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

14 Pages·2016·3.02 MB·English

by  Jinyoung Kim

ARTICLE
Received2Jun 2015 |Accepted 16Feb 2016 |Published 11Apr 2016 DOI:10.1038/ncomms11067 OPEN
Splicing misregulation of SCN5A contributes to
cardiac-conduction delay and heart arrhythmia in
myotonic dystrophy
Fernande Freyermuth1,*,w, Fre´de´rique Rau2,*, Yosuke Kokunai3, Thomas Linke4, Chantal Sellier1, Masayuki Nakamori3,
Yoshihiro Kino5, Ludovic Arandel2, Arnaud Jollet2, Christelle Thibault1, Muriel Philipps1, Serge Vicaire1, Bernard Jost1,
Bjarne Udd6,7,8, John W. Day9, Denis Duboc10, Karim Wahbi10, Tsuyoshi Matsumura11, Harutoshi Fujimura11,
Hideki Mochizuki3, Franc¸ois Deryckere12, Takashi Kimura13, Nobuyuki Nukina14, Shoichi Ishiura15, Vincent Lacroix16,
Amandine Campan-Fournier17, Vincent Navratil18, Emilie Chautard19, Didier Auboeuf19, Minoru Horie20, Keiji Imoto21,
Kuang-Yung Lee22, Maurice S. Swanson23, Adolfo Lopez de Munain24, Shin Inada25, Hideki Itoh20, Kazuo Nakazawa25,
TakashiAshihara20,EricWang23,ThomasZimmer4,DenisFurling2,MasanoriP.Takahashi3&NicolasCharlet-Berguerand1
Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded
CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing
changes.Cardiacalterations,characterizedbyconductiondelaysandarrhythmia,arethesecond
most common cause of death in DM. Using RNA sequencing, here we identify novel splicing
alterationsinDMheartsamples,includingaswitchfromadultexon6Btowardsfetalexon6Ain
thecardiacsodiumchannel,SCN5A.WefindthatMBNL1regulatesalternativesplicingofSCN5A
mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability
comparedwiththecontroladultisoform.Importantly,reproducingsplicingalterationofScn5ain
mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant
featuresofmyotonicdystrophy.Inconclusion,misregulationofthealternativesplicingofSCN5A
maycontributeto asubsetofthecardiacdysfunctionsobservedin myotonicdystrophy.
1DepartmentofTranslationalmedicineandneurogenetics,IGBMC,CNRSUMR7104,INSERMU964,Universite´deStrasbourg,Illkirch67400,France.2SorbonneUniversite´s
UPMCUnivParis06,Inserm,CNRS,CentredeRechercheenMyologieUMRS974/FRE3617,InstitutdeMyologie,GHPitie´-Salpeˆtrie`re,Paris75013,France.3Departmentof
Neurology,OsakaUniversityGraduateSchoolofMedicine,Osaka565-0871,Japan.4DepartmentofPhysiology,FriedrichSchillerUniversityHospital,Jena07743,Germany.
5DepartmentofBioinformaticsandMolecularNeuropathology,MeijiPharmaceuticalUniversity,Kiyose205-8588,Japan.6NeuromuscularResearchCenter,Tampere
UniversityandUniversityHospital,Tampere33520,Finland.7DepartmentofMedicalGenetics,Folkha¨lsanInstituteofGenetics,HelsinkiUniversity,Helsinki00250,Finland.
8DepartmentofNeurology,VaasaCentralHospital,Vaasa65130,Finland.9DepartmentofNeurology,StanfordUniversity,Stanford,California94304,USA.10Servicede
Cardiologie,Universite´Paris-Descartes,HoˆpitalCochin,AP-HP,Paris75014,France.11DepartmentofNeurology,ToneyamaNationalHospital,Toyonaka560-8552,Japan.
12CNRSUMR7175,EcoleSupe´rieuredeBiotechnologiesdeStrasbourg,Illkirch67400,France.13DivisionofNeurology,HyogoMedicalCollege,Nishinomiya663-8501,Japan.
14LaboratoryofStructuralNeuropathology,DoshishaUniversityGraduateSchoolofBrainScience,Kyoto610-0394,Japan.15GraduateSchoolofArtsandSciences,University
ofTokyo,Tokyo153-8902,Japan.16Universite´Lyon1,CNRS,UMR5558LBBE,Villeurbanne69622,France.17HospicescivilsdeLyon,Laboratoiredecytoge´ne´tique
constitutionelle,Bron69500,France.18PoˆleRhoˆneAlpesdeBioinformatique,Universite´Lyon1,BaˆtimentGregorMendel,Villeurbanne69100,France.19CentredeRecherche
enCance´rologiedeLyon,Lyon69373,France.20DepartmentofCardiovascularandRespiratoryMedicine,ShigaMedicalUniversity,Otsu520-2192,Japan.21Departmentof
InformationPhysiology,NationalInstituteforPhysiologicalSciences,Okazaki444-8585,Japan.22DepartmentofNeurology,ChangGungMemorialHospital,Keelung20401,
Taiwan.23DepartmentofMolecularGeneticsandMicrobiology,CenterforNeuroGeneticsandtheGeneticsInstitute,UniversityofFlorida,CollegeofMedicine,Gainesville,
Florida32610,USA.24DepartmentofNeurology,HospitalUniversitarioDONOSTIA,NeuroscienceArea,InstituteBiodonostiaCIBERNEDandUniversityofBasqueCountry
UPV-EHU,SanSebastia´n20014,Spain.25LaboratoryofBiomedicalSciencesandInformationManagement,NationalCerebralandCardiovascularCenterResearchInstitute,
Osaka565-8565,Japan.*Theseauthorscontributedequallytothework.wPresentaddress:MassachusettsGeneralHospital,MassGeneralInstituteforNeurodegenerative
Diseases,Charlestown,Massachusetts02129,USA.CorrespondenceandrequestsformaterialsshouldbeaddressedtoD.F.(email:[email protected])orto
M.P.T.(email:[email protected])ortoN.C-B.(email:[email protected]).
