About OMICS Group Conferences

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1 About OMICS Group OMICS Group International is an amalgamation of Open Access publications and worldwide international science conferences and events. Established in the year 2007 with the sole aim of making the information on Sciences and technology Open Access, OMICS Group publishes 400 online open access scholarly journals in all aspects of Science, Engineering, Management and Technology journals. OMICS Group has been instrumental in taking the knowledge on Science & technology to the doorsteps of ordinary men and women. Research Scholars, Students, Libraries, Educational Institutions, Research centers and the industry are main stakeholders that benefitted greatly from this knowledge dissemination. OMICS Group also organizes 300 International conferences annually across the globe, where knowledge transfer takes place through debates, round table discussions, poster presentations, workshops, symposia and exhibitions.

2 About OMICS Group Conferences OMICS Group International is a pioneer and leading science event organizer, which publishes around 400 open access journals and conducts over 300 Medical, Clinical, Engineering, Life Sciences, Pharma scientific conferences all over the globe annually with the support of more than 1000 scientific associations and 30,000 editorial board members and 3.5 million followers to its credit. OMICS Group has organized 500 conferences, workshops and national symposiums across the major cities including San Francisco, Las Vegas, San Antonio, Omaha, Orlando, Raleigh, Santa Clara, Chicago, Philadelphia, Baltimore, United Kingdom, Valencia, Dubai, Beijing, Hyderabad, Bengaluru and Mumbai.

3 Clinical & Experimental Cardiology OMICS Group Conferences April 15-17, 2013 Hilton Chicago/Northbrook, USA A novel mechanism of an SCN5A mutation causing mixed arrhythmias associated with dilated cardiomyopathy Mohamed Chahine Ph.D. professeur titulaire

4 Dilated Cardiomyopathy (DCM) Definition: Dilatation of cardiac cavities, Impaired contractility and systolic function Aetiology: Idiopathic, secondary, familial in at least 20-30%

5 SCN5A gene: Chronology 1992: Cloning and characterization of SCN5A (Gellens et al) 1995: Mapping SCN5A to chrom 3p21 (George et al) SCN5A: candidate gene for Long QT Syndrome type : SCN5A mutations in Brugada Syndrome (Chen et al) 1999: SCN5A mutations in Progressive Cardiac Conduction Defect (Schott et al) 2003: SCN5A mutations in Congenital Sick Sinus Syndrome (Benson et al) 2004: SCN5A mutation in dilated cardiomyopathy (McNair et al)

6 SCN5A gene is ranked as the sixth most common cause of familial DCM

7 DCM and Na v 1.5 Dilated cardiomypathy

8 Patient with conductance disturbances, AFib, Ventricular fibrillation and DCM I-1, 78 yrs I-2, accident II-1 II-2 II-3 II-4 II-5 II-6 Index patient II-3, II-4, II-5: DCM Not genetically tested III-1 III-2 Gosselin-Badaroudine et al., PlosONE, 2012;7(11):e48690.

9 Genotyping Collecte de sang Extraction de l DNA PCR et dhplc Séquençage

10 Identification of a novel SCN5A mutation CGC Arg (R) CAC His (H) 219 Gosselin-Badaroudine et al., PlosONE, 2012;7(11):e48690.

11 Biophysical properties of SCN5A/R219H mutation Gosselin-Badaroudine et al., PlosONE, 2012;7(11):e48690.

12 The omega current: Chronology

13 The two microelectrode technique V Elec _ + I Elec Succion V Clamp Oocyte

14 SCN5A/R219H revealed an inward current ph o pho Water-injected Nav1.5/WT Water-injected Nav1.5/WT Nav1.5/R219H Nav1.5/R219H 200 na 100 na 50 s 0 pho = 7.40 pho = s TTX (1 M) pho = na 50 ms Na v 1.5/R219H pho TTX (1 M) HP=-80 mv 200 ms V test =-140 mv ph o Nav1.5/R219H 200 na 20 s 200 na 40 s Gosselin-Badaroudine et al., PlosONE, 2012;7(11):e48690.

15 Intracellular ph measurments ph Elec V Elec _ + I Elec Succion V Clamp Oocyte

16 SCN5A/R219H induce a proton current Na v 1.5/R219H Na v 1.5/WT ph o pho ph i 7.5 phi na 200 sec TTX (1 M) 6.6 HP=-80 mv 200 ms 100 na 200 s TTX (1 M) 7.3 V test =-140 mv Gosselin-Badaroudine et al., PlosONE, 2012;7(11):e48690.

