scholarly journals Overexpression of a non-muscle RBFOX2 isoform triggers cardiac conduction defects in myotonic dystrophy

2019 ◽  
Author(s):  
Chaitali Misra ◽  
Sushant Bangru ◽  
Feikai Lin ◽  
Kin Lam ◽  
Sara N. Koenig ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Trinucleotide Repeat ◽  
Dominant Role ◽  
Genetic Disorder ◽  
Heart Tissue ◽  
Cardiac Conduction ◽  
Conduction Delay ◽  
Electrophysiological Properties ◽  
Splice Isoform

SUMMARYMyotonic dystrophy type 1 (DM1) is a multisystemic genetic disorder caused by a CTG trinucleotide repeat expansion in the 3′ untranslated region of DMPK gene. Heart dysfunctions occur in nearly 80% of DM1 patients and are the second leading cause of DM1-related deaths. Despite these figures, the mechanisms underlying cardiac-based DM1 phenotypes are unknown. Herein, we report that upregulation of a non-muscle splice isoform of RNA binding protein RBFOX2 in DM1 heart tissue—due to altered splicing factor and microRNA activities—induces cardiac conduction defects in DM1 individuals. Mice engineered to express the non-muscle RBFOX2 isoform in heart via tetracycline-inducible transgenesis, or CRISPR/Cas9-mediated genome editing, reproduced DM1-related cardiac-conduction delay and spontaneous episodes of arrhythmia. Further, by integrating RNA binding with cardiac transcriptome datasets from both DM1 patients and mice expressing the non-muscle RBFOX2 isoform, we identified RBFOX2-driven splicing defects in the voltage-gated sodium and potassium channels, which can alter their electrophysiological properties. Thus, our results uncover a trans-dominant role for an aberrantly expressed RBFOX2 isoform in DM1 cardiac pathogenesis.

Abstract MP249: Mechanistic Basis And Therapeutic Potential Of Targeting The Non-muscle Rbfox2 Isoform In Myotonic Dystrophy

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Chaitali Misra ◽  
Ullas V Chembazhi ◽  
Sarah Matatov ◽  
Sushant Bangru ◽  
Auinash Kalsotra
Keyword(s):  
Myotonic Dystrophy ◽  
Regulatory Networks ◽  
Rna Binding ◽  
Trinucleotide Repeat ◽  
Therapeutic Potential ◽  
Cardiac Conduction ◽  
Conduction Delay ◽  
Aberrant Expression ◽  
Ctg Repeats ◽  
Cardiac Symptoms

Myotonic Dystrophy type 1 (DM1), the most prevalent form of adult-onset muscular dystrophy, is caused by CTG trinucleotide repeat expansion in the 3’-UTR of the DMPK gene. Heart dysfunctions occur in nearly 80% of DM1 patients, and cardiac arrhythmias or conduction abnormalities are a prominent cause of mortality in affected individuals. Yet, the underlying mechanisms causing such abnormalities are not well understood. We recently demonstrated that aberrant expression of a non-muscle splice isoform of RNA-binding protein RBFOX2 triggers cardiac conduction delay, atrioventricular heart blocks, and spontaneous arrhythmogenesis in DM1 hearts. Here we studied the mechanism(s) by which non-muscle RBFOX2 induces mis-splicing of cardiac conduction genes and tested new therapeutic strategies for treating the lethal cardiac symptoms of this disease. By performing eCLIP and high-resolution RNA-sequencing studies on cardiomyocytes isolated from wild type (expressing the normal muscle-specific RBFOX2 43 isoform), Rbfox2 Δ43/Δ43 (expressing the non-muscle RBFOX2 40 isoform), and RBFOX2 40 overexpressing (OE) mice, we deconstructed the splicing regulatory networks of RBFOX2 43 and RBFOX2 40 isoforms, characterized their respective RNA binding landscapes, and determined the RBFOX2 40 -driven transcriptome alterations in DM1 heart tissue. We acquired induced pluripotent stem cells (iPSC) from healthy, moderate (238 CTG repeats) and severely (1001 CTG repeats) affected DM1 individuals and differentiated them into cardiomyocytes (iPSC-CMs) to generate a human cardiac cell culture model of DM1. Utilizing anti-sense oligonucleotides and RNAi-based approaches, we restored the muscle-specific Rbfox2 splicing pattern and depleted the non-muscle RBFOX2 isoform in the DM1 IPS-CMs. We are currently analyzing the spontaneous electrical phenotypes of normal and DM1 iPSC-CMs. Collectively, our studies provide an in-depth understanding of the molecular basis for DM1-related electrophysiological abnormalities and offer an avenue to test the potential therapeutic utility of targeting the non-muscle RBFOX2 40 isoform in treating cardiac features of DM1.

