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Michael Kühl: in search of the gene architects of the heart

Michael Kühl is investigating the development of the heart using a broad range of different model organisms. The developmental biologist, director of the Institute of Biochemistry and Molecular Biology at the University of Ulm, also uses evolutionary and systems biology approaches for his work. Kühl’s basic research does not follow an art for art’s sake principle, but also addresses the development of new therapies for the treatment of heart diseases.

Prof. Dr. Michael Kühl: The heart is close to his heart. © Pytlik

Michael Kühl’s research interests and motivation are rooted in the field of human medicine. In the Western world, ten percent of all miscarriages are the result of a defective development of the heart. Around one percent of all newborn babies have a congenital heart defect. Kühl believes that these figures also apply to other countries around the globe. 

Another motivation for his interest in cardiogenesis is closely related to the fact that the human heart is unable to regenerate: myocardial infarction results from the interruption of blood supply to a part of the heart, starving the heart muscle of oxygen and causing cardiac cells to die (necrosis). Humans cannot regenerate the dead cells, while zebra fish can. Following myocardial infarction, the cardiomyocytes in the infracted part of the heart are replaced by fibrotic scar tissue, which does not contract. Pumping ability is thus reduced and might have an effect on a person’s lifestyle.

Kühl’s work looks into whether cardiomyocytes can be grown in a culture dish and used for the treatment of mycocardial infarction patients.

Objective: heart muscle cells for clinical application

Kühl is specifically focused on finding answers to the following two issues: on the one hand, he is trying to gain an understanding of how the heart develops normally and elucidate the causes of heart defects. On the other hand, Kühl is seeking to contribute to the joint action of researchers around the world who are aiming to optimize the cultivation of heart muscle cells for use in clinical application.

Kühl and his group of researchers started their work with the South-African clawed frog (Xenopus laevis). Over the last few years, the researchers have also used other popular model organisms, including fruit flies (Drosophila melanogaster), zebra fish (Danio rerio) and mice. All these models have a different degree of complexity and Kühl uses them to obtain detailed insights into the development and evolution of the heart.

The anatomy of the models differs considerably

Kühl’s research group uses numerous animal models to investigate the evolution of the heart. © University of Ulm

The anatomy of the heart differs considerably in the model organisms investigated: the human heart has four chambers, two atria and two ventricles; the ventricles are the discharging chambers and the atria the receiving chambers. The right ventricle pumps blood into the pulmonary circulation for the lungs where the blood takes up oxygen; the left ventricle pumps blood into the systemic circulation where the oxygen is released. The right atrium receives deoxygenated blood and sends it to the lungs for oxygen; the left one receives oxygenated blood and sends it to the left ventricle from where it is taken to the rest of the body.

Mice hearts also have four chambers, the frog has three chambers (two atria and one ventricle, where the blood mixes; frogs also take up oxygen from water, which is why the two cycles do not have to be separated from each other completely). The fish heart consists of two chambers, one atrium and one ventricle; flies only have one chamber, which resembles a contracting tube.

Important mechanisms are highly conserved

Kühl is trying to identify and characterize the mechanisms that are identical in all four species examined as well as those that have been added as the ancestral heart developed into the four-chambered human version. Kühl is interested in both developmental as well as evolutionary aspects and has found that particularly important mechanisms have been conserved throughout evolution.

It is now common knowledge that a network of genes regulates the specialisation of heart muscle cells. This genetic toolbox is conserved in all vertebrates (fish, frogs, mice, and most likely also in humans) because all these organisms possess contracting heart muscle cells. “The different organisms developed different mechanisms to regulate this network, which in turn led to a greater structural complexity,” said Kühl, who is a biochemist by training.

Molecular approach: a common evolutionary origin

Heart muscle cell differentiated from murine embryonic stem cells. The contractile apparatus is stained red, the cell nucleus blue. © Kühl, University of Ulm

Although the different species differ considerably in the anatomy and morphology of the heart, investigations on the molecular level suggest a common evolutionary origin rather than a parallel evolutionary development of the heart. Summarizing state-of-the-art research, Kühl explains that the embryonic development of the heart in different species is regulated by a broad range of orthologous genes. Orthologous genes of the Isl1 transcription factor, which is a major research priority of Kühl’s research group, have been identified in humans; Isl1 has also been identified in mice, chicken, clawed frogs (Brade et al., 2007), zebra fish, lampreys, sea squirt and fruit flies (Mann et al., 2009).

