RNA Binding Motif 20 Research Articles

Abstract P2041: Identification Of The Nuclear Localization Signal In RBM20 And Its Function In The Pathogenesis Of Dilated Cardiomyopathy

Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop an aggressive and arrhythmogenic form of dilated cardiomyopathy (DCM). Five of over 900 genetic mutations in RBM20 have recently been validated in animal models. Four of these five mutations are located in the arginine/serine-rich (RS) domain with the fifth in the RNA recognition motif (RRM). Interestingly, only mutations in the RS domain cause severe DCM, which suggests that disruption of RS domain function is crucial for the pathogenesis of DCM. To test this hypothesis, we generated mice expressing RBM20 with an in-frame deletion of the RS domain ( Rbm20 ΔRS ). We show that Rbm20 ΔRS mice manifest DCM with RBM20 mis-localization and granule formation, similar to that in RS domain mutation knock-in (KI) animals. Conversely, mice expressing RBM20 lacking the RRM do not exhibit RBM20 nucleocytoplasmic transport or granule formation, and, similar to RRM mutation KI mice, do not develop DCM. These data suggest that the critical nuclear localization signal (NLS) in RBM20 is located within the RS domain and that disruption of this sequence leads to RBM20 mis-localization and granule formation. In vitro experiments employing sequence deletion plasmids and immunocytochemical staining revealed that the first nine amino acids in the RS domain constitute the core NLS in RBM20, which is further supported by the fact that all three validated DCM-causing mutations are located in this segment. Analysis of plasmids containing DCM-associated mutations in other regions of RBM20, as well as in the non-NLS RS domain sequence, further confirmed that only mutations in the NLS facilitate re-localization and granule formation. Phosphorylation of residues within the NLS has been shown play an important role in protein nucleocytoplasmic transport. To test this, we mutated all phosphorylatable serine residues in the NLS to unphosphorylatable alanine or phosphomimetic aspartate and provide evidence that phosphorylation is dispensable for nucleocytoplasmic transport. Collectively, our findings identify the critical NLS in RBM20 and demonstrate that mutations in this NLS cause severe DCM through disruption of RS domain-mediated nuclear localization and sarcoplasmic granule formation.

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

Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop a clinically aggressive dilated cardiomyopathy (DCM). Genetic mutation knock-in (KI) animal models imply that altered function of the arginine-serine-rich (RS) domain is crucial for severe DCM. To test this hypothesis, we generated an RS domain deletion mouse model (Rbm20ΔRS). We show that Rbm20ΔRS mice manifest DCM with mis-splicing of RBM20 target transcripts. We found that RBM20 is mis-localized to the sarcoplasm in Rbm20ΔRS mice, which led to the formation of RBM20 granules similar to those detected in mutation KI animals. In contrast, mice lacking the RNA recognition motif (RRM) show similar mis-splicing of RBM20 target genes, but do not develop DCM or exhibit RBM20 granule formation. Using in vitro studies with immunocytochemical staining, we demonstrate that only DCM-associated mutations in the RS domain facilitate RBM20 nucleocytoplasmic transport and promote granule assembly. Further, we defined the core nuclear localization signal (NLS) within the RS domain. Mutation analysis of phosphorylation sites in the RS domain indicate that this modification is dispensable for RBM20 nucleocytoplasmic transport. Collectively, our findings revealed that disruption of RS domain-mediated nuclear localization is crucial for severe DCM caused by NLS mutations.

SR Protein Kinases Regulate the Splicing of Cardiomyopathy-Relevant Genes via Phosphorylation of the RSRSP Stretch in RBM20.

(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.

