Can Scientists Build Muscle Tissue Safely?

Tipping the balance

For most people in mid-adulthood, anabolism and catabolism of muscle are about even. But in aging and muscular dystrophy, catabolism gains the upper hand. Investigators are studying how to tip the scales back toward equilibrium.
by Margaret Wahl on September 1, 2005 - 3:33pm

QUEST Vol. 12, No. 5

During childhood and adolescence, the body's tissues grow, and the average child becomes taller and stronger at a rapid pace. Then, a long period of apparent stability prevails in adulthood, followed by a gradual loss of strength and increased tissue fragility as people age.

In fact, all of life is characterized by the constant competition between anabolism, or tissue building, and catabolism, or tissue breakdown. During childhood, anabolism prevails; in early adulthood, the processes are roughly equal; and in late adulthood, catabolism gains the upper hand.

In muscular dystrophy, muscle catabolism may dominate throughout life.

Since 1986 genes underlying all known forms of muscular dystrophy have been identified, and although more are expected to be uncovered, these findings have already given researchers more knowledge about MD than was gathered in the previous century.

Many MDA grantees have concentrated on strategies to restore functioning genes to muscle, and some are trying to change the way the body interprets abnormal genetic messages.

But others have focused on approaches to help the body build and maintain muscle tissue without concern about which specific genes are abnormal. They're studying anabolic agents that have been identified through studies of how muscles develop, how they grow and how they respond to injury.

Beginning in the 1980s, doctors tried giving muscular dystrophy patients human growth hormone and the male hormone testosterone, both of which are anabolic. These agents increased synthesis of muscle proteins, but they didn't make anyone stronger, and both caused patients to become resistant to the hormone insulin.

Fortunately, some of the same techniques that have enabled scientists to identify some 20 MD genes also have allowed them to study the molecular targets of growth hormone and other substances. Knowing these targets gives investigators a better chance of bringing about specific, desirable changes in muscle.

Boosting anabolic and/or blocking catabolic compounds, they hope, can tip the balance away from muscle breakdown and toward muscle building.

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Deficit Reduction

University of Melbourne (Australia)

Increasing muscle regeneration

Laboratory testing

Gordon Lynch

MDA grantee Gordon Lynch, an associate professor in the Department of Physiology at the University of Melbourne in Australia, says he can trace his interest in muscle research back to his adolescence.

One day, while leafing through an Australian muscle-building magazine, the teenage Lynch was struck by a photo of a body builder wearing a T-shirt that said, "While we're building our muscles, others are wasting away. Help fight muscular dystrophy."

Lynch began to ask himself questions. Why was it that exercise and weight training could build muscles in most people but not in all? What was it about muscular dystrophy that interfered with the process? A few years later, he was asking these questions at La Trobe University in Melbourne, where he would complete an undergraduate degree in physiology with honors.

"During everyday activities, our muscles are being injured slightly," he says. "We're not even aware of these things, but especially when our muscles are performing braking actions, they have a certain degree of stress being placed on them." Normally, Lynch says, repair mechanisms are so efficient that they can quickly compensate for such stresses and any minor damage that arises from them.

But in muscle disease, the damage is sped up, and the situation changes dramatically. "You're getting a lot more breakdown, with insufficient repair, so you're always in a state of deficit."

The aging connection

In 1995, after completing a doctoral degree in skeletal muscle physiology at the University of Melbourne, Lynch secured a C.J. Martin research fellowship from Australia's National Health & Medical Research Council and set off for the University of Michigan in Ann Arbor.

There he worked with muscle physiologist John Faulkner at the Institute of Gerontology (aging studies) and MDA grantee Jeffrey Chamberlain in the Department of Human Genetics. Chamberlain was studying the gene for dystrophin, the protein missing in Duchenne muscular dystrophy (DMD), and gene therapy techniques to restore it to muscle fibers.

For the next two years, Lynch studied aging rats in Faulkner's laboratory and mice lacking dystrophin in Chamberlain's. Those interactions were very fruitful, he says. "As the aging process advances, there becomes a greater similarity between it and severe muscle diseases. Muscle wasting is the thing that links them."

