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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. [ABSTRACT FROM AUTHOR]

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.

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.

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. [ABSTRACT FROM AUTHOR]

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 cardiomyopa)thy patient cohort. We characterized an Rbm20 knockout (KO) mouse model, and show that splicing of key RBM20 targets, car)diac 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 catecholami)nergic 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 cardiomy)opathy 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. [ABSTRACT FROM AUTHOR]

Abstract Introduction 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. Methods 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. Results 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. Conclusion 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.

Aim. To demonstrate two generations of a family with a progressive course of left ventricular non-compaction (LVNC) and the presence of a RBM20 gene variant.Material and methods. Based on the multicenter registry of patients with LVNC, a family with LVNC with a dilated phenotype was selected at the National Medical Research Center for Therapy and Preventive Medicine. Next generation sequencing was performed on a Nextseq 550 systen (Illumina, USA). For clinical interpretation, nucleotide sequence variants in the genes associated with LVNC development were selected according to the available literature data, with frequencies

(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. [ABSTRACT FROM AUTHOR]

Abstract Background Inherited dilated cardiomyopathy (DCM) contributes to approximately 25% of idiopathic DCM cases, and the proportion is even higher in familial DCM patients. Most studies have focused on familial DCM, whereas the genetic profile of sporadic DCM in Chinese patients remains unknown. Methods Between June 2018 and September 2019, 24 patients diagnosed with idiopathic DCM without a family history were included in the present study. All patients underwent genetic screening for 80 DCM-related genes using targeted next-generation sequencing. Results By in silico analysis, 10 of 99 detected variants were considered pathogenic or likely-pathogenic, including seven TTN truncating variants (TTNtv), one in-frame deletion in TNNT2, one missense mutation in RBM20, and one frameshift deletion variant in FLNC. Of these variants, eight are reported for the first time. Conclusions Using targeted next-generation sequencing, potential genetic causes of idiopathic DCM were identified. Sarcomere mutations remained the most common genetic cause of inherited DCM in this cohort of sporadic Chinese DCM.

Abstract Background Dilated cardiomyopathy (DCM) is a disease of the heart muscle characterized by ventricular dilation and a left ventricular ejection fraction of less than 40%. Unlike hypertrophic cardiomyopathy (HCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC), DCM-causing mutations are present in a large number of genes. In the present study, we report a case of the early age of onset of DCM associated with a pathogenic variant in the RBM20 gene in a patient from India. Case presentation A 19-year-old Indian male diagnosed with DCM was suggested for heart transplantation. His ECG showed LBBB and echocardiography showed an ejection fraction of 14%. He had a sudden cardiac death. A detailed family history revealed it to be a case of familial DCM. Genetic screening identified the c.1900C>T variant in the RBM20 gene which led to a missense variant of amino acid 634 (p.Arg634Trp). Conclusion To the best of our knowledge, the variant p.Arg634Trp has been earlier reported in the Western population, but this is the first case of p.Arg634Trp in an Indian patient. The variant has been reported to be pathogenic at an early age of onset; therefore, close clinical follow-up should be done for the family members caring for the variant.

Introduction: 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. Methods: 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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

The study objectives were to determine the prevalence and geographic distribution of a dilated cardiomyopathy (DCM)-associated RNA-binding motif protein 20 (RBM20) variant in canine DNA samples submitted for testing and to evaluate the influence of the genotype on cardiac phenotype and lifespan. Samples from 2136 dogs including 1834 Standard Schnauzers (SSNZ), 266 Giant Schnauzers (GSNZ), and 36 dogs of other breeds. The University of Missouri Canine Genetics Laboratory's sample-accession spreadsheet and Orthopedic Foundation for Animals' database were retrospectively reviewed for samples submitted for RBM20 genotyping from November, 2013, through May, 2018. Data analyzed included breed, date of birth, RBM20 genotype (homozygous wild-type, heterozygous variant [HET], or homozygous variant [HOM]), geographic origin of submission, pedigree, cardiac phenotype, and date of death or current age if alive. The RBM20 variant was only detected in SSNZ and GSNZ. A total of 389 SSNZ were variant-positive (prevalence = 21.2%), with 361 HET (19.7%) and 28 HOM (1.5%). Of the HOM SSNZ, DCM was confirmed in 26 of 28 (92.9%), with the remainder lost to follow-up. The median lifespan of HOM SSNZ (3.06 years) was significantly shorter than that for HET (15.11 years) and wild-type (15.18 years) SSNZ. Twenty-six GSNZ were variant-positive (prevalence = 9.8%), with 23 HET (8.6%) and three HOM (1.1%). Nine GSNZ belonged to one family, including the three HOM GSNZ that all had DCM. The HOM genotype is associated with DCM and premature death in SSNZ and GSNZ. [ABSTRACT FROM AUTHOR]

RNA binding motif 20 (RBM20) cardiomyopathy has been detected in approximately 3% of populations afflicted with dilated cardiomyopathy (DCM). It is well conceived that RBM20 cardiomyopathy is provoked by titin isoform switching in combination with resting Ca2+ leaking. In this study, we characterized the cardiac function in Rbm20 knockout (KO) rats at 3‐, 6‐, 9‐, and 12‐months of age and examined the effect of the ryanodine receptor stabilizer S107 on resting intracellular levels and cardiomyocyte contractile properties. Our results revealed that even though Rbm20 depletion promoted expression of larger titin isoform and reduced myocardial stiffness in young rats (3 months of age), the established DCM phenotype required more time to embellish. S107 restored elevated intracellular Ca2+ to normal levels and ameliorated cardiomyocyte contractile properties in isolated cardiomyocytes from 6‐month‐old Rbm20 KO rats. However, S107 failed to preserve cardiac homeostasis in Rbm20 KO rats at 12 months of age, unexpectedly, likely due to the existence of multiple pathogenic mechanisms. Taken together, our data suggest the therapeutic promises of S107 in the management of RBM20 cardiomyopathy. [ABSTRACT FROM AUTHOR]