Abstract 531: Mechanistic and Functional Differences in Rna Binding and Processing Activities of the Muscle- and Non-muscle Rbfox2 Isoforms

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Chaitali Misra ◽  
Ullas Valiya Chembazhi ◽  
Sarah Matatov ◽  
Sushant Bangru ◽  
Auinash Kalsotra
Keyword(s):  
Regulatory Networks ◽  
Rna Binding ◽  
Trinucleotide Repeat ◽  
Splice Variants ◽  
Heart Tissue ◽  
Conduction System ◽  
Cardiac Conduction ◽  
Conduction Delay ◽  
Aberrant Expression ◽  
Splicing Regulators

Myotonic Dystrophy type 1 (DM1), the most prevalent form of adult onset muscular dystrophy, is caused by CTG trinucleotide repeat expansion in the 3’-UTR of DMPK gene. Over 80% of DM1 patients exhibit heart dysfunctions, which are the second leading cause for DM1-related deaths. Recently, we demonstrated that aberrant expression of a non-muscle splice isoform of RNA-binding protein RBFOX2 triggers cardiac conduction delay, atrioventricular heart blocks, and spontaneous arrhythmogenesis in DM1 heart. RBFOX2 is a master regulator of tissue-specific alternative splicing and a pair of mutually exclusive 43-nucleotide(nt) and 40-nt exons in its C-terminal domain encode the muscle (RBFOX2 43 ) and non-muscle (RBFOX2 40 ) isoforms. The RBFOX2 40 isoform is predominantly expressed in the fetal heart, and is replaced by the RBFOX2 43 isoform in development, specifically within the cardiomyocytes of adult hearts. To deconstruct the splicing regulatory networks of RBFOX2 43 and RBFOX2 40 isoforms, characterize their respective RNA binding landscapes, and determine the RBFOX2 40 -driven transcriptome alterations in DM1 heart tissue, we performed eCLIP and high-resolution RNA-sequencing studies on cardiomyocytes isolated from wild type (expressing the normal muscle-specific RBFOX2 43 isoform), Rbfox2 Δ43/Δ43 (expressing the non-muscle RBFOX2 40 isoform), and RBFOX2 40 overexpressing (OE) mice. By integrating genome-wide RNA binding and processing activities for the two RBFOX2 isoforms, we found that a switch from the muscle-specific (RBFOX2 43 ) to non-muscle (RBFOX2 40 ) isoform provokes DM1-like cardiac pathology by altering the mRNA abundance and splicing of genes encoding components of the conduction system and/or contractile apparatus. Further, through subnuclear fractionation and protein-protein interaction studies, we demonstrate that the higher-order assembly of LASR (large assembly of splicing regulators) complexes formed by the RBFOX2 40 isoform boost its splicing activity and promote the generation of pathogenic splice variants of voltage-gated ion channels and other components of the cardiac conduction system.

scholarly journals Straightjacket/α2δ3 deregulation is associated with cardiac conduction defects in Myotonic Dystrophy type 1

2018 ◽  
Author(s):  
Emilie Plantié ◽  
Masayuki Nakamori ◽  
Yoan Renaud ◽  
Aline Huguet ◽  
Caroline Choquet ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Cardiac Cells ◽  
Cardiac Conduction ◽  
Regulatory Subunit ◽  
Conduction Delay ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Intraventricular Conduction

ABSTRACTCardiac conduction defects decrease life expectancy in myotonic dystrophy type 1 (DM1), a complex toxic CTG repeat disorder involving misbalance between two RNA- binding factors, MBNL1 and CELF1. How this pathogenic DM1 condition translates into cardiac conduction disorders remains poorly understood. Here, we simulated MBNL1 and CELF1 misbalance in the Drosophila heart and identified associated gene deregulations using TU-tagging based transcriptional profiling of cardiac cells. We detected deregulations of several genes controlling cellular calcium levels and among them increased expression of straightjacket/α2δ3 that encodes a regulatory subunit of a voltage-gated calcium channel. Straightjacket overexpression in the fly heart leads to asynchronous heart beating, a hallmark of affected conduction, whereas cardiac straightjacket knockdown improves these symptoms in DM1 fly models. We also show that ventricular α2δ3 expression is low in healthy mice and humans but significantly elevated in ventricular muscles from DM1 patients with conduction defects. Taken together, this suggests that reducing the straightjacket/α2δ3 transcript levels in ventricular cardiomyocytes could represent a strategy to prevent conduction defects and in particular intraventricular conduction delay associated with DM1 pathology.

