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How to explain such a divided heart

The heart’s chambers may be the result of the action of retinoic acid, the same acid used in some cosmetics

A shoal of zebrafish: study model for formation of the atria and ventricles

LÉO RAMOSA shoal of zebrafish: study model for formation of the atria and ventricles LÉO RAMOS

Small fish with horizontal stripes maintained in an aquarium at the Brazilian Biosciences National Laboratory (LNBio), located in Campinas (São Paulo State), are revealing much about the formation and evolution of the human heart. In late June 2014, after spending months examining the gene activation mechanisms in the heart muscles of tiny zebrafish, José Xavier Neto and his team completed a series of experiments reinforcing their hypothesis. They assert that the structure of the human heart, with four inner chambers divided by valves that regulate blood flow, could have appeared at least 500 million years ago, well before the emergence of the human species itself, 2 million years ago. The human heart, therefore, would have emerged before humans themselves.

The implications of this conclusion are a bit disconcerting. “In evolutionary terms, our hearts are practically the same as those of the lamprey,” says Neto. In view of the importance and significance of the human heart, it is uncomfortable to think about this similarity, since the lamprey is a primitive, elongated fish, easily considered quite ugly, with no fins or jaw, and a mouth in the form of a circular suction cup the width of its body. Neto does not seem to mind the close relationship. “From the point of view of cladistics,” he says, referring to the classification system of living things based on the evolutionary relationship among species, “we never stopped being fish. We are modified fish, the fins turned into arms and legs.”

Primitive fish such as the lamprey already have a four-chambered heart, but it is arranged in a sequence rather than in a single block, as is the human heart. In another evolutionarily very old species, the piramboia, which is elongated like a snake and endowed with lungs, the heart is already more refined, with an internal division separating oxygen-rich blood from carbon-dioxide rich blood. It may have appeared about 400 million years ago and is found in the Amazon (a specimen can also be found in the Campinas laboratory). A more significant aspect to Neto, regardless of its shape, is that the path of blood flow in the heart already has a kind of S shape, which is more pronounced in fish and more subtle in humans.

The colors of the heart: of fish...

LÉO RAMOSThe colors of the heart: of fish…LÉO RAMOS

The Campinas team has not only experimented with zebrafish, which are much more friendly than lamprey, but also with mice, chickens and quail, and have examined the formation of their inner heart chambers—the atria and ventricles, which are essential for storage and distribution of the blood circulating throughout the organism. After almost two decades of work, they have concluded that the types of heart chambers are determined by the action of retinoic acid. It is a wave-like action, that is sometimes intense, sometimes less so, at specific moments of embryonic development. According to Neto, when as yet unspecialized cells come in contact with retinoic acid, they are instructed to arrange themselves and form a reservoir for blood, that is, an atrium. When no retinoic acid is detected, they form into a strong driving pump for blood, that is, a ventricle.

The two structures are quite different: the atrium has a smooth surface and acts as an inflatable reservoir to receive blood. The proteins responsible for its contraction, the myosins, are slow. The ventricle has a rough surface and walls that are thicker and higher than those of the atrium, with its fast-acting myosins, and able to contract forcefully to distribute blood to all cells of the body. The human heart—an organ about the size of a fist, weighing 250 grams in adult women and 300 grams in adult men, which beats 100,000 times per day, pumping about five liters of blood—has two atria above two ventricles.

The Campinas team and others pursuing the same line of research are furthering the understanding of the origin of cardiac problems associated with retinoic acid, a vitamin A derivative widely used in cosmetics. “If a woman uses it at the beginning of her pregnancy, malformations are almost a certainty. That’s why doctors request a pregnancy test before prescribing retinoic acid for skin treatments,” says Neto, a Rio de Janeiro native with a degree in medicine from the Federal University of Ceará (UFC). “Human beings are extremely sensitive to retinoic acid, but without it, we would not be here. It all depends on the dose and the location where it is going to act.” For now the possibility of preventing and correcting early fetal heart problems is remote, because retinoic acid acts in the first few weeks of pregnancy, when women in general do not even know they are pregnant.

