Regulation der Expression und Aktivität von RBM20 im gesunden und erkrankten Herzen

• Antragsteller: Professor Dr. Michael Gotthardt
• Fachliche Zuordnung: Kardiologie, Angiologie
• Förderung: Förderung von 2015 bis 2022
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 271048839

• Erstellungsjahr: 2024

Zusammenfassung der Projektergebnisse

In the the proposed work, our main focus was to understand the role of the splicing factor RBM20 in cardiac health and disease, particularly its mutation in a severe form of dilated cardiomyopathy as well as its involvement in heart failure with preserved ejection fraction (HFpEF). Our research was cantered around three main hypotheses aimed at exploring the regulation of RBM20 expression, its mediation of alternative splicing, and the functional consequences of RBM20 mutations associated with dilated cardiomyopathy (DCM). We made ssignificant progress across all objectives despite experimental challenges and limitations posed by sample availability and the constraints of maintaining and breeding mice during the COVID-19 pandemic. A notable achievement was the completion and publication of studies related to the molecular and mechanistic basis of RBM20-dependent splicing, including the identification of PTB4 as a novel regulator of Titin splicing. Additionally, the generation of novel RBM20 knockout mice did provide additional insights into the complex regulation of RBM20 isoform expression. We uncovered several mechanisms through which RBM20 mutations lead to reduced splicing activity, mainly affecting the localization of splicing factors post-phosphorylation. This was illustrated by the analysis of diverse RBM20 mutations, highlighting the diverse impact of these genetic variations. Key findings from the conducted word include: The spatial and temporal regulation of RBM20 significantly influences titin isoform expression during cardiac development and pathology, correlating with stages of cardiac remodelling. This underscores RBM20's pivotal role in cardiac physiology and pathology. We identified critical cis-elements contributing to RBM20's transcriptional regulation and revealed a more complex exon structure than previously known. These findings have implications for the interpretation of patient mutations in the 5’ region of the gene and for therapeutic targeting, especially in conditions associated with aberrant RBM20 expression. Through the use of minigene reporters and deletion constructs, we published a refined picture of RBM20-mediated splicing regulation, offering insights crucial for developing interventions to correct splicing abnormalities in heart diseases. The analysis of RBM20 mutants linked to DCM revealed that mutations leading to clinical phenotypes were associated with differential localization and accumulation outside the nucleus, suggesting that restoring normal nuclear localization could potentially rescue the phenotype. Furthermore, the insights gained into RBM20 expression during embryonic development, the establishment of orthogonal organic phase separation (OOPS) for studying RNA/protein interactions, and the identification of pharmacological interventions to modulate RBM20 expression represent significant advances. In conclusion, our research has substantially advanced our understanding of RBM20's function in the heart and extended the basis to develop novel therapeutic strategies for cardiac diseases. The identification of pharmacological modulators of RBM20 expression and splicing activity, along with antisense approaches, has the potential to improve drug development targeting specific molecular pathways involved in cardiomyopathy and heart failure. Future studies will focus on the regulation of RBM20 and its interconnection with other factors in a cardiac splice regulatory network, aiming to translate these findings into clinical applications and validate therapeutic efficacy in animal models of heart disease.

Projektbezogene Publikationen (Auswahl)

• Reducing RBM20 activity improves diastolic dysfunction and cardiac atrophy. Journal of Molecular Medicine, 94(12), 1349-1358.
  Hinze, Florian; Dieterich, Christoph; Radke, Michael H.; Granzier, Henk & Gotthardt, Michael
  
• Drug discovery with an RBM20 dependent titin splice reporter identifies cardenolides as lead structures to improve cardiac filling. PLOS ONE, 13(6), e0198492.
  Liss, Martin; Radke, Michael H.; Eckhard, Jamina; Neuenschwander, Martin; Dauksaite, Vita; von Kries, Jens-Peter & Gotthardt, Michael
  
• Expanding the map of protein-RNA interaction sites via cell fusion followed by PAR-CLIP. RNA Biology, 15(3), 359-368.
  Hinze, Florian; Drewe-Boss, Philipp; Milek, Miha; Ohler, Uwe; Landthaler, Markus & Gotthardt, Michael
  
• Molecular basis of titin exon exclusion by RBM20 and the novel titin splice regulator PTB4. Nucleic Acids Research, 46(10), 5227-5238.
  Dauksaite, Vita & Gotthardt, Michael
  
• iPSC Modeling of RBM20-Deficient DCM Identifies Upregulation of RBM20 as a Therapeutic Strategy. Cell Reports, 32(10), 108117.
  Briganti, Francesca; Sun, Han; Wei, Wu; Wu, Jingyan; Zhu, Chenchen; Liss, Martin; Karakikes, Ioannis; Rego, Shannon; Cipriano, Andrea; Snyder, Michael; Meder, Benjamin; Xu, Zhenyu; Millat, Gilles; Gotthardt, Michael; Mercola, Mark & Steinmetz, Lars M.
  
• Proteome-wide quantitative RNA-interactome capture identifies phosphorylation sites with regulatory potential in RBM20. Molecular Cell, 82(11), 2069-2083.e8.
  Vieira-Vieira, Carlos Henrique; Dauksaite, Vita; Sporbert, Anje; Gotthardt, Michael & Selbach, Matthias
  
• Loss of Cardiac Splicing Regulator RBM20 Is Associated With Early-Onset Atrial Fibrillation. JACC: Basic to Translational Science, 9(2), 163-180.
  Vad, Oliver B.; Angeli, Elisavet; Liss, Martin; Ahlberg, Gustav; Andreasen, Laura; Christophersen, Ingrid E.; Hansen, Camilla C.; Møller, Sophie; Hellsten, Ylva; Haunsoe, Stig; Tveit, Arnljot; Svendsen, Jesper H.; Gotthardt, Michael; Lundegaard, Pia R. & Olesen, Morten S.