Female carriers of a Duchenne muscular dystrophy (DMD) gene mutation manifest exercise intolerance and metabolic anomalies that may be exacerbated following menopause due to the loss of estrogen, a known regulator of skeletal muscle function and metabolism. Here, we studied the impact of estrogen depletion (via ovariectomy) on exercise tolerance and muscle mitochondrial metabolism in female mdx mice and the potential of estrogen replacement therapy (using estradiol) to protect against functional and metabolic perturbations. We also investigated the effect of estrogen depletion, and replacement, on the skeletal muscle proteome through an untargeted proteomic approach with TMT-labelling. Our study confirms that loss of estrogen in female mdx mice reduces exercise capacity, tricarboxylic acid cycle intermediates, and citrate synthase activity but that these deficits are offset through estrogen replacement therapy. Furthermore, ovariectomy downregulated protein expression of RNA-binding motif factor 20 (Rbm20), a critical regulator of sarcomeric and muscle homeostasis gene splicing, which impacted pathways involving ribosomal and mitochondrial translation. Estrogen replacement modulated Rbm20 protein expression and promoted metabolic processes and the upregulation of proteins involved in mitochondrial dynamics and metabolism. Our data suggest that estrogen mitigates dystrophinopathic features in female mdx mice and that estrogen replacement may be a potential therapy for post-menopausal DMD carriers.

The post-transcriptional regulation of gene expression plays an important role in heart development and disease. Cardiac-specific alternative splicing, mediated by RNA-binding proteins, orchestrates the isoform switching of proteins that are essential for cardiomyocyte organization and contraction. Dysfunctions of RNA-binding proteins impair heart development and cause the main types of cardiomyopathies, which represent a heterogenous group of abnormalities that severely affect heart structure and function. In particular, mutations of RBM20 and RBFOX2 are associated with dilated cardiomyopathy, hypertrophic cardiomyopathy, or hypoplastic left heart syndrome. Functional analyses in different animal models also suggest possible roles for other RNA-binding proteins in cardiomyopathies because of their involvement in organizing cardiac gene programming. Recent studies have provided significant insights into the causal relationship between RNA-binding proteins and cardiovascular diseases. They also show the potential of correcting pathogenic mutations in RNA-binding proteins to rescue cardiomyopathy or promote cardiac regeneration. Therefore, RNA-binding proteins have emerged as promising targets for therapeutic interventions for cardiovascular dysfunction. The challenge remains to decipher how they coordinately regulate the temporal and spatial expression of target genes to ensure heart function and homeostasis. This review discusses recent advances in understanding the implications of several well-characterized RNA-binding proteins in cardiomyopathies, with the aim of identifying research gaps to promote further investigation in this field.

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We showed an association between atrial fibrillation and rare loss-of-function (LOF) variants in the cardiac splicing regulator RBM20 in 2 independent cohorts. In a rat model with loss of RBM20, we demonstrated altered splicing of sarcomere genes (NEXN, TTN, TPM1, MYOM1, and LDB3), and differential expression in key cardiac genes. We identified altered sarcomere and mitochondrial structure on electron microscopy imaging and found compromised mitochondrial function. Finally, we demonstrated that 3 novel LOF variants in RBM20, identified in patients with atrial fibrillation, lead to significantly reduced splicing activity. Our results implicate alternative splicing as a novel proarrhythmic mechanism in the atria.

Sorafenib, a multi-targeted tyrosine kinase inhibitor, is a first-line treatment for advanced solid tumors, but it induces many adverse cardiovascular events, including myocardial infarction and heart failure. These cardiac defects can be mediated by alternative splicing of genes critical for heart function. Whether alternative splicing plays a role in sorafenib-induced cardiotoxicity remains unclear. Transcriptome of rat hearts or human cardiomyocytes treated with sorafenib was analyzed and validated to define alternatively spliced genes and their impact on cardiotoxicity. In rats, sorafenib caused severe cardiotoxicity with decreased left ventricular systolic pressure, elongated sarcomere, enlarged mitochondria and decreased ATP. This was associated with alternative splicing of hundreds of genes in the hearts, many of which were targets of a cardiac specific splicing factor, RBM20. Sorafenib inhibited RBM20 expression in both rat hearts and human cardiomyocytes. The splicing of RBM20\'s targets, SLC25A3 and FHOD3, was altered into fetal isoforms with decreased function. Upregulation of RBM20 during sorafenib treatment reversed the pathogenic splicing of SLC25A3 and FHOD3, and enhanced the phosphate transport into mitochondria by SLC25A3, ATP synthesis and cell survival.We envision this regulation may happen in many drug-induced cardiotoxicity, and represent a potential druggable pathway for mitigating sorafenib-induced cardiotoxicity.