scholarly journals Increased Muscleblind levels by chloroquine treatment improve myotonic dystrophy type 1 phenotypes in in vitro and in vivo models

2019 ◽  
Vol 116 (50) ◽  
pp. 25203-25213 ◽  
Author(s):  
Ariadna Bargiela ◽  
Maria Sabater-Arcis ◽  
Jorge Espinosa-Espinosa ◽  
Miren Zulaica ◽  
Adolfo Lopez de Munain ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Trinucleotide Repeat ◽  
Rna Binding Proteins ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Muscle Area ◽  
In Vivo Models

Myotonic dystrophy type 1 (DM1) is a life-threatening and chronically debilitating neuromuscular disease caused by the expansion of a CTG trinucleotide repeat in the 3′ UTR of the DMPK gene. The mutant RNA forms insoluble structures capable of sequestering RNA binding proteins of the Muscleblind-like (MBNL) family, which ultimately leads to phenotypes. In this work, we demonstrate that treatment with the antiautophagic drug chloroquine was sufficient to up-regulate MBNL1 and 2 proteins in Drosophila and mouse (HSALR) models and patient-derived myoblasts. Extra Muscleblind was functional at the molecular level and improved splicing events regulated by MBNLs in all disease models. In vivo, chloroquine restored locomotion, rescued average cross-sectional muscle area, and extended median survival in DM1 flies. In HSALR mice, the drug restored muscular strength and histopathology signs and reduced the grade of myotonia. Taken together, these results offer a means to replenish critically low MBNL levels in DM1.

scholarly journals Zebrafish mbnl mutants model physical and molecular phenotypes of myotonic dystrophy

2019 ◽  
Author(s):  
Melissa N. Hinman ◽  
Jared I. Richardson ◽  
Rose A. Sockol ◽  
Eliza D Aronson ◽  
Sarah J. Stednitz ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Myotonic Dystrophy ◽  
Protein Function ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Genetic Disorder ◽  
Loss Of Function ◽  
High Fecundity ◽  
Molecular Phenotypes ◽  
Human Genetic Disorder

AbstractThe muscleblind RNA binding proteins (MBNL1, MBNL2, and MBNL3) are highly conserved across vertebrates and are important regulators of RNA alternative splicing. Loss of MBNL protein function through sequestration by CUG or CCUG RNA repeats is largely responsible for the phenotypes of the human genetic disorder myotonic dystrophy (DM). We generated the first stable zebrafish (Danio rerio) models of DM-associated MBNL loss of function through mutation of the three zebrafish mbnl genes. In contrast to mouse models, zebrafish double and triple homozygous mbnl mutants were viable to adulthood. Zebrafish mbnl mutants displayed disease-relevant physical phenotypes including decreased body size and impaired movement. They also exhibited widespread alternative splicing changes, including the misregulation of many DM-relevant exons. Physical and molecular phenotypes were more severe in compound mbnl mutants than in single mbnl mutants, suggesting partially redundant functions of Mbnl proteins. The high fecundity and larval optical transparency of this complete series of zebrafish mbnl mutants will make them useful for studying DM-related phenotypes and how individual Mbnl proteins contribute to them, and for testing potential therapeutics.

scholarly journals Splicing misregulation of SCN5A contributes to cardiac-conduction delay and heart arrhythmia in myotonic dystrophy

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Fernande Freyermuth ◽  
Frédérique Rau ◽  
Yosuke Kokunai ◽  
Thomas Linke ◽  
Chantal Sellier ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Cardiac Conduction ◽  
Conduction Delay ◽  
Heart Arrhythmia

scholarly journals Zebrafish mbnl mutants model physical and molecular phenotypes of myotonic dystrophy

2021 ◽  
Author(s):  
Melissa N. Hinman ◽  
Jared I. Richardson ◽  
Rose A. Sockol ◽  
Eliza D Aronson ◽  
Sarah J. Stednitz ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Myotonic Dystrophy ◽  
Protein Function ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Genetic Disorder ◽  
Loss Of Function ◽  
High Fecundity ◽  
Molecular Phenotypes ◽  
Human Genetic Disorder

