Human beings (Homo sapiens) are touted as topping the totem pole of life on earth. But we can't beat the feats of lowly newts and salamanders. When one of these critters loses a limb, a tail, a fin or other body part, it simply regenerates the missing member.
The tiny 1.5-inch zebrafish (Danio rerio), popular in pet shops, goes these amphibians a giant step better. Apparently unique among the world's vertebrates, the zebrafish can regenerate its heart. Humans can grow back most of a damaged or missing liver, the body's largest visceral organ.
"There's a competition going on in the liver between regenerative capacity and fibrosis," said cell biologist Mark Keating at Harvard-affiliated Children's Hospital (Boston, Massachusetts). "Thereby hangs an apparent paradox. Humans are capable of regenerating liver without forming a scar, yet cirrhosis scarring the liver is a huge clinical problem."
One thing that distinguishes those two processes, he said, "is that liver regeneration in humans occurring after partial hepatectomy is an acute injury in a healthy individual. However, fibrosis, or cirrhosis, occurs with chronic liver damage from hepatitis, viral infections or chronic alcohol use. All of which diminishes the ability of hepatocytes – liver cells – to proliferate and regenerate."
Keating, a research cardiologist at the Howard Hughes Medical Institute (also Boston), is senior author of a report in the Dec. 13 issue of Science.
"The most important aspect of this paper," he told Cardiovascular Device Update's sister publication, BioWorld Today, "is we've shown that a vertebrate organism can completely regenerate heart in a scarless manner through a process that involves the proliferation of cardiomyocytes – heart muscle cells." Noting that "there has not been a definitive study in any organism showing a scarless cardiac regeneration," he said "we were actually able to get at the mechanism of what was happening at the cellular level. The process was that existing cardiomyocytes dedifferentiated and re-entered the cell cycle to proliferate and form new zebrafish heart muscle."
Cardiac regeneration yes, fibrosis no
Keating added, "We think that zebrafish's most important future role will be in regeneration research. It is the only genetic and mainstream model system that has such remarkable regenerative capacity. So if one can enhance cardiomyocyte proliferation in humans, using the zebrafish results, we may be able to enhance heart regeneration and diminish fibrosis, which would be a salutary thing to do."
He recounted his co-authors' in vivo intervention with more than 100 laboratory zebrafish and as many control animals. "What we did first," Keating said, "was to anesthetize the fish. Then we made incisions in the animals' chest, the thorax, at the level of the heart. Next, by putting gentle pressure on the abdomen, we could actually push the heart partially out of its cavity and open up the pericardium. Using scissors, we exposed the beating heart itself, a centimeter in size, and clipped off about 20% of the ventricle, including its apex."
That caused rapid bleeding from the heart, he said, "but with care we were able to get that effusion to clot and then put the clotted ventricle back in the thorax." The surgical site continued to bleed throughout the whole procedure. "Then we had to resuscitate the fish by pushing oxygenated water through their gills, and getting them to swim again."
For the next seven to 10 days, Keating said, "those fish were not happy. They were sluggish and hung around the tank. When they recovered they seemed fine, and their behavior appeared fairly normal. The newly heart-regenerated zebrafish lived out a normal life."
He said the researchers began to examine the fish at various time points after they had had this surgery, to see what was happening with their heart and heart cells over time. "After checking the recovery and reformation of the heart," he said, "we began to look at what was happening to the cells within that organ, during the process of cardiac regeneration."
Keating said the researchers found, by following bromouridine chemical markers for cell division, that the cardiomyocytes "did de-differentiate, then re-entered the cell cycle and formed new daughter cells and new cardiomyocytes. Since we saw that the hearts regenerated beautifully without scar, we wondered whether it was possible that zebrafish don't scar at all or whether they actually could scar if regeneration was blocked." Then, he added, "we wanted to look at this idea of competition between regeneration and scar formation. So we used the Mps1 mutant gene, which we had discovered in previous experiments, to block myocardiocyte proliferation."
Of course, he said, "we found that fibrosis does indeed occur in zebrafish. They proved capable of scarring, but when their regenerative capacity was intact and robust, they just didn't. This suggested that if we could enhance cardiac and cardiomyocyte regeneration in humans, we might be able to reduce scar formation."
To create DNA damage in zebrafish, then screen for mutants that didn't regenerate properly, Keating said, "we did a chemical mutagenesis screen – not reported in the Science paper, but just now published elsewhere. That's how we identified this temperature-sensitive Mps1 mutant gene. We can turn it on and off, depending on the temperature the fish are in. At a higher temperature, it shuts down the intense proliferation of the heart muscle cells, and stops cardiac regeneration. What occurs instead is fibrosis."
From fish to rodents to people – perhaps
Now, Keating said, "we're involved in a number of ongoing projects. We're looking at the zebrafish heart to see if we can develop a model of infarction or ischemia damage, which would be more similar to what happens in humans."
He said the researchers also are looking at the consequences of the cellular mechanisms, and finally the molecular mechanisms. "We're asking: 'What are the molecules that turn this whole process on?'"
Planning preclinical in vivo experiments higher up the chain than in zebrafish, Keating said, "we're interested in testing rats and mice to see if we can enhance cardiomyocyte proliferation in vitro as well as in vivo." He said the research group already is engaged in that process.
"If successful at that level," he said, "the first thing we would do would be to see if we could regenerate heart muscle in a rodent. If that went well, then we'd likely try and go to living human patients." But, he noted, "that will take time."