Aim. To study the prevalence of RBM20 gene polymorphisms and their relationship with the structural and functional left atrial (LA) characteristics in patients with coronary artery disease and heart failure with reduced ejection fraction (HFrEF).Material and methods. The study included 138 men aged 55,8±6,6 years with prior myocardial infarction ³12 months ago and HFrEF (class II-IV heart failure, left ventricular ejection fraction (Simpson’s methods), 25,1±7,2%). The control group consisted of 384 healthy donors. Genotyping of two RBM20 polymorphic variants (rs942077 and rs35141404) was performed by real-time polymerase chain reaction.Results. The prevalence of RBM20 polymorphisms did not differ in the HFrEF cohort and the control group. The GA rs35141404 genotype was more common among patients with a less pronounced increase in LA volume index (LAVI) (p=0,034). The minor A allele rs35141404 was associated with a protective effect on severe LA remodeling. However, this association did not reach the level of significance.Conclusion. For the rs942077 and rs35141404 polymorphic variants of the RBM20 gene, no significant associations were found with the LA size and atrial fibrillation presence in patients with HFrEF and old myocardial infarction. There was a tendency towards the association of the A allele and the GA rs35141404 genotype with a protective effect on LA remodeling. The data obtained confirm the need for further search for genotype-phenotype relationships of a wider population of patients with heart failure and coronary artery disease.

Abstract RNA binding motif 20 (RBM20) cardiomyopathy has been detected in approximately 3% of populations afflicted with dilated cardiomyopathy (DCM). It is well conceived that RBM20 cardiomyopathy is provoked by titin isoform switching in combination with resting Ca2+ leaking. In this study, we characterized the cardiac function in Rbm20 knockout (KO) rats at 3‐, 6‐, 9‐, and 12‐months of age and examined the effect of the ryanodine receptor stabilizer S107 on resting intracellular levels and cardiomyocyte contractile properties. Our results revealed that even though Rbm20 depletion promoted expression of larger titin isoform and reduced myocardial stiffness in young rats (3 months of age), the established DCM phenotype required more time to embellish. S107 restored elevated intracellular Ca2+ to normal levels and ameliorated cardiomyocyte contractile properties in isolated cardiomyocytes from 6‐month‐old Rbm20 KO rats. However, S107 failed to preserve cardiac homeostasis in Rbm20 KO rats at 12 months of age, unexpectedly, likely due to the existence of multiple pathogenic mechanisms. Taken together, our data suggest the therapeutic promises of S107 in the management of RBM20 cardiomyopathy.

Aim. Description of three clinical cases of pediatric patients with dilated cardiomyopathy (DCMP) and an analysis of their genetic causes.Material and methods. Using the method of targeted sequencing, data were obtained on the presence of pathogenic variants of the RBM20 gene in three pediatric patients with DCMP.Results. Three cases of childhood DCMP development, associated with structural disorders in the RBM20 gene, are particularly described. It is known that RBM20 is involved in the splicing of mRNA of the TTN gene encoding the titin protein. A splicing disorder associated with pathogenic variants in the RBM20 gene can lead to a change in biomechanical and signaling processes in myocardial cells, causing pathological dilated remodeling and rhythm disorders.Conclusion. Variants in the RBM20 gene are associated with severe DCMP with a childhood debut. In some cases, the progression of RBM20-associated cardiomyopathies is associated with an infectious disease. Further study of the molecular mechanisms of the pathogenesis of DCMP associated with pathogenic variants in the TTN and RBM20 genes is extremely relevant for both clinical cardiology and fundamental medicine.

Dilated cardiomyopathy (DCM) is a complex, etiologically heterogeneous myocardial disease, which is one of the main causes of heart failure and heart transplantation. In 2016, experts from the European working group proposed a new definition of cardiomyopathy, which includes intermediate variants with a change in phenotype in carriers of mutations from subclinical form to the full manifestations of the disease. The classification of DCM was supplemented with intermediate phenotypes with the inclusion of a hypokinetic form with reduced contractile function without ventricular dilatation and variants with predominant dilation or arrhythmogenicity. Pathological architectonics of DCM consists of many genetic determinants that interact with numerous environmental factors. Clinical manifestations depend not only on the malignancy and penetrance of the gene mutation, but also on a number of other causes — epigenomic factors, age, toxic effects, environmental aggressiveness, pregnancy, and the effects of other acquired diseases. The article summarizes the current epidemiological data and ideas about specific molecular changes with an unfavorable prognosis. For clarity, we present clinical observations of familial DCM with mutations in the RBM20 and LMNA genes.

