Abstract Body: Background: Pathogenic genetic variants in the RBM20 gene, encoding the splicing factor RNA binding motif protein 20 (RBM20), cause an aggressive form of dilated cardiomyopathy (DCM). Many disease-associated variants disrupt RBM20 nuclear localization, leading to the mis-splicing of target genes and accumulation of the protein in cytoplasmic granules. Our recent study showed that cytoplasmic RBM20 granules contribute to DCM; however, whether they are sufficient to drive DCM independent of splicing defects remains unresolved.

Research hypothesis: We tested the hypothesis that cytoplasmic RBM20 granules are sufficient to induce severe DCM in the setting of minimal RBM20 target gene mis-splicing.

Approach: To directly isolate the effects of cytoplasmic RBM20 granules, we generated a novel mouse model with S639G mutant RBM20 inserted in the Rosa26 safe harbor locus downstream of a lox-stop-lox sequence using CRISPR-Cas9. These mice were crossed with Myh6-Cre mice to generate mice with cardiac-specific expression of mutant RBM20 in an otherwise wild-type background (Rosa26^{Rbm20-S639G/+};Myh6-Cre). RBM20 expression and subcellular localization were assessed by qPCR, Western blot, and immunohistochemistry. RBM20-dependent splicing was evaluated by SDS-agarose gel electrophoresis and RT-PCR in Rosa26^{Rbm20-S639G/+};Myh6-Cre mice. Cardiac structure and function were assessed by histology and echocardiography, and global transcriptomic changes were analyzed by RNA-seq.

Results: Rosa26^{Rbm20-S639G/+};Myh6-Cre mice exhibited robust cytoplasmic RBM20 granule formation with only modest alterations in canonical RBM20 target gene splicing, establishing a model that functionally separates granule formation from mis-splicing. Despite relatively preserved splicing, these mice developed severe DCM, characterized by marked ventricular dilation, wall thinning, and reduced systolic function, by 1-month-of-age. This was accompanied by striking premature mortality, with >90% lethality by 100 days-of-age. Transcriptomic profiling revealed gene expression changes consistent with advanced heart failure.

Conclusions: These findings provide direct in vivo evidence that cytoplasmic RBM20 granules are sufficient to drive severe DCM independent of major splicing defects. This establishes cytoplasmic RBM20 granules as a primary, gain-of-function disease mechanism in RBM20 cardiomyopathy caused by variants that disrupt RBM20 nuclear localization, highlighting them as a critical therapeutic target.