Abstract 891: Phosphorylation of RNA Binding Motif 20 is a Novel Target to Reduce Myocardial Stiffness in Diastolic Dysfunction

Introduction: Published work has shown that RNA binding motif 20 (RBM20) can improve ventricular stiffening through altering titin sizes in HFpEF, a syndrome with no effective therapeutic options. Reducing RBM20 activity to treat diastolic dysfunction is promising, but it may cause side effects due to its multiple targets except for titin. RBM20 is one of the serine-arginine (SR) proteins of which the phosphorylation plays a critical role in gene splicing. Here we investigate the phosphorylation status of RBM20 and its potential role in diastolic dysfunction. Methods: Insect cell expression system was utilizedfor RBM20 expression. Purified RBM20 was subjected to the middle-down mass spectrometry (MS) analysis. Identified phosphorylation sites were mutated for in-vitro splicing and dual luciferase splice reporter assay through co-transfection with titin mini-gene. In-vitro kinase and co-IP assays were performed to identify the kinases that phosphorylate RBM20. Since phosphorylation is also important for translocation of RNA binding proteins, the confocal microscope was applied to estimate RBM20 trafficking. Results: Sixteen phosphorylation sites with four of them on SR domain of RBM20 were identified with MS analysis. In-vitro splicing and dual luciferase reporter assays revealed that two phosphorylation sites on SR domain play a major role in titin mini-gene splicing. In-vitro kinase assay indicated that kinases SRPK, CLK, and AKT can all phosphorylate RBM20, and co-IP confirmed the interaction between kinases and RBM20. Co-transfection of these kinases with RBM20 and titin mini-gene revealed that splicing pattern was different from co-transfection of mutated RBM20. Interestingly, two-phosphorylation sites mutation on SR domain facilitated RBM20 trafficking from the nucleus to the cytoplasm and thus, form RNA granules, suggesting the new role of RBM20 phosphorylation in diastolic dysfunction. In-vivo studies with mutation knock-in mouse model will be warranted in near future. Conclusion: These results suggest that RBM20 phosphorylation plays an important role in titin splicing, which is a potential target to treat diastolic dysfunction. RBM20 trafficking could be another new pathway to treat HFpEF.