Windows to the Heart

One big reason Jiandong Liu, PhD, assistant professor in the Department of Pathology and Laboratory Medicine and the McAllister Heart Institute, moved to Chapel Hill this summer was for the fish.  Not the tasty, celebrated ones that are caught off Carolina’s coast and served with butter or tartar sauce, but the extremely tiny, almost invisible ones studied in the Zebrafish Aquaculture Core (ZAC) Facility that forms a part of the McAllister Heart Institute.

The popularity of zebrafish facilities has been increasing throughout the U.S. due to the value of the fish as genetic models for studying a variety of biological processes and diseases.  The core at UNC is one of only a few such facilities that are associated with a major cardiovascular research center. It was created six years ago as part of the package of support offered to two new recruits to UNC.  John Rawls was one of the two and has served as the core’s director since his arrival.

Rawls’ work with the fish has had two foci.  The first is how friendly microorganisms that naturally live in the intestines influence absorption of dietary fat and other nutrients, which can contribute to cardiovascular disease.  Press coverage of his discoveries has recently appeared in Scientific American, The Smithsonian, and Discovery.

The second focus has been on understanding how the vascular system affects the organization and expansion of body fat.  Using the zebrafish as windows into this interaction, he has found that the growth of blood vessels in adipose tissue affects how that tissue grows, which in turn has implications for the development of insulin resistance, Type II diabetes and, ultimately, heart disease.

Zebrafish are a hardy, minnow-like species. As embryos they are transparent and a mere 3/64th of an inch long.  By the time they hatch, they are three times larger and have transparent skin and muscle, making it very easy to see their internal organs.  They grow to about 1.5 to 2.0” as adults and acquire color, sporting long, dark stripes on sleek, silvery sides—whence their name.

As soon as a clutch of zebrafish eggs has been fertilized by a male, they are transparent and their developmental trajectory is swift.  Within 36 hours, all the major organs have appeared in the embryos, including a rudimentary heart, and within another 36 hours they have developed sufficiently to hatch out of the eggs and begin breathing independently in their watery world.  Still, they are essentially dormant, only beginning to swim and feed some 36 hours later.

Their relatively long period of transparency allows real-time in vivo imaging—just one of the features scientists have found that make these fish ideal models of development and disease.

Another is their relatively simple genome, which has been fully sequenced, making it easy to observe and test the behaviors of specific genes under different conditions.  The zebrafish genome is largely preserved in higher vertebrates.  So what is discovered by studying it can be reliably taken as a model of what happens in mice and monkeys—and human beings.

Another advantage of using zebrafish models in research is that, unlike rodents or primates, the fish breed quickly, their upkeep is not very expensive and they are so small that relatively large numbers can be kept in fairly small spaces .

The ZAC, for instance, consists of two rooms totaling a mere 725 sq. ft.  One is a fully functional aquaculture room where ideal conditions are created for the growth and development of approximately 50,000 to 100,000 zebrafish embryos each year.  It includes a large re-circulating aquaculture system, equipment for filtering and conditioning the water, and a means of producing food and new water.  The adjoining room contains three types of microscopy systems for researchers to use in their studies.

Although set up originally to benefit just two researchers, the zebrafish core rapidly became a shared resource.

“As is often the case at UNC,” said Rawls, “there is a spirit of collaboration and openness.  We wanted to set up a mechanism so that people who do not have fish experience could come in and use the fish system for their experiments.  So we set up a training course for them, and then a simple method of billing them for their use of the facility’s resources.

“At this point in time, the ZAC has provided services for researchers from six UNC departments, the Center for Gastrointestinal Biology and Disease, Lineberger Comprehensive Cancer Center, and, of course, the McAllister Heart Institute.  It’s really become a very interdisciplinary enterprise.”

In fact, the demand for zebrafish research capability is growing every day.

“We’re seeking to become even more accommodating,” Rawls explained.  “We need to be able to allow our existing users to grow and expand their programs more readily.  We also need to be able to attract new users, since zebrafish research is only going to increase in importance in the coming years.”

One of the new users who have already been attracted is  Liu, who decided that UNC was the place to pursue his research precisely because it already had an existing zebrafish core.

“My research has two main areas,” says Liu.  “One is to examine how the heart is formed early in development.  The heart is the first organ to form in an embryo.  It starts out as little more than a small tube with the big job of ensuring that oxygen and nutrients get to all parts of the developing organism. It goes on to develop into a complex, fully functional, three-dimensional organ.

“The problem is that cardiac developmental abnormalities occur in 1% of all human births.  The beauty of using zebrafish models to study abnormal cardiac development rather than, say, mouse models, is that because of their very simple physiology, they can survive longer, even with severe cardiac defects.  And since they are transparent, we can see into the living system and examine the problem more carefully.”

Liu is also using a zebrafish model in collaboration with mouse model researchers in order to tease out the genetic mechanisms behind the heart’s pacemaker development.

“The pacemaker is a small concentration of cells in one corner of the heart that is the primary site for electrical regulation of the heart’s contraction,” explained Liu.  “When it’s defective, a patient has to have an artificial pacemaker implanted in order to keep their heart beating.  We’re trying to understand the genetic mechanisms behind the development—and the dysfunction—of cardiac pacemaker tissue.

“We have isolated pacemaker tissue and analyzed it to see what genes are expressed there.  But we’re unsure which of the genes are the ones that actually cause normal cardiac tissue to differentiate during development and become pacemaker tissue.

“So we take this genetic information and go back to the fish.  We systemically knock out those same genes in a zebrafish embryo and observe the effects.  Because we can observe these effects within a living system, we have a much better idea of what each gene controls.  Once we’ve isolated the gene that affects pacemaker development, we can then go back to the mouse model and do more research to find out how to prevent or cure this kind of cardiac dysfunction.”

Liu came to UNC from University of California, San Francisco.  The fact that he could walk into a fully functioning fish lab here was important to both his research projects and his career trajectory.

“Setting up this facility originally took about six months,” recalls Rawls, “which is about normal.  There were institutional permissions to get, space to locate, equipment to purchase and get set up.  Not to mention the time and effort it took to learn how to run a fish research facility.  That was six months that I couldn’t spend actually doing much productive research, as well as time that was ticking against the tenure clock.

“For new hires like Jiandong,” he said, “this is not an issue.  They can come here and get to work right away.”

“It’s true,” said Liu.  “It was a very exciting prospect to think of being a part of the cardiovascular research that is going on here at the McAllister Heart Institute.  That’s one of the things that attracted me to Chapel Hill.  And, of course, the fish!”

This story was originally published in the 2012 McAllister Heart Institute Annual Report.