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Developing Genetic Therapies for Congenital Heart Defects

Journeying With tinman to the Heart of Congenital Cardiac Malformations

I am a Woodman, and made of tin. Therefore I have no heart, and cannot love. I pray you to give me a heart that I may be as other men are. —The Tin Woodman from L. Frank Baum’s The Wonderful Wizard of Oz

When the Tin Woodman in L. Frank Baum’s The Wonderful Wizard of Oz is ushered into the presence of the great and terrible Oz, who is thought to have the power to grant any wish, he asks for a heart so that he can be “as other men are.”

Children with congenital heart anomalies have much the same wish—to be like any other child.

Ironically, the tinman gene, a homeobox (HOX) gene that helps drive embryonic heart development in the fruit fly and that has homologs in vertebrates such as the human and the mouse, could well hold the key to future gene therapies for some of these children. “Understanding the factors and genes that control cardiac development opens up new opportunities for addressing some forms of congenital heart disease through a genetic rather than surgical therapy. We envision someday treating a hole in the heart by turning on a gene that makes heart tissue around the hole grow and close the defect,” explains Andrew M. Atz, M.D., Chief of the Division of Pediatric Cardiology at MUSC Children’s Hospital.

Understanding the part played by the tinman gene and its vertebrate homolog Nkx2-5 in embryonic heart development is the focus of the research of Kyu-Ho Lee, M.D., PhD, a developmental biologist who holds a dual appointment in the Department of Pediatrics, Division of Pediatric Cardiology, and the Department of Regenerative Medicine and Cell Biology at MUSC. He is particularly interested in the splitting of the common outflow tract (the vascular structures associated with movement of blood from the ventricles) into a left side of the heart responsible for systemic circulation and a right side primarily dedicated to pulmonary circulation (Figure 1). The tinman gene appears to regulate progenitors in the second heart field that develop into the right ventricle, pulmonary artery, and aorta, all of which play important roles in balancing the pulmonary and systemic circulation.

tinman fig1
FIGURE 1. Dr Lee’s laboratory tests gene switch function. Pieces of regulatory DNA direct color gene expression in the right-sided heart of mouse embryos.

Almost a third of congenital heart defects in children who survive birth occur as a result of improper splitting of the outflow tract. These defects, according to Dr. Atz, are among the most challenging for surgeons: “Many of the complex anomalies pediatric surgeons deal with are related to the defects in the development of the outflow of blood from the heart, and in particular to the separation of the outflow tract into a left and right side.” Of these anomalies, perhaps the most common is the tetralogy of Fallot, characterized by four defects in the side of the heart associated with pulmonary circulation that result in inadequately oxygenated blood entering systemic circulation. This set of defects often results in blue baby syndrome, in which infants have a blue tinge to their skin because of the poorly oxygenated blood.

Controlling the body’s genetic signaling to trigger the regrowth of heart tissue to repair defective hearts would seem to be the stuff of miracles. And yet, such therapy is now realistically in reach in the foreseeable future. What has brought such a miracle into the realm of reality is not scientific hubris but a revolution in our understanding of genetics and development and evolution that has brought with it some sobering lessons in humility.

Lessons in Humility

Science has dealt humans a couple of hard blows to their collective ego. When Galileo found that the sun did not revolve around the earth and its inhabitants, it came as quite a shock to our human sensibilities and understanding of our position as central to the universe. When Darwin traced our lineage to the apes, he shattered the long-held notion that we had been created as the dominant species in the image of God. Even today’s scientists, chastened by such discoveries, were shocked to have another assumption of human superiority challenged—that our intelligence and other special human traits must mean that we have many more genes than other species.

The sequencing of the human genome brought this generation’s lesson in humility—instead of the 90,000-120,000 genes predicted by many scientists, we actually only have about 23,000, no more than some insects or other “lower forms” of life. And many of the genes are the same, surprisingly conserved across what seem to be widely divergent species.

As it turns out, most creatures with a common ancestor (ie, all tetrapods) share a common genetic toolkit that drives their development. What then accounts for difference, say between a mouse and a human, if we all start with the same or a very similar set of genes?

Drivers of Diversity

Increasing diversity results not from an increasing number of genes, each charged with creating a protein with a specific biological function or the building of a specific tissue, but from the regulation of the timing, intensity, and duration of the expression of genes shared by species with a common toolkit. While on Galapagos, Darwin collected several species of finches, which he later surmised had descended from a common ancestor. He attributed the wide diversity in the beaks of these different finch species to adaptations to their environment—finches on islands where seeds were abundant developed tough beaks able to crack them while those on islands where flowers and their nectar were the main foods developed long and probing beaks. The same gene controlled beak formation in each of these species, but the type of beak that developed depended on the timing, intensity, and duration of that gene’s expression.¹ What, then, controls when and whether a gene is expressed?