The muscleblind RNA binding proteins (MBNL1, MBNL2, and MBNL3) are highly conserved across vertebrates and are important regulators of RNA alternative splicing. Loss of MBNL protein function through sequestration by CUG or CCUG RNA repeats is largely responsible for the phenotypes of the human genetic disorder myotonic dystrophy (DM). We generated the first stable zebrafish (Danio rerio) models of DM-associated MBNL loss of function through mutation of the three zebrafish mbnl genes. In contrast to mouse models, zebrafish double and triple homozygous mbnl mutants were viable to adulthood. Zebrafish mbnl mutants displayed disease-relevant physical phenotypes including decreased body size and impaired movement. They also exhibited widespread alternative splicing changes, including the misregulation of many DM-relevant exons. Physical and molecular phenotypes were more severe in compound mbnl mutants than in single mbnl mutants, suggesting partially redundant functions of Mbnl proteins. The high fecundity and larval optical transparency of this complete series of zebrafish mbnl mutants will make them useful for studying DM-related phenotypes and how individual Mbnl proteins contribute to them, and for testing potential therapeutics.

scholarly journals Pathogenic mechanisms of myotonic dystrophy

2009 ◽  
Vol 37 (6) ◽  
pp. 1281-1286 ◽  
Author(s):  
Johanna E. Lee ◽  
Thomas A. Cooper
Keyword(s):  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Genetic Disorder ◽  
Mrna Translation ◽  
Loss Of Function ◽  
Downstream Effects ◽  
Expanded Allele ◽  
Dystrophia Myotonica Protein Kinase ◽  
Dm Type 2

DM (myotonic dystrophy) is a dominantly inherited genetic disorder that is the most common cause of muscular dystrophy in adults affecting 1 in 8500 individuals worldwide. Different microsatellite expansions in two loci cause different forms of the disease that share similar features: DM1 (DM type 1) is caused by a tri- (CTG) nucleotide expansion within the DMPK (dystrophia myotonica protein kinase) 3′-untranslated region and DM2 (DM type 2) is caused by a tetra- (CCTG) nucleotide expansion within intron 1 of the ZNF9 (zinc finger 9) gene. The pathogenic mechanism of this disease involves the RNA transcribed from the expanded allele containing long tracts of (CUG)n or (CCUG)n. The RNA results in a toxic effect through two RNA-binding proteins: MBNL1 (muscleblind-like 1) and CUGBP1 (CUG-binding protein 1). In DM1, MBNL1 is sequestered on CUG repeat-containing RNA resulting in its loss-of-function, while CUGBP1 is up-regulated through a signalling pathway. The downstream effects include disrupted regulation of alternative splicing, mRNA translation and mRNA stability, which contribute to the multiple features of DM1. This review will focus on the RNA gain-of-function disease mechanism, the important roles of MBNL1 and CUGBP1 in DM1, and the relevance to other RNA dominant disorders.

scholarly journals Straightjacket/α2δ3 deregulation is associated with cardiac conduction defects in myotonic dystrophy type 1

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Emilie Auxerre-Plantié ◽  
Masayuki Nakamori ◽  
Yoan Renaud ◽  
Aline Huguet ◽  
Caroline Choquet ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Cardiac Cells ◽  
Cardiac Conduction ◽  
Regulatory Subunit ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Conduction Disorders ◽  
Decrease Life Expectancy

Cardiac conduction defects decrease life expectancy in myotonic dystrophy type 1 (DM1), a CTG repeat disorder involving misbalance between two RNA binding factors, MBNL1 and CELF1. However, how DM1 condition translates into conduction disorders remains poorly understood. Here we simulated MBNL1 and CELF1 misbalance in the Drosophila heart and performed TU-tagging-based RNAseq of cardiac cells. We detected deregulations of several genes controlling cellular calcium levels, including increased expression of straightjacket/α2δ3, which encodes a regulatory subunit of a voltage-gated calcium channel. Straightjacket overexpression in the fly heart leads to asynchronous heartbeat, a hallmark of abnormal conduction, whereas cardiac straightjacket knockdown improves these symptoms in DM1 fly models. We also show that ventricular α2δ3 expression is low in healthy mice and humans, but significantly elevated in ventricular muscles from DM1 patients with conduction defects. These findings suggest that reducing ventricular straightjacket/α2δ3 levels could offer a strategy to prevent conduction defects in DM1.

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