Background: Dilated cardiomyopathy (DCM) is a disease of the heart muscle characterized by ventricular dilation and a left ventricular ejection fraction of less than 40%. Unlike hypertrophic cardiomyopathy (HCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC), DCM-causing mutations are present in a large number of genes. In the present study, we report a case of the early age of onset of DCM associated with a pathogenic variant in the RBM20 gene in a patient from India. Case presentation: A 19-year-old Indian male diagnosed with DCM was suggested for heart transplantation. His ECG showed LBBB and echocardiography showed an ejection fraction of 14%. He had a sudden cardiac death. A detailed family history revealed it to be a case of familial DCM. Genetic screening identified the c.1900C>T variant in the RBM20 gene which led to a missense variant of amino acid 634 (p.Arg634Trp). Conclusion: To the best of our knowledge, the variant p.Arg634Trp has been earlier reported in the Western population, but this is the first case of p.Arg634Trp in an Indian patient. The variant has been reported to be pathogenic at an early age of onset; therefore, close clinical follow-up should be done for the family members caring for the variant. [ABSTRACT FROM AUTHOR]

Background: Inherited dilated cardiomyopathy (DCM) contributes to approximately 25% of idiopathic DCM cases, and the proportion is even higher in familial DCM patients. Most studies have focused on familial DCM, whereas the genetic profile of sporadic DCM in Chinese patients remains unknown.Methods: Between June 2018 and September 2019, 24 patients diagnosed with idiopathic DCM without a family history were included in the present study. All patients underwent genetic screening for 80 DCM-related genes using targeted next-generation sequencing.Results: By in silico analysis, 10 of 99 detected variants were considered pathogenic or likely-pathogenic, including seven TTN truncating variants (TTNtv), one in-frame deletion in TNNT2, one missense mutation in RBM20, and one frameshift deletion variant in FLNC. Of these variants, eight are reported for the first time.Conclusions: Using targeted next-generation sequencing, potential genetic causes of idiopathic DCM were identified. Sarcomere mutations remained the most common genetic cause of inherited DCM in this cohort of sporadic Chinese DCM. [ABSTRACT FROM AUTHOR]

Mutations in RBM20 encoding the RNA‐binding motif protein 20 (RBM20) are associated with an early onset and clinically severe forms of cardiomyopathies. Transcriptome analyses revealed RBM20 as an important regulator of cardiac alternative splicing. RBM20 mutations are especially localized in exons 9 and 11 including the highly conserved arginine and serine‐rich domain (RS domain). Here, we investigated in several cardiomyopathy patients, the previously described RBM20‐mutation p.Pro638Leu localized within the RS domain. In addition, we identified in a patient the novel mutation p.Val914Ala localized in the (glutamate‐rich) Glu‐rich domain of RBM20 encoded by exon 11. Its impact on the disease was investigated with a novel TTN‐ and RYR2‐splicing assay based on the patients' cardiac messenger RNA. Furthermore, we showed in cell culture and in human cardiac tissue that mutant RBM20‐p.Pro638Leu is not localized in the nuclei but causes an abnormal cytoplasmic localization of the protein. In contrast the splicing deficient RBM20‐p.Val914Ala has no influence on the intracellular localization. These results indicate that disease‐associated variants in RBM20 lead to aberrant splicing through different pathomechanisms dependent on the localization of the mutation. This might have an impact on the future development of therapeutic strategies for the treatment of RBM20‐induced cardiomyopathies. [ABSTRACT FROM AUTHOR]

Purpose of Review: This review aims to give an update on recent findings related to the cardiac splicing factor RNA-binding motif protein 20 (RBM20) and RBM20 cardiomyopathy, a form of dilated cardiomyopathy caused by mutations in RBM20. Recent Findings: While most research on RBM20 splicing targets has focused on titin (TTN), multiple studies over the last years have shown that other splicing targets of RBM20 including Ca2+/calmodulin-dependent kinase IIδ (CAMK2D) might be critically involved in the development of RBM20 cardiomyopathy. In this regard, loss of RBM20 causes an abnormal intracellular calcium handling, which may relate to the arrhythmogenic presentation of RBM20 cardiomyopathy. In addition, RBM20 presents clinically in a highly gender-specific manner, with male patients suffering from an earlier disease onset and a more severe disease progression. Summary: Further research on RBM20, and treatment of RBM20 cardiomyopathy, will need to consider both the multitude and relative contribution of the different splicing targets and related pathways, as well as gender differences. [ABSTRACT FROM AUTHOR]

Alternative splicing is a regulatory mechanism essential for cell differentiation and tissue organization. More than 90% of human genes are regulated by alternative splicing events, which participate in cell fate determination. The general mechanisms of splicing events are well known, whereas only recently have deep-sequencing, high throughput analyses and animal models provided novel information on the network of functionally coordinated, tissue-specific, alternatively spliced exons. Heart development and cardiac tissue differentiation require thoroughly regulated splicing events. The ribonucleoprotein RBM20 is a key regulator of the alternative splicing events required for functional and structural heart properties, such as the expression of TTN isoforms. Recently, the polypyrimidine tract-binding protein PTBP1 has been demonstrated to participate with RBM20 in regulating splicing events. In this review, we summarize the updated knowledge relative to RBM20 and PTBP1 structure and molecular function; their role in alternative splicing mechanisms involved in the heart development and function; RBM20 mutations associated with idiopathic dilated cardiovascular disease (DCM); and the consequences of RBM20-altered expression or dysfunction. Furthermore, we discuss the possible application of targeting RBM20 in new approaches in heart therapies. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Sorafenib down-regulates Rbm20 gene expression in cardiomyocytes. • Sorafenib-induced cardiotoxicity is associated with Rbm20-mediated alternative slicing among others. • Increasing or reversing Rbm20 down-regulation by sorafenib rescues cells from cell death. [ABSTRACT FROM AUTHOR]

