Search

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]

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]

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]

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. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]