In 1997, back in Melbourne, Lynch found there was increasing interest in preserving muscle mass in the elderly by administering anabolic hormones.

"The circulating levels of these hormones certainly decrease as we get older, and that's what has prompted this," Lynch says. "But the question is, do we simply give a top-up of these hormones?"

"There's some merit to that thinking, but how much do we give, and when do we give it? Most people think, 'Well, if I have X amount, then 10 times X should give me 10 times the benefit.' That's when problems start to happen, because the body can't tolerate excessive doses of growth hormone or testosterone."

Moving downstream

Lynch was among those who began focusing their efforts on insulin-like growth factor 1 (IGF1), an anabolic compound that increases in response to growth hormone levels in the bloodstream. It comes in several varieties, including a liver form and a muscle form.

Lynch is concerned not only about which IGF1 form to administer to build muscle, but when to administer it.

"We can be armed with a whole lot of different growth factors and agents, but pinpointing when each of them can be most effectively applied is very tricky. I think the key is understanding the processes of muscle breakdown and repair. It's knowing when to intervene.

"At which point is the muscle cell most accepting of these interventions? It might be that applying a particular growth factor at one stage will have very little effect, but that 24 or 48 hours later, the cell might be primed to be stimulated."

Fighting fibrosis

In 1995, a research group in Tacoma, Wash., published an interesting finding: A substance known as interleukin 15 (IL15), previously thought of mainly in terms of its role in enhancing an immune response, made muscle cells in lab containers grow larger than average and accumulate more myosin, a muscle protein.

After further research showed IL15 also slowed muscle wasting in rats with malignant tumors, Lynch and his team decided to test it in DMD-affected mice. They found that four weeks of IL15 improved strength in their respiratory diaphragms.

But they also got a surprise. The improved diaphragm strength didn't seem to result from a change in the immune system or an increase in muscle protein synthesis. Instead, it came from another effect entirely: scar reduction.

As muscles degenerate, they're eventually replaced by collagen, a fibrous protein found in connective tissue and scars.

"What makes the diaphragm so poor in terms of its regeneration [in DMD]," Lynch says, "is that you get a lot of infiltration of connective tissue, which interrupts the normal muscle regeneration process."

IL15 reduced this collagen replacement, also known as fibrosis, and increased the performance of the diaphragm in the mice.

Combining forces

These days, Lynch and colleagues are thinking of combining anabolic agents and other substances.

"I think the coadministration of something like interleukin-15 in conjunction with something more anabolic, such as IGF1, may be even more effective," he says.

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Lifting the Brake on Muscle Regeneration

John Hopkins University, Baltimore

Blocking myostatin

Clinical trials

Kathyrn Wagner

When Kathryn Wagner's mother wanted some time to herself, she frequently banished her to her father's laboratory at Yale University in New Haven, Conn., where Allan Wagner conducted experiments on animal learning. (He's still a psychobiologist at Yale and a member of the National Academy of Sciences.)

Kathryn and her sister, Krystn (now an infectious disease specialist), spent many happy hours pulling rats out of their cages and occasionally feeding lab animals that weren't supposed to be fed.

Perhaps not surprisingly, by the early 1990s, Kathryn had earned a degree in biology at Yale and was working toward her doctorate in neuroscience in the laboratory of Richard Huganir at Johns Hopkins University in Baltimore.

Huganir's special interest was the transmission of signals among cells, and Wagner set about studying how the muscle cell membrane reacts to nerve signals. In 1993, she identified a protein that would later be called dystrobrevin, a muscle membrane component located near dystrophin, the protein missing in Duchenne MD. (Dystrobrevin is thought to carry signals between the outside and inside of a muscle cell.)

"That brought molecular biology home to me in a human form," Wagner says. "The association with dystrophin and Duchenne meant this wasn't just a protein anymore. It was something associated with things that cause disease.

"At that point I only really knew of muscular dystrophy intellectually, but from that point on, I was pretty much sold on specializing in neuromuscular disease." Meanwhile, Wagner undertook clinical training in neurology and then specialized education in neuromuscular disease and neurogenetics, obtained MDA research funding, and became an MDA clinic co-director at Hopkins.

