Evolving Knowledge

Fred Anopol's analysis of muscle structure may provide a clearer picture of how we move

A recent segment of the PBS program, Scientific American Frontiers, features a computerized device that allows Eric Bellamy to move his paralyzed legs again. Through electrodes implanted in his legs, the device provides electrical impulses to the muscles, replacing nerve function that was lost in a motorcycle accident five years earlier. Now it's time for Eric to try to climb the stairs of a bus on his own.

"Things don't look good," narrates the show's host, Alan Alda. "For some reason, Eric's left knee is not locking up." Eric and the man who programs the computer controllers run through the muscle stimulation sequence.

"Looks like he's got enough power in that left leg, but it doesn't seem to bring him up," the programmer tells Alda.

They try the sequence again. This time it works. "They don't know why," Alda narrates, "but artificially stimulated muscles often seem to change in strength."

Although it hasn't been featured on television yet, Fred Anapol's research on normal muscle function may someday help patients like Eric, by providing the designers of computerized devices like his a more detailed picture of the normal sequence of muscle stimulation.

One problem with computerized and prosthetic devices is that their designers don't fully understand the complexity of normal muscle composition or behavior.

"That's precisely what we're working on in this lab -- trying to uncover that mystery as to what's going on in different parts of a muscle, because we do see that muscles are heterogeneous in composition," says the associate professor in UWM's Department of Anthropology. "This is the kind of work that would lay the foundation for clinicians to fine-tune these kinds of therapies.

"What we're finding is that different parts of a single muscle are active at different times during the activity that's involved."

Anapol's primary research interest is in functional morphology, the relationship between animal behavior and how the neuromuscular-skeletal system is put together to support the behavior.

"I say neuromuscular-skeletal because the skeleton cannot move by itself. It provides a stable base for a system of levers to allow the muscles to do what they do, and of course the muscles are controlled by the nervous system."

"During normal walking there is a sequence of movements that occurs," Anapol explains. "The sequence of movements is dependent upon the sequence of muscle contractions that also occurs. And you can program that (in the computerized devices), and then stimulate the muscle in a designated sequence. But you have to know the sequence during normal walking first before you can program them to do that."

Anapol has found that most research in functional morphology has been limited to analyzing skeletal development in evolution, and that when muscles are considered, discussion is usually limited to relative muscle size and position. Internal muscle composition generally isn't considered to be a variable in interpretations of what the bones are doing, Anapol says. In his lab, the focus is on gaining a greater understanding of the muscle itself.

"Muscle has a greater flexibility of behavior than a bone. A bone is a bone; it doesn't instantly shorten when you need a shorter bone, it doesn't lengthen when you need a longer bone. Nature may select for longer or shorter bones over evolutionary time, but for an individual, a muscle has a much broader range (of behavior) ."

Different Muscle-Fiber Types

To help provide a better understanding of his research, Anapol gives a quick lesson in muscle biology.

For a muscle to contract, it needs ATP, or adenosine triphosphate. "It's the currency of energy and movement," Anapol says. "When your ATP runs out, that's it. It's your energy source." ATP is produced from sugar and fat by mitochondria, found within cells. An enzyme in the muscle fiber hydrolyzes ATP to ADP+P, separating one of the three phosphates from the ATP. The energy of this separation is what makes muscle contract.

Skeletal muscle fibers can be classified into two groups, according to how fast they hydrolyze ATP. Slow-twitch fibers hydrolyze it slowly; they reach maximum tension and relax gradually and are used during most activities. They are highly aerobic, or fatigue-resistant. Fast-twitch fibers hydrolyze ATP quickly, and are reserved for quick, sudden, or extreme activities. Some fast-twitch fibers are fatigue-resistant, like the slow-twitch, and some are non-aerobic, or fatigable.

"You find that these slow-twitch fibers are really at the core of the limbs and at the core of the vertebral structure," Anapol says. "As you go outward from the bone, the fibers become a lot faster and more fatigable.

