Creld1 – a novel key player in cardiac development

Heart valve defects are a common cause of death in newborns. In collaboration with scientists at the University of Bonn, we discovered the “Creld1 gene” in mice as a key regulator for the generation of heart valves. The Creld1 gene in mice and humans is very similar. We were able to show that Creld1 controls the same signaling pathway in the hearts of both mice and humans and how defects in Creld1 signaling pathway affect cardiac development. This discovery is important for the molecular understanding of congenital heart valve defects.

Malformations of the heart are among the most common disorders of newborns and represent the number one cause of death during the first year of life. Defects of the heart valves and cardiac septa are particularly common. One of these malformations is referred to as AVSD (atrioventricular septal defect). The heart valves and septa divide the human heart into four chambers – the two atria and the two ventricles. Patients with AVSD show incomplete separation between the atria and ventricles. This causes the oxygen-rich blood from the pulmonary (lung) circulation to mix with the oxygen-poor blood from the main bloodstream. As a result, the body cannot be adequately supplied with oxygen (Figure 1).

Figure 1: AVSD – a congenital heart defect. The heart is divided into four chambers – the left and right atrium (RA, LA), and the right and left ventricle (RV, LV ). Oxygen-poor blood (blue) from the body’s bloodstream reaches the heart via the right atrium, while the right ventricle pumps it towards the lungs, allowing it to enter the pulmonary circulation. Once there, the blood is enriched with oxygen (red) and returns to the heart into the left atrium and then the left ventricle, which pumps it back into the body. The separation of the right and left sides by a wall called the cardiac septum ensures that oxygen-poor and oxygen-rich blood do not mix. In patients with AVSD (atrioventricular septal defect), this septum is not fully formed. In most of these cases, the heart valves, which direct the flow of the blood from the atria into the ventricles, are also defective. The result is that oxygen-poor and oxygen-rich blood mix, so that less oxygen reaches the body. This means that the body becomes deficient in oxygen, impairing physical performance.

In a cooperative effort within the ImmunoSensation Cluster of Excellence, working together with researchers from the LIMES Institute in Bonn, we were able to identify Creld1 (cysteine-rich with EGF-like domains 1) as a new key protein in the development of the heart. In order to analyze the function of Creld1 in vivo, we produced a knock- out mouse for Creld1 (Creld1KO). This mouse lacks the Creld1gene, so that it does not synthetize the Creld1 protein. Creld1KO mice die of a heart defect on the eleventh day (E11) of embryonic development (Figure 2).

FI GURE 2: Creld1 knock-out embryos (Creld1KO) die on day E11 of their development. Normal and Creld1KO embryos were isolated on different days of embryonic development (E9.5, E10.5, E11.0). Up to day E10.5, the two embryos are indistinguishable; on day E11.0, however, the Creld1KO embryos are already dead.

The heart valve precursors, the so called atrioventricular cushion (AVC) do not develop in Creld1KO mice; the separation into atria and ventricles remains incomplete (Figure 3a). We were able to show that Creld1 controls the calcineurin/NFAT signaling pathway. This signaling pathway is decisive for the separation of the atria and ventricles and controls cell growth in the AVC. Calcineurin is an enzyme, a phosphatase, which controls the activity of NFATc1 – a transcription factor. Transcription factors migrate into the cell nucleus, bind to DNA, and regulate gene expression. Creld1KO mice show marked impairment of this signaling pathway: their calcineurin activity is reduced, preventing the migration of NFATc1 into the cell nucleus (Figure 3b). The translocation of NFATc1 to the nucleus is particularly important in the AVC. In Creld1KO embryos, the cells in the AVC no longer divide and the heart valves are not formed.

FIGURE 3: Creld1 controls the development of the heart. a. Hearts from mouse embryos, isolated on day E10.5 of embryonic development. At this point, the normal heart is already divided into two atria and two ventricles. In contrast, the heart from a Creld1KO embryo is at an immature stage, in which the two ventricles are only partly separated. RA, LA: right and left atrium; RV, LV: right and left ventricle. b. Location of NFATc1 in the atrioventricular cushion (AVC). Left: schematic diagram of a mouse embryonic heart. The region from which the heart valves subsequently develops (the AVC) is highlighted. A: future atria, V: future ventricles. Right: sections through an embryo heart on day E10 of embryonic development. The AVC region is shown. NFATc1 was labeled with an antibody (violet). The DNA in the cell nuclei was stained with DAPI (blue). As can be seen in the magnification, Creld1KO embryos show impaired NFATc1 translocation (migration into the cell nucleus). Scale bar: 20 ?m.

Mutations in the human CRELD1 gene have been associated with the onset of AVSD. To date, however, it has not been clear whether AVSD is actually caused by these mutations. The underlying molecular mechanism also remains unknown. Therefore, we introduced CRELD1 gene mutations found in patients with AVSD into the Creld1gene of the mouse, and used cell culture methods to investigate, whether this impairs the function of the calcineurin/NFAT signaling pathway. Two of the most common mutations reduced the Creld1-dependent migration of NFATc1 into the cell nucleus and, thereby, NFATc1-dependent gene expression. These results suggest that mutations in the CRELD1 gene influence the activity of the calcineurin/NFAT signaling pathway also in humans, and could therefore be involved in the generation of AVSD. In the future, we will gain an even better understanding of the function of Creld1, in order to develop potential treatment approaches for AVSD.

Publication

Mass, E., Wachten, D., Aschenbrenner, A.C., Voelzmann, A. & Hoch, M. (2014)"Murine Creld1 Controls Cardiac Development through Activation of Calcineurin/NFATc1 Signaling" Developmental Cell 28, 711-726 DOI:10.1016/j.devcel.2014.02.012