Abstract Body: Introduction/Background: RBM20 cardiomyopathy is a familial dilated cardiomyopathy (DCM) with severe, often early-onset heart failure. RBM20 is a cardiac splicing regulator, and RS-domain variants mis-localize the protein from nuclear speckles to cytoplasmic granules, disrupting splicing of key structural and calcium-handling genes. These mutation-specific defects offer a model to link altered RNA regulation with human DCM.
Research Questions/Hypothesis: We aim to define the molecular composition and functional consequences of cytoplasmic RBM20 granules caused by RS-domain variants. We hypothesize that RS mutations drive DCM via combined loss of nuclear splicing activity and toxic gain-of-function from granule-associated interactors, explaining the aggressive phenotype.
Goals/Aims: (1) Define RBM20 localization, granule formation, and canonical splicing defects in patient myocardium. (2) Map the protein interactome of cytoplasmic RBM20 granules. (3) Characterize global splicing changes in patient ventricles and their reproduction in patient-derived engineered heart tissue.
Methods/Approach: We performed localization and splicing assays on explanted myocardium from patients with RS-domain mutations. TurboID–RBM20 fusion constructs in neonatal rat ventricular myocytes induced cytoplasmic granules for proximity-labeling proteomics. RNA sequencing of patient ventricles and matched engineered tissues will define transcriptome-wide splicing changes with targeted validation.
Results: Myocytes carrying RS-mutations showed RBM20 relocation from nuclear speckles to cytoplasmic puncta and patient myocardium showed increased N2BA/N2B titin ratios, with enhanced inclusion of TTN PEVK, distal Ig, RYR2, and CAMK2D exons, yielding an RS-specific splicing pattern. In RBM20-deficient myocytes, RS variants formed robust cytoplasmic granules; ongoing proteomics is expected to define their interactome.
Conclusions: RS-domain RBM20 mutations define a mutation-specific DCM mechanism that couples cytoplasmic granule formation with titin and calcium-handling mis-splicing absent in idiopathic DCM. Integrating human myocardium with engineered heart tissue will identify conserved, mutation-linked regulatory hubs connecting RBM20 mis-localization to contractile dysfunction and nominate tractable targets for mechanism-based therapies.