Catching a Heart Disease Before it Happens

By Jason Socrates Bardi

James, a 65-year-old African American man living in Mississippi, walks into his doctor's office complaining that his legs are swollen, he is overly tired, he has difficulty breathing, and he can often feel his heart beating too hard. After taking an echocardiogram, an ultrasound image of the James's heart, the doctor identifies findings consistent with protein deposits inside the heart and determines that these are indicative of cardiac amyloidosis—a common cause of heart failure in the elderly.

While the doctor's diagnosis is correct in this fictitious scenario, what he does not see is that the deposits are composed of protein fibrils made from a protein with a mutation that James has been carrying his whole life. The doctor does not know that James has been predisposed to get these plaques since the day he was born, because of the DNA he inherited from one or both parents.

Hereditary diseases are not the same as congenital ("with birth") defects. While some are manifest birth, many, like the mutation that causes James's heart disease, only become evident later in life. One of the promises of molecular medicine is to find ways to identify genetically determined disorders early in life. The discipline may also lead to new ways for such diseases to be treated and perhaps prevented.

Alzheimer's of the Heart

The amyloidoses are a collection of disorders in which proteins that are secreted from cells into the bloodstream as soluble molecules become insoluble in other tissues. There, they form microscopic fibrils that sometimes aggregate to form larger plaques made up of hundreds of misfolded proteins clustered together. Both fibrils and plaques deposit in organs, interfering with their normal function, and lead to organ failure. In the case of cardiac amyloidosis, the fibrils cause heart disease by building up deposits inside the heart, which decrease the heart's ability to pump blood with congestion of the lungs and swelling of the feet.

"We refer to this as 'Alzheimer's of the heart,'" says Professor Joel Buxbaum of the Department of Molecular and Experimental Medicine at The Scripps Research Institute (TSRI). Both cardiac amyloidosis and Alzheimer's are characterized by deposits of a particular misfolded protein. But the ß protein responsible for Alzheimer's disease does not affect the heart and the transthyretin protein that forms fibril deposits in cardiac amyloidosis are almost never found in the brain.

Cardiac amyloidosis is probably more accurately described as a group of diseases rather than a single illness, because it is strongly influenced by one's genetic makeup in more than one way. A subset of some 80 mutations in the gene that codes for the serum protein transthyretin (TTR), a 127-amino acid protein that is made in the liver and secreted into the bloodstream to carry thyroid hormone and vitamin A, can lead to cardiac amyloidosis. These mutations all cause the protein to misfold and form those characteristic waxy, starch-like deposits in the heart. Even more interesting is that the most common form of cardiac amyloidosis in the elderly occurs when transthyretin without mutations is deposited.

Buxbaum and his colleagues have characterized several of these heart disease-causing TTR mutations, including that form associated with the earliest, most aggressive clinical disease. They have also identified a mutation that is present in about four percent of African Americans (1.5 million individuals) who have ancestral ties to West Africa. The mutation gives rise to amyloid deposits and subsequent heart disease after the age of 60.

The Anatomy of a Disease

Any protein can exist in a variety of conformations, or shapes, and a realistic view of proteins in living tissue is that they regularly explore many of these conformations. However, any protein must adopt its "native" conformation to be active and carry out the biological function for which it was synthesized.

Normally, TTR circulates in the blood as an active "tetramer" made up of four separate copies, or protein subunits, that bind to each other. These subunits are encoded by the same gene on the paired chromosome 18. One of these, from either parent, can carry a mutation. TTR tetramers are composed of identical protein subunits when the genes are identical, but when one of the copies has a heritable defect, hybrid tetramers composed of mutant and normal subunits form.

The inclusion of these mutated subunits can make the tetramers less stable and cause the four subunits to dissociate under conditions in which they are usually stable. Once the misfolded subunits are free, they reassemble into the hair-like amyloid fibrils.

"These [fibrils] do not stay in solution," says Buxbaum.

Instead of remaining dissolved in the blood they form deposits either within the blood vessels or in between the cardiac muscle fibers. These fibrils can then recruit more TTR proteins and keep building until the microscopic plaques become large enough to affect the operation of the organ.

The current best therapy for the disease is a liver transplant, which replaces the mutant gene with a normal copy.

Where Basic Science Meets Public Health

One of the approaches Buxbaum uses to study this disease is epidemiology. He is genotyping the TTR genes in African American participants in two large studies of cardiovascular risk in the African American community.

In these studies, the patients are genotyped using DNA taken from blood samples and they are followed over the course of many years to see whether they develop heart disease, and if so what type. The study also collects and tracks relevant data, such as electrocardiograms, echocardiograms, and chest x-rays, which can all be used to monitor the functional state of the heart.

