New Clue to Heart Defect That Kills Young Athletes

April 21, 2002
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The most common cause of sudden death in young athletes -- a heart condition known as hypertrophic cardiomyopathy -- can develop from a single genetic mutation that disrupts at least two other genes, interfering with the normal beating of the heart, UCSF-led research suggests. All three genes encode contractile proteins that interact in heart muscle.

Hypertrophic cardiomyopathy affects one person in 500, and genetic defects are thought to be responsible for at least half of all cases. Although mutations in a gene for a contractile protein - known as cardiac troponin T, or TNNT2 -- have been identified in 15 percent of these cases, how the mutations cause disease has not been determined.

The gene's function can't be easily explored by traditional "knockout" methods in mice, since this would kill the embryo as soon as the heart begins to develop. But another animal, the inch-long zebrafish, revealed the answers. Using radiation to create random mutations in the zebrafish genome and then tracking their impact in the embryo, the scientists discovered an embryo whose heart does not beat, which they dubbed silent heart.

With genetic techniques they found that the silent heart gene encodes the zebrafish counterpart of TNNT2. A lack of the protein in the embryos, they discovered, leads to reduced expression of two other contractile proteins, which like TNNT2 are necessary for formation of the sarcomere, the fundamental unit of heart contraction. The research shows for the first time that TNNT2 is essential for the heart to beat.

"The finding leads to a new model of what might be going wrong in cardiac sudden death of young athletes. This little fish seems to have a lot to teach us," said Didier Stainier, UCSF associate professor of biochemistry and biophysics and senior author of a paper on the research in Nature Genetics.

"Our research demonstrates the key importance of TNNT2 in the sarcomere and shows, surprisingly, that its expression can affect the expression of two other proteins," said Dr. Amy Sehnert, a pediatric cardiologist at UCSF Medical Center and lead author on the paper. "This suggests new possibilities for the molecular pathways leading to the heart muscle problems seen in hypertrophic cardiomyopathy." Sehnert, UCSF assistant professor of pediatric cardiology, is a physician-scientist studying the molecular causes of heart diseases at the same time she treats them in the clinic.

She pursued zebrafish molecular genetics in collaboration with Stainier. Over the past seven years, Stainier has identified the developmental role of more than 10 vertebrate genes using the random mutagenesis technique in zebrafish. In this approach, adult male zebrafish are mutagenized, mated to female fish, and two generations later their tiny, transparent embryos are screened for abnormalities. Many pivotal early changes that take place in the human embryo can be witnessed in the zebrafish embryo which also is easy to manipulate genetically.

Once they had created a silent heart mutant, the scientists used two techniques to determine that embryos lacking this gene also fail to express two other contractile proteins in the heart. Using antibodies to search for the presence of contractile proteins, they found that one protein, troponin I was totally absent, and a second protein, tropomyosin, was dramatically reduced in the silent heart mutants. Gene level analysis showed a marked decrease in the expression of tropomyosin, suggesting for the first time that the expression of TNNT2 and the other two genes are under similar control in the heart.

Most often, the defect in zebrafish mutations is found within the portion of the gene that encodes the protein. However, the defect in the TNNT2 gene in silent heart mutant embryos was traced to a regulatory region of the gene that is critical for its expression. This finding provides a novel entry point for the researchers to explore what other factors might interact with this region to drive expression of TNNT2.

While the animal is a superb model for teasing apart the function of different genes in the developing embryo, it cannot serve as a perfect model for the human genetic disease, Sehnert points out. The zebrafish can continue to develop for up to one week even if it lacks the genes needed for the heart muscle to contract. But people with the genetic forms of hypertrophic cardiomyopathy, typically have one normal and one mutated copy of the gene. "If a human embryo lacked two copies of TNNT2, it would never survive," says Sehnert.

Still, the researchers are hopeful that the new finding can contribute to future gene therapy strategies that might allow only the normally functioning gene to be expressed, thereby sparing people the potential devastation of the genetic condition.

Co-authors on the paper and collaborators in the research are Anja Huq, staff research associate, UCSF biochemistry and biophysics; Brant Weinstein, Laboratory of Molecular Genetics, National Institute of Child Health and Human Development; Charline Walker, Research Staff, Institute of Neuroscience, University of Oregon; and Dr. Mark Fishman, chief of cardiology, Cardiovascular Research Center, Massachusetts General Hospital.

The research was supported by grants from the National Institutes of Health, the American Heart Association Western States Affiliate, the Pediatric Scientist Development Program, and the Packard Foundation.