Congenital heart defects occur in an astonishing 1% of live births1,2, and fetal heart malformations are implicated in many pregnancies that end in stillbirth or spontaneous abortion2. Why, of all the body's complex organ systems, is the heart so susceptible to malformation during fetal development? Genetic factors are probably central to many congenital heart defects, but complicating factors such as environmental toxins or the diet and age of the parents are likely to affect the severity of these disorders. Despite compelling statistics on their incidence, and in the face of exciting genetic progress3,4 implicating several genes in the developmental programme of the normal heart, we still do not understand biochemically why heart defects are so prevalent. This is mainly due to a lack of the molecular targets that might indicate preventative therapeutic approaches or outline signalling systems for further investigation.
On pages 182 and 186 of this issue, de la Pompa et al.5 and Ranger et al.6 offer tantalizing hints as to how a single transcriptional regulator might link the genetic and environmental causes of one class of congenital heart disorders — birth defects involving valve and septum formation. Both groups specifically mutated a mouse gene that encodes a member of the NF-AT (nuclear factor of activated T cells) family of transcription factors. This gene (NF-ATc) is known to act in the immune system7. But, unexpectedly, as a result of the introduced mutations, the pulmonary and aortic valves completely and specifically failed to form. These critical valves control the flow of blood from the ventricles of the heart into the arteries leading to the lungs and the main circulation, respectively (Fig. 1, overleaf).
Figure 1: Cardiac valves that depend on the activity of fetal NF-ATc.
The structure of the normal human heart is shown: the right atrium and ventricle are in blue; the left atrium and ventricle are in red. Blood from the main circulation enters the right atrium and passes through the tricuspid valve (4, green). The right ventricle pumps blood through the pulmonary valve (2) and into the lungs. Oxygenated blood returns from the lungs into the left atrium and passes through the mitral valve (3) into the left ventricle. From here, the ventricle pumps the blood through the aortic valve (1) and into the main circulation. de la Pompa et al.5 and Ranger et al.6 have found that the aortic and pulmonary valves are not formed in NF-ATc-/- homozygous deletion mice, and de la Pompa et al. found that the mitral and tricuspid valves are also malformed.
High resolution image and legend (111K)The ventricular septal structure, which separates the left and right ventricles, was also rendered defective by the mutations. Less severely affected (although clearly defective in the study of de la Pompa et al.), were the tricuspid and mitral valves, which regulate the flow of blood from the right and left atria into their respective ventricles. The spectrum of malformations found in human hearts more closely resembles the defects seen in the pulmonary and aortic valves of the mouse NF-ATc mutants. Human clinical cases only rarely involve the complete lack of all four valves, consistent with the phenotypes observed in the mice. However, as a result of these combined defects observed in both studies5,6, the mouse embryos died after 14-15 days' gestation.
One of the defects caused by disruption of the mouse NF-ATc gene is strikingly similar to a class of human congenital heart diseases. This class describes defects in formation of the atrioventricular valves and septal structures that, combined, separate the four chambers of the developing heart (Fig. 1). During development, the septal and valve structures originate from endocardial cells that migrate into the 'cardiac cushions' — regional swellings on the inner lining of the developing heart that eventually give rise to the septum and valves. Migration of these endocardial cells results in their transformation into mesenchymal cells, apparently in response to soluble protein signals. This transformation seems to be essential to the participation of endocardial cells in development of the valve leaflets or septal structures. A burst of NF-ATc activation is observed in the developing 'primordial' septal structures of the cardiac cushions in wild-type animals, correlating exactly with initiation of their formation. Thus, disruption of the NF-ATc gene interrupts a normal and important function for NF-ATc in the primordia.
Although de la Pompa et al.5 and Ranger et al.6 find that NF-ATc is expressed in endocardium and valve primordia, we would have predicted from the literature that the main effect of knocking out the gene would be immune dysfunction. This is because the NF-AT holoprotein complex was originally characterized by Crabtree and colleagues as a critical component of the transcriptional regulatory apparatus of interleukin-2 in antigen-stimulated T cells. NF-AT is composed of at least two components. In T cells, a cytoplasmic component pre-exists, containing a DNA-binding domain that is homologous to Rel. So far, four genes are known7 to encode proteins belonging this subgroup — NF-ATc, NF-ATp, NF-AT3 and NF-AT4. Although these genes are expressed in various tissues, their role is best understood in the immune system. After T-cell activation, the Rel component (for example, NF-ATc) translocates to the nucleus, where it can bind to DNA directly, or in a complex with members of the bZIP transcription-factor families7,8 (Fig. 2).
Figure 2: Common paths for activation of NF-ATc in T cells and fetal endocardial cells?a, T-cell activation involving.
NF-ATc. Engagement of the T-cell receptor (and other co-receptors) triggers the activation of the phosphatase calcineurin. Calcineurin dephosphorylates the amino terminus of NF-ATc, causing it to translocate into the nucleus, where it is thought to interact with newly synthesized proteins such as the bZIP protein AP-1 (Fos/Jun) and activate specific genes. b, A comparable activation system may be used in the developing fetal heart. Endocardial cells activate NF-ATc through a calcineurin-specific pathway. Upstream activators and downstream target loci are proposed by analogy to the well-characterized T-cell system.
High resolution image and legend (143K)Amazingly, it seems that key aspects of these NF-ATc signalling processes are also used during early heart development. Moreover, genetic disruption of NF-ATp (which is biochemically and genetically similar in many ways to NF-ATc) leads to no obvious defect in heart development9. But it does disrupt the immune system, as might have been expected9. Because NF-ATp does not seem to be expressed in heart tissue, other transcription factors must control the temporal and tissue-specific expression of NF-ATc, distinct from that of NF-ATp.
How does NF-ATc respond to signals in endocardial and T cells? In part, it is thought to interpret changes in the levels of calcium in the cytoplasm. Calcineurin — a protein that responds to calcium signalling — is known to activate NF-ATc by dephosphorylating residues in its amino terminus. By blocking calcineurin function using pharmacological agents such as FK506 and cyclosporin A, activation of NF-ATc can be blocked. Moreover, de la Pompa et al.5 and Ranger et al.6 showed that movement of NF-ATc from the cytoplasm to the nucleus in cells from these developing heart structures can be inhibited by FK506 and cyclosporin A, respectively.
It is tempting to speculate, therefore, that defects in cardiac valve formation are caused by problems in NF-ATc signalling. These may be genetically determined, or they could be influenced by environmental toxins that deregulate the heart's developmental programme. For example, if inappropriate environmental signals are present during the critical stage of valve and septal development in the human embryo, they could lead to cardiac birth defects. Moreover, other forms of valve defect, such as stenosis (narrowing of the valve orifice) and mitral valve prolapse (which affects many women in later life and is a primary risk for endocardial infection), might result from subtle deregulation of the signalling systems linked to NF-ATc function. So the upstream activators and target loci of NF-ATc in the developing heart should now be investigated, because the activity of these proteins — or their misregulation — could be crucial in eradicating this important class of birth disorders.