The goal of the studies is to relate the presence of the mutation to the occurrence of heart disease and age of onset, as well as to explore possible interactions of amyloidosis with other heart conditions. If the mutation-associated risk for heart disease can be accurately determined, then a simplified genotyping might become a useful diagnostic test, and genotype-positive individuals can be given inhibitory drugs when they become available.

"We already have some data that suggests the allele we are looking at is associated with an increase in mortality," says Buxbaum. "If you look at the prevalence of the allele with age, it goes down—the older [the population,] the fewer people who have it.

There are also more basic questions that Buxbaum is interested in addressing. "What is it that keeps this from happening until late in life?" he asks. "And why these tissues only?"

Somehow the body keeps the heart free from fibrils throughout decades of normal operation. Since the defects in the TTR gene that cause the disease are genetic, the abnormal protein is present in the circulation throughout life, yet doesn't deposit until after age 60.

One possible explanation is that there are mechanisms that take care of the misfolded proteins in the bloodstream, but these mechanisms decline late in life. TTR spontaneously forms misfolded fibrils in vitro in a short amount of time—days or weeks. This does not happen nearly as quickly in vivo, which is good evidence for some as-yet-undetermined misfolding correction mechanism.

Another possibility is that the oxidation of TTR proteins enhances the misfolding, a theory that has as its premise the known fact that oxidative damage increases with age. If TTR oxidation is linked to cardiac amyloidosis, then one's chances of developing those complications would increase with age.

Yet another possibility is that the binding of TTR to other molecules changes with age. It is possible that the affinity for TTR of some proteins in the affected tissues increase with age while the TTR binding of molecules that keep it soluble in the circulation decreases.

Alongside these basic questions of the mechanism of cardiac amyloidosis, there is, of course, the question of what to do about it.

Possible Therapies and a Powerful Model

Recently, Professor Jeffery W. Kelly and his colleagues in the Department of Chemistry and The Skaggs Institute for Chemical Biology discovered a novel technique for dealing with TTR fibrils in another, unrelated amyloid disease. Their strategy is to introduce another protein that interacts with the mutant protein and prevents misfolding by preventing dissociation.

A "suppressor" TTR subunit incorporated into a TTR tetramer with disease-associated destabilizing subunits prevents the tetramer from dissociating into potential fibril-forming monomers. Significantly, they found that incorporating even one of the suppressor subunits into a tetramer where the remainder of the subunits have disease-associated mutations doubles its stability.

This "trans" suppression approach may form the basis for a new therapy for various blood-borne amyloidoses in which the patient would receive an injection of the suppressor protein.

"I'm very excited about pursuing these potential therapeutic opportunities," says Kelly.

Another, more traditional means of treatment involves using inhibitors that block the binding of the misfolded monomer to itself. Kelly and his colleagues have discovered a series of small molecules that inhibit fibril formation in vivo. Using these or similar inhibitors may become a useful strategy for treating amyloid diseases.

Inhibition studies are important, says Buxbaum, "because what you would really like to do is to prevent this genetic disease."

Buxbaum says he came to TSRI in order to strengthen his working relationship with Kelly and increase the rate of progress towards an effective treatment. In order to address this, the Buxbaum laboratory has developed a model for observing the progress of the disease in vivo. Using this model, he can test the efficacy of possible fibril-blocking therapeutics. This provides more insight than simply looking in vitro, since the physiological changes that lead to cardiac amyloidosis take place in the context of many other proteins and signals in the bloodstream.

The transgenics have the human TTR gene inserted into its genome, and a certain percentage develop fibrils with an age-related delay, analogous to that in humans. Furthermore, less structured deposits that appear to be precursors to the amyloid fibrils develop in both the heart and kidney in a larger percentage of cases.

Using this model, Buxbaum and his colleagues can also study the basic process of deposition, observing the buildup of the fibrils in living tissue over time. And they can relate the molecular changes in TTR to what is happening in the heart that may be responsible for the change in behavior of the protein with age.

"We're just beginning to look at [these questions] in our model," says Buxbaum. "The sequential events that happen from the time the protein is synthesized—where it goes and what it does."

Insight into ths in vivo process and how it can be slowed or stopped using the molecules found to work in the test tube is one of the two ultimate goals of the Buxbaum laboratory. Helping to make these molecules available to individuals found to carry the responsible gene before they get sick is the other.


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TSRI Professor Joel Buxbaum studies a type of cardiac amyloidosis, which he describes as "Alzheimer's of the heart." Photo by Biomedical Graphics.










Amyloid deposits in heart muscle caused by a mutation in the transthyretin gene, such as the ones apparent in this micrograph, cause progressive cardiomyopathy.





















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