Titin Splicing Research Articles

The skeletal muscle circadian clock regulates titin splicing through RBM20.

Circadian rhythms are maintained by a cell-autonomous, transcriptional-translational feedback loop known as the molecular clock. While previous research suggests a role of the molecular clock in regulating skeletal muscle structure and function, no mechanisms have connected the molecular clock to sarcomere filaments. Utilizing inducible, skeletal muscle specific, Bmal1 knockout (iMSBmal1-/-) mice, we showed that knocking out skeletal muscle clock function alters titin isoform expression using RNAseq, liquid chromatography-mass spectrometry, and sodium dodecyl sulfate-vertical agarose gel electrophoresis. This alteration in titin's spring length resulted in sarcomere length heterogeneity. We demonstrate the direct link between altered titin splicing and sarcomere length in vitro using U7 snRNPs that truncate the region of titin altered in iMSBmal1-/- muscle. We identified a mechanism whereby the skeletal muscle clock regulates titin isoform expression through transcriptional regulation of Rbm20, a potent splicing regulator of titin. Lastly, we used an environmental model of circadian rhythm disruption and identified significant downregulation of Rbm20 expression. Our findings demonstrate the importance of the skeletal muscle circadian clock in maintaining titin isoform through regulation of RBM20 expression. Because circadian rhythm disruption is a feature of many chronic diseases, our results highlight a novel pathway that could be targeted to maintain skeletal muscle structure and function in a range of pathologies.

Contributions of Titin and Collagen to Passive Stress in Muscles from mdm Mice with a Small Deletion in Titin's Molecular Spring.

Muscular dystrophy with myositis (mdm) is a naturally occurring mutation in the mouse Ttn gene that results in higher passive stress in muscle fibers and intact muscles compared to wild-type (WT). The goal of this study was to test whether alternative splicing of titin exons occurs in mdm muscles, which contain a small deletion in the N2A-PEVK regions of titin, and to test whether splicing changes are associated with an increase in titin-based passive tension. Although higher levels of collagen have been reported previously in mdm muscles, here we demonstrate alternative splicing of titin in mdm skeletal muscle fibers. We identified Z-band, PEVK, and C-terminus Mex5 exons as splicing hotspots in mdm titin using RNA sequencing data and further reported upregulation in ECM-associated genes. We also treated skinned mdm soleus fiber bundles with trypsin, trypsin + KCl, and trypsin + KCL + KI to degrade titin. The results showed that passive stress dropped significantly more after trypsin treatment in mdm fibers (11 ± 1.6 mN/mm2) than in WT fibers (4.8 ± 1 mN/mm2; p = 0.0004). The finding that treatment with trypsin reduces titin-based passive tension more in mdm than in WT fibers supports the hypothesis that exon splicing leads to the expression of a stiffer and shorter titin isoform in mdm fibers. After titin extraction by trypsin + KCl + KI, mdm fibers (6.7 ± 1.27 mN/mm2) had significantly higher collagen-based passive stress remaining than WT fibers (2.6 ± 1.3 mN/mm2; p = 0.0014). We conclude that both titin and collagen contribute to higher passive tension of mdm muscles.

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.

Abstract 10966: Characterization of the Novel Cardiac Splicing Factor Slm2 in Heart Failure, Which Regulates Splicing of Titin

Introduction: The versatility of the human genome is increased by the process of alternative mRNA splicing. Impaired splicing of the cardiac transcriptome is involved in the pathophysiology of heart failure. Especially, mutations in cardiac-specific splicing factors such as RBM20 cause severe forms of cardiomyopathy. Aim: We aimed to identify novel cardiomyopathy-associated splicing factors in the human heart using a score of myocardial tissue specificity including 53 human tissues and disease-associated expression changes in hearts of dilated cardiomyopathy (DCM) patients and controls. Methods & Results: We found the splicing factor Slm2 to be significantly upregulated on the mRNA and protein level in the human myocardium of patients diagnosed with DCM (1.88 and 5.3-fold, both P<0.05). Reduction of Slm2 in vivo resulted in DCM in zebrafish and sarcomere irregularity and altered calcium cycling in human cardiomyocytes. Sequencing of RNAs bound to Slm2 isolated from diseased human hearts, revealed the interaction of Slm2 with important mRNA transcripts encoding for sarcomere constituents, such as troponin I, troponin T, tropomyosin and titin. In the failing human myocardium, Slm2 bound to an alternative titin mRNA variant including retained introns. In detail, Slm2 interacted with exons of the titin mRNA encoding for the PEVK region, which mediates flexibility and contractile properties of the heart muscle. Using a splice reporter assay, which contains parts of titin’s PEVK region, we found Slm2 mediating the splicing process of intron retention. Conclusion: Slm2 is a novel splicing factor in the diseased human myocardium, maintaining cardiomyocyte integrity and function by binding and splicing essential sarcomere constituents including titin.