17 SCN5A/R219H induce an intracellular acidification Water injected Na v 1.5/WT Na v 1.5/R219H ph i *** *** *** ph o ph i *** *** *** ph o Gosselin-Badaroudine et al., PlosONE, 2012;7(11):e48690.

18 Biophysical caracterization of the proton current pho=8.40 pho=7.40 (E H+ = -90 mv) Voltage (mv) na 50 ms pho= mv Δ 5 mv -80 mv 200 ms -140 mv pho=6.00 Normalized Q-V H + Current (na) ph o = 8.40 (E H+ = -88.9mV) ph o = 7.40 (E H+ = -29.7mV) ph o = 6.80 (E H+ = 5.8 mv) ph o = 6.00 (E H+ = 53.1 mv) Gosselin-Badaroudine et al., PlosONE, 2012;7(11):e48690.

19 Intracellular mechanisms

20 Mouse ES cells differentited into cardiac myocytes Day 0 to day 2 of differentiation ES cells Embryoid body Formation Hanging drop Day 2 to day 5 From day 5

21 Homologous recombination: CGA to CAC to obtain the heterozygous R219H mutation Targeting construct Probe (southern blot) LAH ACN SAH SCN5A R219H-ACN R219H * 5 6 Cre NeoR HindIII HindIII HindIII HindIII loxp loxp 7 Na v 1.5 / WT Na v 1.5 / R219H C T T C G A A C T C T T C N N A C T

22 Na v 1.5 / WT Na v 1.5 / R219H

23 ( ) -( Background l=490 ) ( F l=440 ) -( Background l=440 ) R = F l=490 ph = pk a - log R - R ph 5 R ph 8 - R ( ) ( ) FpH 5 l=440 F ph 8 l=440 ( ) ( ) The BCECF ratiometric dye allow to measure the cellular acidification caused by the specific proton leak.

24 Cardiomyocytes expressing Na v 1.5 / WT Cardiomyocytes expressing Na v 1.5 / R219H

25 Na v 1.5 / WT mlc2v ctnt Merge Na v 1.5 / R219H mlc2v ctnt Merge D7 D20 D60 D20 D30 D60

26 CONCLUSION The R219H mutation found at the heterozygous state: - Generate a proton leak - Unbalance the ionic homeostatsis The DCM is not an adaptative phenomena Cardiac myocytes derived from ES cells are good model to study SCN5A mutations linked to DCM

27 THANKS Zurich Dagmar I. Keller M.D. University Hospital Zurich, Zurich, Switzerland Québec Pascal Gosselin-Badaroudine Adrien Moreau Hugo Poulin Valérie Pouliot

28 LES CANAUX IONIQUES Cl -

29 COURANTS IONIQUE ET POTENTIEL D ACTION c v

30 POTENTIELS D ACTION ET ECG

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32 XENOPUS LAEVIS: SYTÈME HÉTÉROLOGUE D EXPRESSION

33 _ I Elec V Elec + V Clamp Oocyte

34 LE PROPRIÉTÉS BIOPHYSIQUE DE BASES DE SCN5A/R219H SONT INCHANGÉES Na v 1.5/WT Na v 1.5/R219H 200 na 200 na 10 ms 10 ms Normalized Conductance Na v 1.5/WT Na v 1.5/R219H Normalized Current Voltage (mv) Recovery time (ms) Gosselin-Badaroudine et al., PlosONE, 2012;7(11):e48690.

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36 H + Current Amplitude (na) R2= Na Current Amplitude ( A) Normalized H + Current pk a = pho Gosselin-Badaroudine et al., PlosONE, 2012;7(11):e48690.

37 DIRECTIONS FUTURES Souris transgénique (Knocking) portant La mutation R219H Différencier les cellules ES en myocytes cardiaques Reprogrammer les fibroblastes du patient en cellules ips puis en myocytes

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39 Conclusion La mutation n affecte pas les caractéristiques biophysiques du pore α On observe un courant H + passant à travers Na v 1.5 «Proton Wire» Ce courant semble être à l origine du phénotype de cardiomyopathie dilatée de nos patients

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41 100 na Specificity of R219H substitution Na v 1.5/R219A Na v 1.5/R219Q pho pho HP=-80 mv 200 ms Na v 1.5/R219K V test =-140 mv Na v 1.5/R219C pho sec pho na 20 sec Gosselin-Badaroudine et al., PlosONE, 2012;7(11):e48690.

42 DCM and SCN5A gene Phenotypes: Conduction disease and DCM Genotype: D1275N SCN5A McNair et al. Circulation 2004 Phenotype: Atrial standstill and DCM Genotype: D1275N SCN5A and Cx40 pol. Groenewagen et al. Cir Res 2003

43 Thanks' for your kind attention!!!!!! 43

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