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M
yotonicdystrophy(DM),themostcommonadult-onset observed in DM. These results suggest that altered splicing of
muscular dystrophy, includes two genetically distinct SCN5A mRNA may participate to the electrical cardiac
forms. DM of type 1 (DM1) and its severe congenital abnormalities observed in DM.
form(CDM1)arecausedbyanexpansionofCTGrepeatsinthe
30-untranslated region (UTR) of the DMPK gene1–3. In contrast,
DMoftype2(DM2)iscausedbyanexpansionofCCTGrepeats Results
withinthefirstintronoftheCNBP(alsoknownasZNF9)gene4. Identification of splicing changes in DM heart samples. To
The pathogenesis of DM involves a RNA gain-of-function determine novel splicingabnormalities in DMheart samples, we
mechanism caused by expression of mutant RNAs containing first used whole-genome microarrays (GeneChip Human Exon
hundred to thousands of CUG or CCUG repeats that interfere 1.0STarray)onpolyadenylatedRNAextractedfromleftventricle
with the splicing of other pre-mRNAs through dysfunction samples of three adult DM1 patients compared with three age-
of two classes of RNA-binding proteins. MBNL proteins matched control individuals. Bioinformatic analyses predicted
(MBNL1, MBNL2 and MBNL3) are sequestered within nuclear significant (Fold Change Z2, Sudent t-test, P value r0.01)
RNA foci formed by expanded CUG and CCUG repeats5,6, changes in the splicing of 24 exons between control and DM1
whereas expression and phosphorylation of CUG-binding samples (Supplementary Table 1), including a misregulation of
protein 1 (CUGBP1, encoded by the CELF1 gene) are increased thealternativesplicingoftheSCN5Apre-mRNA.Toextendthis
in DM1 heart samples7. MBNL and CUGBP1 proteins regulate analysis, we performed paired-end RNA sequencing (RNA-seq)
alternative splicing, and alterations of their functional levels in on the same DM1 and control heart samples, yielding 1,611
myotonic dystrophic tissues results in reversion to fetal million of mapped 100bp reads. DESeq and Cuffdiff were then
splicing patterns for several mRNAs, such as the insulin applied to estimate differential gene expression and over or
receptor (INSR) (ref. 8), the muscle chloride channel (CLCN1) under-expressed mRNAs were selected by using the Benjamini
(refs 9,10), dystrophin (DMD) (refs 11,12) and key components and Hochberg adjusted P values (false discovery rate (FDR)
of the skeletal muscle excitation–contraction coupling r0.1). A total of 9 and 19 upregulated genes were predicted
process, including amphiphysin2 (BIN1) (ref. 13), ryanodine differentially expressed with DESeq and Cuffdiff, respectively,
receptor 1 (RYR1) (ref. 14), sarcoplasmic/endoplasmic reticulum but none were confirmed by quantitative real-time RT-qPCR
Ca2þ-ATPase SERCA1 (ATP2A1) (ref. 14) and the muscle analyses. This low number of differentially expressed mRNAs
calcium channel Ca 1.1 (CACNA1S) (ref. 15). Misregulation of suggests that cardiac pathology in DM is not associated with
V
the alternative splicing of the insulin receptor INSR, CLCN1 and drastic modifications of gene expression levels. In contrast,
DMD mRNAs are associated with the insulin resistance8, DEXSeq(ref.22),whichtestsdifferentialexonusagebetweentwo
myotonia9,10,16 and dystrophic process12, respectively, while conditions, predicted 134 significant (Log2 Fold Change Z1.2,
alterations of the alternative splicing of BIN1, RYR1, ATP2A1 FDR r0.1) alternative splicing changes between control and
and CACNA1S may contribute to the skeletal muscle weakness DM1 heart samples (Supplementary Data 1). Similarly, MISO
observed in DM13–15. (ref. 23) analysis, which computes the fraction of mRNA that
In contrast, the molecular mechanisms underlying the cardiac includes a given cassette alternative exon, predicted 259
defects, which affect 80% of individuals with DM and represent significant (DPSI Z0.3; Z-score Z1.2) alternative splicing
the second most common cause of death in this disease17,18, are changes between control and DM1 heart samples (Fig. 1a and
yettobedefined. CardiacinvolvementsinDMarecharacterized Supplementary Data 2), including a robust misregulation of the
by cardiac-conduction delay that may result in fatal atrio- alternative splicing of SCN5A (Fig. 1b). MISO and DEXSeq pre-
ventricular block, and by atrial or ventricular tachycardia17,18. dictions overlapped, but with some exceptions, such as the
Electrocardiography (ECG) analyses in DM patients indicate skipping of the consecutive exons 18, 19 and 20 of CAMK2B
prolonged conduction time from the sinoatrial node to the predicted by DEXSeq but not by MISO; or the shift of SCN5A
ventricles (PR interval) and elongated ventricular depolarization exon6Btowardsexon6AidentifiedbyMISObutnotbyDEXSeq.
(QRS duration). Interestingly, cardiac dysfunctions in DM are Thesedifferencesareinherenttotheircomputationmodels,since
reminiscent in some aspect to an alteration of the cardiac MISOdoesnotdetectalterationsofsuccessiveexonsandDEXSeq
sodiumcurrent.Thea-subunitofthecardiacvoltage-gatedNaþ does not identify mutually exclusive exons, highlighting that
channel, Na 1.5, is encoded by the SCN5A gene and plays a key MISO and DEXSeq are complementary bioinformatics
v
role in the excitability of cardiomyocytes and for rapid approaches. Next, we tested by PCR with reverse transcription
propagation of the impulse through the cardiac-conduction (RT–PCR)fortycandidatemRNAshavingthehighestprobability
system. Mutations in SCN5A lead to a variety of arrhythmic ofmisregulationinDEXSeqand/orMISOanalyses.Wevalidated
disorders, including long QT3, progressive and non-progressive splicingalterationsfor32ofthem,includingsomethathavebeen
cardiac-conduction disease (also known as Lev-Lene`gre disease), identified in previous studies (TNNT2, TNNT3, ABLIM1, LDB3,
atrial fibrillation, sick sinus syndrome, Brugada syndrome and MBNL1, CAMK2B, MAPT and so on)24–26, and 20 others that
numerous overlapping syndromes19–21. represent, to the best of our knowledge, novel alterations of
Using transcriptomic approaches, we identified various novel alternative splicing (ADD3, GOLGA4, CRTC2, ARHGEF10L,
splicingchangesinheartsamplesofDM1individuals.Analysisof ANK3, DCLK2, EPN2, UNC13B, TECR, ARVCF, SOCS7, CELF1
the RNA motifs enriched in the vicinity of these misregulated and so on) in DM1 heart samples (Fig. 1c). Of interest, some of
exons indicates that sequestration of the MBNL proteins is these splicing alterations may be of pathological consequence
probably the main cause of splicing misregulation in heart of inDM.Forexample,knockoutoftheSocs7geneinmouseresults
individuals with DM. Among these novel splicing alterations, in insulin resistance27. Whether the splicing misregulation of
we focused on misregulation of alternative splicing of the SOCS7 in DM contributes to insulin resistance remains to be
SCN5A pre-mRNA. This splicing alteration results in expression tested. Also, RNA sequencing predicts an increased retention of
of a fetal isoform of SCN5A with altered electrophysiological thepenultimateintronofFCGRT,whichencodestheFcfragment
properties. Of importance, we demonstrate that reproducing of the IgG receptor transporter a (FCRN) protein, involved in
the splicing alteration of Scn5a in mouse is sufficient to cause IGGrecycling28.WhethersplicingalterationofFCGRTinDMis
heartarrhythmiaandcardiac-conduction delaywithelevatedPR responsible to the decreased level of IGG in blood of these
interval, which are key characteristics of the heart alterations patients is an attractive hypothesis that remains to be tested.