Depletion of m6 A reader protein YTHDC1 induces dilated cardiomyopathy by abnormal splicing of Titin.

N 6‐methyladenosine (m6A) is the most prevalent modification in mRNA and engages in multiple biological processes. Previous studies indicated that m6A methyltransferase METTL3 (‘writer’) and demethylase FTO (‘eraser’) play critical roles in heart‐related disease. However, in the heart, the function of m6A ‘reader’, such as YTH (YT521‐B homology) domain‐containing proteins remains unclear. Here, we report that the defect in YTHDC1 but not other YTH family members contributes to dilated cardiomyopathy (DCM) in mice. Cardiac‐specific conditional Ythdc1 knockout led to obvious left ventricular chamber enlargement and severe systolic dysfunction. YTHDC1 deficiency also resulted in the decrease of cardiomyocyte contractility and disordered sarcomere arrangement. By means of integrating multiple high‐throughput sequence technologies, including m6A‐MeRIP, RIP‐seq and mRNA‐seq, we identified 42 transcripts as potential downstream targets of YTHDC1. Amongst them, we found that Titin mRNA was decorated with m6A modification and depletion of YTHDC1 resulted in aberrant splicing of Titin. Our study suggests that Ythdc1 plays crucial role in regulating the normal contractile function and the development of DCM. These findings clarify the essential role of m6A reader in cardiac biofunction and provide a novel potential target for the treatment of DCM.

Deleting Titin's C-Terminal PEVK Exons Increases Passive Stiffness, Alters Splicing, and Induces Cross-Sectional and Longitudinal Hypertrophy in Skeletal Muscle.

The Proline, Glutamate, Valine and Lysine-rich (PEVK) region of titin constitutes an entropic spring that provides passive tension to striated muscle. To study the functional and structural repercussions of a small reduction in the size of the PEVK region, we investigated skeletal muscles of a mouse with the constitutively expressed C-terminal PEVK exons 219–225 deleted, the TtnΔ219–225 model (MGI: TtnTM 2.1Mgot). Based on this deletion, passive tension in skeletal muscle was predicted to be increased by ∼17% (sarcomere length 3.0 μm). In contrast, measured passive tension (sarcomere length 3.0 μm) in both soleus and EDL muscles was increased 53 ± 11% and 62 ± 4%, respectively. This unexpected increase was due to changes in titin, not to alterations in the extracellular matrix, and is likely caused by co-expression of two titin isoforms in TtnΔ219–225 muscles: a larger isoform that represents the TtnΔ219–225 N2A titin and a smaller isoform, referred to as N2A2. N2A2 represents a splicing adaption with reduced expression of spring element exons, as determined by titin exon microarray analysis. Maximal tetanic tension was increased in TtnΔ219–225 soleus muscle (WT 240 ± 9; TtnΔ219–225 276 ± 17 mN/mm2), but was reduced in EDL muscle (WT 315 ± 9; TtnΔ219–225 280 ± 14 mN/mm2). The changes in active tension coincided with a switch toward slow fiber types and, unexpectedly, faster kinetics of tension generation and relaxation. Functional overload (FO; ablation) and hindlimb suspension (HS; unloading) experiments were also conducted. TtnΔ219–225 mice showed increases in both longitudinal hypertrophy (increased number of sarcomeres in series) and cross-sectional hypertrophy (increased number of sarcomeres in parallel) in response to FO and attenuated cross-sectional atrophy in response to HS. In summary, slow- and fast-twitch muscles in a mouse model devoid of titin’s PEVK exons 219–225 have high passive tension, due in part to alterations elsewhere in splicing of titin’s spring region, increased kinetics of tension generation and relaxation, and altered trophic responses to both functional overload and unloading. This implicates titin’s C-terminal PEVK region in regulating passive and active muscle mechanics and muscle plasticity.

Titin splicing regulates cardiotoxicity associated with calpain 3 gene therapy for limb-girdle muscular dystrophy type 2A.