"Generally, you use your slow-twitch fibers for most of your normal activities," Anapol says. As movements become more rapid, the fast-twitch, fatigue-resistant fibers are applied. "It's not until you've got to do something that entails a fast burst of activity that you start to apply the fast-twitch, fatigable fibers."

The different fiber types are seen as light and dark meat in birds.

"If you eat chicken breast, it's all white meat. If you eat duck breast, it's all dark meat," Anapol says. "It's the same muscle -- the pectoralis muscle. These are the flying muscles. Chickens don't fly. Sometimes they use their wings very rapidly to get out of the way of a rambunctious rooster, but they generally don't fly, so they only have the fast-twitch, non-aerobic fibers in there. Ducks, on the other hand, are flying long distances, for long periods of time, so they have much more highly aerobic kinds of fibers."

"Dog limbs have all slow-twitch and fast oxidative because they sort of run at a pretty regular pace even when they're chasing prey. Cats have a lot of slow-twitch and a lot of the fast, non-aerobic fibers -- very few of the fast aerobic. What do cats do? They creep slowly after their prey and then they pounce suddenly. So you see that muscle fiber type composition is highly adaptive to animal behavior."

As another example Anapol cites muscle-fiber composition in the hind limbs of various primate species, which he has studied extensively since beginning his doctoral studies.

"Galagos, which are little primates that just sort of leap all the time -- they run hardly at all -- have almost exclusively fast-twitch fibers. If you look at lorises, which are also primates -- and these are all closely related to lemurs -- they're all slow-twitch fibers and they're just kind of moving stealthily through the trees."

Chewing Muscles

In recent years, Anapol has applied the principles of muscle morphology he used for studying primate locomotion to study chewing muscles. Both of these anatomical regions are the main focal areas of evolutionary morphology, particularly in humans.

"Human evolution is best represented in changes in the head and neck and in the locomotor anatomy," Anapol says. "Locomotor, obviously, because we're bipedal, as opposed to the rest of the animal kingdom, except for penguins, ostriches, and perhaps a few others. And the head and neck because of our relatively large brain size compared to other animals, including other primates, and the fact that we use our hands to feed ourselves and we process our food. So changes have evolved in both hindlimb and feeding structures."

Anapol's early feeding research was in jaws and teeth of non-human primates. One study focused on how several species of monkeys that live in the rainforests of Suriname, in northern South America, adapt to different feeding strategies. For example, the black spider monkey, Ateles pansicus, eats mostly ripe fruit, and therefore has large incisors, or front teeth. The howler monkey, Alouatta seniculus, has large molars, or back teeth, well suited for grinding leaves, which are a major part of its diet. The black-bearded saki, Chiropotes satanas, eats seeds, and has a strong lower jaw for crushing its food.

For insight into human functional morphology, however, monkeys and apes are of limited use because of the particular specializations that have developed in their diets.

Anapol's current research uses pigs. Both pigs and humans are omnivorous -- they eat both plants and animals -- and the design of each animal's main chewing muscle, the masseter, is similar.

"There are a lot of characteristics about their teeth and jaw shape, and how they use their jaws to chew, that are very similar to humans," Anapol says. "The functional information we get from pigs is even more applicable to humans than our closest living relatives, the great apes, which are more specialized for things like fruit and leafy matter. But if you look at pig teeth, they're made for chewing the same kinds of foods we eat."

The pigs Anapol is studying range in age from 2 to 16 weeks. Compared to humans, pigs' feeding structures are more developed at birth and mature more quickly as they age, so this period covers a broad range of development.

"We look at these developmentally, because all mammals develop in a similar fashion. They start off by suckling as infants, then beginning mastication (chewing), juvenile mastication, and go on to adult mastication of foods of various consistencies."

The pigs Anapol uses are miniature, rather than common farm pigs. "They're bred from wild boars, so we get a better indication of how these critters are in the wild than we would by studying a farm pig. They're also preferable because they don't gain that body fat that farm pigs do. When we have a 140-pound pig, we have a solid pig; it's not just a bunch of fat with a pig inside."