Using the most recent experiments, as soon as they are published, Neto wants to reinforce his hypothesis and refute the opposing views of other teams interested in elucidating the mechanisms that define the size, shape and mode of operation of each cardiac chamber. In 2008, an article by Deborah Yelon’s team, currently at the University of California at San Diego, reduced the role of retinoic acid; she associated it only with the size of the heart, and instead assigned greater weight to the protein produced by the HOXB5 gene, which also acts in the formation of the gut and the lungs, based on experiments with zebrafish. “When I saw this paper,” says Neto, “I almost cried.” “Because of where I was in my work in 2008,” says Neto, “I knew it would take some time for me to formulate an answer.”

... and mice

Léo Ramos… and miceLéo Ramos

In 2005, as a researcher with the Heart Institute (Incor) of the University of São Paulo (USP), Neto and his team had presented their hypothesis on the formation and evolution of the blood compartments in vertebrate hearts. Based on experiments with an impressive variety of organisms, for example, Ciona intestinalis, a cylindrical marine invertebrate that is the closest living relative of vertebrates (the formation of the heart of this group, the tunicates, is similar to the early stages of heart formation in vertebrates), the team argued that vertebrate heart chambers could have evolved from changes to an equivalent of the Ciona heart tube. It is capable of movements similar to those that occur in the intestine when a food mass is moved during the process of digestion. “Animals such as lobsters and other crustaceans represent another model for the formation of the cardiac chambers, as they have only one chamber whose movements are much faster  than, for example, the onychophora, worms that have only a simple peristaltic tube,” he says. “The heart chambers are an attribute of vertebrates, with simultaneous contraction and separation by valves, all within a single membrane, the pericardium.” A coherent argument, however, was not enough. “I knew I would still have to prove my hypothesis,” he says. “I had to wait six years to redo the experiments and demonstrate the role of retinoic acid.”

Even now, with more arguments, Neto knows he will have to struggle a lot to make his view prevail; if he fails, it may be disregarded or forgotten. “Hypotheses about evolution can hardly be tested,” he says. In addition, the heart easily deceives those seeking to understand it. The Roman physician Claudius Galen, a founder of western medicine, said the heart was made ​​of a special fabric. Almost 1500 years later, Leonardo da Vinci, after dissecting cadavers, as had Galen, made several drawings of the anatomy of the heart, and declared: “The heart is a muscle of great strength, much stronger than the other muscles.” It was a breakthrough, but other misconceptions persisted. For centuries it was thought that veins carried air, which were empty in animals and dead people. A century after Da Vinci, English physician William Harvey described the circulation of blood in detail, and demonstrated that veins, like arteries, carried blood, not air.

The four chambers of the heart, as seen by Da Vinci: the artist dispelled Galen’s misconception that the heart had only two chambers

SCIENCE PHOTO LIBRARYThe four chambers of the heart, as seen by Da Vinci: the artist dispelled Galen’s misconception that the heart had only two chambersSCIENCE PHOTO LIBRARY

Like other contemporary scientists, Neto also took some wrong turns. Soon after arriving at Harvard University for his postdoctoral research in 1997, he found himself drawn, almost inevitably, to the then fashionable idea that a single gene would be able to determine heart formation. There was a candidate gene, but experiments in mice indicated that heart formation depended on many genes; even without the candidate gene, the mice were born with hearts, although they died soon after. And he surrendered: “It’s much more complicated than we thought.”

After that, Neto was able to pull together the appropriate reagents and transgenic animals—which gradually fell into the hands of the laboratory coordinator, Nadia Rosenthal—to design experiments that would indicate new things about heart formation. “Even if I fail, I thought, it’s enough to observe the development of the embryo,” he recalls. “And as I was getting into the developmental biology, I could see the organ formation process with my own eyes, without being tainted by too much reading of scientific papers.”

Neto then began to examine how expression of the beta-galactosidase enzyme could indicate the action of retinoic acid in different regions of the heart in nine-day-old mouse embryos. “I nearly tore off my retina trying to see what was not in the mouse embryos,” he says. Gradually he was able to clearly see the color pattern defined by activating retinoic acid: “Depending on the enzyme’s expression, regions of the heart appeared green, indicating that retinoic acid was operating in the area, as an activator or repressor of various genes.”