RBM20 regulates muscle hypertrophy through titin and calcium signaling

Study objectiveRNA binding motif 20 (RBM20), an RNA binding protein, regulates alternative splicing of titin and calcium handling genes that mediate hypertrophy signaling. The objective of this study is to investigate whether RBM20 regulates muscle hypertrophy through titin and/or calcium signaling.MethodsSoleus (SOL) and extensor digitorum longus (EDL) were collected from wild‐type (WT) and Rbm20 knockout (KO) rats. Transcriptome profiling was analyzed by RNA sequencing (RNA‐seq). Hypertrophic signaling was examined using HeLa cells as well as differentiated and undifferentiated C2C12 myoblasts. RBM20 was knocked down in C2C12 myoblasts by siRNA transfection. Both immunostaining and western blotting were performed to test expression of hypertrophy associated proteins in both cell lines.ResultsRBM20 KO promoted SOL hypertrophy and EDL atrophy in rats. Hypertrophy‐associated hallmark proteins CaN, Erk and p38 were elevated in KO SOL, but not in KO EDL, while atrophy‐associated hallmark protein p62 was increased in KO EDL, but not in KO SOL. RNA‐seq data identified differentially expressed and spliced genes associated with hypertrophy in both EDL and SOL. Titin was switched to largest isoform as a result of alternative splicing in both KO SOL and EDL muscles. To test whether titin is the major mediator that regulatew hypertrophic signaling in RBM20 KO rats, we transfected Rbm20 gene into HeLa cells that do not express titin. The results revealed that the levels of hallmark proteins did not changed with overexpression of RBM20. To further test the role of titin in RBM20‐mediated hypertrophy, Rbm20 was knocked down in undifferentiated C2C12 cells which do not express titin as well, and no change was observed for hallmark proteins. C2C12 myoblasts were then differentiated to myotubes and reduced RBM20 in differentiated C2C12 cells promoted reduced expression of hypertrophic hallmark proteins.ConclusionOur results suggest that RBM20 regulates muscle hypertrophy in a muscle type‐dependent manner, and RBM20‐mediated hypertrophy is regulated via titin, a hypertrophic mediator.

RBM20 phosphorylation and its role in nucleocytoplasmic transport and cardiac pathogenesis.

Arginine–serine (RS) domain(s) in splicing factors are critical for protein–protein interaction in pre‐mRNA splicing. Phosphorylation of RS domain is important for splicing control and nucleocytoplasmic transport in the cell. RNA‐binding motif 20 (RBM20) is a splicing factor primarily expressed in the heart. A previous study using phospho‐antibody against RS domain showed that RS domain can be phosphorylated. However, its actual phosphorylation sites and function have not been characterized. Using middle‐down mass spectrometry, we identified 16 phosphorylation sites, two of which (S638 and S640 in rats, or S637 and S639 in mice) were located in the RSRSP stretch in the RS domain. Mutations on S638 and S640 regulated splicing, promoted nucleocytoplasmic transport and protein‐RNA condensates. Phosphomimetic mutations on S638 and S640 indicated that phosphorylation was not the major cause for RBM20 nucleocytoplasmic transport and condensation in vitro. We generated a S637A knock‐in (KI) mouse model (Rbm20S637A) and observed the reduced RBM20 phosphorylation. The KI mice exhibited aberrant gene splicing, protein condensates, and a dilated cardiomyopathy (DCM)‐like phenotype. Transcriptomic profiling demonstrated that KI mice had altered expression and splicing of genes involving cardiac dysfunction, protein localization, and condensation. Our in vitro data showed that phosphorylation was not a direct cause for nucleocytoplasmic transport and protein condensation. Subsequently, the in vivo results reveal that RBM20 mutations led to cardiac pathogenesis. However, the role of phosphorylation in vivo needs further investigation.

