Freyermuth F , Rau F , Kokunai Y , Linke T , Sellier C , Nakamori M , Kino Y , Arandel L , Jollet A , Thibault C , Philipps M , Vicaire S , Jost B , Udd B , Day JW , Duboc D , Wahbi K , Matsumura T , Fujimura H , Mochizuki H , Deryckere F , Kimura T , Nukina N , Ishiura S , Lacroix V , Campan-Fournier A , Navratil V , Chautard E , Auboeuf D , Horie M , Imoto K , Lee KY , Swanson MS , de Munain AL , Inada S , Itoh H , Nakazawa K , Ashihara T , Wang E , Zimmer T , Furling D , Takahashi MP , Charlet-Berguerand N .

310659 European Research Council, DP5 OD017865 NIH HHS , P01 NS058901 NINDS NIH HHS , ERC_310659 European Research Council

	Figure 1. Identification of novel splicing misregulations in DM1 heart samples.(a) Δ-PSI versus Z-score plot of exon cassettes misregulations predicted by MISO analysis. (b) Exons structure and coverage of RNA-seq reads across SCN5A exons 5–7 show increased inclusion of exon 6A and decreased inclusion of exon 6B in heart samples of three DM1 patients (bottom, blue) versus three control samples (top, red). (c) Validation by RT–PCR of RNA-seq predictions in human heart samples of normal adult individuals (CTL, black) versus adult DM1 patients (DM1, red). Molecular size markers in bps are reported to the left of each RT–PCR gels. bp, base pair.
	Figure 2. MBNL-binding motifs are enriched in vicinity of exons misregulated in DM1.Sequence and binomial test P values of 4-mer RNA motifs enriched downstream, within and upstream of exons misregulated in DM1 heart samples. Sequences enriched in exons excluded in DM are indicated in red, while sequences enriched in exons included in DM are indicated in blue.
	Figure 3. Splicing of SCN5A exon 6A is altered in DM heart samples.(a) Schematic representation of mutually exclusive exons 6A and 6B of SCN5A. SCN5A mRNA includes exon 6A (red) in fetal heart, while SCN5A mRNA expresses exon 6B (blue) in adult heart. (b) Schematic representation of SCN5A topology expressing exon 6A (red). Exons 6A or 6B encodes part of segment 3, connecting loop between S3 and S4 and most part of the voltage-sensitive segment 4 of domain 1 of the sodium channel SCN5A. (c). Representative BstBI-digested RT–PCR analysis of endogenous SCN5A mRNA from human heart samples of normal adult (CTL), adult ALS, non-DM fetuses (20, 24 and 35 weeks), congenital DM1 fetuses (CDM1 of 22, 25 and 28 weeks), adults DM1 and DM2 individuals. Molecular size marker is indicated in bp. (d) Graphical representation of RT–PCR analysis depicting the percentage of SCN5A mRNA including exon 6A in left ventricular heart samples from fetal and adult control, ALS, DCM, DMD, CDM1 and adult DM1 and DM2 individuals. (e) Graphical representation of quantitative real-time RT-qPCR depicting the mRNA expression of SCN5A relative to RPLP0 in control normal adults (n=5) versus adult DM1 (n=5) heart samples. Bars indicate s.e.m. bp, base pairs.
	Figure 4. MBNL1 regulates alternative splicing of SCN5A.(a) Upper panel, RT–PCR analysis of endogenous SCN5A mRNA from differentiated primary muscle cell cultures derived from biopsies of control or DM1 individuals. (lower) Quantification of the percentage of SCN5A mRNA including exon 6B. (b, upper) RT–PCR analysis of endogenous SCN5A mRNA from human differentiated cultures of control primary muscle cells transfected with a scrambled siRNA (siCTL) or a siRNA targeting MBNL1 mRNA (siMBNL1). (lower) Percentage of SCN5A mRNA including exon 6B. (c, upper) RT–PCR analysis of endogenous Scn5a mRNA in heart samples of wild-type and compound Mbnl1−/−, Mbnl2+/− double knockout mice. (lower) Percentage of Scn5a mRNA including exon 6A. (d, upper) RT–PCR analysis of exogenous SCN5A mRNA from differentiated C2C12 muscle cells co-transfected with a SCN5A minigene containing exons 6A and 6B bordered by their introns and with either a plasmid expressing 960 CTG repeats, MBNL1, CUGBP1 or with a siRNA directed against Mbnl1 (siMbnl1) or Celf1 (encoding Cugbp1; siCelf1). # Indicates usage of a cryptic splice site inherent to the minigene. (lower) Percentage of SCN5A mRNA including exon 6B. (e, upper) Schematic representation of SCN5A minigene, including the UGC-rich sequence used for binding assays. (lower) Gel-shift assays were performed using 5–1,000 nM of purified bacterial recombinant GST-MBNL1Δ101 and a uniformly 32P-CTP labelled RNA. (f, upper) Schematic representation of mutant SCN5A minigene, including the mutant sequence, used for binding assays. (lower) Gel-shift assay performed as in e. (g, upper) RT–PCR analysis of exogenous SCN5A mRNA from differentiated C2C12 muscle cells co-transfected with mutant SCN5A minigene and with a plasmid expressing 960 CTG repeats or MBNL1 or with a siRNA directed against Mbnl1 (siMbnl1). (lower) Percentage of SCN5A mRNA including exon 6B. All transfection and gel-shift experiments were repeated three to five times. Molecular size markers are indicated in bp. Bars indicate s.e.m. Student test, ** indicates P<0.01, *** indicates P<0.001. bp, base pairs.
