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

Periodic Reporting for period 1 - Titin Signals (Molecular dissection of titin-based mechanisms in charge of cardiomyocyte dysfunction in terminal feart failure)

Reporting period: 2016-07-01 to 2018-06-30

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 and it is leading cause of hospitalization in people over the age of 65. 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 model 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). Also, the Chen laboratory recently demonstrated that inactivation of the CARP gene could protect from HF in a murine MLP gene-based HF failure model. 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 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.
In 2016-2018 I was working in Dr. Ju Chen´s lab at UCSD. His world-leading group is specializing in murine models for the 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. I have got mouse line with the proper insertion of one LoxP site and will use this line for the introduction of second LoxP site to obtain N2A region deficient mouse line with potential for cardiac-specific deletion.
While being at UCSD, I have also 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 monomer of N2A region displaces dimer of CARP to form 1:1 complex. In contrast to previous reports, we have shown that N2A region is not intrinsically unfolded entropic spring but adopts exceptionally thermostable helical fold. Moreover, we have demonstrated that adjacent I81 Ig domain is required for the formation of the N2A-CARP complex. We have solved the high-resolution crystal structure of I81 and revealed unique loop extensions contributing to complex formation. Results of this study have been published. 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. Results of these experiments are being integrated with biomolecular NMR data from the University of Konstanz. These results will help to elucidate N2A-CARP binding surface at the atomic level, that will serve for targeted development interaction inhibitors. At UCSD, I performed a virtual screen for small molecules inhibiting N2A-CARP interaction based on the provided model and obtained 72 hit compounds in physical form.
In my return phase in Europe, I have received office and lab space at ZI, where I have started to experimentally validate the efficiency of compounds from N2A-CARP interaction screen and master mouse phenotyping methods using advanced imaging available in Mannheim. Working at ZI, I have access to a small animal imaging facility equipped with a 9.4T Bruker BioSpec MRI scanner. Besides, at the ZI I am using state-of-the-art animal house facility where transgenic colonies are bred in an SPF-environment. Finally, at the ZI, I am using state-of-the-art molecular biology equipped labs, suitable for recombinant protein expression and small molecule screens.
ZI helps 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 will be performed in ZI and Vilnius University.
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. This collaboration resulted in 4 high impact publications.
Titin Signals is a first comprehensive study of clinically relevant N2A-CARP interactions at different levels of biological complexity. We advanced our fundamental understanding of N2A-CARP interaction, and by the end of this project, we will be able to exploit this information for the targeted design of small molecule N2A-CARP interaction inhibitors. The promising hits then will be 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 best MSc thesis in biological sciences in the competition organized by Lithuanian Academy of Sciences. This award and co-authorship was a vital factor 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.
Titin Signals in one photo