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Molecular dissection of titin-based mechanisms in charge of cardiomyocyte dysfunction in terminal feart failure

Periodic Reporting for period 2 - Titin Signals (Molecular dissection of titin-based mechanisms in charge of cardiomyocyte dysfunction in terminal feart failure)

Reporting period: 2018-07-01 to 2019-06-30

Cardiovascular diseases (CVD) cause over 1.8 million deaths in the European Union yearly, resulting in the overall estimated burden of 210 billion Euros in 2016. Half of the costs are due to health care expenses, one quarter due to productivity losses, and the remaining amount is associated with the informal care of people with CVD. Among CVDs, the burden of heart failure (HF) is substantial and likely to grow: at present, 15 million people living with heart failure in Europe. HF is the leading cause of hospitalization in people over the age of 65. The current management of HF mainly targets the secondary pathophysiological adaptations, but not the myocardial dysfunction itself and is not efficient. Overall, 50% of patients are dead within four years. Thus, there is a massive demand for new therapy and diagnosis methods. Identification of new HF therapy targets within the cardiomyocyte contractile apparatus would contribute to better management of HF and would lead to the reduction of CVD associated costs.
Titin molecule is a central player in cardiovascular health and disease. Titin is a giant protein, longer than one micrometer, that functions as tightly regulated molecular spring, governing biomechanical properties of striated muscle, including the heart. Titin not only contributes to biomechanical properties of cardiac muscle but is a core site for the functional integration of sarcomeric signaling. Numerous animal models and patient biopsy studies have reported the induction of proteins binding to the titin N2A spring region under myocardial stress conditions, including members of the CARP family (Cardiac Ankyrin Repeat proteins). Therefore, the titin N2A-CARP signaling axis may represent a target for HF therapeutic strategies. This multidisciplinary project aims to develop novel HF therapeutic strategies based upon modulating the N2A-CARP signaling axis. This principle goal is pursued by different methodological approaches, including the development and phenotypic characterization of the N2A deficient mouse model, the development of Adeno-associated viral vectors for cardiac-specific CARP overexpression, screens for small molecule modulators of N2A-CARP interaction, and finally a structural characterization of the titin N2A-CARP complex. This multidisciplinary approach will provide me with in-depth training in translational molecular cardiology and provide novel translational insights into heart failure therapy.
During my outgoing phase, I was working at Dr. Ju Chen´s lab at UCSD. His world-leading group is specializing in murine models for cardiovascular research, where I obtained here high-end training in mouse genetics and cardiovascular phenotyping. Using advanced CRISPR/Cas9 technology, I have made significant progress in developing the N2A region deficient mouse line. During my outgoing phase, I worked closely with Dr. Mayans on structural characterization of N2A-CARP interaction. We have developed well-behaved constructs of CARP and N2A region amenable for structural studies. We have demonstrated that the monomer of the N2A region displaces the homodimer of CARP to form 1:1 complex. In contrast to previous reports, we have shown that the N2A region is not intrinsically unfolded entropic spring but adopts exceptionally thermostable helical fold. We have solved the high-resolution crystal structure of adjacent I81 domain and revealed unique loop extensions contributing to complex formation. The results of this study have been published.
During my return phase in Europe, I have received office and lab space at ZI, where I further studied N2A-CARP interaction and mastered mouse phenotyping methods using advanced small animal imaging facility available in Mannheim. Besides, at the ZI, I am using state-of-the-art animal house facility and molecular biology equipped labs, suitable for recombinant protein expression and small molecule screens.
ZI helped to set up a small research group at Vilnius University with additional funding from Lithuanian Research Council and Leducq Foundation-funded 13CVD research network. I am currently supervising Ph.D. student Ieva Rinkūnaitė at Vilnius University. Further phenotypic characterization of mouse models and testing of N2A-CARP interaction modulators have to be performed in ZI and Vilnius University.
In collaboration with Olga Mayans, I have performed deuterium/hydrogen exchange experiments using mass spectroscopy, to further characterize the 3D structure of single proteins and the binding surface of the N2A-CARP complex. These results helped to elucidate the N2A-CARP binding surface at the atomic level, which served for targeted development interaction inhibitors. Moreover, we discovered that the N2A-CARP complex interacts with an unexpected ligand that is essential for cardiac function.
In summary, my outgoing phase research period successfully contributed to research in Dr. Chen´s lab on the molecular mechanisms that couple mechanical cardiomyocyte strain and cardiomyocyte loss during the heart failure involving the mechanosensing titin filament. During my secondment to UCSD, I have acquired new technical qualifications for the generation of genetic mouse models for cardiovascular research and murine cardiovascular phenotyping. I brought these newly obtained competencies back to the EU, where I implemented them while working at ZI and setting up my research group at Vilnius university. The results of my work have been published in 9 scientific publications, presented in numerous scientific conferences, and two more manuscripts are currently in preparation. In the framework of promoting public awareness of science, the results of this project were communicated to the general audience as short communications, interviews, and podcasts.
Competences acquired during this fellowship were a key component allowing me to become a mentor for a Ph.D. student and initiate a transgenic core facility at Vilnius University.
Titin Signals is the first comprehensive study of clinically relevant N2A-CARP interactions at different levels of biological complexity. We advanced our fundamental understanding of N2A-CARP interaction. At the end of the project, we have obtained structural information that would form a basis for the targeted design of small molecule N2A-CARP interaction inhibitors. The promising hits have been characterized biophysically, and lead compounds will be tested in murine models as novel therapeutic agents for HF.
Work on Titin Signals resulted in two successfully defended MSc thesis at Vilnius University. MSc thesis of Egidijus Šimoliūnas (2016) and Ieva Rinkūnaitė (2017) were awarded the best MSc thesis in biological sciences. This award and co-authorship were vital factors in their successful acceptance to Ph.D. programs at Vilnius University. Moreover, Ieva Rinkūnaitė has chosen me as a primary mentor, and she is the core personnel of my research unit at Vilnius University.
Obtained competencies inspired me to have gathered a team of specialists as a workforce for future animal transgenics core facility. Currently, highly motivated student is being trained in mouse transgenics in Heidelberg, Germany. I have presently submitted two proposals to establish a transgenic animal core facility specializing in non-murine mammalian disease models. If this proposal gets funded, it will create new market opportunities and strengthen the competitiveness of the EU market.