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

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.

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

Circulation Research ◽

10.1161/res.129.suppl_1.mp249 ◽

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.

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

10.1101/431569 ◽

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.

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Profound cardiac conduction delay predicts mortality in myotonic dystrophy type 1

Journal of Internal Medicine ◽

10.1111/j.1365-2796.2010.02213.x ◽

2010 ◽

Cited By ~ 2

Author(s):

S. Mörner ◽

P. Lindqvist ◽

C. Mellberg ◽

B.-O. Olofsson ◽

C. Backman ◽

...

Keyword(s):

Myotonic Dystrophy ◽

Cardiac Conduction ◽

Conduction Delay ◽

Myotonic Dystrophy Type 1 ◽

Myotonic Dystrophy Type

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Faculty Opinions recommendation of Reversible model of RNA toxicity and cardiac conduction defects in myotonic dystrophy.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1040694.489717 ◽

2006 ◽

Author(s):

David Brook

Keyword(s):

Myotonic Dystrophy ◽

Cardiac Conduction ◽

Rna Toxicity

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807-4 CUG expansions in a myotonic dystrophy mouse model cause cardiac conduction abnormalities and pathologic electrophysiology findings

Journal of the American College of Cardiology ◽

10.1016/s0735-1097(04)90511-6 ◽

2004 ◽

Vol 43 (5) ◽

pp. A122 ◽

Cited By ~ 1

Author(s):

Cordula M Wolf ◽

Megan C Sherwood ◽

Dorothy Branco ◽

Sita Reddy ◽

Charles I Berul

Keyword(s):

Mouse Model ◽

Myotonic Dystrophy ◽

Cardiac Conduction ◽

Conduction Abnormalities

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Prenatal ethanol exposure impairs the conduction delay at the atrioventricular junction in the looping heart

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00107.2021 ◽

2021 ◽

Author(s):

Shan Ling ◽

Michael W Jenkins ◽

Michiko Watanabe ◽

Stephanie M Ford ◽

Andrew M Rollins

Keyword(s):

Heart Development ◽

Molecular Mechanisms ◽

Early Stage ◽

Heart Defects ◽

Ethanol Exposure ◽

Cardiac Conduction ◽

Conduction Delay ◽

Prenatal Ethanol Exposure ◽

Endocardial Cushions ◽

Atrioventricular Junction

The etiology of ethanol-related congenital heart defects has been the focus of much study, but most research has concentrated on cellular and molecular mechanisms. We have shown with optical coherence tomography (OCT) that ethanol exposure led to increased retrograde flow and smaller atrioventricular (AV) cushions compared to controls. Since AV cushions play a role in patterning the conduction delay at the atrioventricular junction (AVJ), this study aims to investigate whether ethanol exposure alters the AVJ conduction in early looping hearts and whether this alteration is related to the decreased cushion size. Quail embryos were exposed to a single dose of ethanol at gastrulation, and Hamburger-Hamilton stage 19 - 20 hearts were dissected for imaging. Cardiac conduction was measured using an optical mapping microscope and we imaged the endocardial cushions using OCT. Our results showed that, compared with controls, ethanol-exposed embryos exhibited abnormally fast AVJ conduction and reduced cushion size. However, this increased conduction velocity (CV) did not strictly correlate with decreased cushion volume and thickness. By matching the CV map to the cushion size map, we found that the slowest conduction location was consistently at the atrial side of the AVJ, which had the thinner cushions, not at the thickest cushion location at the ventricular side as expected. Our findings reveal regional differences in the AVJ myocardium even at this early stage in heart development. These findings reveal the early steps leading to the heterogeneity and complexity of conduction at the mature AVJ, a site where arrhythmias can be initiated.

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