Dilated cardiomyopathy (DCM) is a leading cause of heart failure, sudden cardiac death and heart transplant. DCM is inherited in approximately 50% of cases, in which the most frequent genetic defects are truncation variants of the titin gene (TTNtv). TTN encodes titin, which is the largest protein in the body and is an essential component of the sarcomere. Titin serves as a biological spring, spanning half of the sarcomere and connecting the Z-disk to the M-line, with scaffold and signaling functions. Truncations of titin are believed to lead to either haploinsufficiency and loss-of-function, or to a “poison peptide” effect. However, other titin mechanisms are postulated to influence cardiac function including post-translational modifications, in particular changes in titin phosphorylation that alters the stiffness of the protein, and diversity of alternative splicing that generates different titin isoforms. In this article, we review the role of TTN mutations in development of DCM, how differential expression of titin isoforms relate to DCM pathophysiology, and discuss how post-translational modifications of titin can affect cardiomyocyte function. Current research efforts aim to elucidate the contribution of titin to myofibril assembly, stability, and signal transduction, and how mutant titin leads to cardiac dysfunction and human disease. Future research will need to translate this knowledge toward novel therapeutic approaches that can modulate titin transcriptional and post-translational defects to treat DCM and heart failure.HIGHLIGHTS- Titin (TTN) truncation variants are the most frequent cause of dilated cardiomyopathy, one of the main causes of heart failure and heart transplant. Titin is a giant protein, and the mechanisms causing the disease are both complex and still incompletely understood.- This review discusses the role of titin in myocardial function and in disease. In particular, we discuss TTN gene structure, the complexity of genotype-phenotype correlation in human disease, the physiology of TTN and the role of post-translation modification.- Additional studies will be required to clarify whether missense variants are associated with cardiac disease. While initial studies suggested a role of non-synonymous variants in arrhythmogenic cardiomyopathy, confirmatory investigations have been hampered by the complexity of the protein structure and function.

Pre-mRNA splicing plays an important role in muscle function and diseases. The RNA binding motif 20 (RBM20) is a splicing factor that is predominantly expressed in muscle tissues and primarily regulates pre-mRNA splicing of Ttn, encoding a giant muscle protein titin that is responsible for muscle function and diseases. RBM20-mediated Ttn splicing has been mostly studied in heart muscle, but not in skeletal muscle. In this study, we investigated splicing specificity in different muscle types in Rbm20 knockout rats and hormonal effects on RBM20-mediated splicing both in cellulo and in vivo studies. The results revealed that RBM20 is differentially expressed across muscles and RBM20-mediated splicing is muscle-type specific. In the presence of RBM20, Ttn splicing responds to hormones in a muscle-type dependent manner, while in the absence of RBM20, Ttn splicing is not affected by hormones. In differentiated and undifferentiated C2C12 cells, RBM20-mediated splicing in response to hormonal effects is mainly through genomic signaling pathway. The knowledge gained from this study may help further understand muscle-specific gene splicing in response to hormone stimuli in different muscle types.

Dilated cardiomyopathy (DCM) is a leading cause of heart failure, sudden cardiac death and heart transplant. DCM is inherited in approximately 50% of cases, in which the most frequent genetic defects are truncation variants of the titin gene (TTN tv). TTN encodes titin, which is the largest protein in the body and is an essential component of the sarcomere. Titin serves as a biological spring, spanning half of the sarcomere and connecting the Z-disk to the M-line, with scaffold and signaling functions. Truncations of titin are believed to lead to either haploinsufficiency and loss-of-function, or to a "poison peptide" effect. However, other titin mechanisms are postulated to influence cardiac function including post-translational modifications, in particular changes in titin phosphorylation that alters the stiffness of the protein, and diversity of alternative splicing that generates different titin isoforms. In this article, we review the role of TTN mutations in development of DCM, how differential expression of titin isoforms relate to DCM pathophysiology, and discuss how post-translational modifications of titin can affect cardiomyocyte function. Current research efforts aim to elucidate the contribution of titin to myofibril assembly, stability, and signal transduction, and how mutant titin leads to cardiac dysfunction and human disease. Future research will need to translate this knowledge toward novel therapeutic approaches that can modulate titin transcriptional and post-translational defects to treat DCM and heart failure. HIGHLIGHTS: - Titin (TTN) truncation variants are the most frequent cause of dilated cardiomyopathy, one of the main causes of heart failure and heart transplant. Titin is a giant protein, and the mechanisms causing the disease are both complex and still incompletely understood. - This review discusses the role of titin in myocardial function and in disease. In particular, we discuss TTN gene structure, the complexity of genotype-phenotype correlation in human disease, the physiology of TTN and the role of post-translation modification. - Additional studies will be required to clarify whether missense variants are associated with cardiac disease. While initial studies suggested a role of non-synonymous variants in arrhythmogenic cardiomyopathy, confirmatory investigations have been hampered by the complexity of the protein structure and function. [ABSTRACT FROM AUTHOR]

Titin functions as a molecular spring, and cardiomyocytes are able, through splicing, to control the length of titin. We hypothesized that together with diastolic [Ca2+], titin‐based stretch pre‐activates cardiomyocytes during diastole and is a major determinant of force production in the subsequent contraction. Through this mechanism titin would play an important role in active force development and length‐dependent activation. Mutations in the splicing factor RNA binding motif protein 20 (RBM20) result in expression of large, highly compliant titin isoforms. We measured single cardiomyocyte work loops that mimic the cardiac cycle in wild‐type (WT) and heterozygous (HET) RBM20‐deficient rats. In addition, we studied the role of diastolic [Ca2+] in membrane‐permeabilized WT and HET cardiomyocytes. Intact cardiomyocytes isolated from HET left ventricles were unable to produce normal levels of work (55% of WT) at low pacing frequencies, but this difference disappeared at high pacing frequencies. Length‐dependent activation (force–sarcomere length relationship) was blunted in HET cardiomyocytes, but the force–end‐diastolic force relationship was not different between HET and WT cardiomyocytes. To delineate the effects of diastolic [Ca2+] and titin pre‐activation on force generation, measurements were performed in detergent‐permeabilized cardiomyocytes. Cardiac twitches were simulated by transiently exposing permeabilized cardiomyocytes to 2 µm Ca2+. Increasing diastolic [Ca2+] from 1 to 80 nm increased force development twofold in WT. Higher diastolic [Ca2+] was needed in HET. These findings are consistent with our hypothesis that pre‐activation increases active force development. Highly compliant titin allows cells to function at higher diastolic [Ca2+]. [ABSTRACT FROM AUTHOR]

