How Zebrafish Hearts Regenerate


After a heart attack, the human heart suffers from the loss of heart muscle that has been replaced with a non-contracting scar. Replacing lost heart muscle is something that human hearts do not do terribly well. However the zebrafish heart can easily replace lost cells. New research from Duke University has discovered the properties of the outer layer of the heart known as the epicardium that explains the fish’s incredible ability to regrow heart tissue.

After injury to the heart, the cells in the zebrafish epicardium begin to divide and form new cells that will cover the wound. The epicardial cells also secrete chemicals that prompt muscle cells to grow and divide. These cells also support the production of new blood vessels to carry oxygen to new heart tissues.

The Duke study was published in the May 4 edition of the journal Nature, and reported that when the epicardium is damaged, the entire repair process is delayed until the epicardium heals itself before tending to the rest of the heart. Epicardium-based healing of the heart is dependent on the production of a small, secreted protein called sonic hedgehog. In fact, applying Sonic Hedgehog to the surface of the heart drives the epicardial response to injury.

These results provide a new target to exploit in the quest to help heart patients repair the damage caused by a heart attack, which is a major cause of death and disability in the United States. Over five million Americans are currently in the throes of heart failure, and over 900,000 suffer a heart attack each year.

“The best way to understand how an organ regenerates is to deconstruct it. So for the heart, the muscle usually gets all the attention because it seems to do all the work,” said Kenneth D. Poss, Ph.D., senior author of the study and professor of cell biology at Duke University School of Medicine. “But we also need to look at the other components and study how they respond to injury. Clearly, there is something special about the epicardium in zebrafish that makes it possible for them to regenerate so easily.”

Poss and his coworkers have been studying heart regeneration in zebrafish for the last 13 years. When he worked as a postdoctoral research fellow, he was the first to show that zebrafish could regrow severed pieces of heart tissue. Since that time, his laboratory has found that this regeneration involves the input from the epicardium, that thin layer of cells that covers the heart surface.
“The epicardium is underappreciated, but we think it is important because similar tissues wrap up most of our organs and line our organ cavities,” Poss said. “Some people think of it as a stem cell because it can make more of its own, and can contribute all different cell types and factors when there is an injury. The truth is we know surprisingly little about this single layer of cells or how it works. It is a mystery.”

Poss and his colleagues attempted to identify the characteristics of the epicardium that make it so good at regenerating the heart. Duke postdoctoral fellow Jinhu Wang performed open-heart surgery on live zebrafish, and removed approximately one fifth of the heart muscle. Wang also used genetic tricks to kill off 90 percent of the epicardial cells. Then he measured how well the heart healed at various time points. Wang discovered that removing the epicardium created a clear delay in regeneration, but that regeneration eventually caught up to that of zebrafish with an intact epicardium. Wang’s results suggested that the 10 percent of epicardial cells left were able to rebuild the epicardial layer before moving on to heart muscle.

Intrigued by the finding, Jingli Cao, another postdoctoral fellow in the Poss laboratory, devised a technique to remove hearts from zebrafish and grow them in laboratory culture. In culture, the tiny two-chambered fish hearts continue to beat and behave as if they were still tucked inside the organism.

As before, Cao destroyed most of the epicardial layer of the heart and then they placed the cultured hearts under the microscope every day to capture heart regeneration in action. Cao noticed that the epicardium regenerated rapidly, covering the heart like a wave from the base of one chamber to the tip of the other in just a week or two.

The outer layer of the zebrafish heart (shown in green) is regenerated rapidly after damage, covering the heart like a wave from the base of one chamber to the tip of the other. Researchers have discovered properties of this mysterious outer layer -- known as the epicardium -- that could help explain the aquarium denizen’s remarkable ability to regrow cardiac tissue. Photo credit: Jingli Cao
The outer layer of the zebrafish heart (shown in green) is regenerated rapidly after damage, covering the heart like a wave from the base of one chamber to the tip of the other. Researchers have discovered properties of this mysterious outer layer — known as the epicardium — that could help explain the aquarium denizen’s remarkable ability to regrow cardiac tissue. Photo credit: Jingli Cao

The Poss laboratory then searched for small molecule compounds or drugs that would affect the ability to regenerate. In particular, they screened molecules known to be involved in the development of embryos, like fibroblast growth factors and sonic hedgehog, and discovered that sonic hedgehog was essential for heart regeneration. Poss and others plan to extend such a screen to molecules that could enhance heart repair in zebrafish, and perhaps one day give clues for new treatments in humans with heart conditions.

In a second paper that was published in the April 1, 2015, edition of the journal eLife, Poss and his colleagues found that the epicardium produces a molecule called neuregulin1 that makes heart muscle cells divide in response to injury. When Poss and his coworkers artificially boosted levels of neuregulin1, even without injury, the heart started building more and more heart muscle cells.

“Studies of the epicardium in various organisms have shown that this tissue is strikingly similar between fish and mammals, indicating that what we learn in zebrafish models has great potential to reveal methods to stimulate heart regeneration in humans,” said Poss.

