For Angela Dulhunty, the main draw of studying cells’ electrical properties was the reward of instantaneous data. Rather than having to wait sometimes days to get the results of a biochemistry experiment, with electrophysiology “you see what is happening in an individual cell in the moment,” says the muscle biology researcher and now emeritus professor at Australian National University in Canberra.
Dulhunty was attracted to learning how muscle works as an undergraduate student studying physiology and biochemistry at the University of Sydney. “Biochemistry in those days was a lot of learning metabolic cycles, which was not as intuitive for me as understanding how the micro-components of tissues and organs inform their functions,” she says.
Dulhunty’s first real lab experience was during her final year as an undergraduate. She was completing her honors thesis, studying how hearing is registered by the ear and translated into electrical signals through the nervous system.
She liked the hands-on lab work as much as the intellectual exercise of cogitating on a biological problem. “I don’t think you can separate the lab work from the thinking part of research. I’ve always enjoyed putting the two together,” she says. This was among the reasons Dulhunty continued to do her own hands-on experiments in the lab, long after she started her own laboratory back in 1975.
In 2000, she made what she calls a “completely unexpected and serendipitous discovery.” Dulhunty and her colleagues were studying how the ryanodine receptor, a type of protein receptor, functions in muscle cells. Ryanodine is an ion channel, embedded in an internal membrane within the muscle cell, that surrounds a pocket of calcium ions. The channel regulates the changes in calcium ion concentration that control the muscle contractile apparatus and, in turn, muscle movement.
Dulhunty had set up electrophysiology experiments on a receptor from mammalian cardiac muscle fiber to measure its activity, and her initial measurements on the receptor’s activity were going nicely. On a whim, she decided to add the enzyme glutathione transferase to the muscle cells’ medium, just because the chemical was sitting on the lab bench next to her. “I had an extra 20 minutes to play, and I thought, ‘What would happen if I added the enzyme? Would it interact with the ryanodine receptor?’” Dulhunty recalls.
She did not expect anything to happen, but the addition of the enzyme blocked the cardiac ryanodine receptor’s function. “I could see immediately that the glutathione transferase began to inhibit the cardiac muscle receptor’s activity,” she recalls. Because the effect was unexpected, she didn’t believe it initially and repeated the experiment several more times to make sure the results were real. Within a few months, Dulhunty and her colleagues published their first paper on the role of the omega class glutathione S-transferase, GSTO1-1, in inhibiting the ryanodine receptor in cardiac muscle and in increasing the activity of the skeletal muscle ryanodine receptor. The team continued the work: finding the part of the GSTO1-1 molecule responsible for inhibiting the ryanodine receptor, they modified the molecule for maximum efficacy. They also explored its potential as a therapeutic drug for use in preventing cardiac arrhythmias. Three years after the initial discovery, they found that another protein structurally related to glutathione transferases, a chloride intracellular ion channel, CLIC-2, could also dampen the activity of the ryanodine receptor in the heart. This was the start of Dulhunty’s work to discover the significance of these proteins in muscle.
Throughout her career, Dulhunty has been driven by her curiosity to know how the underlying physiology of the body works, and, as a result, has made important discoveries about how skeletal and heart muscle contractions are generated and regulated.
An early start
Dulhunty was born in 1946 in Sydney, Australia, an only child. Her father was a geologist at the University of Sydney, and her mother had been a sheep farmer and breeder prior to getting married. Dulhunty was a “tomboy,” who couldn’t wait to get home from school, change into shorts, and run around and climb trees in the Australian bush with her friends, she says. Thanks to her father, Dulhunty was also interested in science from an early age, which led her to study biochemistry when she entered the University of Sydney in 1965. She also began riding horses as a youngster, and every summer during university she was a counselor and instructor at a horse riding camp in the Snowy Mountains, close to where she now lives just outside of Canberra.
When wrapping up her undergraduate studies, Dulhunty thought she wanted to go to medical school so that she could do medical research, but a professor of biochemistry advised her to attend graduate school instead if research was her main interest. After seeking advice from other professors, she ended up in Peter Gage’s membrane physiology and neuroscience lab at the University of New South Wales in Sydney. “He was exciting to talk to and an inspiring scientist,” Dulhunty says.
Dulhunty began her PhD in 1969, getting straight to work on her thesis, as no coursework was required for a graduate degree in Australia at the time. She initially worked on characterizing a deadly toxin secreted by the Southern blue-ringed octopus (Hapalochlaena maculosa), which lives in tidal rock pools along the southern coast in Australia. Using live octopuses, Dulhunty found that the water-soluble maculotoxin inhibits action potentials in motor nerves and muscle fibers, preventing the transmission of neuromuscular signals.
Dulhunty also began her initial work on the biophysical properties of muscle fibers, which contract in milliseconds based on the translation of signals from the brain to the muscle. “The basic question,” she says, “was, how does the electrical signal on the surface of the muscle fiber get translated into a muscle contraction? And that is still what I am working on today!”
In the 1970s and 1980s, Dulhunty—and most labs studying muscle—used individual toad or frog muscle fibers for electrophysiology experiments because the animals have robust muscle fibers that can be dissected and separated relatively easily. “Mammalian fibers are much harder to work with. It is very difficult to separate out an individual fiber because they are much longer, and there is so much surrounding connective tissue,” Dulhunty explains.
Beyond the limit
After completing her PhD in 1973, Dulhunty applied for and was awarded a postdoctoral fellowship from the Muscular Dystrophy Association of America and had her first overseas adventure. Dulhunty worked at the University of Rochester in New York in the lab of Clara Franzini-Armstrong, who is now an emeritus professor at the University of Pennsylvania. At the time, Armstrong had already made her name in science by demonstrating how the structure of the muscle fiber membrane facilitates its function.