Here we report the identification of two novel mutations in a previously asymptomatic young man who suffered an out-of-hospital sudden cardiac arrest. During following evaluation, diagnosis of early stage dilated cardiomyopathy was established, while electrocardiogram monitoring showed frequent complex ventricular arrhythmias, incomplete right bundle branch block and prolonged QT duration. No reversible causes explaining the clinical presentation were established and an automatic implantable cardioverter defibrillator was therefore implanted. Heterozygous mutations in human protein coding genes NKX2-5 and RBM20 are associated with a wide array of pathological phenotypes some of which are sudden cardiac death, unexplained syncope and either combined or isolated congenital heart diseases such as dilated cardiomyopathy. [ABSTRACT FROM AUTHOR]

Abstract Regulation of alternative splicing events is an essential step required for the expression of functional cytoskeleton and sarcomere proteins in cardiomyocytes. About 3% of idiopathic dilated cardiomyopathy cases present mutations in the RNA binding protein RBM20, a tissue specific regulator of alternative splicing. Transcripts expressed preferentially in skeletal and cardiac muscle, including TTN , CAMK2D , LDB3 , LMO7 , PDLIM3 , RTN4 , and RYR2, are RBM20-dependent splice variants. In the present study, we investigated the RBM20 involvement in post-transcriptional regulation of splicing variants expressed by Formin homology 2 domain containing 3 (FHOD3) gene. FHOD3 is a sarcomeric protein highly expressed in the cardiac tissue and required for the assembly of the contractile apparatus. Recently, FHOD3 mutations have been found associated with heart diseases. We identified novel FHOD 3 splicing variants differentially expressed in human tissues and provided evidences that FHOD3 transcripts are specific RBM20 and PTBP1 targets. Furthermore, we demonstrated that the expression of RBM20 and PTBP1 promoted the alternative shift, from inclusion to exclusion, of selected FHOD3 exons. These results indicate that RBM20 and PTBP1 play a role in the actin filament functional organization mediated by FHOD3 isoforms and suggest their possible involvement in heart diseases. [ABSTRACT FROM AUTHOR]

Objective: We present the genotypic and phenotypic characterization of a family displaying dilated cardiomyopathy (DCM). Methods: The proband and his relatives underwent full cardiological assessment. Genetic analysis of the proband was performed with the use of next-generation sequencing technology. Results: In this study, we present 6 members of a family carrying the RBM20 mutation NM_001134363.2:c.1900C>T. The proband was initially diagnosed with DCM at the age of 18 years and received an implantable cardioverter defibrillator (ICD) due to ventricular arrhythmias. His brother, carrier of the mutation, has been diagnosed with borderline left ventricular function. The mutation was shown to be of paternal origin, but their father remains asymptomatic with a mild DCM, while his electrocardiogram at the initial evaluation showed a right bundle branch block pattern. The mutation was also detected in the index case's aunt who was resuscitated from sudden cardiac death. Her echocardiography revealed early stages of DCM and a bicuspid aortic valve. Her children are both carriers of the mutation. Her daughter is unaffected, but her son has an ICD implanted due to sustained ventricular tachycardia and presents early signs of DCM. Conclusion: Our findings are the first report of co-segregation of the mutation in 6 family members, supporting its pathogenic role. [ABSTRACT FROM AUTHOR]

RBM20 is a vertebrate-specific RNA-binding protein with two zinc finger (ZnF) domains, one RNA-recognition motif (RRM)-type RNA-binding domain and an arginine/serine (RS)-rich region. RBM20 has initially been identified as one of dilated cardiomyopathy (DCM)-linked genes. RBM20 is a regulator of heart-specific alternative splicing and Rbm20ΔRRM mice lacking the RRM domain are defective in the splicing regulation. The Rbm20ΔRRM mice, however, do not exhibit a characteristic DCM-like phenotype such as dilatation of left ventricles or systolic dysfunction. Considering that most of the RBM20 mutations identified in familial DCM cases were heterozygous missense mutations in an arginine-serine-arginine-serine-proline (RSRSP) stretch whose phosphorylation is crucial for nuclear localization of RBM20, characterization of a knock-in animal model is awaited. One of the major targets for RBM20 is the TTN gene, which is comprised of the largest number of exons in mammals. Alternative splicing of the TTN gene is exceptionally complicated and RBM20 represses >160 of its consecutive exons, yet detailed mechanisms for such extraordinary regulation are to be elucidated. The TTN gene encodes the largest known protein titin, a multi-functional sarcomeric structural protein specific to striated muscles. As titin is the most important factor for passive tension of cardiomyocytes, extensive heart-specific and developmentally regulated alternative splicing of the TTN pre-mRNA by RBM20 plays a critical role in passive stiffness and diastolic function of the heart. In disease models with diastolic dysfunctions, the phenotypes were rescued by increasing titin compliance through manipulation of the Ttn pre-mRNA splicing, raising RBM20 as a potential therapeutic target.