Anapol and his graduate assistants first insert electrodes into the pigs to record the electrical activity during normal chewing to determine what parts of the muscle the pig uses for different foods. The pigs are fed pig chow -- a soft, easily crushed food, and cracked corn -- a tough, rigorous food.

"You see how the EMG activity really shoots up when it goes to the corn. And it also does this in some parts of the muscle and not others. It seems to do this in the part of the muscle that has more fast-twitch fibers. It's almost like there's a shunt; its normal chewing is in the slow-twitch range and then all of a sudden you give it something hard and it shifts the responsibility to the fast-twitch."

Anapol also is trying to see how much of masseter development is genetic and how much is determined by the animal's use of the muscle over time. To help make the distinction, one group of pigs is fed liquefied forms of the pig chow and cracked corn, so no stress is applied to the chewing muscles. Theoretically, only genetically controlled growth will occur in these pigs.

After using electrodes on the living pigs, Anapol analyzes enzyme activity in pig masseters by removing muscle samples when the animals are terminated. If the muscle is removed and flash-frozen to below -180�C within two hours of the animals' death, the enzymes can be preserved.

After the muscle is removed, it is cut into thin sections, put through washes and buffers, and then incubated in a solution with ATP. This prompts the muscle enzymes to start hydrolyzing the ATP again, as it did in the living animal. The amount of inorganic phosphate left over after the ATP is hydrolyzed to ADP+P reveals the muscle fiber types: The fast-twitch fibers produce more than the slow-twitch fibers.

"We can take the muscles out and we can emulate the same biochemical activity that's going on that allows this physiologic response to occur. We can mimic this in the lab. These enzymes that are causing this ATP hydrolysis are going to still be there if you take the muscle out and freeze it very quickly in liquid nitrogen. The enzymes will maintain their structure."

Anapol's work goes beyond identification and analysis of different tissues, or histology; he is studying chemical activity occurring in the muscle, or histochemistry.

"You actually have a chemical reaction going on in there that we can visualize -- a natural biochemical reaction that occurs in that tissue during the life of that animal. Rather than just coloring it, we're actually tickling it to do something. We can see how many fast twitch fibers there are and how many slow-twitch fibers there are and where they're located.

"Some of our muscles are pure -- one or the other -- or close to pure. This one isn't," he says of the pig masseter. "But that's part of the interest: Why different muscles have different fiber-type composition -- different relative percentage of slow and fast -- why the same muscle in different animals doesn't have the same composition, and how these fiber types are distributed throughout a whole muscle."

Anapol recently discovered a surprising phenomenon while analyzing the pigs' muscle fibers.

The ATP incubation solution is alkaline, with a pH of 9.4. If the tissue is first immersed in a higher alkaline (pH=10.35) solution, the slow-twitch enzyme is disabled, causing the fast-twitch fibers to be stained dark. Conversely, immersion in an acidic solution disables the fast-twitch enzyme, and the slow-twitch fibers stain dark. This inversion is known as acid reversal, and is observed in adult muscle fibers. While working with samples from the infant pigs, however, Anapol has discovered that some fibers turn dark under both acid and alkaline conditions, indicating properties of both fast- and slow-twitch fibers.

Exactly what this means is not yet clear. "That's one problem we're trying to find out here. Do we have the enzymes for both fast-and slow-twitch co-existing in the same fibers in early (young) animals? Is the fast-twitch enzyme actually being transformed to the slow-twitch enzyme during development?"

One fact that may explain this phenomenon is that pig masseter muscle starts out as predominantly fast-twitch, highly aerobic in infants. As animals increase use of their muscles, slow-twitch and fast-twitch non-aerobic characteristics gradually develop. "What we get is high percentages of fast-twitch fibers, about 90 percent, in the infants and then in the adults you start getting a significant percentage of slow-twitch fibers, about 30 percent."

