The river that shapes the heart
The Seyfried group revises our view of cardiac development.
Our most intricate organs begin as much simpler structures in the early embryo. An animal heart, for example, originates from a tube of cells that grow to build chambers of different sizes. This involves two different tissues: an inner layer of cells called the endocardium and an outer layer of muscle (the myocardium). Their growth has to be coordinated while the early tube undergoes a massive expansion in size. This occurs at different rates in various regions of the heart tube, a process called cardiac ballooning. (Think of the way a long, thin balloon bulges if you pinch it while continuing to inflate it.) The mechanisms that control and coordinate the development of this shape have been a mystery. Now a recent study by Salim Seyfried’s group, published in the August issue of Developmental Cell, shows that the development of cardiac structure is linked to the beginning of blood flow during early embryonic development. This affects molecular signals in the endocardium and guides the construction of heart chambers of different sizes.
Salim, Ann-Christin Dietrich, Veronica Lombardo, and their colleagues carried out their work in the zebrafish, a small organism that is particularly suited for studies of the development of the heart and other internal organs. The small size and high transparency of the fish allows scientists to observe internal structures such as the endocardium that are difficult to study using other methods. Another advantage involves one of the methods used in the current project, a photoconversion technique that uses light to track the fate of specific cells. Finally, the zebrafish can survive quite a while without a functioning heart, which permits scientists to study the effects of genetic defects or manipulations over longer phases of development. In many other species these interventions would be quickly lethal.
Answering questions about heart architecture requires tracing the origins of its cells and decoding the molecular signals that guide their development and functions. While zebrafish hearts have just two chambers, compared to the four found in humans and higher vertebrates, they all begin as a single tube. The evolutionary relationship between fish and mammals means that the first steps of heart-building probably depend on many of the same mechanisms. So discoveries in this model organism likely have implications for understanding healthy human cardiac development and the way it is disturbed by genetic defects.
In the zebrafish, cardiac ballooning transforms the narrow linear tube into a large atrium (the chamber which collects blood) and a smaller ventricle (which pumps it out). Cardiac cushions form between them to separate the two chambers. Both compartments are rounded, with a larger outer myocardium surrounding the inner endocardial layer.
Ann-Christin and her colleagues determined that during ballooning, the number of endocardial cells doubles. Where do the extra cells come from? The myocardium was known to grow through an addition of cells from outside the heart. This was not the case for the endocardium, the team found, using methods that followed the fates of cells and analyzed their functions.
Instead, existing cells divided as the chambers ballooned. This process was stimulated by a protein called Bmp. When the scientists blocked this signal, cell division in the endocardium was severely reduced.
Other signals might play a role as well. “The endocardium is a specialized endothelial tissue – the cells that line the interior of blood vessels,” Salim says. “In other contexts a signal called VEGF is crucial to the proliferation of endothelial cells, so we examined its activity in the early heart.” Blocking this signal, however, turned out to have no discernible effect on the growth of endocardial cells or the developing structure.
But disturbing the flow of blood, which begins when muscle cells begin to push fluid through the tube, had dramatic effects. The scientists examined strains of fish in which mutations disrupted this flow and found that the two chambers of the zebrafish heart remained about the same size. Cells in the endocardium failed to divide often enough. Similar changes were found in fish that produced no red blood cells, which altered the forces produced by fluid moving through the heart.
What accounted for the fact that the atrium achieved a size three times that of the ventricle? “We discovered that the two chambers contain about the same number of cells – both before and after ballooning,” Ann-Christin says. “This meant that difference in size couldn’t be attributed to a difference in the rate at which cells were reproducing. Instead, the cells in the ventricle were simply smaller.”
Blocking or reducing the passage of blood caused ventricle cells to become rounder than their shapes in healthy hearts. The pressure of normal blood flow puts the cells under a certain amount of mechanical stress, and in the ventricle this causes them to shrink and become more elongated.
“There was evidence that cells under mechanical stress produce larger quantities of a protein called Klf2 ‚” Salim says. “So our next question was whether this molecule was somehow influencing cell growth and shape.”
Eliminating Klf2 through molecular techniques once again produced ventricular cells that were larger and rounder. “These effects on cell form were virtually identical to the mutants with a disruption in blood flow,” Salim says. “If the cells produced too much Klf2, they shrank in size. We interpret this to mean that blood flow acts through Klf2 to create smaller, elongated cells in this chamber.”
Because endocardial cells in the two chambers respond differently to the passage of blood, they create compartments of unequal proportions. Achieving the proper curved shape of the chambers requires that cells in particular regions be smaller than in other areas. The proportions of these shapes in the endocardium are exactly reflected by the surrounding myocardium. Its structure was also known to be influenced by blood flow; Salim and his colleagues have now offered an explanation.
“We have demonstrated that there is a one-to-one match of endocardial and myocardial cell shapes in different regions of the chambers,” Salim says. “Our work suggests that the endocardial response to blood flow is the trigger that gets things started and shapes myocardial structure as well.” The end product is an organ whose proper architecture provides the underlying pulse that governs the life of a small fish – and a human being.
