Humans have very limited ability to regenerate heart muscle cells, which is a key reason why heart attacks that kill cells and scar heart tissue are so dangerous.

But damaged heart muscles in the amazing, highly regenerative zebrafish have given Duke University Medical Center scientists a few ideas that may lead to new directions in clinical research and better therapy after heart attacks.

"Our hearts don't seem so complex that they shouldn't have the capacity to regenerate," said Kenneth Poss, Ph.D., senior author of the study in Nature and professor of cell biology at Duke. The data in this study showed that the major contributors to the regeneration of surgically removed heart muscle came from a subpopulation of heart muscle cells (cardiomyocytes) near the area where the removal occurred.

The study appears in the March 25 issue of Nature.

The team labeled cells in the heart and found that cells that activated the gata4 gene upon injury ultimately contributed to regenerating the heart muscle.

The team first used a labeled "fluorescent reporter" fish that shows the presence of gata4, a gene required for heart formation in the developing embryo. They found that fluorescence was undetectable in uninjured zebrafish ventricles, but when they clipped a small section of the heart, a subpopulation of cardiac muscle cells along the outer wall of the ventricles began to fluoresce. Some of these cells near the removal site ultimately proliferate and integrate into the wound, replacing the injury clot.

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"We don't know the instructions or the mechanisms yet that mobilize these cells or cause them to proliferate, but we now know that they are the cells that are participating in new muscle growth," said Poss, who is also an investigator in the Howard Hughes Medical Institute.

Finding a key origin of heart muscle provides a target for studies that will help scientists understand cardiac muscle regeneration, said lead author Kazu Kikuchi, a postdoctoral fellow in the Poss lab. "By studying this important cell population, we expect results that could help in the repair of diseased human hearts."

The team uncovered other interesting findings.

They still needed to know whether the new cardiomyocytes were connecting in a useful way to the muscle that was spared by injury. They found that within two weeks of the injury, the new heart cells started to show normal electrical coupling needed to keep the heart beating in rhythm. A month later, the electric coupling was the same as in the uninjured heart, Poss said. "This is the first evidence that I know of that the new cardiomyocytes do become electrically coupled. It's exciting because the new muscle has to approach the functional level of the existing muscle to be of use." This experiment was done in collaboration with colleagues at Brigham and Women's Hospital in Boston.

Another finding was that the fish heart muscle found a way to work around scar tissue, a finding with interesting implications for the human heart, which stubbornly scars after heart muscle dies during a heart attack. Poss and the team found a genetic way to manipulate the zebrafish to slow down the regenerative process and form cardiac scars after tissue removal, which they normally don't do.

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The scientists were able to make the tissue form a scar by blocking a certain genetic signaling pathway. Then they returned the activity of the pathway to the animal to learn whether regeneration could occur after scar tissue formed.

The manipulation worked, and the researchers saw the gata4 label expressed in cells near the scar. They could also see a wall of new muscle forming around the scarred tissue removal site.

"I think this experiment is relevant to a lot of heart attack victims who have established scars," Poss said. "We would like to know ultimately to what extent regenerative therapy of the heart could help people who have lived with scars for a long time. When we allowed the regenerative signaling pathway to remain active after the scar was formed, we didn't see removal of scar tissue, but we did see improvements in the tissue near those injuries."

Poss said there is more to learn from the zebrafish. "We want to know the sources of all of the different cell types in the regenerated heart tissue, and the molecular events responsible for activating those sources," he said. "There is a lot left to learn about the mysterious regenerative abilities of animals like zebrafish and salamanders."

Other authors include Jennifer E. Holdway, Yi Fang and Gregory F. Egnaczyk of the Duke Departments of Cell Biology and the Howard Hughes Medical Institute (Gregory Egnacyzk is also in the Duke Dept. of Medicine); Andreas A. Werdich and Calum A. MacRae of the Cardiovascular Division, Brigham and Women's Hospital in Boston; Ryan M. Anderson and Didier Y. R. Stainier of the Department of Biochemistry and Biophysics, University of California -- San Francisco; and Todd Evans of the Department of Surgery, Weill Cornell Medical College, Cornell University in New York, NY.

The study was funded by postdoctoral fellowships from American Heart Association, the Juvenile Diabetes Research Foundation, the Japan Society for the Promotion of Science (JSPS), NIH training grants, NHLBI grants, the National Institute for General Medical Sciences, the March of Dimes, and grants from the AHA, Pew Charitable Trusts and the Whitehead Foundation.