Stopping sudden cardiac death, one molecule at a time
Volume 8, Issue 2
Thomas P. Burghardt, Ph.D., uses whiz-bang microscopy techniques to compare single protein molecules from damaged hearts to healthy specimens in hopes of finding ways to stop this leading killer of young athletes.
Thomas Burghardt, Ph.D., calls himself a builder — his workbench is the molecular structure of the heart.
Maybe you've heard of sudden cardiac death — the unexpected death of an apparently healthy person, usually a teenager or young adult, without any warning. Many of these sudden deaths are caused by an inability of the heart to beat properly because of faulty genes.
One in 500 babies is born with one of these genes, causing hypertrophic cardiomyopathy, a disease of the heart muscle resulting in its enlargement. People affected by hypertrophic cardiomyopathy may seem perfectly healthy — even athletic — but out of the blue, when they're kicking a ball or making a jump shot, they collapse. That's because their heart muscle cells increase in size (hypertrophy), causing the heart wall to thicken, which makes it harder to pump blood efficiently. Also, enlarged cells become misaligned, disrupting the heart's electrical impulses. Some people with hypertrophic cardiomyopathy experience shortness of breath, chest pain or fainting. But when children with hypertrophic cardiomyopathy get sick, they often develop severe forms of the disease.
Investigators such as Mayo Clinic biochemist Thomas P. Burghardt, Ph.D., are looking for ways to stop these symptoms before they start. With its team research approach, Mayo Clinic is ideally suited to both treat patients and piece together the complex genetics underlying hypertrophic cardiomyopathy.
"People walk into Mayo when they're sick," Dr. Burghardt says. "When we suspect hypertrophic cardiomyopathy, we test their genes. We want to get clear on exactly what's going wrong."
Heart genes and their proteins
Heart muscle is composed of protein filaments. The heart's thick filaments are made up of a protein called myosin, and its thin filaments are made up of another protein called actin. When the heart beats, the myosin filaments grab onto the actin filaments and pull them along, sort of like hauling in a rope.
The heart makes a variety of myosin and actin proteins, each encoded by its own gene. In people with hypertrophic cardiomyopathy, one of these genes is mutated and therefore makes a myosin or actin protein that doesn't work quite right. These genes can be passed from parent to child.
One of the remarkable things about hypertrophic cardiomyopathy is the number of faulty genes out there — more than 900 mutations have been found in 20 different genes. Unrelated families with hypertrophic cardiomyopathy usually have different mutations. And even within families, the disease manifests in different people in different ways and at different times. This seems to be the case regardless of which gene is faulty.
This means that specific mutations can't necessarily be used to predict how sick carriers might become, only that they're more liable to become sick. On the other hand, understanding why a mutant protein doesn't work as it should could lead to preventive treatments, such as medications or gene therapy.
Pediatric cardiologist Michael J. Ackerman, M.D., Ph.D., a colleague of Dr. Burghardt's at Mayo Clinic's campus in Rochester, Minn., treats children who have hypertrophic cardiomyopathy. For almost half of these children, the genetic problem was not inherited at all but occurred as a spontaneous mutation, so their parents and siblings are not at risk of this condition. However, the new mutation is heritable, so a patient's future children could be at risk.
Dr. Ackerman evaluates the myosin and actin genes in these youngsters to find their mutations. He shares the sequences of these mutant genes with Dr. Burghardt, who generally chooses to investigate the ones from patients with serious illness. Even though much progress has been made elucidating the genetic basis of hypertrophic cardiomyopathy, the molecular events that lead to the disease's symptoms are still unclear.
"We've got to know why a child's heart muscle behaves this way to predict the outcome, and treat him as he grows up," Dr. Burghardt says. So he looks at the heart closely, zooming in to observe the action of one muscle filament at a time.
What makes a heart beat?
Every heartbeat begins with the binding of a molecule called adenosine triphosphate (ATP) to myosin's globular head. ATP transports energy in cells — it makes things happen. It's kind of like a rechargeable battery: Clipping one of its three phosphate groups off powers a reaction; sticking a new phosphate group back on prepares it to fuel the next.
When ATP binds to myosin, the myosin releases the actin. Myosin clips off one of ATP's phosphate groups and the little zap of energy released powers the flexion of the myosin head. As the phosphorous group is released, myosin's globular head changes shape and becomes powerfully attracted to the adjacent actin unit. The myosin head extends, making its power stroke and tugging the actin along.
"It's a little motor," Dr. Burghardt explains. "The mechanism is that the myosin strongly binds actin then rotates its lever-arm to impel actin."
Myosin's lever-arm walks along actin filaments a lot like a caterpillar scoots along a twig. The swinging steps of multitudes of myosin lever-arms ultimately generate a heartbeat.