He noted that by the seventh day of gestation, which takes 21 days, the heart had not yet formed, nor was there any sign of retinoic acid action in cardiac tissues. Two days later the heart had already been delineated as a tube, then a discharge of retinoic acid occurred and the atrium formed. Soon after, the retinoic acid disappeared and the ventricle formed. Other experiments in quail indicated that without retinoic acid the atrium remained unformed and, in addition, an excess of this substance prevented formation of the ventricle. “Retinoic acid is an actor who enters and exits the stage, playing different roles in the same performance,” says Neto.

“Retinoic acid is indeed a key player in the formation of the cardiac chambers,” says Didier Stainier, coordinator of a team from the University of California in San Francisco (UCSF), who studies heart formation in zebrafish. In 2002, Stainier and Deborah Yelon, who worked in his laboratory, saw the role of retinoic acid in an earlier stage of development: with other molecules, it could induce the formation of a primordial embryonic tissue called the endoderm (heart formation comes from another tissue, the mesoderm). According to Stainier, Neto “has been in the forefront of this research, which will undoubtedly lead to additional insights into the process of heart development.”

Chicken embryo: under the action of retinoic acid

Léo RamosChicken embryo: under the action of retinoic acidLéo Ramos

Even after the heart has formed, this versatile actor is still on the scene. In 2011, researchers at Duke University in the United States showed that retinoic acid, because of its ability to induce cell proliferation, facilitated regeneration of the endocardium, the inner layer of the heart. Once again, the experimental model was the zebrafish; this species has been used for decades because females produce many eggs, which can be easily collected, and the embryo is formed from a single cell, just one day after fertilization.

After two years at Harvard University, Neto happily returned to USP’s Heart Institute ready to assemble a group to research the genetics of embryonic development and continue the work for which he had spent two years at Harvard. His first challenge was acquiring mice, which did not arrive in the anticipated amount or time frame. He did not give up, but instead went around asking where he could buy fertilized chicken eggs and an oven, so as not to stop his work. Many years before, with the same avidity for science, he had hunted frogs to conduct the experiments planned for his first year of medical school at the Federal University of Ceará. “Ever since my undergraduate days, I’ve wanted to be a researcher,” he says, adding that he really liked the basic sciences such as biochemistry, which are usually snubbed by aspiring doctors. He has always enjoyed the world of science, accompanying his father, who was a biochemistry professor at the university, to laboratories and greenhouses. “Remember the science kits from the 1970s? I had them all. From an early age I’ve lived in that world.”

Neto took five years to assemble his own team and the laboratory he needed to resume the pace of work he wanted. “If left alone, it is lost,” he concludes. Networking is everything.” Through testing in chickens and quail, he found that the action of retinoic acid, in turn, was regulated by the RALDH2 enzyme. “I detailed what happened and when,” he says. He never ceased to take advantage of studies of other animals—marine worms, snails, lobsters and others—to examine the evolutionary heart formation process and, since 2010, when he moved to the Biosciences National Laboratory (LNBio), he has continued to produce transgenic animal strains, most customized for the experiments of other researchers and for his own group. Unable to sit still, in August 2014 he traveled again to the Chapada do Araripe (plateau in northeastern Brazil forming the boundary of Ceará and Pernambuco states), in search of fish fossils with an average age of 120 million years, which when examined by tomography could reveal a little more about the evolution of the heart.

Evolution and development of cardiac chambers (nº 06/50843-0); Grant mechanism Regular Research Grant; Principal investigator José Xavier Neto (LNBio); Investment R$ 311,558.83 (FAPESP).

Scientific articles
SIMÕES-COSTA M. S. et al. The evolutionary origin of cardiac chambers.Developmental Biology. v. 277, n. 1, p. 1-15. 2005.
MOSS, J. B. et al. Dynamic patterns of retinoic acid synthesis and response in the developing mammalian heart. Developmental Biology. v. 199, p. 55-71. 1998.
WAXMAN, J. S. et al. Hoxb5b acts downstream of retinoic acid signaling in the forelimb field to restrict heart field potential in zebrafish. Developmental Biology.v. 15, n. 6, p. 923-34.
YELON D. e STAINIER, D.Y. Pattern formation: swimming in retinoic acid. Current Biology. v. 12, n. 20, p. 707-9.