Mutations in the LMNA gene, which encodes for the nuclear lamina proteins lamins A and C, are responsible for a diverse group of diseases known as laminopathies. One type of laminopathy is Dilated Cardiomyopathy (DCM), a heart muscle disease characterized by dilation of the left ventricle and impaired systolic function, often leading to heart failure and sudden cardiac death. LMNA is the second most commonly mutated gene in DCM. In addition to LMNA, mutations in more than 60 genes have been associated with DCM. The DCM-associated genes encode a variety of proteins including transcription factors, cytoskeletal, Ca2+-regulating, ion-channel, desmosomal, sarcomeric, and nuclear-membrane proteins. Another important category among DCM-causing genes emerged upon the identification of DCM-causing mutations in RNA binding motif protein 20 (RBM20), an alternative splicing factor that is chiefly expressed in the heart. In addition to RBM20, several essential splicing factors were validated, by employing mouse knock out models, to be embryonically lethal due to aberrant cardiogenesis. Furthermore, heart-specific deletion of some of these splicing factors was found to result in aberrant splicing of their targets and DCM development. In addition to splicing alterations, advances in next generation sequencing highlighted the association between splice-site mutations in several genes and DCM. This review summarizes LMNA mutations and splicing alterations in DCM and discusses how the interaction between LMNA and splicing regulators could possibly explain DCM disease mechanisms.

Alternative splicing is an important mechanism used by the cell to generate greater transcriptomic and proteomic diversity from the genome. In the heart, alternative splicing is increasingly being recognised as an important layer of post-transcriptional gene regulation. Driven by rapidly evolving technologies in next-generation sequencing, alternative splicing has emerged as a crucial process governing complex biological processes during cardiac development and disease. The recent identification of several cardiac splice factors, such as RNA-binding motif protein 20 and 24, not only provided important insight into the mechanisms underlying alternative splicing but also revealed how these splicing factors impact functional properties of the heart. Here, we review our current knowledge of alternative splicing in the heart, with a particular focus on the factors controlling cardiac alternative splicing and their role in cardiomyopathies and subsequent heart failure. [ABSTRACT FROM AUTHOR]

Mutations in the LMNA gene, which encodes for the nuclear lamina proteins lamins A and C, are responsible for a diverse group of diseases known as laminopathies. One type of laminopathy is Dilated Cardiomyopathy (DCM), a heart muscle disease characterized by dilation of the left ventricle and impaired systolic function, often leading to heart failure and sudden cardiac death. LMNA is the second most commonly mutated gene in DCM. In addition to LMNA, mutations in more than 60 genes have been associated with DCM. The DCM-associated genes encode a variety of proteins including transcription factors, cytoskeletal, Ca2+-regulating, ion-channel, desmosomal, sarcomeric, and nuclear-membrane proteins. Another important category among DCM-causing genes emerged upon the identification of DCM-causing mutations in RNA binding motif protein 20 (RBM20), an alternative splicing factor that is chiefly expressed in the heart. In addition to RBM20, several essential splicing factors were validated, by employing mouse knock out models, to be embryonically lethal due to aberrant cardiogenesis. Furthermore, heart-specific deletion of some of these splicing factors was found to result in aberrant splicing of their targets and DCM development. In addition to splicing alterations, advances in next generation sequencing highlighted the association between splice-site mutations in several genes and DCM. This review summarizes LMNA mutations and splicing alterations in DCM and discusses how the interaction between LMNA and splicing regulators could possibly explain DCM disease mechanisms. [ABSTRACT FROM AUTHOR]

Titin (TTN) is a major disease-causing gene in cardiac muscle. Titin (TTN) contains 363 exons in human encoding various sizes of TTN protein due to alternative splicing regulated mainly by RNA binding motif 20 (RBM20). Three isoforms of TTN protein are produced by mutually exclusive exons 45 (Novex 1), 46 (Novex 2), and 48 (Novex 3). Alternatively splicing in Novex isoforms across species and whether Novex isoforms are associated with heart disease remains completely unknown. Cross-species exon comparison with the mVISTA online tool revealed that exon 45 is more highly conserved across all species than exons 46 and 48. Importantly, a conserved region between exons 47 and 48 across species was revealed for the first time. Reverse transcript polymerase chain reaction (RT-PCR) and DNA sequencing confirmed a new exon named as 48' in Novex 3. In addition, with primer pairs for Novex 1, a new truncated form preserving introns 44 and 45 was discovered. We discovered that Novex 2 is not expressed in the pig, mouse, and rat with Novex 2 primer pairs. Unexpectedly, three truncated forms were identified. One TTN variant with intron 46 retention is mainly expressed in the human and frog heart, another variant with co-expression of exons 45 and 46 exists predominantly in chicken and frog heart, and a third with retention of introns 45 and 46 is mainly expressed in pig, mouse, rat, and chicken. Using Rbm20 knockout rat heart, we revealed that RBM20 is not a splicing regulator of Novex variants. Furthermore, the expression levels of Novex variants in human hearts with cardiomyopathies suggested that Novexes 2 and 3 could be associated with dilated cardiomyopathy (DCM) and/or arrhythmogenic right ventricular cardiomyopathy (ARVC). Taken together, our study reveals that splicing diversity of Novex exons across species and Novex variants might play a role in cardiomyopathy. [ABSTRACT FROM AUTHOR]

