I536T variant of RBM20 affects splicing of cardiac structural proteins that are causative for developing dilated cardiomyopathy

Abstract

RBM20 is one of the genes predisposing to dilated cardiomyopathy (DCM). Variants in the RS domain have been reported in many DCM patients, but the pathogenicity of variants within the RNA-recognition motif remains unknown. Two human patients with the I536T-RBM20 variant without an apparent DCM phenotype were identified in sudden death cohorts. A splicing reporter assay was performed, and an I538T knock-in mouse model (Rbm20I538T) was generated to determine the significance of this variant. The reporter assay demonstrated that the human I536T variant affected the TTN splicing pattern compared to wild-type. In the mouse experiments, Rbm20I538T mice showed different splicing patterns in Ttn, Ldb3, Camk2d, and Ryr2. The expressions of Casq1, Mybpc2, and Myot were upregulated in Rbm20I538T mice, but Rbm20I538T mice showed neither DCM nor cardiac dysfunction on histopathological examination and ultrasound echocardiography. The I536T-RBM20 (I538T-Rbm20) variant changes gene splicing and affects gene expression, but the splicing and expression changes in Ttn and Ca handling genes such as Casq1, Camk2d, and Ryr2 do not cause DCM morphology in the mouse model. Key messages • Two human patients with the I536T-RBM20 variant without a DCM phenotype were identified. • A splicing reporter assay demonstrated that the variant affected the TTN splicing. • Rbm20I538T mice showed neither DCM nor cardiac dysfunction. • Rbm20I538T mice showed different splicing patterns and the gene expressions.

Disruption of the nuclear localization signal in RBM20 is causative in dilated cardiomyopathy

“…[4][5][6][7][8] However, it remains unclear which of these mechanisms is the driving force behind the development of DCM. Notably, multiple studies have now shown that mice with disrupted splicing of RBM20 target transcripts do not develop an overt DCM phenotype, 17,18 indicating that altered splicing alone does not significantly contribute to the development of RBM20 cardiomyopathy. Herein, employing a novel RBM20 RS domain deletion mouse model in combination with in vitro experiments, we show that loss of RBM20 nuclear localization, rather than disrupted splicing, is the driving mechanism in DCM.…”

“…17 Similarly, it was recently demonstrated that mice harboring the I536T variant, which is located within the RRM, develop neither DCM nor cardiac dysfunction despite altered splicing of RBM20 target transcripts. 18 Given that the RBM20 RS domain is a hotspot for DCM-linked mutations (along with the RRM and E-rich regions), 19,20 in this study we sought to (1) determine the function of the RBM20 RS domain in DCM and (2) examine the underlying mechanism(s) of its regulation in DCM-related mis-localization.…”

Mechanisms of RBM20 Cardiomyopathy: Insights From Model Systems

“…This variant was previously identified in an apparent familial case of sudden cardiac death 76 ; however, Rbm20 I538T mice did not develop cardiomyopathy or exhibit sudden death until late in life. 89 We have also previously demonstrated that Rbm20 ΔRRM mice display splicing defects in major RBM20 target genes but do not show evidence of pathological remodeling. 24 Moreover, systolic dysfunction was only detected in homozygous Rbm20 ΔRRM mice, 24 whereas the majority of patients with DCM-associated variants in RBM20 carry the variants in a heterozygous state.…”

“…At the molecular level, RBM20 splicing deficiency produced by genetic ablation or loss of nuclear localization induces a characteristic shift in titin isoform expression from the dominant N2B isoform to the longer and more compliant N2BA isoforms. [9][10][11][12]20,22,89 This shift decreases the passive tension of the sarcomere and can, via the effect of passive tension on calcium sensitivity, 24,107 explain impaired systolic function in Rbm20 knockout rodents. 108…”

“…In addition to titin, RBM20 animal models also exhibit disrupted splicing of Ca 2+ -handling genes. [9][10][11][12]20,22,30,89,109 One such change is the exclusion of exon 14 and the inclusion of exons 15 and 16 in Camk2d (Figure 5). At the protein level, these changes result in a near-complete switch from the CaMKII-δB isoform to the CaMKII-δA and CaMKII-δ9 isoforms.…”