Reducing Granules Without Splicing Restoration Alleviates RBM20 Cardiomyopathy.

BACKGROUND RBM20 (RNA binding motif protein 20) cardiomyopathy is a severe form of dilated cardiomyopathy (DCM). Genetic variants in the nuclear localization signal of Rbm20 hinder its nuclear import and promote cytoplasmic pathogenic RNP (ribonucleoprotein) granules. We aimed to investigate whether reducing RNP granules by inhibiting Rbm20 expression could alleviate the DCM phenotype in Rbm20 S639G (Rbm20S639G) knock-in mice. METHODS We downregulated Rbm20, utilizing antisense oligonucleotides (ASOs) that specifically inhibit Rbm20 expression. We administered Rbm20-ASOs in Rbm20S639G mice that carry a serine-to-glycine substitution in the nuclear localization signal of RBM20. The Rbm20-ASOs were administered subcutaneously at 25 mg/kg once a week for 8 weeks in both young (14-day-old) mice before the onset of DCM and adult (3-month-old) mice with established DCM phenotype. In vivo cardiac function was assessed by echocardiography. RNP granules were identified through fluorescent immunohistochemical staining, and the number and size of RNP granules were quantified using Cell Profiler software. Alternative splicing of RBM20 target genes was determined by reverse transcription polymerase chain reaction, and titin isoform expression was analyzed by gel electrophoresis. Cardiomyocyte Ca2+ release-reuptake kinetics and mouse electrocardiography were also studied. RESULTS The results revealed that reducing the level of Rbm20 expression through treatment with ASOs significantly decreased the cytoplasmic RNP granules within the Rbm20S639G cardiomyocytes. ASO treatment reduced the severity of DCM developed when treatment was initiated before the onset of the disease. Importantly, ASO treatment reversed cardiac dysfunction and remodeling when treatment was commenced in mice with established DCM as shown by a significant improvement in ejection fraction and a decrease in the severity of left ventricular chamber dilation. Treatment with ASOs also effectively mitigated left ventricular hypertrophic remodeling and improved ECG parameters observed as normalized P wave and QRS durations. These beneficial effects occur without the restoration of mis-splicing of RBM20 target genes, including the primary target gene Ttn, and other genes such as Camk2d, Ryr2, and Ank3. CONCLUSIONS The findings of this study demonstrated that RNP granules serve as a critical driver for RBM20 cardiomyopathy, and reduction of RNP granules through treatment with ASOs is a possible therapeutic option for RBM20 cardiomyopathy in patients carrying RBM20 genetic variants in the nuclear localization signal region.