Cardiomyopathy, also known as heart muscle disease, is an unfavorable condition leading to alterations in myocardial contraction and/or impaired ability of ventricular filling. The onset and development of cardiomyopathy have not currently been well defined. Titin is a giant multifunctional sarcomeric filament protein that provides passive stiffness to cardiomyocytes and has been implicated to play an important role in the origin and development of cardiomyopathy and heart failure. Titin-based passive stiffness can be mainly adjusted by isoform switching and post-translational modifications in the spring regions. Recently, genetic mutations of TTN have been identified that can also contribute to variable passive stiffness, though the detailed mechanisms remain unclear. In this review, we will discuss titin isoform switching as it relates to alternative splicing during development stages and differences between species and muscle types. We provide an update on the regulatory mechanisms of TTN splicing controlled by RBM20 and cover the roles of TTN splicing in adjusting the diastolic stiffness and systolic compliance of the healthy and the failing heart. Finally, this review attempts to provide future directions for RBM20 as a potential target for pharmacological intervention in cardiomyopathy and heart failure. [ABSTRACT FROM AUTHOR]

Aims In this study, we aimed to clinically and genetically characterize LVNC patients and investigate the prevalence of variants in known and novel LVNC disease genes. Introduction Left ventricular non-compaction cardiomyopathy (LVNC) is an increasingly recognized cause of heart failure, arrhythmia, thromboembolism, and sudden cardiac death. We sought here to dissect its genetic causes, phenotypic presentation and outcome. Methods and results In our registry with follow-up of in the median 61 months, we analysed 95 LVNC patients (68 unrelated index patients and 27 affected relatives; definite familial LVNC = 23.5%) by cardiac phenotyping, molecular biomarkers and exome sequencing. Cardiovascular events were significantly more frequent in LVNC patients compared with an age-matched group of patients with non-ischaemic dilated cardiomyopathy (hazard ratio = 2.481, P = 0.002). Stringent genetic classification according to ACMG guidelines revealed that TTN, LMNA, and MYBPC3 are the most prevalent disease genes (13 patients are carrying a pathogenic truncating TTN variant, odds ratio = 40.7, Confidence interval = 21.6-76.6, P < 0.0001, percent spliced in 76-100%). We also identified novel candidate genes for LVNC. For RBM20, we were able to perform detailed familial, molecular and functional studies. We show that the novel variant p.R634L in the RS domain of RBM20 co-segregates with LVNC, leading to titin mis-splicing as revealed by RNA sequencing of heart tissue in mutation carriers, protein analysis, and functional splice-reporter assays. Conclusion Our data demonstrate that the clinical course of symptomatic LVNC can be severe. The identified pathogenic variants and distribution of disease genes--a titin-related pathomechanism is found in every fourth patient--should be considered in genetic counselling of patients. Pathogenic variants in the nuclear proteins Lamin A/C and RBM20 were associated with worse outcome. [ABSTRACT FROM AUTHOR]

The CACNA1C gene encodes for the CaV1.2 protein, which is the pore subunit of cardiac l-type voltage-gated calcium (Ca2+) channels (l-channels). Through alternative splicing, CACNA1C encodes for various CaV1.2 isoforms with different electrophysiological properties. Splice variants of CaV1.2 are differentially expressed during heart development or pathologies. The molecular mechanisms of CACNA1C alternative splicing still remain incompletely understood. RNA sequencing analysis has suggested that CACNA1C is a potential target of the splicing factor RNA-binding protein motif 20 (RBM20). Here, we aimed at elucidating the role of RBM20 in the regulation of CACNA1C alternative splicing. We found that in neonatal rat cardiomyocytes (NRCMs), RBM20 overexpression promoted the inclusion of CACNA1C’s exon 9*, whereas the skipping of exon 9* occurred upon RBM20 siRNA knockdown. The splicing of other known alternative exons was not altered by RBM20. RNA immunoprecipitation suggested that RBM20 binds to introns flanking exon 9*. Functionally, in NRCMs, RBM20 overexpression decreased l-type Ca2+ currents, whereas RBM20 siRNA knockdown increased l-type Ca2+ currents. Finally, we found that RBM20 overexpression reduced CaV1.2 membrane surface expression in NRCMs. Taken together, our results suggest that RBM20 specifically regulates the inclusion of exon 9* in CACNA1C mRNA, resulting in reduced cell-surface membrane expression of l-channels in cardiomyocytes.

RNA binding motif 20 (RBM20) is a key regulator of pre-mRNA splicing of titin and other genes that are associated with cardiac diseases. Hormones, like insulin, triiodothyronine (T3), and angiotensin II (Ang II), can regulate gene-splicing through RBM20, but the detailed mechanism remains unclear. This study was aimed at investigating the signaling mechanism by which hormones regulate pre-mRNA splicing through RBM20. We first examined the role of RBM20 in Z-, I-, and M-band titin splicing at different ages in wild type (WT) and RBM20 knockout (KO) rats using RT-PCR; we found that RBM20 is the predominant regulator of I-band titin splicing at all ages. Then we treated rats with propylthiouracil (PTU), T3, streptozotocin (STZ), and Ang II and evaluated the impact of these hormones on the splicing of titin, LIM domain binding 3 (Ldb3), calcium/calmodulin-dependent protein kinase II gamma (Camk2g), and triadin (Trdn). We determined the activation of mitogen-activated protein kinase (MAPK) signaling in primary cardiomyocytes treated with insulin, T3, and Ang II using western blotting; MAPK signaling was activated and RBM20 expression increased after treatment. Two downstream transcriptional factors c-jun and ETS Transcription Factor (ELK1) can bind the promoter of RBM20. A dual-luciferase activity assay revealed that Ang II, but not insulin and T3, can trigger ELK1 and thus promote transcription of RBM20. This study revealed that Ang II can trigger ELK1 through activation of MAPK signaling by enhancing RBM20 expression which regulates pre-mRNA splicing. Our study provides a potential therapeutic target for the treatment of cardiac diseases in RBM20-mediated pre-mRNA splicing.