Only about 2% of the human genome is made up of protein-coding genes. Other genes do not code proteins but regulate the expression of the genes that do. Gene switches, also known as transcription factor binding sites, are pieces of regulatory DNA that flank the protein-coding parts of the gene and control whether and when the gene is turned on. A specific protein called a transcription factor binds to that site to tell that gene how much protein to make and when to make it, determining the direction development will take. Because the same protein-coding genes are associated with similar function across a genetic toolkit—for example, the distal-less/DLX gene with a variety of animal appendages—such switches are key to ensuring the integrity of a species’ developmental program. They control the timing, extent, and duration of the gene’s expression to ensure that a human develops an arm and not a wing or a claw.

If diversity across the members of a given taxon is driven by the regulation of the timing and intensity of gene expression by gene switches, it is not surprising that switch-regulated gene expression is also responsible for the embryonic development of diverse tissues and structures within a species. For instance, the decapentaplegic gene, which is reported to activate tinman expression in the fruit fly,² is also responsible for the body axis, epidermal patterning, gut formation, and patterning of wings, legs and other appendages.³ Dr. Lee thinks that this “makes sense evolutionarily—if you had to have a separate and unique signal for each gene, your genome would be huge, but this way, by using common elements over and over again, but in slightly different contexts, you minimize the number of genes needed to establish compartmental or cellular identities.”

But what regulates the gene switches? The discovery of the HOX genes in 1984 began to provide an answer to this question. These “master genes,” which help determine the axis and body plan of tetrapods (ie, what will be head, trunk, arms, fingers, legs, toes and where these develop), also regulate the gene switches so that the right genes are expressed at the right time at the right intensity to ensure that a given species develops according to program.¹ If something goes wrong and the switches do not turn on or silence the correct genes, then a body part can suddenly appear in an inappropriate place. Most famously, if there are loss-of-function mutations in the HOX gene Antennapedia that controls leg development in the fruit fly, the set of legs that should have appeared on the fly’s thorax instead manifest where its antennae should be.

Nkx2-5 and Human Embryonic Heart Development

The patterning of the embryonic heart is complete early, usually by ten to twelve weeks of gestation. The segments of the heart developing out of the second heart field are last to develop, well after the left-sided structures of the heart, suggesting that separate regulatory systems drive the development of the left and right sides of the heart.

Dr. Kyu-Ho Lee Dr. Kyu-Ho Lee

Dr. Lee’s work focuses on the regulation by Nkx2-5, the vertebrate homolog of the tinman gene in the fruit fly, of right-sided heart development (Figure 2). Studies have shown that the right-sided heart structures associated with pulmonary circulation fail to develop properly in embryos deficient in Nkx2-5: the outflow tract is hypoplastic and, instead of well-differentiated left and right ventricles, a single amorphous chamber develops that has some characteristics of the left ventricle.⁴,⁵ Restoring some Nkx2-5 expression results in less severe defects, showing that there is a directly proportionate relationship between the degree of Nkx2-5 expression and proper right-sided heart development. Dr. Lee has shown that this critical regulation of Nkx2-5 expression in second heart field cells is quite intricate, with its initial expression controlled by SMAD transcription factors activated by bone morphogenetic protein (interestingly a homolog of fruit fly Decapentaplegic) and its later expression sustained by other members of a complex genetic network of other heart-specific transcription factors interacting with the same control element.⁶

How does Nkx2-5 regulate right-sided heart development? It is a HOX gene that produces a transcription factor that throws the appropriate gene switches at the right time, telling other protein-coding genes when and how much protein to make. For example, Dr. Lee has identified 11 genes showing sensitivity to variations in the levels of Nkx2-5 in the pharyngeal arches of the second heart field, which appear to be under direct control of Nkx2-5 during right-sided heart development, and whose protein products may play multiple roles in the specification, growth, and structural development of embryonic right heart cells.⁷,Nkx2-5 is very likely to control many other gene targets, the identity and role of which are still being elucidated.

Nkx2-5 illustrates another of the tenets of modern genetics: that the same genes can be used to drive development in different body compartments depending upon the intensity and timing of their expression. A surprise finding by Dr. Lee and his colleagues in Obstetrics & Gynecology, including Eugene Y. Chang, M.D., Associate Professor in the Division of Maternal-Fetal Medicine at MUSC, implicated Nkx2-5, which drives the development not only of the right-sided heart but also of amniotic structures, in the genesis of preeclampsia. Intriguingly, this finding also extends to the expression of Nkx2-5 target genes that Dr. Lee found to be regulated in the developing heart; these target genes may also be relevant to preeclampsia.⁹ While these studies are still in the early stages, their preliminary findings raise intriguing questions not only about the conservation of genetic pathways, but also regarding previously unknown linkages between maternal health and child development.

Genetic therapies could hold the key to helping patients with congenital heart defects
FIGURE 2. Dr. Lee’s research focuses on second heart field development. Reprinted from the Journal of Clinical Investigations, Online, with permission.

Potential Clinical Applications

Regulation by Nkx2-5 is crucial to right-sided heart development, and children in whom it is inadequately expressed are prone to defects that involve improper splitting of the outflow tract and underdevelopment or abnormal development of the right ventricle, pulmonary artery, and aorta. Inadequate expression of other genes regulating the formation of other areas of the heart would likewise lead to different sets of cardiac malformations. Better understanding which gene networks are associated with which malformations will help predict the course of disease and provide the basis for tailored gene therapies that could begin to heal or prevent some of those defects without the need to resort to invasive surgery.