Limb-girdle muscular dystrophy type 2A (LGMD2A or LGMDR1) is a neuromuscular disorder caused by mutations in the calpain 3 gene (CAPN3). Previous experiments using adeno-associated viral (AAV) vector-mediated calpain 3 gene transfer in mice indicated cardiac toxicity associated with the ectopic expression of the calpain 3 transgene. Here, we performed a preliminary dose study in a severe double-knockout mouse model deficient in calpain 3 and dysferlin. We evaluated safety and biodistribution of AAV9-desmin-hCAPN3 vector administration to nonhuman primates (NHPs) with a dose of 3 × 1013 viral genomes/kg. Vector administration did not lead to observable adverse effects or to detectable toxicity in NHP. Of note, the transgene expression did not produce any abnormal changes in cardiac morphology or function of injected animals while reaching therapeutic expression in skeletal muscle. Additional investigation on the underlying causes of cardiac toxicity observed after gene transfer in mice and the role of titin in this phenomenon suggest species-specific titin splicing. Mice have a reduced capacity for buffering calpain 3 activity compared to NHPs and humans. Our studies highlight a complex interplay between calpain 3 and titin binding sites and demonstrate an effective and safe profile for systemic calpain 3 vector delivery in NHP, providing critical support for the clinical potential of calpain 3 gene therapy in humans.

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 440: The Cardiomyocyte Diastolic Stiffness and Contractility are Inversely Related to the Size of Titin: A Cellular Work Loop Study of Single Intact Cardiomyocytes

Studying cardiac titin under physiological conditions can be at the level of the left ventricular (LV) chamber and the multicellular muscle level. However, those preparations are influenced by neurohormonal reflex, preload-afterload status and extracellular matrix. In this study, cellular work loop force measurement of the single intact cardiomyocyte was conducted under physiological conditions. Intact cell experiments were performed at 37°C under field-stimulation at 2 Hz frequency. The cell was attached to the force transducer and the piezo length controller which modulates the cell length by the feedback control system based on developed force. The force length relation (cellular work loop) of the single cell was generated. The end diastolic force length relation (EDFLR), the end systolic force length relation (ESFLR) and the preload recruitable stroke work (PRSW) were obtained. The Ttn ΔIAjxn -/- ( IA -/- ) mice, in which the I-band/A-band region of titin’s spring has been deleted were studied in comparison to α-MHC-Cre ; cRbm20 ΔRRM +/- ( cRbm20 +/- ) mice which are deficient in titin splicing and express large titin isoforms in the heart. The cellular diastolic stiffness (slope of EDFLR) was increased in IA -/- and was reduced in cRbm20 +/- (151.3, 104.0 and 91.5 μN/mm 2 in IA -/- , WT and cRbm20 +/- respectively). The contractility (slopes of ESFLR and PRSW) were 233.5 and 1215.6 uN/mm 2 in WT, and were increased in IA -/- (371.2 and 2076 μN/mm 2 ) but attenuated in cRbm20 +/- (202.9 and 907.7 μN/mm 2 ). Cellular work loop studies were also conducted in intact cells isolated from the mice that underwent transverse aortic constriction (TAC) surgery with deoxycorticosterone acetate (DOCA) implantation. TAC DOCA mice developed diastolic dysfunction at the LV chamber level. The result showed that the cells from TAC DOCA hearts had increased slopes of EDFLR, ESFLR and PRSW ( 157.9, 378.9 and 1699.0 μN/mm 2 ) compared to the cells from sham mice (98.6, 230.3, and 1203.9 μN/mm 2 ). Conclusions: Cellular work loops recapitulated the cardiac physiology at the LV chamber level. The result showed that the cellular diastolic stiffness and contractility were inversely related to the size of titin and that titin stiffness plays an important roles in both diastolic and systolic function.

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.

Z‐band and M‐band Titin Splicing and Regulation by RBM20 in Striated Muscles

Titin (TTN) has multifunctional roles in sarcomere assembly, mechanosignaling transduction and muscle stiffness. TTN splicing generates variable protein sizes that are associated with its functions. Therefore, understanding TTN splicing is important to develop novel treatment for titin‐based diseases. The I‐band TTN splicing and regulation by RBM20 have been extensively studied. However, the Z‐ and Mband splicing and regulation remain poorly understood. Herein, we aimed to define the Z‐ and M‐band splicing in striated muscles, and also determine whether RBM20 regulates the Z‐ and M‐band splicing. Our data discovered four new Z‐band TTN splicing variants, and one of them dominates in mouse, rat, sheep and human heart. Frog and chicken hearts express only one variant. In skeletal muscles, three new Z repeats were detected, and variant with Zr4–6 skipping 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 intron362. However, only sheep heart expresses two new variants, while other species express only one variant with Mex5+. Skeletal muscles express all three M‐band variants with altered ratios of Mex5+ to Mex5−. Lastly, 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.Support or Funding InformationThis work was supported by the National Institute of Health/National Institute of General Medical Sciences [NIGMSP20GM103432]; the BGIA from the American Heart Association [16BGIA27790136 to WG]; the USDA National Institute of Food and Agriculture [Hatch project 1009266 to WG]; NICHD 1R01HD070096‐01A1 (SPF and PWN). Ying Ge would like to acknowledge NIH R01 HL109810 and HL096971.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The complexity of titin splicing pattern in human adult skeletal muscles