Abstract 13431: Gain-of-Function Mutations in RNA Binding Motif 20 Result in Cardiac Remodelling and Pathogenesis in Mice

Gain-of-function mutations of a gene result in new molecular function. Genetic studies show that mutations in a gene named as RNA binding motif 20 (RBM20) are associated with dilated cardiomyopathy (DCM). To test whether Rbm20 mutations are responsible for cardiac remodelling and potential underlying mechanisms, we selected two mutations (S637A and S639G) located in arginine/serine-rich domain of RBM20 and generated two knock-in mouse models ( Rbm20 S637A and Rbm20 S639G ) respectively. Both mouse lines were subjected to histological and anatomical analysis as well as cardiac function assessment with echocardiography. The results revealed that heterozygous (HT) and homozygous (HM) male and female mice from both lines displayed DCM phenotype with enlarged left ventricular (LV) chamber, thinner LV walls and reduced ejection fraction. Interestingly, we observed that Rbm20 S639G mice manifested more severe phenotype than that in Rbm20 S637A mice. Rbm20 S639G HM mice exhibited higher mortality rate (~50%) than that (~34%) in Rbm20 S637A HM mice at an early age (<100 days). Differentially expressed and spliced genes from RNA-seq data are involved in arrhythmia, cardiomyopathy, and sudden death based on Human Phenotype Ontology enrichment analysis (FDR≤0.05) in both lines. Immunofluorescent analysis in tissues and single cardiomyocytes respectively showed both mutations promote RBM20 nucleocytoplasmic trafficking and protein condensates, suggesting they are gain-of-function mutations, leading to high mortality rate at younger age. Both mutations also interrupted the alternative splicing of TTN and calcium handling genes. Force measurements and Ca 2+ transient evaluation of intact myocytes showed that the reduced cellular diastolic stiffness and impaired contractility in HT and HM mice. Studies suggested that phosphorylation of RS domain is critical for protein trafficking. We then performed mass spectrometry on the in vitro expressed RBM20 and identified phosphorylation on S637 and S639. Phosphomimetic assay showed that phosphorylation was not responsible for RBM20 trafficking and condensates. Together, we reported a new mechanism by which cardiac genetic mutations causes protein trafficking and aggregation in cardiac remodelling.

The ryanodine receptor stabilizer S107 ameliorates contractility of adult Rbm20 knockout rat cardiomyocytes.

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.

RBM20-Mediated Pre-mRNA Splicing Has Muscle-Specificity and Differential Hormonal Responses between Muscles and in Muscle Cell Cultures

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.

Angiotensin II Influences Pre-mRNA Splicing Regulation by Enhancing RBM20 Transcription Through Activation of the MAPK/ELK1 Signaling Pathway.

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.

Abstract 891: Phosphorylation of RNA Binding Motif 20 is a Novel Target to Reduce Myocardial Stiffness in Diastolic Dysfunction

Introduction: Published work has shown that RNA binding motif 20 (RBM20) can improve ventricular stiffening through altering titin sizes in HFpEF, a syndrome with no effective therapeutic options. Reducing RBM20 activity to treat diastolic dysfunction is promising, but it may cause side effects due to its multiple targets except for titin. RBM20 is one of the serine-arginine (SR) proteins of which the phosphorylation plays a critical role in gene splicing. Here we investigate the phosphorylation status of RBM20 and its potential role in diastolic dysfunction. Methods: Insect cell expression system was utilizedfor RBM20 expression. Purified RBM20 was subjected to the middle-down mass spectrometry (MS) analysis. Identified phosphorylation sites were mutated for in-vitro splicing and dual luciferase splice reporter assay through co-transfection with titin mini-gene. In-vitro kinase and co-IP assays were performed to identify the kinases that phosphorylate RBM20. Since phosphorylation is also important for translocation of RNA binding proteins, the confocal microscope was applied to estimate RBM20 trafficking. Results: Sixteen phosphorylation sites with four of them on SR domain of RBM20 were identified with MS analysis. In-vitro splicing and dual luciferase reporter assays revealed that two phosphorylation sites on SR domain play a major role in titin mini-gene splicing. In-vitro kinase assay indicated that kinases SRPK, CLK, and AKT can all phosphorylate RBM20, and co-IP confirmed the interaction between kinases and RBM20. Co-transfection of these kinases with RBM20 and titin mini-gene revealed that splicing pattern was different from co-transfection of mutated RBM20. Interestingly, two-phosphorylation sites mutation on SR domain facilitated RBM20 trafficking from the nucleus to the cytoplasm and thus, form RNA granules, suggesting the new role of RBM20 phosphorylation in diastolic dysfunction. In-vivo studies with mutation knock-in mouse model will be warranted in near future. Conclusion: These results suggest that RBM20 phosphorylation plays an important role in titin splicing, which is a potential target to treat diastolic dysfunction. RBM20 trafficking could be another new pathway to treat HFpEF.