Unlike the Wizard of Oz who proved powerless to grant the wishes of the Tin Woodman and his companions, cardiovascular developmental biology research, informed by the principles of modern genetics, promises to one day offer new hope to children with some congenital heart anomalies who want nothing more than to be as other children are.


¹ NOVA. “What Darwin Never Knew,” NOVA website, (accessed September 9, 2013).

² Frasch M. Induction of visceral and cardiac mesoderm by ectodermal Dpp in the early Drosophila embryo. Nature 1995 Mar 30;374(6521):464-7.

³ Carroll SB. Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution. Cell 2008; 134:25-36.

⁴ Lints TJ, Parsons LM, Hartley L, et al. Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development 1993; 119(3):969.

⁵ Tanaka M, Chen Z, Bartunkova S, et al. The cardiac homeobox gene Csx/Nkx2-5 lies genetically upstream of multiple genes essential for heart development. Development 1999; 126:1269-1280.

⁶ Barth JL, Clark CD, Fresco VM, Knoll EP, Lee B, Agraves WS, Lee K-H. Jarid2 is among a set of genes differentially regulated by Nkx2-5 during outflow tract morphogenesis. Developmental Dynamics 2010; 239:2024-2033.

⁷ Prall OW, Menon MK, Solloway MJ,et al. An Nkx2–5/Bmp2/Smad1negative feedback loop controls heart progenitor specification and proliferation. Cell 2007;128:947-959.

⁸ Lee KH, Evans S, Ruan TY, Lassar AB. SMAD-mediated modulation of YY1 activity regulates the BMP response and cardiac-specific expression of a GATA4/5/6-dependent chick Nkx2-5 enhancer. Development 2004 Oct;131(19):4709-23.

⁹ Lee K-H, Robinson CJ, Rivers E, Horton AJ, Clark CD. Molecular basis for racial disparity in preeclampsia. Pediatric Academy Societies Annual Meeting. Washington, DC. May 7, 2013. Abstract number 4503.112.

This article originally appeared in the November 2013 issue of Progressnotes.


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  • All species descended from a common ancestor share a similar genetic toolkit for development.
  • Diversity results not from the addition of new genes but via regulation of the timing, duration, and intensity of the expression of protein-coding genes by gene switches and homeobox genes (the “master genes” throwing the genetic switches).
  • The tinman gene and its homolog Nkx2-5 regulate embryonic heart development in fruit flies and vertebrates like humans and mice, respectively, by throwing the gene switches that in turn control expression of protein-coding genes and their synthesis of proteins needed to build anatomic structures.
  • If adequate Nkx2-5 is not expressed, the right-sided structures of the heart associated with pulmonary circulation (ie, right ventricle, pulmonary artery) do not develop properly, leading to congenital anomalies like the tetralogy of Fallot.
  • Surgery is currently the best option for children with such anomalies, but, once Nkx2-5’s regulatory role in gene expression is better understood, gene therapies could be developed that could conceivably trigger the body to prevent or repair congenital heart defects like holes in this part of the heart.

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The Children’s Heart Program of South Carolina is a collaborative effort by all children’s hospitals in South Carolina to provide the state’s children with the best clinical cardiac care while at the same time fostering research that could revolutionize the treatments of tomorrow. Local pediatric cardiologists’ offices serve as the medical home for these children, ensuring consistency of care and follow-up, but all procedures and catheterizations take place at the MUSC Children’s Hospital. As a result of this concentration of surgical volume, MUSC’s pediatric cardiac surgeons have vast experience in treating a variety of congenital heart anomalies and other pediatric heart conditions, allowing them to achieve an impressive 99% survival rate for pediatric cardiac surgery in each of the past five years (as recognized by U.S. News and World Report), especially impressive as the MUSC Children’s Hospital treats some of the most challenging and complex of cases.


Over the course of the past 20 years, the Cardiovascular Developmental Biology Center (CDBC) has promoted institutional, statewide, national, and international research collaborations on fundamental studies of heart and vascular birth defects that have evolved to include two translationally relevant missions: the developmental basis of adult cardiovascular diseases and the application of the principles of developmental biology to stem cell–based, cardiovascular tissue engineering. The CDBC is a home to a cadre of world-class researchers in the field, including Roger Markwald, PhD, a pioneer of heart valve development and extracellular matrix protein biology who serves as the Chair of the Department of Regenerative Medicine and Cell Biology at MUSC; Arno (Andy) Wessels, PhD, an internationally known expert in morphological heart development, and Corey H. Mjaatvedt, PhD; as well as scientists who study the embryonic formation of blood vessels, such as Chris Drake, PhD, and the biochemistry of extracellular matrix, like W. Scott Argraves, PhD. This ground-breaking research is facilitated by MUSC’s expertise in immunohistochemistry (ie, knowing which antibody to use to look for embryonic myocytes) and a strong molecular morphology and imaging core, the Josh Spruill Imaging Facility, headed by Tom Trusk, PhD, which includes a large confocal microscopy facility that allows for digital analysis and quantification of small structures (needed for embryonic research).