	Figure 5. Electrophysiological properties of hNav1.5 and hNav1.5e channels.(a) Representative Na+ currents generated in Xenopus oocytes by hNav1.5 (encoded by SCN5A containing the adult exon 6B), hNav1.5e (encoded by SCN5A including the fetal exon 6A), and simultaneously expressed Nav1.5 and Nav1.5e channels at a 1:1 ratio. (b) Peak current amplitudes at the test potential of −10 mV in Xenopus oocytes injected with equimolar amount of cRNA encoding hNav1.5, hNav1.5e or 1:1 combination of Nav1.5 and Nav1.5e channels. (c) Current–voltage relationships. (d) Steady-state activation curves. (e) Inactivation time constants τh (ms) at different test pulses. (f) Steady-state inactivation curves. (g) Fractional recovery curves. Data were obtained from 11 different batches of oocytes. To illustrate steady-state activation, steady-state inactivation and recovery from inactivation, we used 3–5 representative measurements. For total number of measurements (n=25–27) and for statistical data evaluation (Vm, s) see the Supplementary Table 2. Bars indicate s.e.m. Student test, *** indicates P<0.001.
	Figure 6. Alteration of Scn5a splicing causes heart conduction defects and arrhythmias.(a) Schematic representation of mutually exclusive exons 6A and 6B of Scn5a and of antisense sequences driven by optimized U7-snRNAs (U7-ASScn5a) to force fetal exon 6A inclusion in adult wild-type mouse heart. (b, upper) RT–PCR analysis of the alternative splicing of endogenous Scn5a mRNA from heart samples of mice injected with AAV2/9 expressing U7-ASScn5a compared with control injected mice. Molecular size marker is indicated in bp. (lower) Percentage of Scn5a mRNA including exon 6A. (c) Real-time RT-qPCR quantification of the expression of Scn5a, Scn1b and GJja1 (connexin 43) mRNAs in heart samples of mice expressing U7-ASScn5a (n=6) compared with control injected mice (n=6). (d) Representative ECG traces show prolongation of the PR interval in U7-ASScn5a-injected mice compared with control mice. (e) ECG measures of PR interval, QRS and QT intervals in 4-month-old mice injected with AAV2/9 expressing U7-ASScn5a (n=25) compared with age-matched control mice (n=17). (f) Representative ECG traces reveal atrial fibrillation in U7-ASScn5a-injected mice compared with control mice. (g) Variation of the RR interval indicates evidences of heart arrhythmias in U7-ASScn5a-injected mice (n=25) compared with control mice (n=17). (h) Representative image of six analysed heart samples showing mild fibrosis revealed by Red Sirius histology staining in AAV-U7-ASScn5a-injected mice. Scale bar, 100 μm. (i) Real-time RT-qPCR quantification of the expression of Cola1a, Col3a1 and Tgfb mRNAs in heart of control (n=6) or AAV-U7-ASScn5a-injected mice (n=6). Bars indicate s.e.m. Student test, * indicates P<0.5, ** indicates P<0.01, *** indicates P<0.001. bp, base pair.
	Figure 7. Computer simulation predicts cardiac conduction abnormalities in DM.(a) Simulations of ECG (upper) and action potential (AP) alterations (middle and lower) caused by the switch from adult SCN5A exon 6B towards fetal exon 6A, using a modified human ventricular ORd model. (b) Simulations of the atrio-ventricular changes caused by inclusion of SCN5A fetal exon 6A instead of adult exon 6B, employing an atrio-ventricular nodal model. APs of atrial and AV node cells were simulated and atrium-His interval was measured as the difference in the latency of APs between atrial and nodal-His cells. (c) Model of splicing alteration of the cardiac sodium channel, SCN5A, in DM. MBNL proteins regulate the switch from SCN5A exon 6A in fetal heart to exon 6B in adult. In DM, titration of MBNL proteins by mutant RNA containing expanded CUG repeats leads to expression of a fetal splicing form of SCN5A, inappropriate to adult heart physiology, ultimately resulting in cardiac-conduction delay and heart arrhythmias, which are two keys features of DM.