A New Way to Mend Broken Hearts


Salk Institute researchers have discovered a way to heal injured hearts by reactivating long dormant molecular machinery found in the heart cells. This significant finding could open the door to new therapies for heart disorders in humans.

These new results were published in the November 6th, 2014 edition of the journal Cell Stem Cell. Although adult mammals don’t normally regenerate damaged tissue, they seem to retain a latent ability to do so. When the Salk team inhibited four different molecules that suppress genetic programs that lead to organ regeneration, they observed a dramatic improvement in heart regeneration and healing in laboratory mice.

These experiments provide proof-of-concept for a new type of clinical treatment in the fight against heart disease, which, according to the US Centers for Disease Control and Prevention, kills about 600,000 people each year in the United States alone.

“Organ regeneration is a fascinating phenomenon that seemingly recapitulates the processes observed during development. However, despite our current understanding of how embryogenesis and development proceeds, the mechanisms preventing regeneration in adult mammals have remained elusive,” says the study’s senior author Juan Carlos Izpisua Belmonte, holder of the Roger Guillemin Chair and primary investigator in the Gene Expression Laboratory and the Salk Institute.

We have within every cell of our bodies, the genes for organ regeneration. For several years, Izpisua Belmonte and his coworkers have attempted to clarify the genes that organism uses during embryonic development and during tissue healing highly regenerative organisms such as the zebrafish.

An injured zebrafish heart showing proliferating cells in the wounded area of the heart (red) and cardiac muscle cells (green).
An injured zebrafish heart showing proliferating cells in the wounded area of the heart (red) and cardiac muscle cells (green).

In 2003, Izpisua Belmonte’s laboratory first identified the signals that precede zebrafish heart regeneration, which they followed-up with a 2010 Nature paper, in which scientists from Izpisua Belmonte’s laboratory described how regeneration occurred in the zebrafish. Rather than stem cells invading injured heart tissue, the cardiac cells themselves reverted to a precursor-like state (a process called ‘dedifferentiation’). Dedifferentiation allowed the cells to proliferate within the damaged tissue.

n a dish, heart muscle cells return to a precursor-like state after pro-regenerative treatment with microRNA inhibitors. Green shows a disorganized cardiomyocyte cytoskeleton indicative of cell dedifferentiation; red shows mitochondrial organization.
In a dish, heart muscle cells return to a precursor-like state after pro-regenerative treatment with microRNA inhibitors. Green shows a disorganized cardiomyocyte cytoskeleton indicative of cell dedifferentiation; red shows mitochondrial organization.

They next determined if mammals retained any of the molecular players responsible for this kind of regenerative reprogramming. However, such an experiment came with some risks, recalls Ignacio Sancho-Martinez, a postdoctoral researcher in Izpisua Belmonte’s lab.

“When you speak about these things, the first thing that comes to peoples’ minds is that you’re crazy,” he says. “It’s a strange-sounding idea, since we associate regeneration with salamanders and fish, but not mammals.”

What are the things that cause a heart to regenerate in these smaller animals? Extensive work on the regenerating hearts of fish and salamanders failed to reveal anything concrete. Therefore, the laboratory changed its tack. “Instead, we thought, ‘If fish know how to do it, there must be something they can teach us about it,’” says the study’s first author Aitor Aguirre, a postdoctoral researcher in Izpisua Belmonte’s group.

The team focused on microRNAs, which control the expression of many genes. They used an extensive genetic screen for microRNAs that changed their expression levels during the healing of the zebrafish heart and that were found in the mammalian genome.

Their studies uncovered four molecules in particular–MiR-99, MiR-100, Let-7a and Let-7c–that fit their criteria. All were heavily repressed during heart injury in zebrafish and they were also present in rats, mice and humans.

However, in studies of mammalian cells in a culture dish and studies of living mice with heart damage, the group saw that the levels of these molecules were high in adults and failed to decline after the heart experienced injury. Therefore, Izpisua Belmonte’s team used adeno-associated viruses that could specifically infect the heart to target each of those four microRNAs and experimentally suppress their expressing levels.

Injecting these inhibitors into the hearts of mice that had suffered a heart attack triggered the regeneration of cardiac cells, and improved numerous physical and functional aspects of the heart, such as the thickness of its walls and its ability to pump blood. The scarring caused by the heart attack was significantly reduced with treatment compared to controls.

The improvements were still obvious three and six months after treatment–a long time in a mouse’s life. “The good thing is that the success was not limited to the short-term, which is quite common in cardiac regenerative biology,” Sancho-Martinez says.

The new study focused only on a handful of 70 some microRNA candidates that turned up in their initial screen. These other molecules might also play a part in heart cell proliferation, healing scars and promoting the formation of new blood vessels–all processes critical for heart repair, Sancho-Martinez says. The data are available so that other research groups can focus on molecules that interest them.

The next step for Izpisua Belmonte’s team is to move into larger animals and see whether “regenerative reprogramming” can work in larger hearts, and for extended periods after treatment, says Sancho-Martinez. And, although the virus packaging disappeared from the animals’ bodies by 2 weeks after treatment, the scientists are working on a new way to deliver the inhibitors to avoid the need for viruses altogether.