Z-band and M-band titin splicing and regulation by RNA binding motif 20 in striated muscles.

Titin (TTN) has multifunctional roles in sarcomere assembly, mechanosignaling transduction, and muscle stiffness. TTN splicing generates variable protein sizes with different functions. Therefore, understanding TTN splicing is important to develop a novel treatment for TTN-based diseases. The I-band TTN splicing regulated by RNA binding motif 20 (RBM20) has been extensively studied. However, the Z- and M-band splicing and regulation remain poorly understood. Herein, we aimed to define the Z- and M-band splicing in striated muscles and determined whether RBM20 regulates the Z- and M-band splicing. We discovered four new Z-band TTN splicing variants, and one of them dominates in mouse, rat, sheep, and human hearts. But only one form can be detected in frog and chicken hearts. In skeletal muscles, three new Z repeats (Zr) were detected, and Zr4 to 6 exclusion dominates in the fast muscles, whereas Zr4 skipping dominates in the slow muscle. No developmental changes were detected in the Z-band. In the M-band, two new variants were discovered with alternative 3' splice site in exon363 (Mex5) and alternative 5' splice site in intron 362. However, only the sheep heart expresses two new variants rather than other species. Skeletal muscles express three M-band variants with altered ratios of Mex5 inclusion to Mex5 exclusion. Finally, we revealed that RBM20 does not regulate the Z- and M-band splicing in the heart, but does in skeletal muscles. Taken together, we characterized the Z- and M-band splicing and provided the first evidence of the role of RBM20 in the Z- and M-band TTN splicing.

Abstract 269: RBM20 Knockout Ameliorates Angiotensin II Induced Hypertension and Cardiac Hypertrophy

Introduction: RNA binding motif 20 (RBM20) is a muscle specific splicing factor that has been implicated in heart failure (HF). RBM20 regulates mRNA splicing of titin, a giant muscle protein that is expressed in cardiac and smooth muscles and plays a major role in passive stiffness of them. RBM20 knockout (KO) results in less myocardial stiffness by inducing large titin isoform expression. Hence, we hypothesize that RBM20 decreases arterial stiffness through large titin isoform expression, and thus reduces blood pressure (BP) in hypertension. The aim of this study was to investigate whether RBM20 could be a therapeutic target to reduce BP in patients with hypertension and prevent HF progression. Methods: Male wild type (WT) (Sprague-Dawley х Brown Norway) and Rbm20 KO rats (n=10 each) were subcutaneously implanted with osmotic mini-pump to continuously release angiotensin II (Ang II) at a rate of 400 ng/kg/min for 28 days. BP was measured by noninvasive tail-cuff system weekly. Pulse wave velocity (PWV) was measured by Doppler system. Cardiac remodeling was evaluated by echocardiography. Biochemistry and histological analyses were performed on aorta and heart tissues. Results: Larger titin isoform was increased and no difference of elastin and collagen expression was observed in RBM20 KO blood vessels when compared to WT. Basal BPs were the same in WT and KO rats (diastolic BP, KO 91.75 ± 1.83 vs. WT 94.33±1.88 mmHg; systolic BP, KO 127.6 ± 0.97 vs. WT 128.0 ± 1.35 mmHg). Ang II increased BP in both WT and KO rats, but KO had a significantly lower BP than WT (diastolic BP, KO 141.7 ± 5.00 vs. WT 182.0±4.84 mmHg; systolic BP, KO 195.4 ± 3.671 vs. WT 226.4 ± 3.093 mmHg, P<0.0001). PWV is lower in KO rats than in WT (KO 4.035 ± 0.21 mm/s vs. WT 5.128±0.37 mm/s, P<0.05). Cardiac hypertrophy was developed in both WT and KO hearts treated with Ang II, but it was alleviated in the KO heart (left ventricular wall, KO 0.324 ± 0.01 cm vs. WT 0.368 ± 0.013 cm, P<0.05). Conclusions: RBM20 KO reduces arterial stiffness through increased larger titin isoform expression, decreases Ang II-induced hypertension, and prevents HF progression. These findings suggest that RBM20 could be a potential novel target for the treatment of hypertension and hypertension-induced cardiac hypertrophy.

