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Hi Magdalena, hi Frank! Congratulations on the European ERA-CVD grant, the national DCVA grant, and the internal UMCU Circulatory Health/UU Jubileumfonds grants! I would like to give you space to introduce yourself, your background, and discuss your projects and visions within these grants.

Magdalena: I am an Assistant Professor at the Department of Cardiology at the UMCU. My research focus can be summarized by the question “Why is DNA not our destiny?”. We still do not understand completely why one family member carrying a pathogenic DNA mutation develops a cardiac disease (cardiomyopathy) in early adulthood while another develops the disease in senior age or even remains healthy. There are several factors that can influence the effect of a pathogenic DNA mutation: it could be an additional mutation in another gene or a non-coding DNA region, post-transcriptional modifications; we even see an effect of non-genetic factors, such as pregnancy, hypertension, chemotherapy treatment, or obesity. This phenomenon is called genome-exposome interaction.

Frank: I am an Assistant Professor at the faculty of Veterinary Medicine at Utrecht ľ�ϸ���Ӱ�� and at the Department of Cardiology at the UMCU. I am working on molecular genetics with patients – cats and dogs – that suffer from cardiomyopathies. These patients exhibit the same clinical features as their human counterparts. These animals are at the same time patients who we try to help, as well as models in which it is easier to keep track of molecular changes and disease progression, with the final goal to find treatments for both humans and pets. The factors influencing genome-exposome interaction contribute to drawing the line between health and disease and by combining our expertise we want to use different models to learn from each other and to better understand this complex interaction in the context of cardiomyopathies in multiple species. Besides, we aim to build a biobank of all cardiomyopathy patients, regardless of their species, which is something completely new.

What are you going to focus specifically on in the three projects?

Frank: All three projects - SCALE, Double-Dose, and MINI-ME - are complementary from the point of studying the genome-exposome interaction in cardiomyopathies. Together with whole teams of additional researchers, we are going to study the genome-exposome interaction in (perfused) cardiomyocytes. Endothelial cells are known to be gate-keepers for substances to reach cardiomyocytes: this interaction is similar to what happens in vivo. We aim to mimic this physiological situation in a custom bioreactor system and focus on genetic mutations that cause cardiomyopathies.

Magdalena: The SCALE grant stands for Single Cardiomyocyte ALlelic imbalancE in hypertrophic cardiomyopathy. It has been granted by the European Research Area Network for Cardiovascular Diseases (ERA-CVD), part of the Horizon program. This specific call was focusing on transnational cardiovascular research projects driven by Early Career Scientists. This is a very nice opportunity for us, we have our own consortium and conduct independent research. We have partners from Germany (Judith Montag), France (Eric Villard), and Spain (Fernando Dominguez). I am the consortium leader, Frank joined with a bioreactor he designed. We are really proud of this one, the grant was in the top 10 of the European-funded projects in this call. SCALE is about a deeper understanding of the allelic imbalance phenomena. In most cases, patients bear a mutation only on one chromosome, having only one copy of the mutated gene. However, on the RNA level, we observe allelic imbalance: the two copies of the gene can be differentially expressed, instead of 50:50 ratio we see it ranging from 0 to 100. The amount of protein translated because of allelic imbalance could determine disease: this might be one of the reasons why DNA is not always our destiny. Jiayi Pei, a junior postdoc from our lab, is going to tackle this problem using various exciting techniques.

Frank: Within MINI-ME, a project supported by the Circulatory Health focus area of the UMCU and the UU Jubileumfonds, we will explore the genome-exposome interaction more broadly. Here we are teaming up with two other UMCU researchers, Annette Baas and Pieter Vader. We will use the same set-up to study the effect of the presence/absence of additional mutations by editing the genome. We can add additional mutations found in patients or remove them, exploring the effect of different genetic backgrounds. We will test the exposome influence on gene expression and phenotypes by using different media to mimic a series of conditions, for example, fasting, specific diets, obesity. With this grant, we will build a system to study any possible clinically relevant condition.

Magdalena: Last week, a large Double-Dose consortium was officially launched. Double-Dose is part of the Dutch Cardiovascular Alliance (DCVA) initiative supported by the Dutch Heart Foundation. It is led by Jolanda van der Velden and Rudolf de Boer together with other researchers from Amsterdam, Utrecht, Groningen, Maastricht, and Rotterdam. For UMCU, Folkert Asselbergs and Peter van Tintelen are senior principal investigators. Annette Baas, Frank, and I are among the junior principal investigators. The main aim of the consortium is to lower the burden of cardiomyopathies and investigate the metabolic aspect of the exposome on cardiomyopathy mutations. Our PhD candidate will start in autumn and we hope to share some exciting first updates with you in the next year.