Doxorubicin (DOX) is a widely used treatment for human cancers, but increases the risk of life-threatening congestive heart failure (CHF). DOX-induced mitochondrial damage is cumulative and persistent, similar to that observed clinically for risk of CHF. Recent evidence suggests the persistent nature of this injury is caused by altered regulation of genes important to normal cardiac functioning. We hypothesize that chronic DOX therapy is associated with epigenetic modifications of DNA methylation status, particularly in critical regulators of mitochondrial function and capacity. Cardiac tissue from Sprague Dawley rats receiving injections of DOX (2 mg/kg, s.c.) or saline once a week for 6 weeks, followed by 5 weeks of drug-free holiday was used for Reduced Representation Bisulfite Sequencing to map specific sites of DNA methylation. Comparison of these methylomes indicated DOX exposure alters DNA methylation landscapes, and identified 14 genes with highly altered methylation status. Preliminary functional effects of DNA methylation changes were characterized by quantifying mRNA expression of selected targets (Rbm20, Nmnat2, Klhl29, Cacna1c, Scn5a.) Gene expression of Rbm20, Klhl29, and Nmnat2 were significantly altered in DOX treated animals; Klhl29 and Nmnat2 demonstrated significant decreases in protein expression corresponding to gene expression. Through an epigenotype-to-phenotype approach, this study identifies potential markers and molecular regulators of irreversible DOX-induced cardiovascular toxicity associated with clinically limiting CHF. However, none of the most prevalent genes identified directly relate to mitochondrial structure or function. Thus, the investigation fails to demonstrate a direct association between this altered methylome and persistent mitochondrionopathy associated with chronic doxorubicin cardiac toxicity. [ABSTRACT FROM AUTHOR]

Titin, a giant sarcomeric protein, is largely responsible for the diastolic properties of the heart. It has two major isoforms, N2B and N2BA due to pre-mRNA splicing regulated mainly by a splicing factor RNA binding motif 20 (RBM20). Mis-splicing of titin pre-mRNA in response to external stimuli may lead to altered ratio of N2B to N2BA, and thus, impaired cardiac contractile function. However, little is known about titin alternative splicing in response to external stimuli. Here, we reported the detailed mechanisms of titin alternative splicing in response to insulin. Insulin treatment in cultured neonatal rat cardiomyocytes (NRCMs) activated the PI3K-Akt-mTOR kinase axis, leading to increased N2B expression in the presence of RBM20, but not in NRCMs in the absence of RBM20. By inhibiting this kinase axis with inhibitors, decreased N2B isoform was observed in NRCMs and also in diabetic rat model treated with streptozotocin, but not in NRCMs and diabetic rats in the absence of RBM20. In addition to the alteration of titin isoform ratios in response to insulin, we found that RBM20 expression was increased in NRCMs with insulin treatment, suggesting that RBM20 levels were also regulated by insulin-induced kinase axis. Further, knockdown of p70S6K1 with siRNA reduced both RBM20 and N2B levels, while knockdown of 4E-BP1 elevated expression levels of RBM20 and N2B. These findings reveal a major signal transduction pathway for insulin-induced titin alternative splicing, and place RBM20 in a central position in the pathway, which is consistent with the reputed role of RBM20 in titin alternative splicing. Findings from this study shed light on gene therapeutic strategies at the molecular level by correction of pre-mRNA mis-splicing. [ABSTRACT FROM AUTHOR]

Aim Mutations in the RS-domain of RNA-binding motif protein 20 (RBM20) have recently been identified to segregate with aggressive forms of familial dilated cardiomyopathy (DCM). Loss of RBM20 in rats results in missplicing of the sarcomeric gene titin (TTN). The functional and physiological consequences of RBM20 mutations outside the mutational hotspot of RBM20 have not been explored to date. In this study, we investigated the pathomechanism of DCM caused by a novel RBM20 mutation in human cardiomyocytes. Methods and results We identified a family with DCM carrying a mutation (RBM20E913K/+) in a glutamate-rich region of RBM20. Western blot analysis of endogenous RBM20 protein revealed strongly reduced protein levels in the heart of an RBM20E913K/+ carrier. RNA deep-sequencing demonstrated massive inclusion of exons coding for the spring region of titin in the RBM20E913K/+ carrier. Titin isoform analysis revealed a dramatic shift from the less compliant N2B towards the highly compliant N2BA isoforms in RBM20E913K/+ heart. Moreover, an increased sarcomere restinglength was observed in single cardiomyocytes and isometric force measurements revealed an attenuated Frank- Starling mechanism (FSM), which was rescued by protein kinase A treatment. Conclusion A mutation outside the mutational hotspot of RBM20 results in haploinsufficiency of RBM20. This leads to disturbed alternative splicing of TTN, resulting in a dramatic shift to highly compliant titin isoforms and an impaired FSM. These effects may contribute to the early onset, and malignant course of DCM caused by RBM20 mutations. Altogether, our results demonstrate that heterozygous loss of RBM20 suffices to profoundly impair myocyte biomechanics by its disturbance of TTN splicing. [ABSTRACT FROM AUTHOR]