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.

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.

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.

Development of the cardiac conduction system

ESC CardioMed ◽  
2018 ◽  
pp. 49-52
Author(s):  
Jan Hendrik van Weerd ◽  
Vincent M. Christoffels
Keyword(s):  
Regulatory Networks ◽  
Atrioventricular Node ◽  
Ventricular Myocardium ◽  
Conduction System ◽  
Cardiac Conduction ◽  
Cardiac Conduction System ◽  
Cellular Mechanisms ◽  
Fibre Network ◽  
Electrical Impulses ◽  
And Function

The contraction of the heart is orchestrated by the components of the cardiac conduction system (CCS), which initiate and propagate the electrical impulses to coordinately activate the cardiac chambers. In the adult heart, the impulse is generated in the sinoatrial node and activates the atrial myocardium. Slow conduction of the impulse through the atrioventricular node allows for emptying of the atria and filling of the ventricles prior to ventricular contraction. Subsequent fast conduction through the atrioventricular bundle, bundle branches, and Purkinje fibre network activates the ventricular myocardium and causes the ventricles to contract. The development and function of the CCS involves complex regulatory networks of transcription factors acting in stage-, tissue-, and dose-dependent manners. Disrupted function or expression of these factors might lead to impaired development or function of the CCS components, associated with heart failure and sudden death. It is therefore crucial to understand the molecular and cellular mechanisms controlling the complex regulation of CCS development. This chapter summarizes current insight in the development and function of the different compartments of the CCS, and discusses the transcriptional networks underlying these processes.

Development of the cardiac conduction system

ESC CardioMed ◽  
2018 ◽  
pp. 49-52
Author(s):  
Jan Hendrik van Weerd ◽  
Vincent M. Christoffels
Keyword(s):  
Regulatory Networks ◽  
Atrioventricular Node ◽  
Ventricular Myocardium ◽  
Conduction System ◽  
Cardiac Conduction ◽  
Cardiac Conduction System ◽  
Cellular Mechanisms ◽  
Fibre Network ◽  
Electrical Impulses ◽  
And Function

The contraction of the heart is orchestrated by the components of the cardiac conduction system (CCS), which initiate and propagate the electrical impulses to coordinately activate the cardiac chambers. In the adult heart, the impulse is generated in the sinoatrial node and activates the atrial myocardium. Slow conduction of the impulse through the atrioventricular node allows for emptying of the atria and filling of the ventricles prior to ventricular contraction. Subsequent fast conduction through the atrioventricular bundle, bundle branches, and Purkinje fibre network activates the ventricular myocardium and causes the ventricles to contract. The development and function of the CCS involves complex regulatory networks of transcription factors acting in stage-, tissue-, and dose-dependent manners. Disrupted function or expression of these factors might lead to impaired development or function of the CCS components, associated with heart failure and sudden death. It is therefore crucial to understand the molecular and cellular mechanisms controlling the complex regulation of CCS development. This chapter summarizes current insight in the development and function of the different compartments of the CCS, and discusses the transcriptional networks underlying these processes.

scholarly journals Screening for circulating miR-208a and -b in different cardiac arrhythmias of dogs

2018 ◽  
Vol 62 (3) ◽  
pp. 359-363 ◽  
Author(s):  
Agnieszka Noszczyk-Nowak ◽  
Maciej Zacharski ◽  
Marcin Michałek
Keyword(s):  
Cardiac Arrhythmias ◽  
High Sensitivity ◽  
Tissue Expression ◽  
Heart Tissue ◽  
Treatment Monitoring ◽  
Conduction System ◽  
Cardiac Conduction ◽  
Canine Heart ◽  
Serum Samples ◽  
Novel Mirna

AbstractIntroductionIn recent years, the high sensitivity and specificity of novel miRNA biomarkers have been utilised for early diagnosis and treatment monitoring of various diseases. Previous reports showed that abnormal expression of miR-208 in mice resulted in the development of an aberrant cardiac conduction system and consecutive arrhythmias. On the other hand, a study on infarcted human heart tissue showed upregulation of miR-208a in subjects with ventricular tachyarrhythmias compared to healthy controls. We prospectively investigated the expression of miR-208a and -208b in the serum of dogs presenting different cardiac arrhythmias.Material and MethodsA total of 28 dogs with atrial fibrillation (n = 8), ventricular premature contractions (n=6), conduction system disturbances (n = 7), and free of heart conditions (as controls) (n = 7) were enrolled in the study. Total RNA was extracted from serum samples and miR-208a and -b, miR-16 as well as a cel-miR-39-5p spike-in were analysed with qPCR and ddPCR.ResultsmiR-208a and miR-208b were not expressed in any of the samples. The calculated ddPCR miR-16 relative expression (normalised with cel-miR-39 spike-in) showed a good correlation (r = 0.82; P < 0.001) with the qPCR results.ConclusionThis outcome warrants further investigation, possibly focusing on tissue expression of miR-208 in the canine heart.

scholarly journals Impulse Delay in the Cardiac Conduction System

2020 ◽  
pp. 29-47
Author(s):  
Nzerem F. E ◽  
Ugorji H. C
Keyword(s):  
Conduction System ◽  
Cardiac Cells ◽  
Cardiac Conduction ◽  
Conduction Delay ◽  
Graph Theoretic ◽  
System A ◽  
Input Control ◽  
Cardiac System ◽  
Time Invariant ◽  
Theoretic Description

The physiology or otherwise of blood circulation is predicated on the electrical conduction of the heart. As a rule electrical impulse suffusing the cardiac cells, just like all time-dependent phenomena, transmits with a modicum of time delay. Such delay may be physiological (benign) or pathological; the later is seen as a cardiac liability. This paper treated impulse conduction delay in the cardiac system. A set of matrices resulting from the graph theoretic description of the conduction system was generated and fitted into a continuous time invariant state-space delay equation, and a state-transition matrix solution was sought. An input control-based minimization scheme by which ensuing deleteriousness of pathological delay could be assuaged was proposed.

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.

Origin and development of the cardiac conduction system

2018 ◽  
Author(s):  
Lucile Miquerol
Keyword(s):  
Regulatory Networks ◽  
Conduction System ◽  
Cardiac Conduction ◽  
Cardiac Conduction System ◽  
Transcriptional Regulatory Networks ◽  
Cardiac Progenitors ◽  
Electrophysiological Properties ◽  
Cellular Origin ◽  
System P ◽  
Transcriptional Regulatory

The cardiac conduction system represents the ‘wiring’ of the heart and orchestrates the propagation of the electrical activity to synchronize heartbeats. It is built from specialized cardiomyocytes expressing a subset of ion channels and gap junctions indispensable for their electrophysiological properties. Although representing only a very small volume of the heart, the conduction system plays a crucial role in the appearance of cardiac arrhythmias. The cells forming the conduction system are derived from the same cardiac progenitors as the working cardiomyocytes, and the choice between these two fates is acquired during embryonic development. The components of the conduction system are progressively established during cardiac morphogenesis and converge to form an integrated electrical system in the definitive heart. This chapter will discuss recent advances using mouse genetic approaches which have improved understanding of the cellular origin and the transcriptional regulatory networks involved in the development of the conduction system.

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