Which techniques are you going to use in your projects?

Magdalena: Cardiomyocytes from patient hearts or in our in vitro models present a great degree of mosaicism on all levels, including allelic imbalance. We will use a variety of novel techniques to investigate this. Firstly, we are going to use multiple single-cell techniques to map these differences transcriptome-wide, this part is going to be led by Jiayi Pei. We are also looking at localization-specific information, to understand how neighboring cells behave. Currently, we are building a high-throughput image analysis pipeline that would allow us to investigate these differences among neighboring cells in health and cardiomyopathy. We are very happy about our experienced head technician Christian Snijders Blok who is constantly improving the staining combinations suitable for high-throughput image analysis. Our focus will be also on long-read single transcriptome sequencing allowing us to reconstruct whole DNA/RNA haplotypes. Our strength is the unusual multi-species approach paired with a methodological toolset that is currently beyond the state of the art.

Frank: I have been designing a bioreactor that can be perfused on which I also base my VIDI application. I developed this system in which endothelial cells create a vascular network. This can be applied also to cardiac tissue, and I am now working on induced pluripotent stem cells (IPSCs)-derived cardiomyocytes interacting with endothelial cells. We are very excited to see vascular network formation also in this setting. The end goal would be to combine cardiomyocytes, cardiac fibroblasts, and endothelial cells that truly resemble the cardiac endothelium, forming a perfusable model system for the cardiac tissue. This is going to enable us to study the effect of external factors that normally will not get into direct contact with cardiomyocytes. For instance, we can model obesity by providing external fats in the perfusion system through the endothelium, and study the interactions and effects on cardiomyocytes. This system will be applied to study the genome-exposome interaction in cardiomyocytes, in canine, feline and human cells, taking advantage of the iPSC technology widely used at the RMCU.

Magdalena: We are very happy about our local embedding for these projects. Among others, we are working closely with the Epigenomic facility run by Michal Mokry at the UMCU allowing us to be beta-testers for multiple novel techniques to study allele-specific transcriptional regulation. In addition, we are building on the amazing iPSC-cardiomyocyte work that Renee Maas, Alain van Mil, Jan Willem Buikema, Linda van Laake, and many other researchers working with Joost Sluijter have piloted at RMCU. Through cardiogenetics Annette Baas and Peter van Tintelen we have access to a large retrospective and prospective cohorts of genetic cardiomyopathy patients. Last but not least, thanks to the comprehensive UNRAVEL repository led by Folkert Asselbergs, our main supervisor, we have access to the clinical information of the individuals donating heart tissue or other biopsies for iPSC-models. This will allow us a much quicker translation into clinics.

How do you think that the RMCU contributed to these projects?

Magdalena: We have known each other for a long time, since our PhD training. For a certain period after our PhD, we did not work together but we kept meeting regularly, discussing what we could work on as collaborators. We eventually had this chance thanks to the RMU, which served as a hub to enhance our collaboration. As a researcher, you adapt to your scientific environment. In a certain way, our vision is the result of us adapting to the RMCU. The proximity of people from different departments, faculties, and even institutions working together on the same floor is a great and peculiar value of the RMCU. We could have been collaborators in another setting, but thanks to the RMCU we are actual colleagues. In our projects, we have a regenerative medicine approach, aiming to mimic in vivo interactions: the mission consists of creating models to get closer to what happens in vivo. This environment and the RMCU’s mission drove us to this project.

Frank: In the same building, researchers from the Hubrecht Institute, Utrecht ľ�ϸ���Ӱ��, and UMC Utrecht work interconnected with each other: this whole network is one of Utrecht’s strengths, and the RMCU empowers it even further. We join more and more meetings that belong to each other’s field, increasing the interactions between us. Also, the network of collaborators expands. This last year, in particular, this was even accelerated during the COVID-19 pandemic, because we were forced to move these meetings online. We all benefit from this extensive participation in online meetings: if a new mutation is discovered in dogs, we can stain our human materials and perform tests quickly. We are now automatically considering the other species as a control and as a validation cohort. This is something new, that we did not have before: everything is in place now.

What do you think is the biggest advantage of your multispecies approach, also compared to “regular” animal testing?