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a b
Exons excluded in DM Exons included in DM SCN5A
3 5 6A 7
SCN5A 6B
RPKM321000
12Z score RPKMRPKMRPKM321321321000000000 CTL
RPKM321000 DM1
RPKM321000
–1 –0.5 0 0.5 1
38662462 38658666 38654924 38651227
Δ PSI Genomic coordinate (chr3), “-” strand
c
CTL DM1 CTL DM1 CTL DM1 CTL DM1
320000 6B 200 + 14 320000 + 8 435000000 ++ 1189..1290.20
100 6A 100 – 14 100 – 8 120000 – 18.19.20
SCN5A ABLIM1 COPZ2 CAMK2B
CTL DM1 CTL DM1 CTL DM1 CTL DM1
300 300 200 200
200 + 16 + 6 + 24 + 10
– 16 200 – 6 100 – 24 – 10
100 100
ADD3 CLTB GOLGA4 SUN1
CTL DM1 CTL DM1 CTL DM1 CTL DM1
200 300 300 400
+ 6 + 13 + 4 300 + 8
– 6 200 – 13 200 – 4 200 – 8
100 100 100
MYH11 CRTC2 MXRA7 ARHGEF10L
CTL DM1 CTL DM1 CTL DM1 CTL DM1
300 300 200
300 + 47 200 + 20 200 + 40 100 + 5
200 - 47 – 20 – 40 – 5
100 100
NCOR2 NUMA1 ANK3 DCLK2
CTL DM1 CTL DM1 CTL DM1 CTL DM1
300 400 300
+ 6B 300 + 5 200 + 38 200 + 5
200 – 6A 200 – 5 – 38 100 – 5
100 100
TPM2 EPN2 UNC13B SOCS7
CTL DM1 CTL DM1 CTL DM1 CTL DM1
200 300 200
+ 2 + 6 200 + 19 + 2
200
100 – 2 100 – 6 100 – 19 100 – 2
TECR ZFYVE21 ARVCF CELF1
Figure1|IdentificationofnovelsplicingmisregulationsinDM1heartsamples.(a)D-PSIversusZ-scoreplotofexoncassettesmisregulationspredicted
byMISOanalysis.(b)ExonsstructureandcoverageofRNA-seqreadsacrossSCN5Aexons5–7showincreasedinclusionofexon6Aanddecreased
inclusionofexon6BinheartsamplesofthreeDM1patients(bottom,blue)versusthreecontrolsamples(top,red).(c)Validationby
RT–PCRofRNA-seqpredictionsinhumanheartsamplesofnormaladultindividuals(CTL,black)versusadultDM1patients(DM1,red).Molecularsize
markersinbpsarereportedtotheleftofeachRT–PCRgels.bp,basepair.
Finally,RNAsequencingpredictsmisregulationofthealternative these RNA motifs are indeed present in the vicinity of exons
splicing of a cardiac-specific exon located in the 50-UTR of misregulated in DM, we determined all 4-mer RNA motifs
CELF1, which encodes CUGBP1. Whether this alternative enrichedwithin,upstreamordownstreamoftheexonspredicted
splicing may contribute to the increase levels of CUGBP1 asmisregulated byMISOinDM1heartsamples,comparedwith
protein observed in DM1 hearts remains also to be evaluated. 2,000 control exons (Fig. 2). Most RNA motifs significantly
enriched (binomial test, P value o1.0 10(cid:2)7) contained YGC
sequences, while none were found to contain UGU sequences.
Splicing altered in DM are enriched for MBNL-binding sites. Furthermore, YGC sequences were enriched upstream of
Mutants RNAs containing expanded CUG or CCUG repeats exons abnormally included in DM1, while YGC motifs were
interfere with the functional levels of CUGBP1 and MBNL enriched downstream of exons repressed in DM1. These results
proteins. Earlier studies determined thatMBNLproteins bind to matchedtheMBNLsplicingregulatorymapdeterminedbyCLIP
YGCRNAmotifs(whereYisapyrimidine)29–32,whileCUGBP1 experiments,wherebindingofMBNLupstreamofanexontends
binds to UGU-enriched sequences33,34. To determine whether toinhibitexoninclusionwhereasbindingofMBNLdownstream
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UGCU (2×10–26)
GCUU (2×10–19)
CUGC (2×10–15)
UUGC (2×10–9) CUGC (2×10–11)
Exons included in DM GAAG (5.1 10–7) CGCU (4×10–9) UGCU (3×10–9)
GCUC (7×10–7) GCUG (6×10–8)
CCCC (2×10–18)
Exons excluded in DM
CUGC (9×10–15) CCGC (2×10–22)
UGCU (2×10–14) CGCC (8×10–17)
CUAA (5×10–10) CGCU (9×10–10)
UUGC (5×10–8) UCGC (4×10–7)
CCUG (4×10–7) GCGC (7×10–7)
UGCC (7×10–7) CCCG (8×10–7)
Figure2|MBNL-bindingmotifsareenrichedinvicinityofexonsmisregulatedinDM1.SequenceandbinomialtestPvaluesof4-merRNAmotifs
enricheddownstream,withinandupstreamofexonsmisregulatedinDM1heartsamples.SequencesenrichedinexonsexcludedinDMareindicatedinred,
whilesequencesenrichedinexonsincludedinDMareindicatedinblue.
of an exon generally stimulates exon inclusion35,36. In contrast, alternative splicing of SCN5A resulting in expression of a fetal
we found no enriched motifs for other RNA-binding proteins, form of this channel in adult DM heart. These results are
includingCUGBP1,rbFOX1,hnRNPHorStaufen.Theseresults, consistent with previous studies where alternative splicing
as well as previous data36–38, support a model in which titration changes in DM resume a MBNL-dependent fetal splicing
of MBNL proteins is the main cause of splicing change in DM1 pattern that persist in adult tissues26,37.
heart, while misregulation of other RNA-binding proteins may
contribute to a subset of splicing alterations.