Mass Spectrometry Analysis of RBM20 Phosphorylation and Its Role in Titin Splicing

ObjectiveThe SR protein family has a characteristic domain which is rich in arginine and serine residues. They play significant roles in constitutive pre‐mRNA splicing and the phosphorylation status of them is important in the regulation of alternative splicing. RNA binding motif 20 (RBM20), a newly identified SR protein, mainly regulates splicing of a giant muscle gene titin, a major heart disease‐causing gene. However, the mechanism of RBM20 modulation on titin splicing is not well defined. We hypothesize that RBM20 phosphorylation plays an important role in titin splicing. In this study, we employed middle‐down mass spectrometry and in vitro splicing assay to explore the phosphorylation status of RBM20 and its role in titin splicing.MethodsBoth bacteria and insect cell expressed RBM20 were used for ProQ diamond staining and middle down mass spectrometry analysis. In vitro approaches with co‐transfection of mutated RBM20 and titin minigene, and a dual luciferase splice reporter assay were performed to explore the role of RBM20 phosphorylation in titin splicing.ResultsInsect cell expressed RBM20 was stained by ProQ diamond, a dye which is specifically used for phosphorylated protein staining in SDS‐PAGE gel, whereas ProQ diamond stained bands were not observed with bacteria expressed RBM20. Mass spectrometry analysis on the insect cell expressed RBM20 identified 16 phosphorylation sites with four of them located in RS domain. 16 mutated RBM20 plasmids on individual phosphorylation sites co‐transfected with titin minigene in HeLA cells, and the results indicated that only phosphorylation in RS domain affects titin splicing, and splice report assay further confirmed the results.ConclusionRBM20 is massively phosphorylated with 16 phosphorylation sites, and only RS domain phosphorylation plays a role in titin splicing. These results reveal that the phosphorylation sites on RS domain residues could be potential therapeutic targets for titin splicing‐induced heart disease.Support or Funding InformationNIFA‐USDA 1009266, NIH NIGMSP20GM103432, AHA BGIA and Faculty‐Grant‐in‐Aid from University of WyomingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Splicing Variation of TTN Novex Isoforms Across Species and RBM20 Does Not Regulate Splicing of Novex Isoforms in Cardiac Muscle.

TTN is a major disease‐causing gene in cardiac muscle. 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 alternative exon usages of exon 45 (Novex 1), exon 46 (Novex 2) and exon 48 (Novex 3). It is unknown how these exons are alternatively used in Novex isoforms across species and whether Novex isoforms are associated with heart disease. In this study, we compared alternative exon usage between species with the mVISTA online tool, and found that exon 45 is conserved across all species, but exons 46 and 48 only found in some species. Interestingly, we found a conserved region between exons 47 and 48 across species which has never been characterized before. RT‐PCR and DNA sequencing confirmed that this is a new exon named as 48′ in Novex 3. In addition, with primer pairs for Novex 1, we found two new splicing forms, which one has intron 44 retention and the other has intron 45 retention. With Novex 2 primer pairs, we found that Novex 2 is lack of expression in pig, mouse and rat. A new truncated isoform with intron 46 retention is mainly expressed in human and frog heart, and another new truncated isoform with exon 45 and 46 are co‐expressed predominantly in chicken and frog heart. Using RBM20 knockout heart, we revealed that RBM20 doesn't regulate splicing of Novex isoforms. Finally, we examined the expression levels of Novex isoforms in human heart with cardiomyopathies, and we didn't find any difference. These results suggest that exon usage of Novex isoforms varies across species which is not associated with cardiomyopathies. Hence, their functional role in the heart need be further studied in the future, which is beyond the scope of this study.Support or Funding InformationThis work was supported by the NIFA‐USDA 1009266, National Institutes of Health NIGMSP20GM103432, AHA BGIA and Faculty‐Grant‐in‐Aid from University of Wyoming.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of TTN Novex Splicing Variants across Species and the Role of RBM20 in Novex-Specific Exon Splicing.