Frank: The widely accepted in vivo model commonly used for cardiomyopathies is a murine model. If you look at sex differences, sizes, heart-bit rate: there are many differences compared to humans. Cats and dogs are more similar to us, and in addition, are treated as family members. This means that sometimes cats and dogs even eat the same food as their owners. Another relevant point of studying these patients is prevalence. For humans, 1 out of 250 people has a cardiac problem. For cats, this number is 1 out of 7!

Magdalena: Animals live in different homes, different gardens, different environments. The exposome in different individuals is not the same, even if they are genetically similar because of a certain breed. In addition, they are actual patients, with naturally occurring cardiac disease. There are specialized veterinary cardiologists that take care of them. The multispecies level is something we did not even think about before. Now we think this way, and it is really unique.

Frank: We are also working on the database and biobank integrating human, cat, and dog patients. We are going to collect blood for producing IPSCs, make bioreactors, and eventually go back to the clinic, for example improving diagnosis. We can use the plasma from the same patients in our model, for instance after treatment, or after exercise. There are so many possibilities, it is the beginning of something with a universal value, that we could do thanks to the RMU and it is a multidisciplinary environment.

Magdalena: In addition, there is a difference in care that reflects on our research. Cats and dogs are euthanized when no other options are available. Transplantation is not performed on pets. We can collect samples from euthanized animals, at an early disease stage compared to humans. We can make observations on cardiac tissue that we could not do in humans. It is also easier to get fresh material to be used in research. We are learning from any angle.

What makes your research so interesting to you?

Frank: The whole multispecies approach intrigues me in particular. I have been working in canine genetics for over ten years now, so I know the power of this inbred genetic ‘model’. In this collaboration, the translational aspect of genetic diseases is studied from different angles: physiological consequences measured at the cell level and disease progression across species. Both patient groups are helping each other.

Magdalena: My PhD was on Mendelian disorders in which having the mutation directly translated into being a patient. Now, I study something way more complicated, which is challenging for a geneticist, because it is not only a mutation causing a phenotype. And it is not only limited to mutations in DNA, but the whole cell, whole organism, and their environment. It is really complex and fascinating, I often have headaches after our brainstorming meetings! I find it very challenging, much more than finding a mutation and linking it to a phenotype.

How does regenerative medicine link to you?

Magdalena: We are using induced stem cells and bioreactors, but it is not only this. We have mutation carriers that present (or not) the phenotype: how can we keep healthy carriers still healthy for their whole life? How can we eradicate chances of developing a certain disease, given a specific genetic background? We want to focus on chromatin remodeling and transcription factors regulators as prevention. Our end goal is to prevent mutation carriers from ever becoming patients. And we want to do it in any patient, regardless of their species.

Frank: Of course in pets, with existing breeding programs, the most important step to make towards a healthy population is understanding the genetic background of diseases and selecting the best breeding strategies. In complex genetics, however, this will not be entirely feasible. And then again, this would provide a multispecies opportunity. With currently incurable diseases like cardiomyopathies, new approaches could be tested in the preclinical pet model.

What do you expect to be the future in this research field?

Magdalena: What I think will be the future of the field is to have personalized models for every patient. Even pets from the same breed have differences in their DNA, so personalized models will give the freedom to test genome-exposome interactions also for these patients. The field is going towards developing in vitro models that mimic the organism physiology as much as possible, and this is our goal in studying the genome-exposome interactions. In the future, genetic engineering is likely to be developed and feasible to treat patients, removing genetic mutations linked to diseases. But even at that moment – that patients are waiting for –, our approach is still going to be valid: we only identified mutations in 30% of cases of cardiomyopathies in humans. For the rest 70% of patients, mutations are not yet identified. Our model will allow us to test genome-exposome interactions in these cases.

Frank: For veterinary patients, identifying mutations will help to prevent carriers from breeding, but the genetic background has to be understood in order to do this. With our research, we aim to make this real. Additionally, the perfusable heart-on-a-chip truly has lots of potential in studying the genome-exposome interaction in genetic cardiomyopathies by combining genetics with microfluidics, stem cell models, genome editing, state of the art imaging, multi-OMICs … I think Utrecht and specifically RMCU has the perfect environment for a strong foundation.

Thank you both for sharing details on your work and vision with us. Good luck with these intriguing projects, and to all your patients, whatever their species is!

This blog is written by Alessandro Cutilli, PhD candidate in Regenerative Medicine (2019-current).