Alternative splicing of SCN5A is regulated by MBNL1. To
determine the mechanisms underlying misregulation of SCN5A
Splicing of SCN5A is misregulated in DM heart samples. Both splicing,wefirstdetermineditssplicingpatternincellmodelsof
microarray and RNA-seq predicted misregulation of alternative DM.SinceSCN5Aisexpressedatlowlevelincultureofimmature
splicingofSCN5Apre-mRNAinDM1heartsamples.Splicingof skeletal muscle cells, we investigated its splicing in primary
SCN5A is developmentally regulated, such that exon 6A is cultures of differentiated skeletal muscle cells originating from
included in fetal heart but rapidly replaced by exon 6B after muscle biopsies of control and DM1 individuals. RT–PCR
birth39.Consequently,SCN5Aexon6Aisnamedasembryonicor experiments determined a switch of exon 6B towards exon 6A
fetal, while exon 6B is known as adult. Exons 6A and 6B are in DM1 muscle cells compared with control, reproducing the
mutually exclusive exons encoding part of the voltage sensor, splicing alteration observed in cardiac tissue (Fig. 4a). Of
segments3and4locatedinthedomainIofthesodiumchannel technical interest, the basal level of exon 6A inclusion was
(Fig. 3a,b). These are key segments for the electrical activity of higher in muscle cell cultures than in adult heart samples
the sodium channel, and inclusion of either fetal exon 6A or (compareFig.4atoFig.3c),whichprobablyreflecttheimmature
adult exon 6B results in channel isoforms, named, respectively, aspect of cell cultures. Since mutant RNAs containing expanded
hNa 1.5e and hNa 1.5, with different electrophysiological CUGorCCUGrepeatsinterferewithalternativesplicingthrough
v v
properties39–41. We confirmed our microarray and RNA-seq titration of MBNLproteins, we testedwhetherMBNL1 regulates
predictions by RT–PCR and found that adult SCN5A exon 6B is SCN5A splicing. Reduction of MBNL1 expression through a
partlyreplacedbyitsfetalexon6Ainheartsamplesofindividuals siRNA-mediated approach in human control primary muscle
with DM, including adult DM1 and adult DM2 cases (Fig. 3c). cellsmimickedtheeffectofCUGrepeatsandpromotedaswitch
Note that to differentiate exon 6B from exon 6A that have from adult exon 6B towards fetal exon 6A (Fig. 4b). Western
the exact same length of 92bp, we took advantage of a BstbI blotting analysis confirmed the successful depletion of MBNL1
restriction site present only in exon 6A, which thus appears as a expression(SupplementaryFig.3A).Next,weassessedalternative
BstbI-digested doublet band in Fig. 3c. These results are splicing of Scn5A in heart samples of Mbnl knockout mice43.
consistent with the recent report of a splicing misregulation of RT–PCRanalysisshowsthatinclusionoftheexon6AofScn5ais
SCN5AinoneDM1heartsample42.AlthoughsplicingofSCN5A increased in heart samples of mice with no Mbnl1 and reduced
ismisregulatedinDM1,weobservednocorrelationbetweenthe level of Mbnl2 (Mbnl1(cid:2)/(cid:2), Mbnl2þ/(cid:2))(Fig. 4c). The increased
percentage of SCN5A exon 6A inclusion and the increased inclusion of Scn5a exon 6A in Mbnl knockout mice is
duration of the PR interval and only a very limited, if any, significant (Student t-test, P value r0.01) but rather mild,
correlation between misregulation of SCN5A exon 6A splicing probablyreflectingdifferenceinregulationofalternativesplicing
and alteration of the QRS duration in individuals with DM1 betweenhumanandmouseorthecompensatoryeffectofresidual
(R2 of 0.2 with six DM1 samples; Supplementary Fig. 1). Mbnl2 expression43. This hypothesis is consistent with the mild
Misregulation of SCN5A splicing was specific to DM, as we did splicingalterationofScn5AobservedinthesoleMbnl1knockout
not observe inclusion of exon 6A in heart samples from mice44.Overall,theseresultssuggestthatMBNLproteinsregulate
individual affected with Duchenne muscular dystrophy (DMD), thealternativesplicingofSCN5Aexons6Aand6B.Todetermine
amyotrophic lateral sclerosis (ALS) or dilated cardiomyopathy whether this regulation is direct or indirect, we constructed a
(DCM) (Fig. 3d). Moreover, misregulation of splicing in DM1 minigene containing exons 6A and 6B of SCN5A bordered by
wasspecificandnotglobal,asweobservednosplicingchangesof their intronic regions. Expression of this construct in mouse
SCN5A alternative exon 18, of CACNA1C mutually exclusive C2C12 myoblasts reproduced a fetal pattern with mainly
exons 8A and 8B, of KCNAB1 alternative exons 2 and 11, or of inclusion of exon 6A (Fig. 4d). Since inclusion of exon 6B was
KCNQ1alternativeexons2and5(SupplementaryFig.2).Finally, repressed, reduction of Mbnl1 activity through siRNA or
we observed no significant alteration of the expression level of expression of expanded CUG repeats had no further repressive
SCN5A mRNA by quantitative real-time RT-qPCR (Fig. 3e). effectonexon6B.Incontrast,expressionofMBNL1promoteda
Overall, these results indicate a specific misregulation of switch from fetal exon 6A towards adult exon 6B, while
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a b
I II III IV
Fetal
5 6A 6B 7 12345 6 12345 6 12345 6 12345 6
BstBI Adult NH2 OH
O
C
c
bp Adult CTL Adult ALS Fetal CTLCongenital DM1 Adult DM1 Adult DM2
300
Exon 6B
200
Exon 6A
100
d e
100
A
% Exon 6Ainclusion 24680000 CN5A mRNexpression 0.51
S
0 0
Fetal CATdLult CTL ALS DCCMongDeMniDtal DAMd1ult DAMd1ult DM2 CTL DM1
Figure3|SplicingofSCN5Aexon6AisalteredinDMheartsamples.(a)Schematicrepresentationofmutuallyexclusiveexons6Aand6BofSCN5A.
SCN5AmRNAincludesexon6A(red)infetalheart,whileSCN5AmRNAexpressesexon6B(blue)inadultheart.(b)SchematicrepresentationofSCN5A
topologyexpressingexon6A(red).Exons6Aor6Bencodespartofsegment3,connectingloopbetweenS3andS4andmostpartofthevoltage-sensitive
segment4ofdomain1ofthesodiumchannelSCN5A.(c).RepresentativeBstBI-digestedRT–PCRanalysisofendogenousSCN5AmRNAfromhumanheart
samplesofnormaladult(CTL),adultALS,non-DMfetuses(20,24and35weeks),congenitalDM1fetuses(CDM1of22,25and28weeks),adultsDM1
andDM2individuals.Molecularsizemarkerisindicatedinbp.(d)GraphicalrepresentationofRT–PCRanalysisdepictingthepercentageofSCN5AmRNA
includingexon6Ainleftventricularheartsamplesfromfetalandadultcontrol,ALS,DCM,DMD,CDM1andadultDM1andDM2individuals.(e)Graphical
representationofquantitativereal-timeRT-qPCRdepictingthemRNAexpressionofSCN5ArelativetoRPLP0incontrolnormaladults(n¼5)versusadult
DM1(n¼5)heartsamples.Barsindicates.e.m.bp,basepairs.