Isl1 was also identified in adult cardiac stem cells of mice, rats and humans. These findings might open up new possibilities for the development of therapies to treat heart failure. In addition, Isl1 was identified in areas of the postnatal heart that developed from the second heart field. It is believed that the cardiac stem cells are remnants of Isl1-positive embryonic cardiac progenitor cells that are active during embryonic heart development (Pandur et al., 2012). 

Michael Kühl carries out comparative investigations into cardiogenesis in different model organisms with the objective of finding answers to his key questions: how does a heart muscle cell develop and how a complex organ? It was only around 10 years ago that molecular biology approaches were combined with anatomical and morphological investigations. 

Everything begins with a special pile of cells

The basic assumption that cardiogenesis starts with a specific cell population is still generally agreed. As humans belong to the Bilateria, it is assumed that there were two piles of cells that moved towards each other during evolution and initially formed a tube like the one found in flies. This tube then underwent morphological changes: a process known as cardiac looping gave rise to an S-shaped structure, which corresponds largely to the structure of a fish heart. This developmental step is known as cardiac jogging in fish: a morphogenetic movement occurred, two chambers were formed and this stopped the development of further chambers.

The heart underwent further morphogenetic changes in higher organisms; the contractile tube was reshaped, giving rise to three (frog) and four chambers (mice). The heart then matured, grew and its pumping capacity increased. This anatomical-morphological theory was valid until early 2000 when researchers discovered that the heart tube was not a unit from which the heart developed, but that it grew and that more and more cells were recruited into the heart. Molecular biology provided further insights with its views into the genetic component of heart evolution.

Mutagenesis screens discover genetic toolbox

Mutagenesis screens soon showed that the core of this genetic network is already present in fruit flies. Molecular biology has since provided evidence that although the hearts of flies and mice have a different anatomy and morphology, their hearts nevertheless have the same (orthologous) genes.

In the meantime, human genetics has shown that the same genes are also active in humans and that patients with congenital heart disease have mutations in these genes. It appears that the anatomical process of cardiogenesis is based on a molecular process governed by a highly conserved gene network. Current research is focused on elucidating this gene network. Michael Kühl explains that mutagenesis and expression screening experiments have led to the identification of the most important players: “We basically know all the genes that are expressed in an organ at a specific point in time.”

However, the evo devo researchers do not yet know how relevant these genes are and in which cell types they are expressed. It is worth noting that the heart not only consists of contracting heart muscle cells, but also of fibroblasts that coat the inner walls of the heart and vascular cells that provide the heart muscle with nutrients and oxygen. But the situation is most likely more complex as not all cell types involved in the development of the heart are known; in addition, the researchers do not know in which cell types which genes are switched on.

Isl1: a key gene in cardiogenesis

Isl1 (Islet1) is a key component of cardiogenesis. Isl1 is a transcription factor that is a characteristic marker for the so-called second heart field, i.e. those cells that are “added” to the heart as it grows. Isl1 is a key component of the network of transcription factors that drives early cardiogenesis in animals of the protostome and deuterostome clades (both belong to the bilatarians, which also include Homo sapiens). “Subsequently, it came to the individual accumulation of cardiac progenitor cells, including Isl1-expressing cells which form in the different heart lineages during the embryogenesis of vertebrates, but not in other animal lineages,” said Michael Kühl giving a highly simplified sketch of the current state of knowledge, which he also recently summarized in a review article. The researchers from Ulm have also characterized frog (Brade et al., 2007) and fruit fly Isl1 (Mann et al., 2009).

Scientific data suggest that Isl1 has two major tasks during cardiogenesis: during the early phase of cardiogenesis, Isl1 is part of a gene regulatory network that is required by the entire population of cardiac progenitor cells. Fruit fly and clawed frog data suggest the presence of a highly conserved network of regulatory interaction partners, including Isl1 as well as members of the GATA, NKx and Tbx transcription factor families. It is likely that a similar network is also present in higher vertebrates (e.g. mice). Isl1 also plays a key role in the development of deuterostome hearts.

