Abstract Wed043: Genetic Variants in RNA Binding Motif Protein 20 Cause Early-onset Heart Failure by Disrupting Post-Transcriptional Process

Introduction: RNA binding motif protein 20 (RBM20) cardiomyopathy is a severe form of dilated cardiomyopathy (DCM), leading to early-onset heart failure (HF). The condition has been linked to cytoplasmic RBM20 granules induced by RBM20 genetic variants, although the precise mechanism remains unclear. Methods: We identified the protein components of RBM20 granules using proximity labeling proteomics analysis. TurboID biotin ligase was fused to wild-type (WT) or Rbm20 with a S640G variant ( Rbm20 S640G ) and transfected into H9c2 cells, followed by biotin treatment. Biotinylated proteins were captured using streptavidin and analyzed with mass spectrometry. Identified proteins were validated by co-immunofluorescence (Co-IF) and co-immunoprecipitation (Co-IP). RT-PCR was performed to assess gene splicing. Anti-Rbm20 antisense oligonucleotides (ASOs) were administered to Rbm20 S639G knock-in mice to inhibit Rbm20 expression. Results: Proximity labeling identified 32 proteins in nuclear and 13 proteins in cytoplasmic RBM20 granules. Gene ontology analysis revealed that nuclear granule proteins were primarily involved in splicing and transcriptional regulation, while cytoplasm granule proteins were associated with cytoplasmic granule assembly. Notably, two RNA binding proteins (RBPs), CELF1 and MBNL2, were found in both nuclear and cytoplasmic RBM20 granules, as confirmed by Co-IF and Co-IP, suggesting their sequestration in cytoplasmic granules. RT-PCR analysis demonstrated mis-splicing of CELF and MBNL target genes in the heart muscle of Rbm20 S639G mice, but not in Rbm20 knockout mice. Importantly, disassembly of cytoplasmic RBM20 granules via ASO treatment alleviated cardiac function and mitigated disease severity. Conclusion: Our findings suggest that cytoplasmic RBM20 granules sequester essential RBPs, leading to abnormal post-transcriptional regulation, which may contribute to the pathogenesis of severe DCM and HF. Targeting and disassembling cytoplasmic RBM20 granules presents a promising therapeutic approach for alleviating disease progression.