expression or siRNA-mediated depletion of CUGBP1 had no (Fig. 5a and Supplementary Table 2). Injection of RNA
effect (Fig. 4d). Western blotting analysis confirmed that siRNA encoding hNav1.5e, which is the splicing isoform of SCN5A
transfection efficiently reduced endogenous Mbnl1 or Cugbp1 found in DM, indicated a significant reduction of the sodium
expression (Supplementary Fig. 3B and C). Next, gel-shift assays current amplitude of 45%, compared with hNa 1.5, the normal
v
determined that recombinant purified GST-tagged MBNL1 adult SCN5A exon 6B form (Fig. 5b,c). Since, the extent of
bound to UGC RNA motifs located upstream of exon 6A splicing misregulation varies among DM individuals, which
(Fig.4e).Ofinterest,thisUGCsequenceisabsentfromthemouse typicallyexpressamixofSCN5Asplicingformscontainingeither
genome,whichmayexplainthemildsplicingalterationofScn5A exon 6A or exon 6B (cf. Fig. 3c), we analysed sodium currents
observedinmiceknockoutforMbnlproteins.Mutationof these generated by a mix of both SCN5A isoforms (Fig. 5a). Injecting
UGC motifs abolished MBNL1 binding (Fig. 4f), as well as the Xenopus oocytes with an equimolar mix of RNA encoding each
regulatory effect of MBNL1 on a mutant SCN5A minigene channel, namely 50% of hNa 1.5e (SCN5A containing fetal exon
v
(Fig. 4g). Overall, these results establish that MBNL1 regulates 6A) and 50% of hNa 1.5 (SCN5A expressing adult exon 6B),
v
directly alternative splicing of SCN5A exons 6A/6B. resultedinareductionof30%ofthecurrentamplitudecompared
with the control hNa 1.5 (Fig. 5b,c). Next, two-electrode voltage
v
clamp recording experiments revealed that the steady-state
SCN5A splicing forms present different electrical properties. activation of the fetal hNa 1.5e was shifted by 7mV towards
v
SCN5A encodes Na 1.5, the main cardiac voltage-gated sodium depolarized potential compared with the control adult hNa 1.5
v v
channel, and loss-of-function mutations in SCN5A lead to a form(Fig.5dandSupplementaryTable2).Thisshiftisconsistent
variety of arrhythmic disorders, which share some common with the shift observed previously in transfected mammalian
pathological features with DM. Furthermore, exons 6A and 6B cells39–41, thus validating our approach in Xenopus oocytes. To
differatsevenaminoacidpositions,resultinginchannelvariants better reproduce the situation observed in DM, we injected in
with different electrophysiological properties39–41. To investigate Xenopus oocytes an equimolar mix of DM (hNa 1.5e, fetal exon
v
the consequences of the switch from SCN5A exon 6B towards 6A) and control (hNa 1.5, adult exon 6B) RNA isoforms of
v
exon 6A observed in DM, we first examined in Xenopus oocytes SCN5A. Importantly, this mix of splicing forms also presented a
the sodium currents generated by either hNa 1.5e, the splice significant shift of steady-state activation towards depolarized
v
variant of SCN5A containing the fetal exon 6A, or hNa 1.5, potentials by 3.8mV, compared with the control hNa 1.5 form
v v
encoded by SCN5A containing the adult control exon 6B (Fig.5d,SupplementaryTable2).Correspondingly,asimilarshift
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a b c
Mbnl1–/–
bp CTL DM1 bp siCTL siMBNL1 bp CTL Mbnl2+/–
300
6B 6B
6B 300
200
200
200
6A 6A
100 6A 100
100
B 60 B 60 A30
n 6 40 n 6 40 n 620 **
o o o
x x x
% E 20 *** % E 20 *** % E10
0 0 0
d e
CMV PolyA
bp Control CUG 960xMBNL1 siMbnl1 CUGBP1siCelf1 5 6A 6B
200 UUUGCUAUGCUGUGCUAUGCCUUGCAG
6B
SCN5A minigene WT
# MBNL1
100 6A
Bound
B 60
6
n 40 ***
o
Ex 20 Free
% 0
f g
CMV PolyA
5 XXX 6A 6B bp Control CUG 960xMBNL1 siMbnl1
200
UUU_CUAU_CUGU_CUAU_CCUU_CAG 6B
SCN5A minigene MUT
MBNL1
100 6A
Bound
B60
6
n 40
o
Free Ex20
% 0
Figure4|MBNL1regulatesalternativesplicingofSCN5A.(a)Upperpanel,RT–PCRanalysisofendogenousSCN5AmRNAfromdifferentiatedprimary
musclecellculturesderivedfrombiopsiesofcontrolorDM1individuals.(lower)QuantificationofthepercentageofSCN5AmRNAincludingexon6B.
(b,upper)RT–PCRanalysisofendogenousSCN5AmRNAfromhumandifferentiatedculturesofcontrolprimarymusclecellstransfectedwithascrambled
siRNA(siCTL)orasiRNAtargetingMBNL1mRNA(siMBNL1).(lower)PercentageofSCN5AmRNAincludingexon6B.(c,upper)RT–PCRanalysisof
endogenousScn5amRNAinheartsamplesofwild-typeandcompoundMbnl1(cid:2)/(cid:2),Mbnl2þ/(cid:2) doubleknockoutmice.(lower)PercentageofScn5amRNA
includingexon6A.(d,upper)RT–PCRanalysisofexogenousSCN5AmRNAfromdifferentiatedC2C12musclecellsco-transfectedwithaSCN5Aminigene
containingexons6Aand6Bborderedbytheirintronsandwitheitheraplasmidexpressing960CTGrepeats,MBNL1,CUGBP1orwithasiRNAdirected
againstMbnl1(siMbnl1)orCelf1(encodingCugbp1;siCelf1).#Indicatesusageofacrypticsplicesiteinherenttotheminigene.(lower)PercentageofSCN5A
mRNAincludingexon6B.(e,upper)SchematicrepresentationofSCN5Aminigene,includingtheUGC-richsequenceusedforbindingassays.(lower)Gel-
shiftassayswereperformedusing5–1,000nMofpurifiedbacterialrecombinantGST-MBNL1D101andauniformly32P-CTPlabelledRNA.(f,upper)
SchematicrepresentationofmutantSCN5Aminigene,includingthemutantsequence,usedforbindingassays.(lower)Gel-shiftassayperformedasin
e.(g,upper)RT–PCRanalysisofexogenousSCN5AmRNAfromdifferentiatedC2C12musclecellsco-transfectedwithmutantSCN5Aminigeneandwitha
plasmidexpressing960CTGrepeatsorMBNL1orwithasiRNAdirectedagainstMbnl1(siMbnl1).(lower)PercentageofSCN5AmRNAincludingexon6B.