But it's too early to tell exactly what is happening in the infant pigs, Anapol says. "We haven't gone far enough to see what's really going on with this. Right now we're just trying to characterize this change using immunocytochemistry. With this technique, we label the different varieties of enzymes with specific antibodies. This enables us to both verify and fine-tune our histochemistry results."

Anapol's work may not grab the headlines, but his research may clear the way for important advances in designs of rehabilitative regimens, prosthetic devices, or the computerized devices featured on Scientific American Frontiers.

"You can't re-program muscle activity unless you know what it's supposed to be in a normal case," Anapol says. "This is what we call basic science; we're studying how things work, how things are put together. . . . Then others devise their more practical application on our basic work."

For his research, Anapol has attracted extramural funding from the National Institutes of Health of nearly $400,000, as well as a grant from the National Science Foundation, in a competitive field where researchers without teaching and service responsibilities often have an advantage.

Forensics Work

Besides working on his current research projects, Anapol is involved in forensic anthropology. He got his start in forensics while in graduate school at the University of Illinois-Chicago in the late '70s. Anapol met the late Charles Warren, a forensic anthropologist who was a consultant for the Cook County medical examiner's office. Warren was assigned in December 1978 to analyze the remains of the 29 victims found buried in the basement of serial killer John Wayne Gacy. Anapol volunteered to help him with the project.

This forensics experience has led to similar work since then, most recently for the Milwaukee County Medical Examiner. Milwaukee County often assists investigators in neighboring counties, and last year Anapol consulted on the widely publicized "Jane Doe" case in Racine County. The body of a female, estimated to be in her teens or early 20s, had been discovered in February 1997 in a marsh near Burlington. She was buried after a seven-month inquiry into her identity.

Anapol and Trudy Turner, chair of the UWM Anthropology Department, have been working to establish a program in forensic anthropology for several years. Anapol says his expertise in analysis of human remains, combined with Turner's research in DNA "fingerprinting," would provide a "uniquely complete coverage of forensic anthropology in a single department." Through these efforts, UWM now offers an undergraduate, multidisciplinary course titled, "Dead Men Do Tell Tales: Bones, Bodies, and Forensic Science."

The new College of Letters & Science course, which attracted about 90 students in the spring semester, is offered as Natural Sciences 532-299. It includes faculty from the departments of Anthropology, Biology, Chemistry, Criminal Justice, and Geography, and covers topics such as: treating forensic sites as archeological sites, forensic and historical archeology, molecular techniques for DNA analysis, forensic chemistry, chemical analysis, and soil analysis.

Anapol is also part of a new interdisciplinary graduate studies program in neuroscience. With faculty from the departments of Anthropology, Biological Sciences, Chemistry, and Psychology, the program provides a neuroscience emphasis for graduate students in the natural and social sciences.

Anthropology Was a Late Discovery

Despite the respect Anapol has earned for his research, anthropology was not his first passion. He first heard about anthropology when he was 29, after a bachelor's degree in accounting, several years of teaching elementary school in the ghettos of Chicago, and a master's in inner-city studies.

"I really didn't even know what anthropology was," Anapol recalls. "I looked it up in the dictionary and it said, 'The study of man.' I thought, 'Well, that sounds sort of interesting.' It had a picture of Rhodesian man and Neanderthal man and Java Man, and they looked almost identical except the hair was different. It was a 1959 dictionary."

He found it interesting enough to visit the University of Illinois-Chicago's anthropology department, where he discovered the world of academia. "I never really had thought about it before. I thought, 'Gee, they pay these people to study cave men.' "

Anapol enrolled as a non-degree student at UIC. "It was the most natural thing to me. School had always been such a struggle for me. This was a whole other world. I got really interested in biology and biological anthropology, and did really well. I hadn't done particularly well in accounting at all."

Anapol was unusual among his classmates at UIC because of his non-scientific background, but a conversion like his is not unheard of. "Anthropology tends to attract people from a wide variety of disciplines," he says.

After a master's in anthropology in 1979 from UIC, Anapol received a fellowship to study at the State University of New York at Stony Brook, where he earned his Ph.D. in anatomy in 1984.