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.

Muscle-Specific Mis-Splicing and Heart Disease Exemplified by RBM20.

Alternative splicing is an essential post-transcriptional process to generate multiple functional RNAs or proteins from a single transcript. Progress in RNA biology has led to a better understanding of muscle-specific RNA splicing in heart disease. The recent discovery of the muscle-specific splicing factor RNA-binding motif 20 (RBM20) not only provided great insights into the general alternative splicing mechanism but also demonstrated molecular mechanism of how this splicing factor is associated with dilated cardiomyopathy. Here, we review our current knowledge of muscle-specific splicing factors and heart disease, with an emphasis on RBM20 and its targets, RBM20-dependent alternative splicing mechanism, RBM20 disease origin in induced Pluripotent Stem Cells (iPSCs), and RBM20 mutations in dilated cardiomyopathy. In the end, we will discuss the multifunctional role of RBM20 and manipulation of RBM20 as a potential therapeutic target for heart disease.

Splicing Factor RBM20 Regulates Transcriptional Network of Titin Associated and Calcium Handling Genes in The Heart.

RNA binding motif 20 (RBM20) regulates pre-mRNA splicing of over thirty genes, among which titin is a major target. With RBM20 expression, titin expresses a larger isoform at fetal stage to a smaller isoform at adult resulting from alternative splicing, while, without RBM20, titin expresses exclusively a larger isoform throughout all ages. In addition to splicing regulation, it is unknown whether RBM20 also regulates gene expression. In this study, we employed Rbm20 knockout rats to investigate gene expression profile using Affymetrix expression array. We compared wild type to Rbm20 knockout at day1, 20 and 49. Bioinformatics analysis showed RBM20 regulates fewer genes expression at younger age and more at older age and commonly expressed genes have the same trends. GSEA indicated up-regulated genes are associated with heart failure. We examined titin binding partners. All titin direct binding partners are up-regulated and their increased expression is associated with dilated cardiomyopathy. Particularly, we found that genes involving calcium handling and muscle contraction are changed by RBM20. Intracellular calcium level measurement with individual cardiomyocytes further confirmed that changes of these proteins impact calcium handling. Selected genes from titin binding partners and calcium handling were validated with QPCR and western blotting. These data demonstrate that RBM20 regulates gene splicing as well as gene expression. Altered gene expression by RBM20 influences protein-protein interaction, calcium releasing and thus muscle contraction. Our results first reported gene expression impacted by RBM20 with heart maturation, and provided new insights into the role of RBM20 in the progression of heart failure.

Insulin regulates titin pre-mRNA splicing through the PI3K-Akt-mTOR kinase axis in a RBM20-dependent manner

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.