Continuing to work on frog skeletal muscles, Dulhunty combined her expertise in electrophysiology with Armstrong’s skills in electron microscopy. “This in itself was remarkable at the time, as the two vastly different techniques were generally not combined into a single study,” she says. In 1975, Armstrong and Dulhunty uncovered a key function of caveolae, Latin for “little caves,” tiny indented structures in the surface membrane of individual muscles that are also found in fat tissue and in blood vessels. The pair found that the thousands of caveolae in frog muscle open, enabling the muscle to survive “enormous stretches to more than double its rest length and without significant damage to the cells,” says Dulhunty. Their findings revealed just how well-equipped muscle is to adapt to stress and resist cell death.
Dulhunty stayed in the US for a year and a half. When she wasn’t in the lab, she took time to travel, visiting Woods Hole in the summer, and taking a three-week road trip from the East Coast to the West Coast and back to Rochester in a “little Mustang car.” Still, she wanted to go back to Australia. And, she wanted to find out more about how mammalian muscles worked, which other researchers were not yet attempting.
After leaving Rochester in 1974, Dulhunty started her own lab at the University of Sydney. Her goal was to better understand how the electrical signal on the surface membrane of a muscle cell is translated into the release of calcium ions that initiates muscle contraction. Dulhunty homed in on how ion channels in the muscle affect membrane potential—the difference in electric potential between the interior and the exterior of a living cell. Her first achievement on this front, in 1977, was authoring a Nature paper that revealed details of mammalian muscle contraction. She found that the threshold of response to potassium ions is higher in rat muscle than in amphibian muscle. Dulhunty then went on to demonstrate the importance of chloride ions and membrane-bound chloride transporter proteins in setting the electrical membrane potential in mammalian skeletal muscle.
“It was assumed that chloride functioned the same way in amphibians and in mammals, but we found chloride ion function was very different from that in amphibian muscle,” Dulhunty explains. The studies helped uncover what goes wrong in several human childhood-onset genetic neuromuscular disorders, including myotonia congenita and myotonic dystrophy. Both occur when chloride channel presence is altered in muscle. Largely following from Dulhunty’s work, researchers have shown that the removal of chloride ions from the muscle membrane results in overexcitable muscle fibers that contract involuntarily.
Building a lab
In 1984, Dulhunty moved her laboratory to Australian National University in Canberra. By then she and Gage were a couple, and he also moved his lab to the university, where the two continued to collaborate until his death in 2005.
Once her lab was established, Dulhunty continued to develop techniques to study mammalian muscle. There was a gradual transition in the field from examining the biophysical properties of amphibian muscles—which are much easier to study—to mammalian muscle, in large part led by Dulhunty.
Her group was the first to measure the “asymmetrical charge movement” in mammalian muscle fibers. The phenomenon—initially identified in nerve and then muscle cells—arises when a charge moves about a millionth of a millimeter inside the cell membrane, which is a critical part of normal muscle fiber contraction. To make this exquisite measurement in the mammalian system, Dulhunty’s and Gage’s labs first had to design and build the equipment for doing so, putting three tiny, glass pipette electrodes into the end of an individual rat muscle fiber and painstakingly dissecting the minute electrical signals of asymmetrical charge movement from much larger signals and noise in the system. The signals had been predicted in the 1950s by the 1963 Nobel Prize winners Alan Hodgkin and Andrew Huxley, but it had taken more than 20 years to develop the technology required to measure so tiny a charge movement. “It was very exciting to be a part of that development,” Dulhunty says.
Researchers in Dulhunty’s lab also began to study the ryanodine receptor ion channel. Among the first to characterize the receptor, Dulhunty’s lab extracted the essential protein from muscle fibers, embedding it into an artificial lipid bilayer to study its function. The receptor works to initiate the final step of muscle contraction. In 1994 and 1995, the team found that a small, multifunctional protein that binds an immunosuppressant drug, FK506, was critical for normal ryanodine receptor function. They also discovered unsuspected effects of high calcium ion concentrations and oxidizing reagents on the activity of the receptor in cardiac muscle. “Everything that the lab has done since then has been partly focused on this ion channel and the way in which it operates, including its role in human muscle diseases,” Dulhunty says.
The study of the transferases and related proteins is an example. In 2012, she and her colleagues found that a mutation in the gene for human CLIC-2, the type 2 chloride intracellular ion channel, could result in X-linked intellectual disability, congestive heart failure, and seizures. “The mutation altered the effect of CLIC-2 on the ryanodine receptor, so altering normal ryanodine receptor activity could explain the characteristics of individuals carrying this mutation,” Dulhunty says. More recently, in still unpublished work, the lab identified additional disease-associated CLIC-2 mutations.
An emeritus professor since 2016, Dulhunty is wrapping up a few key projects, including identifying additional proteins that interact with the ryanodine receptor. She also has been working with a foundation in the US to develop animal models for ryanodine receptor–associated muscle diseases. The lab, which comprises one technician and one graduate student, contributes by exploring how the activity of the ryanodine receptor is affected by genetic alterations. “It’s important to study these human mutations in animal models, not just in cell culture, because there are major differences of how muscle proteins are modified in a whole animal compared to in isolated cell cultures,” Dulhunty says.
She is optimistic about further advances in the field. “When I started my PhD, muscle contraction was a black box, and now we generally know how it works, although not all of the most important details,” she says. The next generation of scientists, she says, will have to develop new techniques to reveal these details.
Dulhunty says that in retirement she hopes to help researchers in her lab and to set aside time to tend her horses. “As soon as I had enough money, I bought my own horse,” Dulhunty says. That was 30 years ago. She now lives on farm with two Jack Russell terriers and two horses, which she tries to ride five times a week.