Alltransfectionandgel-shiftexperimentswererepeatedthreetofivetimes.Molecularsizemarkersareindicatedinbp.Barsindicates.e.m.Studenttest,**
indicatesPo0.01,***indicatesPo0.001.bp,basepairs.
was observed for the time constant of inactivation (Fig. 5e). 6A,presentsareducedexcitabilitycomparedwithhNa 1.5,which
v
Consistent with previous electrophysiological studies39–41, no is the adult control SCN5A isoform containing exon 6B.
significant differences were observed between hNa 1.5 and
v
hNa 1.5e regarding steady-state inactivation and recovery from
v
inactivation (Fig. 5f,g). Overall, our results are consistent with AlterationofSCN5Asplicingleadstoheartconductiondefects.
previousstudies39–41,anddemonstratethathNa 1.5e,thesplicing Misregulation of thealternative splicingof SCN5A in DMisone
v
form of SCN5A expressed in DM and containing the fetal exon alteration identified among many others, thus questioning the
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NATURECOMMUNICATIONS|DOI:10.1038/ncomms11067
a
hNav1.5 (SCN5A exon 6B) hNav1.5e (SCN5A exon 6A) 50% hNav1.5 + 50% hNav1.5e
1 μA
2 ms
b c
Voltage (mV)
4 –60 –40 –20 0 20 40 60
A) 0
μnt ( 3 *** –1
e ***
eak curr 12 μnt (A) –2
P e
urr –3
0 C hNa1.5
hNav1.5 hNav1.55e0% h% NhaNv1.a5v1.5e ––45 h55N00%%avv 1hh.NN5aaevv 11..55 e+
50
d e
1.0 ms) 14
Steady-state activation 00000.....02468 hh55NN00%%aavv 11hh..NN55aaevv11..55 e+ nactivation time constant ( 110246802 h55hNN00%%aavv 11hh..NN55aa evv 11..55 e+
–60–50–40–30–20–10 0 10 20 I –40 –30 –20 –10 0 10
Voltage (mV) Voltage (mV)
f g
n 1.0 1.0
o
Steady-state inactivati 0000....2468 5h5hNN00%%aavv 11hh..NN55aaevv11..55 e+ Fractional recovery 0000....2468 5h5hNN00%%aavv 11hh..NN55aaevv11..55 e+
0.0 0.0
–130 –110 –90 –70 –50 –30 0 20 40 60 80 100
Voltage (mV) Recovery interval Δt (ms)
Figure5|ElectrophysiologicalpropertiesofhNa1.5andhNa1.5echannels.(a)RepresentativeNaþ currentsgeneratedinXenopusoocytesbyhNa1.5
v v v
(encodedbySCN5Acontainingtheadultexon6B),hNa1.5e(encodedbySCN5Aincludingthefetalexon6A),andsimultaneouslyexpressedNa1.5and
v v
Nav1.5echannelsata1:1ratio.(b)Peakcurrentamplitudesatthetestpotentialof (cid:2)10mVinXenopusoocytesinjectedwithequimolaramountofcRNA
encodinghNa1.5,hNa1.5eor1:1combinationofNa1.5andNa1.5echannels.(c)Current–voltagerelationships.(d)Steady-stateactivationcurves.
v v v v
(e)Inactivationtimeconstantsth(ms)atdifferenttestpulses.(f)Steady-stateinactivationcurves.(g)Fractionalrecoverycurves.Datawereobtained
from11differentbatchesofoocytes.Toillustratesteady-stateactivation,steady-stateinactivationandrecoveryfrominactivation,weused3–5
representativemeasurements.Fortotalnumberofmeasurements(n¼25–27)andforstatisticaldataevaluation(Vm,s)seetheSupplementaryTable2.
Barsindicates.e.m.Studenttest,***indicatesPo0.001.
contribution of SCN5A misregulation to the cardiac symptoms producedadeno-associatedvirus(AAV2/9)expressingoptimized
observedinDM.Totest thephysiologicalimportanceof SCN5A U7-snRNA fused to Scn5a antisense sequences (U7-ASScn5a).
splicingmisregulation,weartificiallyforcedtheswitchfromadult Splicing analysis revealed that combination of two U7-AS
exon 6B towards fetal exon 6A into wild-type adult mouse heart constructs, spanning intron 6/exon 6B junction and exon
using an exon-skipping strategy (Fig. 6a). To insure efficient 6B of Scn5A, promoted a switch from inclusion of adult exon
transduction of the cardiac muscle and continuous expression 6B towards inclusion of the fetal exon 6A (Supplementary
of nuclear antisense oligonucleotides, we engineered and Fig. 4A). Thus, AAV2/9 expressing both U7-AS constructs
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a
Fetal Adult
5 6A 6B 7 5 6A 7
Forced inclusion of
Scn5A antisense
exon 6A in adult
sequences
b c
bp CTL U7-ASScn5a CTL
300
6B U7-ASScn5a
200 2
6A A1.5
100 N
on 6A4600 *** mR0.51
Ex20 0
% 0 Scn5a Scn1b Gja1
d e
CTL 50 20 0.058 50
P P ***
40 15 40
QRS QRS ms 30 10 30
U7-ASScn5a 20 20
P P 10 5 10
0 0 0
QRS QRS 25 ms PR QRS QT
CTL U7-ASScn5a
f g
CTL
225
R150
R
75 CTL
0
U7-ASScn5a 0 0.5 1 1.5 2
225
R150
R 75 U7-ASScn5a
0
0 0.5 1 1.5 2
mn
h i
Control U7-ASScn5a
CTL
U7-ASScn5a
2 **
1.5 *
A
RN 1
m
0.5
0
Cola1a Col3a1 Tgfb1
Figure6|AlterationofScn5asplicingcausesheartconductiondefectsandarrhythmias.(a)Schematicrepresentationofmutuallyexclusiveexons6A
and6BofScn5aandofantisensesequencesdrivenbyoptimizedU7-snRNAs(U7-ASScn5a)toforcefetalexon6Ainclusioninadultwild-typemouseheart.
