Methawasin Mei, Zhang Yanghai, Gregorich Zachery R, He Yaqin, Liu Chunling, Muldoon Julia, Hourani Zaynab, Smith John E, Granzier Henk, Guo Wei
Department 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.).
Department of Medical Pharmacology and Physiology, University of Missouri, Columbia (M.M.).
Circ Res. 2025 May 9;136(10):1134-1146. doi: 10.1161/CIRCRESAHA.124.324781. Epub 2025 Apr 17.
RBM20 (RNA binding motif protein 20) cardiomyopathy is a severe form of dilated cardiomyopathy (DCM). Genetic variants in the nuclear localization signal of hinder its nuclear import and promote cytoplasmic pathogenic RNP (ribonucleoprotein) granules. We aimed to investigate whether reducing RNP granules by inhibiting expression could alleviate the DCM phenotype in S639G () knock-in mice.
We downregulated , utilizing antisense oligonucleotides (ASOs) that specifically inhibit expression. We administered -ASOs in mice that carry a serine-to-glycine substitution in the nuclear localization signal of RBM20. The -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 Ca release-reuptake kinetics and mouse electrocardiography were also studied.
The results revealed that reducing the level of expression through treatment with ASOs significantly decreased the cytoplasmic RNP granules within the 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 , and other genes such as , , and .
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 genetic variants in the nuclear localization signal region.
RBM20（RNA结合基序蛋白20）心肌病是扩张型心肌病（DCM）的一种严重形式。其核定位信号中的基因变异阻碍了它的核输入，并促进了细胞质致病性核糖核蛋白（RNP）颗粒的形成。我们旨在研究通过抑制其表达来减少RNP颗粒是否能减轻RBM20 S639G（RBM20 S639G）基因敲入小鼠的DCM表型。
我们利用特异性抑制RBM20表达的反义寡核苷酸（ASO）下调RBM20的表达。我们在RBM20核定位信号中携带丝氨酸到甘氨酸替代的小鼠中给予RBM20 -ASO。在DCM发病前的年轻（14日龄）小鼠和具有已确立DCM表型的成年（3月龄）小鼠中，以25 mg/kg的剂量每周皮下注射一次RBM20 -ASO，持续8周。通过超声心动图评估体内心脏功能。通过荧光免疫组织化学染色鉴定RNP颗粒，并使用Cell Profiler软件对RNP颗粒的数量和大小进行定量。通过逆转录聚合酶链反应确定RBM20靶基因的可变剪接，并通过凝胶电泳分析肌联蛋白异构体的表达。还研究了心肌细胞钙释放-再摄取动力学和小鼠心电图。
结果显示，通过ASO治疗降低RBM20表达水平可显著减少RBM20 S639G心肌细胞内的细胞质RNP颗粒。在疾病发作前开始治疗时，ASO治疗降低了DCM的严重程度。重要的是，当在已确立DCM的小鼠中开始治疗时，ASO治疗逆转了心脏功能障碍和重塑，表现为射血分数显著改善和左心室腔扩张严重程度降低。ASO治疗还有效减轻了左心室肥厚性重塑，并改善了观察到的心电图参数，如P波和QRS波持续时间正常化。这些有益效果在未恢复RBM20靶基因（包括主要靶基因TTN）以及其他基因（如FLNC、LMNA和MYBPC3）的错误剪接的情况下发生。
本研究结果表明，RNP颗粒是RBM20心肌病的关键驱动因素，通过ASO治疗减少RNP颗粒是核定位信号区域携带RBM20基因变异的患者RBM20心肌病的一种可能治疗选择。