Modeling and Experiment to Determine the Role of Passive Stiffness on Mechanical (Strain Rate) Control of Relaxation

Diastolic dysfunction is increasingly linked to exercise intolerance and heart failure-like symptoms. The serendipitous discovery of a rat with a spontaneous mutation in the RNA Binding Motif-20 (RBM20) has led to new studies showing the effects of reduced titin-based passive tension. Decreased passive stiffness in the heart is associated with increased exercise tolerance, but also slowed crossbridge attachment and detachment rates. The influence on relaxation, and specifically mechanical control of relaxation by fast end systolic stretch, is not yet known. We utilized MyoSim, a computational modeling environment, and experiments using intact trabeculae to investigate the relationship between titin-based passive stiffness and mechanical control of relaxation. We have previously obtained physiological myofilament parameters by fitting a 2-state crossbridge model to experimental data using MyoSim. These model parameters were held fixed except for passive stiffness, which was reduced by 50 and 75% to match passive stiffness in heterozygous and homozygous RBM20 mutant tissues. Isometric relaxation was slowed by 4 and 5%, respectively, but relaxation rate became 19 and 23% more sensitive to mechanical control of relaxation (slope of relationship between relaxation rate and end systolic strain rate). These data predict that titin based stiffness may modify relaxation rate. Trabeculae experiments using wild-type, heterozygous, and homozygous RBM20 mutant rats, show no significant change in the mechanical control of relaxation. However, reduced titin stiffness was associated with a later time to peak contraction (shortening). Reducing crossbridge attachment and detachment rates in the computational model parameters replicates the experimental results. These data suggest that passive stiffness may play a role on mechanical control of relaxation and confirm that crossbridge properties significantly contribute to early diastolic relaxation.

Experimentally Increasing the Compliance of Titin Through RNA Binding Motif-20 (RBM20) Inhibition Improves Diastolic Function In a Mouse Model of Heart Failure With Preserved Ejection Fraction.

Left ventricular (LV) stiffening contributes to heart failure with preserved ejection fraction (HFpEF), a syndrome with no effective treatment options. Increasing the compliance of titin in the heart has become possible recently through inhibition of the splicing factor RNA binding motif-20. Here, we investigated the effects of increasing the compliance of titin in mice with diastolic dysfunction. Mice in which the RNA recognition motif (RRM) of one of the RNA binding motif-20 alleles was floxed and that expressed the MerCreMer transgene under control of the αMHC promoter (referred to as cRbm20ΔRRM mice) were used. Mice underwent transverse aortic constriction (TAC) surgery and deoxycorticosterone acetate (DOCA) pellet implantation. RRM deletion in adult mice was triggered by injecting raloxifene (cRbm20ΔRRM-raloxifene), with dimethyl sulfoxide (DMSO)-injected mice (cRbm20ΔRRM-DMSO) as the control. Diastolic function was investigated with echocardiography and pressure-volume analysis; passive stiffness was studied in LV muscle strips and isolated cardiac myocytes before and after elimination of titin-based stiffness. Treadmill exercise performance was also studied. Titin isoform expression was evaluated with agarose gels. cRbm20ΔRRM-raloxifene mice expressed large titins in the hearts, called supercompliant titin (N2BAsc), which, within 3 weeks after raloxifene injection, made up ≈45% of total titin. TAC/DOCA cRbm20ΔRRM-DMSO mice developed LV hypertrophy and a marked increase in LV chamber stiffness as shown by both pressure-volume analysis and echocardiography. LV chamber stiffness was normalized in TAC/DOCA cRbm20ΔRRM-raloxifene mice that expressed N2BAsc. Passive stiffness measurements on muscle strips isolated from the LV free wall revealed that extracellular matrix stiffness was equally increased in both groups of TAC/DOCA mice (cRbm20ΔRRM-DMSO and cRbm20ΔRRM-raloxifene). However, titin-based muscle stiffness was reduced in the mice that expressed N2BAsc (TAC/DOCAcRbm20ΔRRM-raloxifene). Exercise testing demonstrated significant improvement in exercise tolerance in TAC/DOCA mice that expressed N2BAsc. Inhibition of the RNA binding motif-20-based titin splicing system upregulates compliant titins, which improves diastolic function and exercise tolerance in the TAC/DOCA model. Titin holds promise as a therapeutic target for heart failure with preserved ejection fraction.