now that they have shown proof-of-concept, the researchers hope to conduct additional animal studies on THI and other compounds that protect the body’s supply of sphingosine 1-phosphate necessary for muscle cell regeneration. If THI continues to show promise as a nutraceutical or food-based drug, medical scientists will head into pre-clinical studies of effectiveness and safety before advancing to human trials.  In addition to muscular dystrophy treatment research, similar studies might also be conducted in the future on loss of muscle strength during normal or accelerated aging.

While excited about the preliminary findings, the scientists cautioned that they are still at the very earliest stages of research, and that much more work needs to be done before any conclusions can be drawn about the potential of THI as a muscular dystrophy treatment.

Other members of the research team were Aislinn L. Hays, 91̽��Pathology; Timothy L. Dose, Junli Qi, Karin A. Fischer, all of 91̽��Biochemistry and the 91̽��Institute for Stem Cell and Regenerative Medicine; Andrew N. Hoofnagle, 91̽��Laboratory Medicine; Martin Sadilek, 91̽��Chemistry; and Jeffrey S. Chamberlain of 91̽��Neurology and the UW’s Sen. Paul D. Wellstone Muscular Dystrophy Cooperative Research Center.

The researchers have filed a federal patent on sphingosine 1-phosphate-promoting therapies for muscular dystrophy. They have not received any benefits from any organization with a financial stake in the research and have no competing financial interests in analyzing and reporting their findings.

The work was supported by the 91̽�� Department of Pathology and Department of Laboratory Medicine, a Provost Bridge grant, a Nathan Shock Center of Excellence in the Basic Biology of Aging, Genetic Approaches to Aging Training Grant, and the American Recovery and Reinvestment Act of 2009 Challenge Grants 5RC1AR058520, R01GM083867, R01GM097372, and 1P01GM081619.

The scientists also received funding from the Washington Research Foundation, the Duchenne Alliance, RaceMD, and Ryan’s Quest.

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