Algalarrondo, Abnormal sodium current properties contribute to cardiac electrical and contractile dysfunction in a mouse model of myotonic dystrophy type 1. 2015, Pubmed

Algalarrondo, Abnormal sodium current properties contribute to cardiac electrical and contractile dysfunction in a mouse model of myotonic dystrophy type 1. 2015, Pubmed
Anders, Detecting differential usage of exons from RNA-seq data. 2012, Pubmed
Banks, Deletion of SOCS7 leads to enhanced insulin action and enlarged islets of Langerhans. 2005, Pubmed
Brook, Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. 1992, Pubmed
Charlet-B, Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. 2002, Pubmed
Chen, Genetic basis and molecular mechanism for idiopathic ventricular fibrillation. 1998, Pubmed , Xenbase
Childs-Disney, Induction and reversal of myotonic dystrophy type 1 pre-mRNA splicing defects by small molecules. 2013, Pubmed
Dixon, Loss of muscleblind-like 1 results in cardiac pathology and persistence of embryonic splice isoforms. 2015, Pubmed
Fu, An unstable triplet repeat in a gene related to myotonic muscular dystrophy. 1992, Pubmed
Fugier, Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy. 2011, Pubmed
Goers, MBNL1 binds GC motifs embedded in pyrimidines to regulate alternative splicing. 2010, Pubmed
Goodwin, MBNL Sequestration by Toxic RNAs and RNA Misprocessing in the Myotonic Dystrophy Brain. 2015, Pubmed
Gorog, A cautionary tale: the risks of flecainide treatment for myotonic dystrophy. 2005, Pubmed
Goyenvalle, Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping. 2004, Pubmed
Groh, Electrocardiographic abnormalities and sudden death in myotonic dystrophy type 1. 2008, Pubmed
Groh, Familial clustering of muscular and cardiac involvement in myotonic dystrophy type 1. 2005, Pubmed
Inada, One-dimensional mathematical model of the atrioventricular node including atrio-nodal, nodal, and nodal-his cells. 2009, Pubmed
Kalsotra, A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart. 2008, Pubmed
Kalsotra, The Mef2 transcription network is disrupted in myotonic dystrophy heart tissue, dramatically altering miRNA and mRNA expression. 2014, Pubmed
Kanadia, Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy. 2006, Pubmed
Kanadia, A muscleblind knockout model for myotonic dystrophy. 2003, Pubmed
Katz, Analysis and design of RNA sequencing experiments for identifying isoform regulation. 2010, Pubmed
Kim, TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. 2013, Pubmed
Kimura, Altered mRNA splicing of the skeletal muscle ryanodine receptor and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase in myotonic dystrophy type 1. 2005, Pubmed
Kino, Muscleblind protein, MBNL1/EXP, binds specifically to CHHG repeats. 2004, Pubmed
Koshelev, Heart-specific overexpression of CUGBP1 reproduces functional and molecular abnormalities of myotonic dystrophy type 1. 2010, Pubmed
Kuyumcu-Martinez, Increased steady-state levels of CUGBP1 in myotonic dystrophy 1 are due to PKC-mediated hyperphosphorylation. 2007, Pubmed
Lazarus, Long-term follow-up of arrhythmias in patients with myotonic dystrophy treated by pacing: a multicenter diagnostic pacemaker study. 2002, Pubmed
Lee, Compound loss of muscleblind-like function in myotonic dystrophy. 2013, Pubmed
Leoni, Variable Na(v)1.5 protein expression from the wild-type allele correlates with the penetrance of cardiac conduction disease in the Scn5a(+/-) mouse model. 2010, Pubmed
Lin, Failure of MBNL1-dependent post-natal splicing transitions in myotonic dystrophy. 2006, Pubmed
Liquori, Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. 2001, Pubmed
Mahadevan, Myotonic dystrophy mutation: an unstable CTG repeat in the 3' untranslated region of the gene. 1992, Pubmed
Mankodi, Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy. 2002, Pubmed
Marquis, CUG-BP1/CELF1 requires UGU-rich sequences for high-affinity binding. 2006, Pubmed , Xenbase
Maury, Prevalence of type 1 Brugada ECG pattern after administration of Class 1C drugs in patients with type 1 myotonic dystrophy: Myotonic dystrophy as a part of the Brugada syndrome. 2014, Pubmed
Miller, Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. 2000, Pubmed
