Repairing the heart: If zebrafish can do it, why not humans?

Humans can't regenerate heart muscle damaged by disease, but scientists have long known that some animals, such as zebrafish, can.

Researchers have now identified a set of genes in zebrafish that reactivate after damage to the heart and patch it up like new, pointing the way to therapies that could reactivate similar genes in humans and jump-start repair of the heart and perhaps other tissues after injury.

The scientists from the University of California, Berkeley, and California Institute of Technology are still working to uncover which upstream gene or genes trigger reactivation of this gene circuit, which normally operates only during development in the embryo. But, once they do, it may be possible to use CRISPR tools to reactivate similar genes in humans after heart damage, since we employ the same set of genes as zebrafish to build the heart during embryonic development.

"Zebrafish and humans are comparable in their cell types and how these cell types form during development, but a major difference in evolution is that adult zebrafish can regenerate many different structures, including their heart, after substantial injury, whereas humans can't," said Megan Martik, a UC Berkeley assistant professor of molecular and cell biology. "How can we harness what nature's already figured out how to do in the zebrafish and apply it in a human context?"

Martik and Marianne Bronner, a Caltech professor of biology and director of the Beckman Institute, are senior authors of a paper about the findings that appeared June 18 in the journal Proceedings of the National Academy of Sciences. The research was led by UC Berkeley graduate students Rekha Dhillon-Richardson and Alexandra Haugan, who are co-first authors of the paper.

The heart is made up of many kinds of cells that comprise muscle, nerve and blood vessel tissue. A portion of these heart cells — in zebrafish, around 12 to 15% — originate from a specific population of stem cells called neural crest cells. Humans have analogous neural crest cells that give rise to varied cell types in almost every organ of the body, ranging from the facial skeleton to the nervous system. Disruption of neural crest cells during development leads to heart defects similar to those found in common congenital heart disorders.

For some reason, zebrafish and a few other animals retain the ability as adults to rebuild tissues derived from the neural crest — the jaw, skull and heart, for example — while humans have lost that ability. These animals are not merely repairing damaged tissue, however. In the heart, cells around an injury revert to an undifferentiated state and then go through development again to make new heart muscle, or cardiomyocytes.

"In both humans and zebrafish, we know that neural crest cells contribute to the heart and that they develop very similarly. But something about them is inherently different on the gene regulatory network level, because the neural crest-derived cardiomyocytes in the zebrafish can respond to injury by regenerating and the same cells in humans can't," Martik said.

CRISPR therapy

In the newly reported research, the scientists used single-cell genomics to profile all the genes expressed by developing neural crest cells in zebrafish that will differentiate into heart muscle cells. They then pieced together the genes expressed after they snipped away about 20% of the fish's heart ventricle. This procedure seemed not to affect the fish, and after about 30 days their hearts were whole again.

By knocking out specific genes with CRISPR, they identified a handful of genes that were essential to reactivation after injury, all of which are utilized during embryonic development to build the heart. One in particular, called egr1, seems to activate the circuit first and perhaps triggers the others, suggesting a potential role in regeneration.

"Differentiated cell types revert back to more of an embryonic gene expression profile and then go through development again," she said. "What we've shown in this paper is that when they do that, they activate this set of genes we know is really important for development of this population of cardiomyocytes."

The researchers also identified the enhancers that turn on these genes. Enhancers are promising targets for CRISPR-based therapies, since they can be manipulated to dial up or down the expression of the gene.

Martik continues to explore the gene circuit involved in regeneration in zebrafish, and has also developed CRISPR techniques to target gene enhancers in heart-like organoids derived from human heart cells. The tiny organoids, called cardioids, are grown in a dish and develop scars similar to normal heart muscle, allowing her team to manipulate the genes involved in repair.

Should she and her colleagues come up with a therapeutic approach, she has a vision that other cells derived from neural crest cells — such as in the jaw or the peripheral nervous system, among others — could be kicked into high gear to stimulate repair.

"There are so many advances, especially here on campus, in terms of CRISPR therapeutics that if we find the switch that can activate the necessary gene programs to drive regeneration in an organism that can regenerate, then I think it'd be completely feasible to develop a CRISPR therapeutic to drive regeneration in a human-derived context," Martik said. "I think Berkeley is the only place something like this can be done."

UC Berkeley assistant specialist Luke Lyons and graduate student Joseph McKenna are also coauthors of the paper. The work was supported by an American Heart Association Career Development Award, the Shurl and Kay Curci Foundation, the National Institutes of Health (K99/R00HD100587, DP2HL173858, T32GM132022, F31HL17614, T32GM148378) and the National Science Foundation (2023360725).