RBM20 cardiomyopathy is an arrhythmogenic form of dilated cardiomyopathy caused by mutations in the splicing factor RBM20. A recent study found a more severe phenotype in male patients with RBM20 cardiomyopathy patients than in female patients. Here, we aim to determine sex differences in an animal model of RBM20 cardiomyopathy and investigate potential underlying mechanisms. In addition, we aim to determine sex and gender differences in clinical parameters in a novel RBM20 cardiomyopathy patient cohort. We characterized an Rbm20 knockout (KO) mouse model, and show that splicing of key RBM20 targets, cardiac function, and arrhythmia susceptibility do not differ between sexes. Next, we performed deep phenotyping of these mice, and show that male and female Rbm20-KO mice possess transcriptomic and phosphoproteomic differences. Hypothesizing that these differences may influence the heart\'s ability to compensate for stress, we exposed Rbm20-KO mice to acute catecholaminergic stimulation and again found no functional differences. We also replicate the lack of functional differences in a mouse model with the Rbm20-R636Q mutation. Lastly, we present a patient cohort of 33 RBM20 cardiomyopathy patients and show that these patients do not possess sex and gender differences in disease severity. Current mouse models of RBM20 cardiomyopathy show more pronounced changes in gene expression and phosphorylation of cardiac proteins in male mice, but no sex differences in cardiac morphology and function. Moreover, other than reported before, male RBM20 cardiomyopathy patients do not present with worse cardiac function in a patient cohort from Germany and the Netherlands.NEW & NOTEWORTHY Optimal management of the cardiac disease is increasingly personalized, partly because of differences in outcomes between sexes. RBM20 cardiomyopathy has been described to be more severe in male patients, and this carries the risk that male patients are more scrutinized in the clinic than female patients. Our findings do not support this observation and suggest that treatment should not differ between male and female RBM20 cardiomyopathy patients, but instead should focus on the underlying disease mechanism.

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.

(1) Background: RNA binding motif 20 (RBM20) regulates mRNA splicing specifically in muscle tissues. Missense mutations in the arginine/serine (RS) domain of RBM20 lead to abnormal gene splicing and have been linked to severe dilated cardiomyopathy (DCM) in human patients and animal models. Interestingly, many of the reported DCM-linked missense mutations in RBM20 are in a highly conserved RSRSP stretch within the RS domain. Recently, it was found that the two Ser residues within this stretch are constitutively phosphorylated, yet the identity of the kinase(s) responsible for phosphorylating these residues, as well as the function of RSRSP phosphorylation, remains unknown. (2) Methods: The ability of three known SR protein kinases (SRPK1, CLK1, and AKT2) to phosphorylate the RBM20 RSRSP stretch and regulate target gene splicing was evaluated by using both in vitro and in vivo approaches. (3) Results: We found that all three kinases phosphorylated S638 and S640 in the RSRSP stretch and regulated RBM20 target gene splicing. While SRPK1 and CLK1 were both capable of directly phosphorylating the RS domain in RBM20, whether AKT2-mediated control of the RS domain phosphorylation is direct or indirect could not be determined. (4) Conclusions: Our results indicate that SR protein kinases regulate the splicing of a cardiomyopathy-relevant gene by modulating phosphorylation of the RSRSP stretch in RBM20. These findings suggest that SR protein kinases may be potential targets for the treatment of RBM20 cardiomyopathy.

Dilated cardiomyopathy (DCM) is characterized by the dilation and impaired contraction of 1 or both ventricles and can be caused by a variety of disorders. Up to 50% of idiopathic DCM cases have heritable familial diseases, and the clinical screening of family members is recommended. Identifying a genetic cause that can explain the DCM risk in the family can help with better screening planning and clinical decision-making. Whole-exome sequencing (WES) has aided significantly in the detection of causative genes in many genetically heterogeneous diseases. In the present study, we applied WES to identify the causative genetic variant in a family with heritable DCM.
WES was applied to identify genetic variants on a 26-year-old man as the proband of a family with DCM. Subsequently, Sanger sequencing was performed to confirm the variant in the patient and all the available affected and unaffected family members. The pathogenicity of the variant was evaluated through co-segregation analysis in the family and employment of in silico predictive software.
WES demonstrated the missense pathogenic heterozygous nucleotide variant, c.1907G > A, (p.Arg636His, rs267607004, NM_0011343), in exon 9 of the RBM20 gene in the proband. The variant was co-segregated in all the affected family members in a heterozygous form and the unaffected family members. The in silico analysis confirmed the variant as pathogenic.
Pathogenic RBM20 nucleotide variants are associated with arrhythmogenic DCM. We believe that our report is the first to show an RBM20 variant in Iranian descent associated with DCM.

Cellular mRNA-binding proteins (mRBPs) are major posttranscriptional regulators of gene expression. Although many posttranslational modification sites in mRBPs have been identified, little is known about how these modifications regulate mRBP function. Here, we developed quantitative RNA-interactome capture (qRIC) to quantify the fraction of mRBPs pulled down with polyadenylated mRNAs. Combining qRIC with phosphoproteomics allowed us to systematically compare pull-down efficiencies of phosphorylated and nonphosphorylated forms of mRBPs. Almost 200 phosphorylation events affected pull-down efficiency compared with the unmodified mRBPs and thus have regulatory potential. Our data capture known regulatory phosphorylation sites in ELAVL1, SF3B1, and UPF1 and identify potential regulatory sites. Follow-up experiments on the splicing regulator RBM20 revealed multiple phosphorylation sites in the C-terminal disordered region affecting nucleocytoplasmic localization, association with cytoplasmic ribonucleoprotein granules, and alternative splicing. Together, we show that qRIC in conjunction with phosphoproteomics is a scalable method to identify functional posttranslational modification sites in mRBPs.