(b,upper)RT–PCRanalysisofthealternativesplicingofendogenousScn5amRNAfromheartsamplesofmiceinjectedwithAAV2/9expressing
U7-ASScn5acomparedwithcontrolinjectedmice.Molecularsizemarkerisindicatedinbp.(lower)PercentageofScn5amRNAincludingexon6A.(c)Real-
timeRT-qPCRquantificationoftheexpressionofScn5a,Scn1bandGJja1(connexin43)mRNAsinheartsamplesofmiceexpressingU7-ASScn5a(n¼6)
comparedwithcontrolinjectedmice(n¼6).(d)RepresentativeECGtracesshowprolongationofthePRintervalinU7-ASScn5a-injectedmicecompared
withcontrolmice.(e)ECGmeasuresofPRinterval,QRSandQTintervalsin4-month-oldmiceinjectedwithAAV2/9expressingU7-ASScn5a(n¼25)
comparedwithage-matchedcontrolmice(n¼17).(f)RepresentativeECGtracesrevealatrialfibrillationinU7-ASScn5a-injectedmicecomparedwith
controlmice.(g)VariationoftheRRintervalindicatesevidencesofheartarrhythmiasinU7-ASScn5a-injectedmice(n¼25)comparedwithcontrolmice
(n¼17).(h)RepresentativeimageofsixanalysedheartsamplesshowingmildfibrosisrevealedbyRedSiriushistologystaininginAAV-U7-ASScn5a-injected
mice.Scalebar,100mm.(i)Real-timeRT-qPCRquantificationoftheexpressionofCola1a,Col3a1andTgfbmRNAsinheartofcontrol(n¼6)orAAV-U7-
ASScn5a-injectedmice(n¼6).Barsindicates.e.m.Studenttest,*indicatesPo0.5,**indicatesPo0.01,***indicatesPo0.001.bp,basepair.
(AAV-U7-ASScn5a) were injected systemically into newborn aconcomitant30–40%increaseoftheinclusionofexon6A,thus
wild-type mice and cardiac functions were investigated 4 and 6 reproducingthesituationobservedinDM(Fig.6b).Quantitative
monthspostinjection.Controlanimalsinjectedeitherwithsaline RT–PCR demonstrated no changes in the expression of Scn5a
oremptyAAV2/9presentednosplicingalterationsofScn5aand mRNA or of its associated subunit Scn1b between control-
normal cardiac functions. In contrast, mice injected with AAV- and AAV-U7-ASScn5a-injected mice (Fig. 6c). Importantly,
U7-ASScn5apresentedadecreasedinclusionofadultexon6Bwith AAV-U7-ASScn5a-injected mice reproduce some of the key
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pathological features of DM, including conduction defects and predicted a change of the QRS duration from 72ms with
heart arrhythmias. Indeed, ECG performed 4 months post control adult hNa 1.5to 88mswith fetalhNa 1.5e,hence a 22%
v v
injection revealed a significant prolongation of the PR intervals increase(Fig.7aandSupplementaryFig.5).Furthermore,wealso
(Student t-test, P value r0.001) in AAV-U7-ASScn5a-injected tested extent of atrio-ventricular change50. Mathematical
micecomparedwithcontrolinjectedmice(Fig.6d,e).Incontrast, simulation predicted a change of the atrium-His interval from
QTintervalwasnotsignificantlyaltered,andweidentifiedonlya 81ms with control hNa 1.5 to 143ms with fetal hNa 1.5e
v v
trendtowardsincreasedQRSduration(Studentt-test,Pvalueof (Fig. 7b). Overall, these results support our mouse results and
0.058 with 8 AAV-U7-ASScn5a-injected mice on 25 presenting a provide additional evidences that misregulation of SCN5A
QRS higher than 19ms versus 16.5ms in control mice) (Fig. 6e alternative splicing causes cardiac-conduction abnormalities,
andSupplementaryFig.4B).Similarly,analysisofheartfunctions which is a key pathological feature of DM (Fig. 7c).
in 6-month-old animals showed that AAV-U7-ASScn5a-injected
mice present a consistent increase of the PR interval compared
with control injected mice (40.5ms versus 34.8ms respectively; Discussion
Student t-test, P value r0.05), without significant changes of Cardiacdefectsaffect80%ofindividualswithDMandrepresent
theQRSandQTintervals(SupplementaryFig.4B).Ofinterest,a the second most common cause of death in this disease17,18.
similarelongation of thePRinterval wasobserved inScn5aþ/(cid:2) However, the molecular mechanisms responsible for
mice, which are hemizygote for Scn5a expression and represent cardiac-conduction delay and ventricular tachycardia in DM are
an established model for cardiac-conduction disease45–47. unclear. Using RNA sequencing we identified various novel
Furthermore, ECG analyses also revealed that 44% of splicing misregulation events in DM1 heart samples. Among
AAV-U7-ASScn5a-injected mice develop significant (Student these changes, the splicing switch from adult exon 6B to fetal
t-test, Po0,001) heart arrhythmia at 4 months post injection exon 6A in SCN5A mRNA is of particular interest. Previous
withanaverageoffivearrhythmicevents,definedasvariationof studies39–41 as well as ours indicate that hNa 1.5e, the splicing
v
the RR interval, per minute whereas control injected animals variant of SCN5A found in DM and that contains the fetal exon
showed no alterations (Fig. 6f,g). We did not detect 6A, possesses a reduced excitability compared with the normal
ventricular fibrillations or second and third-degree heart adult splicing form of SCN5A containing the exon 6B.
blocks in any injected animals. In contrast, we observed Consequently, the switch from the hNa 1.5 to the hNa 1.5e
v v
supraventricular premature contractions and atrial fibrillation in channel in DM may cause a slower upstroke velocity of the
AAV-U7-ASScn5a-injected mice (Fig. 6f), and five of these cardiac action potential, leading to conduction slowing.
