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Reducing Granules Without Splicing Restoration Alleviates RBM20 Cardiomyopathy
Mei Methawasin1,2, Yanghai Zhang3, Zachery R Gregorich3
1Department of Cellular and Molecular Medicine and Sarver Molecular Cardiovascular Research Program, The University of Arizona, Tucson (M.M., J.M., Z.H., J.E.S., H.G.).
Insights
Reducing RNA binding motif protein 20 (RBM20) expression with antisense oligonucleotides (ASOs) decreased pathogenic RNP granules and alleviated RBM20 cardiomyopathy in mice. This approach offers a potential therapy for RBM20-related heart conditions.
Area of Science:
- Cardiovascular Biology
- Molecular Medicine
- Genetic Cardiology
Background:
- RBM20 cardiomyopathy is a severe DCM linked to nuclear import defects of RBM20.
- Defective RBM20 import causes pathogenic cytoplasmic ribonucleoprotein (RNP) granules.
- RBM20 variants in the nuclear localization signal are implicated in disease pathogenesis.
Purpose of the Study:
- To investigate if reducing RNP granules by inhibiting RBM20 expression can ameliorate DCM in RBM20 S639G knock-in mice.
- To assess the therapeutic potential of antisense oligonucleotides (ASOs) targeting RBM20 expression.
Main Methods:
- Administration of RBM20-specific ASOs to RBM20 S639G mice at different disease stages (pre-symptomatic and established).
- Assessment of cardiac function via echocardiography, RNP granule quantification, alternative splicing analysis, and electrocardiography.
- Analysis of titin isoform expression and cardiomyocyte calcium handling.
Main Results:
- ASO treatment significantly reduced cytoplasmic RNP granules in cardiomyocytes.
- Early ASO administration attenuated DCM severity; late administration reversed established cardiac dysfunction and remodeling.
- Beneficial effects included improved ejection fraction, reduced chamber dilation, mitigated hypertrophy, and normalized ECG parameters, independent of splicing restoration.
Conclusions:
- Cytoplasmic RNP granules are key drivers of RBM20 cardiomyopathy.
- Reducing RNP granules via ASO therapy is a promising therapeutic strategy for RBM20 cardiomyopathy.
- This approach may benefit patients with genetic variants affecting RBM20 nuclear import.
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.
