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 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.

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.

Abstract P104: Reactivation of Embryonic Gene Program due to CUG Repeat RNA Expression in Myotonic Dystrophy

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Auinash Kalsotra ◽  
Ravi Singh ◽  
Chad Creighton ◽  
Thomas Cooper
Keyword(s):  
Gene Expression ◽  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Patterns ◽  
Causative Mutation ◽  
Inherited Disease ◽  
Aberrant Expression ◽  
Organ Systems ◽  
General Response

Myotonic dystrophy type 1 (DM1) is a dominantly inherited disease that affects multiple organ systems. Cardiac involvement, which is characterized by conduction defects and arrhythmias, is the second leading cause of death in DM1 patients. The causative mutation is a CTG expansion in the 3' untranslated region of DMPK gene resulting in aberrant expression of CUG repeat RNA that accumulates into nuclear foci and causes misregulation in alternative splicing. Here we show that heart-specific and inducible expression of CUG repeat RNA in a DM1 mouse model results in global reactivation of embryonic gene expression program in adult heart that is distinct from a general hypertrophic stress response. Using q-PCR TaqMan arrays, we identified 54 miRNAs that were differentially expressed in DM1 mouse hearts one week following induction of CUG repeat RNA. Interestingly, 83% (45/54) of them exhibited a developmental shift in expression towards the embryonic pattern. Because over 90% (41/45) of them were down regulated within 72 hr after induction of repeat RNA and only 2/22 examined decreased in two unrelated mouse models of heart disease, we conclude their reduced expression is specific to DM1 and not simply a general response to cardiac injury. Microarray studies revealed a developmental switch not only in the miRNA expression patterns but also a pervasive shift in mRNA steady state levels of a number of genes to embryonic stage. Intriguingly, we found that loss of MBNL1 or gain of CELF1 activity, two major RNA binding proteins disrupted in DM1, are not driving the miRNA misregulation since their expression is indistinguishable between wild type, MBNL1 knock out and CELF1 over expressing mice. Moreover, comparable decrease in ten out of ten primary miRNA transcripts examined suggests loss of expression is not due to a processing defect. Instead, we discovered that adult-to-embryonic shift in expression of select micro- and messenger RNAs in DM1 heart occurs due to specific inactivation of a Mef2 transcriptional program. We are currently determining causal contributions of this Mef2-miRNA circuitry in the developmental reprogramming of gene expression in DM1 as well as its direct role in cardiac manifestations of this disease.

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 High-throughput kinome-RNAi screen identifies protein kinase R activator (PACT) as a novel genetic modifier of CUG foci integrity in myotonic dystrophy type 1 (DM1)

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0256276
Author(s):  
Nafisa Neault ◽  
Sean O’Reilly ◽  
Aiman Tariq Baig ◽  
Julio Plaza-Diaz ◽  
Mehrdad Azimi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Genetic Modifier ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Aberrant Expression ◽  
Significant Correction

Myotonic Dystrophy Type 1 (DM1) is the most common form of adult muscular dystrophy (~1:8000). In DM1, expansion of CTG trinucleotide repeats in the 3’ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene results in DMPK mRNA hairpin structures which aggregate as insoluble ribonuclear foci and sequester several RNA-binding proteins. The resulting sequestration and misregulation of important splicing factors, such as muscleblind-like 1 (MBNL1), causes the aberrant expression of fetal transcripts for several genes that contribute to the disease phenotype. Previous work has shown that antisense oligonucleotide-mediated disaggregation of the intranuclear foci has the potential to reverse downstream anomalies. To explore whether the nuclear foci are, to some extent, controlled by cell signalling pathways, we have performed a screen using a small interfering RNA (siRNA) library targeting 518 protein kinases to look at kinomic modulation of foci integrity. RNA foci were visualized by in situ hybridization of a fluorescent-tagged (CAG)10 probe directed towards the expanded DMPK mRNA and the cross-sectional area and number of foci per nuclei were recorded. From our screen, we have identified PACT (protein kinase R (PKR) activator) as a novel modulator of foci integrity and have shown that PACT knockdown can both increase MBNL1 protein levels; however, these changes are not suffcient for significant correction of downstream spliceopathies.

scholarly journals Aberrant Expression of a Non-muscle RBFOX2 Isoform Triggers Cardiac Conduction Defects in Myotonic Dystrophy

2020 ◽  
Vol 52 (6) ◽  
pp. 748-763.e6 ◽  
Author(s):  
Chaitali Misra ◽  
Sushant Bangru ◽  
Feikai Lin ◽  
Kin Lam ◽  
Sara N. Koenig ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Cardiac Conduction ◽  
Aberrant Expression

Congenital myotonic dystrophy pathology and somatic mosaicism

Neurology ◽  
1997 ◽  
Vol 49 (5) ◽  
pp. 1457-1460 ◽  
Author(s):  
J. T. Joseph ◽  
C. S. Richards ◽  
D. C. Anthony ◽  
M. Upton ◽  
A. R. Perez-Atayde ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Skeletal Muscles ◽  
Trinucleotide Repeat ◽  
Clinical Course ◽  
Molecular Genetic ◽  
Somatic Mosaicism ◽  
Molecular Genetic Analysis ◽  
Ctg Repeats ◽  
Cardiac Tissues ◽  
Congenital Myotonic Dystrophy

We present the pathology and molecular genetic analysis of an infant with congenital myotonic dystrophy. The proband/infant, born at 35 weeks' gestational age to a mother with myotonic dystrophy and 750 CTG repeats, was markedly hypotonic and had severe cardiomyopathy. She died after 16 days of life. At autopsy, skeletal and heart muscles were immature and had a decrease in contractile elements. DNA CTG trinucleotide repeat analysis of the proband demonstrated 2,480 repeats in blood and a slightly greater number of repeats in skeletal muscles, viscera, and gray matter. Corresponding to the clinical course and pathology, cardiac tissues displayed somatic mosaicism, with repeats ranging from 2,760 to 3,220.

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 Correction of RNA-Binding Protein CUGBP1 and GSK3β Signaling as Therapeutic Approach for Congenital and Adult Myotonic Dystrophy Type 1

2019 ◽  
Vol 21 (1) ◽  
pp. 94 ◽  
Author(s):  
Lubov Timchenko
Keyword(s):  
Myotonic Dystrophy ◽  
Complex Disease ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Ctg Repeats ◽  
Toxic Rna

Myotonic dystrophy type 1 (DM1) is a complex genetic disease affecting many tissues. DM1 is caused by an expansion of CTG repeats in the 3′-UTR of the DMPK gene. The mechanistic studies of DM1 suggested that DMPK mRNA, containing expanded CUG repeats, is a major therapeutic target in DM1. Therefore, the removal of the toxic RNA became a primary focus of the therapeutic development in DM1 during the last decade. However, a cure for this devastating disease has not been found. Whereas the degradation of toxic RNA remains a preferential approach for the reduction of DM1 pathology, other approaches targeting early toxic events downstream of the mutant RNA could be also considered. In this review, we discuss the beneficial role of the restoring of the RNA-binding protein, CUGBP1/CELF1, in the correction of DM1 pathology. It has been recently found that the normalization of CUGBP1 activity with the inhibitors of GSK3 has a positive effect on the reduction of skeletal muscle and CNS pathologies in DM1 mouse models. Surprisingly, the inhibitor of GSK3, tideglusib also reduced the toxic CUG-containing RNA. Thus, the development of the therapeutics, based on the correction of the GSK3β-CUGBP1 pathway, is a promising option for this complex disease.

Sign in / Sign up

Export Citation Format

Share Document