BackgroundMutations in the titin gene (TTN) cause a large spectrum of diseases affecting skeletal and/or cardiac muscle. TTN includes 363 coding exons, a repeated region with a high degree of complexity, isoform-specific elements, and metatranscript-only exons thought to be expressed only during fetal development. Although three main classes of isoforms have been described so far, alternative splicing events (ASEs) in different tissues or in different developmental and physiological states have been reported.MethodsTo achieve a comprehensive view of titin ASEs in adult human skeletal muscles, we performed a RNA-Sequencing experiment on 42 human biopsies collected from 12 anatomically different skeletal muscles of 11 individuals without any skeletal-muscle disorders.ResultsWe confirmed that the skeletal muscle N2A isoforms are highly prevalent, but we found an elevated number of alternative splicing events, some at a very high level. These include previously unknown exon skipping events and alternative 5′ and 3′ splice sites. Our data suggests the partial inclusion in the TTN transcript of some metatranscript-only exons and the partial exclusion of canonical N2A exons.ConclusionsThis study provides an extensive picture of the complex TTN splicing pattern in human adult skeletal muscle, which is crucial for a proper clinical interpretation of TTN variants.

The Skeletal Muscle Molecular Clock Regulates Titin Splicing and Protein Expression

The circadian factor BMAL1 is a fundamental transcriptional regulator of cell time keeping and expression of cell specific genes important for homeostasis. Our lab developed the inducible, skeletal muscle specific Bmal1 knockout (iMSBmal1-/-) mouse to determine the role of this protein in adult skeletal muscle. Skeletal muscles from these mice exhibit decreased specific tension and reduced unstimulated tension developed during a fatigability test. Since this change could be caused by changes in the elastic properties of the muscle, we investigated whether titin isoform expression changes in these mice. The tibialis anterior muscle of the iMSBmal1-/- mice exhibits a significant shift in expression from a short to long isoform of titin protein (38% long in iMSBmal1-/- vs. 19% in iMSBmal1+/+). RNA-Seq from these muscles indicates increased inclusion of titin exons 150-155 and 190-225, which are found within titin's PEVK region. Because this region is highly extensible and acts as a spring, we performed immunohistochemistry to test whether changes to this protein could have an effect on sarcomere length. An antibody to α-actinin was used to demarcate Z-lines in longitudinal sections of the tibialis anterior muscle and sarcomere length was measured as the distance between Z-lines. While average sarcomere length was not different between iMSBmal1+/+ and iMSBmal1-/- muscle, variation in sarcomere length increased following Bmal1 knockout. To test if increased variability of titin isoform expression similar to the iMSBmal1-/- mouse could lead to variable sarcomere lengths, we are currently transfecting C2C12 myoblasts with U7 snRNPs targeted to titin PEVK regions. Sarcomere length variability will be measured in these cells following 7 days of differentation. Current studies with this model are ongoing and will lead to increased knowledge of titin's role in regulating sarcomere length in a skeletal muscle at rest.

Aberrant developmental titin splicing and dysregulated sarcomere length in Thymosin β4 knockout mice

Sarcomere assembly is a highly orchestrated and dynamic process which adapts, during perinatal development, to accommodate growth of the heart. Sarcomeric components, including titin, undergo an isoform transition to adjust ventricular filling. Many sarcomeric genes have been implicated in congenital cardiomyopathies, such that understanding developmental sarcomere transitions will inform the aetiology and treatment. We sought to determine whether Thymosin β4 (Tβ4), a peptide that regulates the availability of actin monomers for polymerization in non-muscle cells, plays a role in sarcomere assembly during cardiac morphogenesis and influences adult cardiac function. In Tβ4 null mice, immunofluorescence-based sarcomere analyses revealed shortened thin filament, sarcomere and titin spring length in cardiomyocytes, associated with precocious up-regulation of the short titin isoforms during the postnatal splicing transition. By magnetic resonance imaging, this manifested as diminished stroke volume and limited contractile reserve in adult mice. Extrapolating to an in vitro cardiomyocyte model, the altered postnatal splicing was corrected with addition of synthetic Tβ4, whereby normal sarcomere length was restored. Our data suggest that Tβ4 is required for setting correct sarcomere length and for appropriate splicing of titin, not only in the heart but also in skeletal muscle. Distinguishing between thin filament extension and titin splicing as the primary defect is challenging, as these events are intimately linked. The regulation of titin splicing is a previously unrecognised role of Tβ4 and gives preliminary insight into a mechanism by which titin isoforms may be manipulated to correct cardiac dysfunction.