Murphy, Developmentally regulated SCN5A splice variant potentiates dysfunction of a novel mutation associated with severe fetal arrhythmia. 2012, Pubmed
Nakamori, Splicing biomarkers of disease severity in myotonic dystrophy. 2013, Pubmed
Nakamori, Altered mRNA splicing of dystrophin in type 1 myotonic dystrophy. 2007, Pubmed
Nakamori, Aberrantly spliced alpha-dystrobrevin alters alpha-syntrophin binding in myotonic dystrophy type 1. 2008, Pubmed
O'Hara, Simulation of the undiseased human cardiac ventricular action potential: model formulation and experimental validation. 2011, Pubmed
Onkal, Alternative splicing of Nav1.5: an electrophysiological comparison of 'neonatal' and 'adult' isoforms and critical involvement of a lysine residue. 2008, Pubmed
Otten, Arrhythmia exacerbation after sodium channel blockade in myotonic dystrophy type 1. 2009, Pubmed
Pambrun, Myotonic dystrophy type 1 mimics and exacerbates Brugada phenotype induced by Nav1.5 sodium channel loss-of-function mutation. 2014, Pubmed
Pambrun, Unmasked Brugada pattern by ajmaline challenge in patients with myotonic dystrophy type 1. 2015, Pubmed
Philips, Disruption of splicing regulated by a CUG-binding protein in myotonic dystrophy. 1998, Pubmed
Probst, Progressive cardiac conduction defect is the prevailing phenotype in carriers of a Brugada syndrome SCN5A mutation. 2006, Pubmed
Rau, Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy. 2015, Pubmed
Rau, Misregulation of miR-1 processing is associated with heart defects in myotonic dystrophy. 2011, Pubmed
Royer, Mouse model of SCN5A-linked hereditary Lenègre's disease: age-related conduction slowing and myocardial fibrosis. 2005, Pubmed
Rudnik-Schöneborn, Brugada-like cardiac disease in myotonic dystrophy type 2: report of two unrelated patients. 2011, Pubmed
Savkur, Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. 2001, Pubmed
Schoser, Sudden cardiac death in myotonic dystrophy type 2. 2004, Pubmed
Schott, Cardiac conduction defects associate with mutations in SCN5A. 1999, Pubmed
Tan, A sodium-channel mutation causes isolated cardiac conduction disease. 2001, Pubmed
Tang, Muscle weakness in myotonic dystrophy associated with misregulated splicing and altered gating of Ca(V)1.1 calcium channel. 2012, Pubmed
Teplova, Structural insights into RNA recognition by the alternate-splicing regulator CUG-binding protein 1. 2010, Pubmed
Teplova, Structural insights into RNA recognition by the alternative-splicing regulator muscleblind-like MBNL1. 2008, Pubmed
Trapnell, Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. 2010, Pubmed
Tsuji-Wakisaka, Identification and functional characterization of KCNQ1 mutations around the exon 7-intron 7 junction affecting the splicing process. 2011, Pubmed , Xenbase
Udd, Proximal myotonic dystrophy--a family with autosomal dominant muscular dystrophy, cataracts, hearing loss and hypogonadism: heterogeneity of proximal myotonic syndromes? 1997, Pubmed
Wahbi, Brugada syndrome and abnormal splicing of SCN5A in myotonic dystrophy type 1. 2013, Pubmed
Walzik, Alternative splicing of the cardiac sodium channel creates multiple variants of mutant T1620K channels. 2011, Pubmed
Wang, Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins. 2012, Pubmed
Wang, Clinical, genetic, and biophysical characterization of SCN5A mutations associated with atrioventricular conduction block. 2002, Pubmed
Wani, Familial hypercatabolic hypoproteinemia caused by deficiency of the neonatal Fc receptor, FcRn, due to a mutant beta2-microglobulin gene. 2006, Pubmed
Wheeler, Correction of ClC-1 splicing eliminates chloride channelopathy and myotonia in mouse models of myotonic dystrophy. 2007, Pubmed
Wheeler, Targeting nuclear RNA for in vivo correction of myotonic dystrophy. 2012, Pubmed
Yadava, RNA toxicity in myotonic muscular dystrophy induces NKX2-5 expression. 2008, Pubmed
Yuan, Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs. 2007, Pubmed
Zhang, Sodium channel kinetic changes that produce Brugada syndrome or progressive cardiac conduction system disease. 2007, Pubmed
van Veen, Impaired impulse propagation in Scn5a-knockout mice: combined contribution of excitability, connexin expression, and tissue architecture in relation to aging. 2005, Pubmed