Mighty mice

In 1997, while training at Hopkins to become a neurologist, Wagner saw something that would become a focus for many years to come.

"I'd like to say that I read the scientific paper," she says, "but in fact I first saw it on the evening news. I saw Dan Rather showing pictures of what they were calling mighty mice, and I said, 'That has to be helpful for muscular dystrophy.'"

Mice lacking the myostatin protein are more muscular than mice with myostatin.
Mice lacking the myostatin protein (left) are more muscular than mice with myostatin (right).

The mighty mice had been genetically modified so that they lacked a recently identified protein called myostatin, which puts a brake on muscle growth and regeneration. Without that brake, muscles grew larger, and animals grew stronger.

The 1997 paper reporting the myostatin experiments was published in the prestigious journal Nature, and its authors, Se-Jin Lee, Ann Lawler and Alexandra McPherron, were located at Hopkins, but in different departments from Wagner's. The investigators had identified a protein that influenced muscle growth and dubbed it GDF8. When its function as a growth inhibitor became clear, they suggested a new name for it: myostatin, from the Greek words for muscle and standing still.

"When I completed my clinical training, I went to work with Se-Jin Lee, an incredibly innovative thinker, in the Department of Molecular Biology and Genetics at Hopkins, and we began to investigate whether not having myostatin was going to be helpful for muscular dystrophy."

Lee and Wagner, both of whom would later become MDA research grantees, bred mice lacking myostatin with dystrophin-deficient (mdx) mice, which have a Duchenne-like dystrophy.

The mdx mice lacking myostatin not only had more muscle, as one would anticipate, Wagner says, but they had greater strength. "Probably the most surprising finding was that they had less fibrosis [scar tissue formation]. And that suggested to us that, in the absence of myostatin, there might be better muscle regeneration.

"We've gone on to show now that muscle does regenerate better in the absence of myostatin."

Myostatin moves from mouse to man

In the late 1990s, Neil Wolfman was a biochemist at Genetics Institute, a small biotech company in Cambridge, Mass., that would soon be acquired by Wyeth BioPharma.

If Se-Jin Lee is the father of myostatin, then Neil Wolfman is the father of anti-myostatin, Wagner says. "He really moved the field forward."

In the winter of 2001, Wagner, along with MDA grantee Louis Kunkel of Children's Hospital of Boston, traveled to Genetics Institute and presented their case for developing an anti-myostatin treatment to increase anabolism in muscular dystrophy.

"We gave them a history of the disease, and we impressed upon them the need for novel therapeutics in muscular dystrophy. They really did appreciate the total absence of other therapies to stimulate muscle growth and regeneration."

Around that time, Wyeth bought Genetics Institute, and Se-Jin Lee and Johns Hopkins licensed the rights of myostatin to the company.

The first study drug that they have developed for modulating myostatin is MYO-029, a neutralizing antibody, Wagner says. Antibodies are proteins made by the immune system, usually to fight bacteria and viruses. Wyeth made antibodies that could neutralize myostatin.

Today, Wyeth is testing the antibody-based MYO-029 in adults with Becker, some forms of limb-girdle, and facioscapulohumeral muscular dystrophy at several MDA clinics, including Wagner's at Hopkins.

"It's primarily a safety study, but there are several measures of efficacy as well," she says, adding, "MYO-029 is the first, but hopefully not the last, of the myostatin blockers.

"There are several other ways to inhibit myostatin. I think about expanding the applications of myostatin inhibitors, not only with more inhibitors but for different applications. But," Wagner says, "Even if it only helped one muscular dystrophy, I'd be thrilled."

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Calpain Combat

University of California at Los Angeles

Counteracting calpains

Clinical trials

Melissa Spencer

One night in April 1969, biochemist Darrel Goll (then at Iowa State University in Ames) and graduate student Wayne Busch were studying the effects of calcium on rabbit muscle cells. About 2 a.m., the tired scientists left the muscle cells in a calcium-containing solution overnight.