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.

Increased Titin Compliance Reduced Length-Dependent Contraction and Slowed Cross-Bridge Kinetics in Skinned Myocardial Strips from Rbm (20ΔRRM) Mice.

Titin is a giant protein spanning from the Z-disk to the M-band of the cardiac sarcomere. In the I-band titin acts as a molecular spring, contributing to passive mechanical characteristics of the myocardium throughout a heartbeat. RNA Binding Motif Protein 20 (RBM20) is required for normal titin splicing, and its absence or altered function leads to greater expression of a very large, more compliant N2BA titin isoform in Rbm20 homozygous mice (Rbm20ΔRRM) compared to wild-type mice (WT) that almost exclusively express the stiffer N2B titin isoform. Prior studies using Rbm20ΔRRM animals have shown that increased titin compliance compromises muscle ultrastructure and attenuates the Frank-Starling relationship. Although previous computational simulations of muscle contraction suggested that increasing compliance of the sarcomere slows the rate of tension development and prolongs cross-bridge attachment, none of the reported effects of Rbm20ΔRRM on myocardial function have been attributed to changes in cross-bridge cycling kinetics. To test the relationship between increased sarcomere compliance and cross-bridge kinetics, we used stochastic length-perturbation analysis in Ca2+-activated, skinned papillary muscle strips from Rbm20ΔRRM and WT mice. We found increasing titin compliance depressed maximal tension, decreased Ca2+-sensitivity of the tension-pCa relationship, and slowed myosin detachment rate in myocardium from Rbm20ΔRRM vs. WT mice. As sarcomere length increased from 1.9 to 2.2 μm, length-dependent activation of contraction was eliminated in the Rbm20ΔRRM myocardium, even though myosin MgADP release rate decreased ~20% to prolong strong cross-bridge binding at longer sarcomere length. These data suggest that increasing N2BA expression may alter cardiac performance in a length-dependent manner, showing greater deficits in tension production and slower cross-bridge kinetics at longer sarcomere length. This study also supports the idea that passive mechanical characteristics of the myocardium influence ensemble cross-bridge behavior and maintenance of tension generation throughout the sarcomere.

Alternative Splicing of Titin Restores Diastolic Function in an HFpEF-Like Genetic Murine Model (TtnΔIAjxn).

Patients with heart failure with preserved ejection fraction (HFpEF) experience elevated filling pressures and reduced ventricular compliance. The splicing factor RNA-binding motif 20 (RBM20) regulates the contour length of titin's spring region and thereby determines the passive stiffness of cardiomyocytes. Inhibition of RBM20 leads to super compliant titin isoforms (N2BAsc) that reduce passive stiffness. To determine the therapeutic potential of upregulating compliant titin isoforms in an HFpEF-like state in the mouse. Constitutive and inducible cardiomyocyte-specific RBM20-inhibited mice were produced on a Ttn(ΔIAjxn) background to assess the effect of upregulating compliant titin at the cellular and organ levels. Genetic deletion of the I-band-A-band junction (IAjxn) in titin increases strain on the spring region and causes a HFpEF-like syndrome in the mouse without pharmacological or surgical intervention. The increased strain represents a mechanical analog of deranged post-translational modification of titin that results in increased passive myocardial stiffness in patients with HFpEF. On inhibition of RBM20 in Ttn(ΔIAjxn) mice, compliant titin isoforms were expressed, diastolic function was normalized, exercise performance was improved, and pathological hypertrophy was attenuated. We report for the first time a benefit from upregulating compliant titin isoforms in a murine model with HFpEF-like symptoms. Constitutive and inducible RBM20 inhibition improves diastolic function resulting in greater tolerance to exercise. No effective therapies exists for treating this pervasive syndrome; therefore, our data on RBM20 inhibition are clinically significant.