One molecule at a time
Researchers use infrared light to generate second harmonics (a visible light range) in order to see the myosin in a cardiac muscle fiber and evaluate if the fiber is damaged. Second harmonic generation also enables them to detect and differentiate connective tissue.
Cardiac myosin filaments consist of one heavy protein chain and two light protein chains. One of the light chains is hypothesized to stabilize the lever-arm of Dr. Burghardt's little motor. If the lever-arm were too flexible, it would not move actin effectively. It may be that the mutant light chains from Dr. Ackerman's patients don't stabilize the lever-arm properly.
To see if this is true, Dr. Burghardt is using super-resolution, high-precision microscopy to determine lever-arm orientation at various points during muscle contraction. He isolates a single muscle fiber from the heart and mounts it in a specially designed study chamber. He then painstakingly studies its contractility by measuring the tension that develops when he stimulates it with calcium and ATP under the high-powered microscopes.
Dr. Burghardt can use this system to study mutant proteins. He does this by synthesizing a large amount of the protein in bacteria and introducing it into a mounted muscle fiber — usually from a pig heart that has been pre-treated to flush out its own version of the protein being studied. Amazingly — even though a component of its molecular machinery is replaced by an entirely synthetic protein — the muscle fiber can still contract, allowing Dr. Burghardt to determine how the mutation discovered in the patient impacts heart contractions.
In the past, researchers couldn't observe native myosin behavior in heart muscle because the filaments are so densely packed. Dr. Burghardt gets around this problem by using something called a photoactivatable probe. These molecules can be attached specifically to different regions of myosin. They are especially handy because they have an on switch: They shine bright green when they're exposed to blue light.
"Single molecule studies on proteins like myosin were really not possible until photoactivatable probes came along," Dr. Burghardt says. "Now it's possible to excite a sparse population of probes in the very concentrated myosin."
When Dr. Burghardt looks at heart muscle under the microscope, the probe only shines for a little while. It lasts longer when it's placed in a stable position on the myosin. It burns out more quickly when it's in a spot that's flexible.
"The little motor has stresses and strains," Dr. Burghardt explains. "We can pick those up depending on where the probe is placed on the myosin."
He can measure these differences in space and time and then crunch the numbers, calculating the force of the normal versus the mutant lever-arms.
The microfluidic device developed in Dr. Burghardt’s lab. It holds a small bundle of papillary fibers (roughly 50 um or 0.05 mm in diameter) while researchers perform single-molecule experiments on the myosin light chain in the fibers. The two flanking pipettes — the inlet and outlet for solution exchange — allow researchers to take the fibers through physical states of relaxation, rigor or contraction. All of this is visible only under the microscope.
Dr. Burghardt began his career as a physicist, pursuing research simply to advance knowledge. His work was exploratory and often motivated by his curiosity and intuition without any practical end in mind. But at Mayo Clinic, "research is funneled into themes," Dr. Burghardt says. "This focuses my attention. I have to think about a clear goal — heart disease — and use all my basic research tricks to address that. It's gotten me interested in new techniques, new tools that suit the problem, and I've found it very stimulating."
Beyond microscopy, one of Dr. Burghardt's goals involves creating microfluidic chips that will eventually allow health care providers in less well-equipped clinics to perform diagnostic tests for hypertrophic cardiomyopathy. "I'm basically a toolmaker — I build things," he says.
These labs-on-a-chip would provide a cheap way to assay for mutant proteins. Health care providers could take tiny samples from their patients' leg postural muscles, which express the same genes that cardiac muscles do. Just like heart muscle, postural muscle doesn't get tired. Dr. Burghardt explains that "it doesn't pump iron, it just holds your body steady. Nature manages to put the right kind of myosin in the right spots."
Dr. Burghardt is also interested in using some type of natural mechanism to make stronger muscles. "It seems like a long way off," Dr. Burghardt says, "but protein engineering is here now. I just need to know what to change."
Drug therapies used to treat hypertrophic cardiomyopathy relieve some of its symptoms but do not target hypertrophic cardiomyopathy-specific pathways or alter the natural history of the disease. One goal is to find medications that target the pathways, the mechanisms of the disease. Another goal is using a type of gene therapy in which a mutated gene is turned off by preventing the production of the dangerous protein it encodes.
Whether it's drawing a connection between illness and a specific mutation, engineering a new myosin protein to solve a problem, or determining how a faulty myosin needs to be fixed, it's crucial that investigators understand the molecular pathways that translate a mutation in a muscle protein into the observable properties of hypertrophic cardiomyopathy.
Research is a slow process completed one molecule at a time. "We need to get busy now learning how the disease starts," Dr. Burghardt says, "so we stand a better chance of figuring out a way to fix it."