injected mice died suddenly between 4 and 6 months post Importantly, this hypothesis is supported by mathematical
injections (none of the control mice died). These electrical simulation as well by animal model, since imposing a switch
alterations were specific and not caused by global cardiac from inclusion of the control adult exon 6B towards using the
remodelling since we observed neither systolic nor diastolic fetal exon 6A of Scn5A in adult mouse heart led to
alterationsbydopplerechocardiography(SupplementaryTable3) cardiac-conduction delay and heart arrhythmias, two key
and no change in heart/body weight ratio (4.4±0,1mgg(cid:2)1 features of DM. Moreover, clinical evidence also supports an
in control, n¼9, versus 4,7±0,2mgg(cid:2)1 in AAV-U7-ASScn5a- alteration of the sodium current in DM. Indeed, the
injected animals, n¼14). As further control, H&E-staining electrophysiological features39–41 of the fetal isoform of SCN5A
revealed normal heart structures with no evident expressed in DM are similar to the electrophysiological
cardiomyopathy or dilation at 6 months post AAV injections characteristics observed with loss-of-function mutations of
(Supplementary Fig. 4C). Similarly, quantitative RT–PCR SCN5A causing cardiac-conduction disease51–53. Also, there are
experiments show no alteration in the expression levels of somesimilaritiesofECGrecording,includingprolongationofthe
Nppa, Nppb (encoding Anp and Bnp, respectively) and PR interval and of the QRS duration, between individuals with
Myh7 mRNAs (Supplementary Fig. 4D), suggesting no overt DM and individuals affected by cardiac-conduction disease
cardiacremodellinginantisenseAAV-U7-ASScn5a-injectedmice. caused by loss-of-function mutations in SCN5A42,54. Finally, the
Moreover,SiriusRedstainingconfirmednormalheartstructures inductionof abnormalECGpatterninDMpatientstreatedwith
but also revealed some mild fibrosis (Fig. 6h), which was ajmaline55,56,aclassIaantiarrhythmicagentactingonthecardiac
confirmed by increased expression of collagen Cola1a and Tgfb1 sodium channel andtheabnormalsodium currentobserved ina
mRNAs (Fig. 6i). Interestingly, mild fibrosis is also observed in mousemodelofDM57,arealsoevocativeofadysfunctionofthe
DM cardiac samples17,18, as well as in individuals and mice sodium channel in DM. Overall, our results suggest that
models with loss-of-function mutations of the SCN5A misregulation of the splicing of SCN5A participates in a subset
gene20,21,46,47. Overall, heart arrhythmias and prolonged PR of electrical cardiac alterations observed in DM, namely the
interval in AAV-U7-ASScn5a-injected animals demonstrate that cardiac-conductiondelayandtheheartarrhythmias.However,it
inclusion of the fetal exon 6A of Scn5a is inappropriate to adult is likely that other alternative splicing alterations and/or
mouse heart physiology. However, while we found a clear mechanisms58–61 are participating to the full pattern of cardiac
elongation of the PR interval, we did not detect a significant alterations in DM since knockout of Mbnl1 and Mbnl2 in mice
alteration of the QRS duration as only a third of AAV-U7- leads to only mild alteration of Scn5A splicing, while these mice
ASScn5a-injectedmicepresentincreasedQRSduration(419ms). show severe conduction disease and cardiac dilatation43,44.
Interestingly, similar findings have been described in Scn5aþ/(cid:2) In conclusion, this work may also have some clinical
mice, which all show elongation of the PR interval, while only a importance such as considering with caution the treatments of
subset of Scn5aþ/(cid:2) animals present a prolongation of the QRS DM patients with pharmaceutical agents that reduce the activity
interval45. Hence, elongation of the PR interval is not of the cardiac sodium channel, including mexiletine, flecainide
systematically associated with increased duration of the QRS in and other antiarrhythmic drugs of class I. In that aspect, this
mousemodelofScn5adysfunction.Thus,tostrengthenourdata, studymayprovideamolecularexplanationtotheadversecardiac
we mathematically tested whether human cardiac parameters reactionofsomepatientswithmyotonicdystrophictotreatment
wouldbealteredbytheelectrophysiologicaldifferencescausedby withdrugsreducingactivityofSCN5A(refs62,63). Involvement
the switch from adult exon 6B towards fetal exon 6A of SCN5A. of the cardiac sodium channel in DM might also highlight the
Simulation based on a modified O’Hara-Rudy model48,49 importance ofconsideringpolymorphismin theSCN5A gene,as
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a b
Atrium
hNav1.5e hNav1.5e
V
ECG m
0
0
1
200 ms
hNa1.5
v
Nodal-His hNa1.5e
v
APs Endo M hNav1.5
Epi
hNa1.5e
v 200 ms
APs
c
G CUG
Control Myotonic dystrophy U MBNL1C
C U
– + UG GC
C MBNL1 U
5 MBNL1 6A 6B 7 5 MBNL1 6A 6B 7 UG GC
Adult Fetal UGC MBNL1 UGC
C U
G MBNL1 G
U C
I II III IV I II III IV
12345 6 12345 6 12345 6 12345 6 12345 6 12345 6 12345 6 12345 6
hNa1.5 hNa1.5e
v v
Normal adult heart conduction Conduction delay and heart arrhythmia
Figure7|ComputersimulationpredictscardiacconductionabnormalitiesinDM.(a)SimulationsofECG(upper)andactionpotential(AP)alterations
(middleandlower)causedbytheswitchfromadultSCN5Aexon6Btowardsfetalexon6A,usingamodifiedhumanventricularORdmodel.(b)Simulations
oftheatrio-ventricularchangescausedbyinclusionofSCN5Afetalexon6Ainsteadofadultexon6B,employinganatrio-ventricularnodalmodel.APsof
atrialandAVnodecellsweresimulatedandatrium-HisintervalwasmeasuredasthedifferenceinthelatencyofAPsbetweenatrialandnodal-Hiscells.
(c)Modelofsplicingalterationofthecardiacsodiumchannel,SCN5A,inDM.MBNLproteinsregulatetheswitchfromSCN5Aexon6Ainfetalheartto
exon6Binadult.InDM,titrationofMBNLproteinsbymutantRNAcontainingexpandedCUGrepeatsleadstoexpressionofafetalsplicingformofSCN5A,
inappropriatetoadultheartphysiology,ultimatelyresultingincardiac-conductiondelayandheartarrhythmias,whicharetwokeysfeaturesofDM.
wellasinothergenessuchasSCN10A,asapossiblecauseofthe alteration of the alternative splicing of SCN5A, one may hope
clustering of cardiac alterations in some families of DM64, an that correction of splicing misregulations through approaches
hypothesis supported by the recent report that DM exacerbates releasing MBNL1 from CUG expanded repeats or through
Brugada syndrome caused by a mutation in SCN5A (ref. 65). approaches reducing the expression of pathogenic CUG
Inversely, it remains to be tested whether mutations in intronic RNA66,67 may alleviate the cardiac symptoms of DM.
regions regulating alternative splicing of SCN5A exon
6A/6B might be considered as a cause of cardiac-conduction
Methods
abnormalities in patients in whom no mutations were identified
Humansamples.Allsampleswereheartleftventriclesthatweresampledwiththe
in the coding sequence of SCN5A. Also, if dysfunction of the
informedconsentofindividualsandapprovedbytheInstitutionalReviewBoardof
cardiacsodiumchannelinDMiscomparableinsomeaspectsto thePitie´-Salpeˆtrie`rehospital,oftheNeuromuscularResearchCenteroftheTam-
cardiac-conduction disease caused by mutations in SCN5A, an pereUniversityHospital,oftheHospitalDonostiaandoftheToneyamaNational
Hospital.Non-affectedheartsamples(CTL#1to#3)werepurchasedatAmbion
attractive supposition would be that therapeutic approaches
andStratagene,respectively.DCM#1and#2werepatientssufferingfromDCMof
developedforthesediseasescouldbeconsideredforDM.Finally,
uncharacterizedgeneticorigin.DMD#1to#4werepatientsaffectedwithDMD.
if cardiac-conduction defects in DM are associated with an ALS#1to#3werepatientswithALSdescribedpreviously68.DM1samples#1and
10 NATURECOMMUNICATIONS|7:11067|DOI:10.1038/ncomms11067|www.nature.com/naturecommunications