The total number of genes that are regulated by a single transcription factor are relatively unknown, and difficult to find out more about. In principle, this would require combining all available methods, which is a Herculean task needing the involvement of many groups of researchers. On the other hand, highly powerful gene sequencing methods mean that this task is no longer totally unthinkable.

Two groups of molecules are key in cardiogenesis

Cardiogenesis is mainly controlled by two groups of molecules, the transcription and growth factors. “Many hundreds of transcription factors are active in the heart muscle cell, but the function and position inside the network of only a handful is known,” said the specialist from Ulm. “In principle, it is like a big jigsaw puzzle. We have put a few pieces that we know in place and are trying to fit any of the larger number of unknown pieces into the right place.” In addition, these gene switches are regulated by extracellular influences.

Michael Kühl has been focused on a large group of growth factors for a long time. The so-called Wnt proteins are signalling molecules that play a key role in embryogenesis. They are also involved in cardiogenesis. Kühl is investigating the signalling pathways that are activated by these Wnt proteins and which signalling pathway is switched on or off at which point in time during cardiogenesis. He is also focused on finding out the signalling pathways that promote and those that inhibit cardiogenesis. Over the last few years, Kühl and his colleagues have made important contributions to finding out how the Wnt proteins act at different points in time during cardiogenesis and how they need to be switched on or off in order for the heart to be able to develop normally.

The knowledge about Wnt signalling can also be adopted to stem cell cultures, to name but one example. Kühl and his group of researchers succeeded in transferring the temporal patterns of embryonic development to stem cells and were able to control the number of cardiomyocetes with specific growth factors. Kühl and his colleagues have worked for around ten years to refine the protocol and in 2002 they succeeded in controlling the number of cardiomyocetes with the growth factor Wnt11. Recently, Kühl’s team of researchers also succeeded in doing the same thing with the transcription factor TBX5 (Herrmann et al., 2011) and embryonic stem cells of mice and Xenopus.

Only growth factors have the potential to be used in clinical application

Although research into growth and transcription factors is important with regard to obtaining information on the cross-species development of the heart, Kühl believes that only growth factors have the potential to be used in clinical application, as the use of transcription factors, which implies the implantation of genetically modified stem cells into patients, would mean having to take into account an incalculable risk.

Michael Kühl hopes that his group, along with the 150 or so basic research oriented research groups around the world, will over the next ten years gain an understanding about the gene regulatory network that controls cardiogenesis that is as complete as possible. Then Kühl would have completed his puzzle. Based on embryogenetic cardiogenesis, Kühl and other researchers around the world will in the meantime focus on identifying those regulatory nodes of the network that are lacking in less complex models. The researchers will seek answers to the following questions: has the number of genes increased during evolution, has the number of interactions increased, or have both things happened?

References and further reading

a) the role of Wnt signalling in cardiogenesis:

Gessert S, Maurus D, Brade T, Walther P, Pandur P, Kühl M (2008) DM-GRASP/ALCAM/CD166 is required for cardiac morphogenesis and maintenance of cardiac identity in first heart field derived cells, Dev. Biol. 321, 150-61

Gessert S and Kühl M (2010) The multiple phases and faces of Wnt signaling during cardiac differentiation and development, Circ. Res., 107, 186-99

Herrmann F, Bundschu K, Kühl SJ, Kühl  M (2011) Tbx5 overexpression favors a first heart field lineage in murine embryonic stem cells and in Xenopus laevis embryos, in: Development  
Dynamics, p. 2634-45. DOI: 10.1002/dvdy.22776

Pandur P, Läsche M, Eisenberg L, Kühl M (2002) Wnt-11 stimulation of a non-canonical Wnt-pathway is required for cardiogenesis, Nature 418, 636-641

b) the role of Islet in cardiogenesis:

Brade T, Gessert S, Kühl M, Pandur P (2007) The amphibian second heart field: Xenopus islet-1 is required for cardiovascular development, Dev. Biol. 311, 297-310

Mann T, Bodmer R, Pandur P (2009) The Drosophila homolog of vertebrate Islet1 is a key component in early cardiogenesis, Development, 136, 317-26

Pandur P, Sirbu IO, Kühl SJ, Philipp M, Kühl M (2012) Islet1 expressing cardiac progenitor cells: A comparison across species, Dev. Genes Evol. DOI: 10.1007/s00427-012.0400-1

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