'Cardiofunk' Mutation: Probable Source Of Congenital Heart Defects

The surprising discovery, in genetic studies of transparent zebrafish embryos, suggests that the cause of some human congenital heart valve defects may lie not in the genes for the formation of the valve itself, but in genes used in the heart muscle that pumps the blood. About 1,600 valve defects are diagnosed each year in the U.S. and few of the genes responsible for these defects have been identified.

The research team found that a mutation in a single gene involved in heart contraction interfered with the development of the heart valve. While the mutation would kill a human embryo, the discovery shows that normal heart contraction is required for normal valve formation, so even temporary glitches in the early heartbeat might cause valve defects.

"I expected that the early stages of heart valve formation would be completely hard wired, rather than regulated by the beating of the heart," said Didier Stainier, PhD, professor of biochemistry and biophysics at UCSF and senior author of a paper on the discovery published online May 11 by Public Library of Science (PloS) Biology.

"It now seems likely that some heart valve defects in newborn children may be caused by a temporary interruption of normal heart contraction early in the life of the embryo, rather than by genetic defects in the valves themselves. This could lead to a new way to look at congenital heart defects," he said.

If proven true in humans, the finding could allow physicians to treat or prevent heart defects before birth by focusing on fetal heart formation instead of having to cope with the consequences of heart malformations in infants after birth, the researchers said.

The full effect of the mutation on valve formation could not be discovered in a mouse or human because the developing embryo would quickly die if the valve failed to allow blood out of the heart, starving the growing embryo of oxygen. But the zebrafish embryo is so small that it can survive without blood circulation for several days -- long enough for researchers to determine that the single mutation in the gene involved in heart contraction led to defective heart valve formation.

The zebrafish embryo is not only transparent, but it develops outside the mother's body, providing an open window on vertebrate development. Stainier's team could easily detect heart valve defects in the developing fish and examine them as the embryos developed. Scientists studying zebrafish have identified many vertebrate development genes, most of which have now been identified in humans.

The researchers used "forward genetics" to identify the key gene: Several dozen zebrafish males were exposed to a chemical mutagen, and hundreds of thousands of their progeny were screened for developmental abnormalities. This allows researchers to identify genes involved in development that had never been previously considered.

The team identified a mutant they called cardiofunk (cfk), lacking the ability to develop a valve required for blood to exit the heart. Normally, blood is pumped from the atrium to the ventricle; then the valve between them closes so that when the ventricle contracts, blood flows forward into the embryo's circulatory system instead of backward into the atrium. But in the mutants, blood flowed back and forth between the atrium and ventricle, never leaving the heart.

The researchers expected the mutation responsible for the valve defect would be in a gene expressed in the developing valve. But genetic mapping of the cfk mutation revealed a change in a gene for a previously unknown actin molecule, related to actins found in muscle cells. Using techniques to detect RNA, they showed the cfk gene is expressed in the heart muscle, and not in tissue that gives rise to the heart valve.

What is it in the beating heart that triggers normal valve formation?

"I think it will turn out to be a combination of precursor valve cells sensing blood flow and sensing signals sent by muscle cells -- and maybe other factors," Stainier said. "Identifying other mutants with a similar defects should provide the answer."

The study offers a new approach for early detection of human heart defects, he said. It is the first genetic demonstration that function is required for form to mature.

Lead author on the research was Dr. Thomas Bartman, M.D., a former postdoctoral scientist in Stainier's lab and now an assistant professor of neonatology at Cincinnati Children's Hospital Medical Center. Co-authors are Emily Walsh, PhD and Jonathan Alexander, MD, PhD, both former graduate students in the Stainier lab; Peter A. Rubenstein, PhD, professor of biochemistry at the University of Iowa College of Medicine; and Kuo-Kuang Wen, PhD, a research scientist; Melissa McKane, a senior research assistant; and Jihui Ren, a graduate student, all working in Rubenstein's Iowa lab.

The research was supported by NIH, the Packard Foundation and the Howard Hughes Medical Institute.

NOTE: Videos of the normal and mutant embryonic heart can be accessed at: http://home.earthlink.net/~bartmanlab