When they removed them the next morning, they noticed the cells were unusually fragile. Under the microscope, Busch saw immediately that cellular structures known as Z-disks had completely disappeared.

Goll guessed that the extra calcium in the solution had triggered cellular enzymes to cut some of the proteins in the Z-disks, a hypothesis that turned out to be correct.

Promiscuous proteins

By 1989, Goll (then at the University of Arizona in Tucson) and colleagues had found that these enzymes, proteins now called calpains, came in at least two forms, and that they could be suppressed by another protein, calpastatin.

At the time, Melissa Spencer was a graduate student in physiology at the University of California at Los Angeles, studying skeletal muscle remodeling in the laboratory of James Tidball. Others in the same lab were working with the mdx mouse, an animal model of the human disease Duchenne MD.

"I was still in the early phase of deciding what I was going to do for my Ph.D. thesis," Spencer recalls. She had already learned something about calpains and their possible role in remodeling skeletal muscle, but it wasn't until 1991, when she attended a seminar on calcium and calpains in muscular dystrophy-affected cells, that she began to think along these lines.

"It was a light bulb," Spencer says of the seminar. "I thought, 'Why isn't anybody looking at this?' These proteins are likely to be involved in dystrophy."

She considers Goll, a longtime MDA grantee, the founder of this field. "He's an excellent scientist, and he had done a lot of the purification of the different forms of calpain and how they might be activated by calcium, but he hadn't looked at their role in dystrophy. Those were the days before e-mail, and I called Darrel a lot on the phone."

Spencer finished her doctorate in 1994, nearly convinced that, in Duchenne MD, excess calcium flows into muscle cells through leaky, dystrophin-deficient cell membranes, and that it triggers the calpains to destroy muscle proteins in their midst.

"They start chewing around and cleaving proteins indiscriminately," Spencer says. "We call it promiscuous behavior."

Clues from farmers, body builders

In October 2002, Spencer published results of MDA-funded experiments showing that, when mdx mice were bred to make extra calpastatin, they sustained less than the expected amount of muscle damage. But calpastatin inhibits calpain activity in all kinds of cells, so it wasn't likely that a drug could be derived from it.

Spencer knew there were other chemicals that might be exploited for their anti-calpain, apparently anabolic properties. For instance, she knew that farm animals given a drug called clenbuterol developed larger muscles.

Clenbuterol was also in use (illicitly) by body builders, as Spencer found out on the Internet. The body builders pulsed their doses, taking the drug for a while, then stopping, and then resuming it. Otherwise, the buzz was, its effects seemed to wear off.

Fortunately, clenbuterol belongs to a class of compounds called beta-2 agonists, which includes medications such as albuterol, which has a good track record in treating humans. Spencer began to think about trying it in DMD.

Drugs build muscle, then dash hopes

In 2002, with MDA funding and the results from the high-calpastatin mice experiments, Spencer began giving albuterol Repetabs, made by Schering-Plough, to nine boys with DMD. The results were, to say the least, encouraging. Side effects were minimal (some restlessness in two children), and, in contrast to most clinical trial results in DMD, the children actually got stronger.

Spencer excitedly prepared to conduct a 25-subject trial of what looked like a good anabolic agent. Ultimately, she was able to recruit and study only 14 DMD-affected boys, and she faced a still more serious problem: Schering-Plough had stopped making the Repetabs, and the investigators had to look elsewhere for a source of oral albuterol.

They chose VoSpire (made by Odyssey Pharmaceuticals), which provides a fairly stable blood level of albuterol when taken by mouth. Unfortunately, the results didn't live up to the high hopes generated by the pilot trial. The boys gained some muscle mass, but their strength didn't increase.

Spencer thought back to the body builders and their intermittent use of clenbuterol. The Repetabs, she learned, provided the same kind of pulsing, but VoSpire kept blood levels fairly constant.

By stopping the beta-2 agonist for a while in between dosage periods, the body builders were allowing their muscles to again become sensitive to the drug.

Spencer is now proposing a third albuterol study, using alternative dosing schedules.

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