Cardiomyocytes Derived from Pluripotent Stem Cells ... - Circulation

DOI: 10.1161/CIRCULATIONAHA.111.066092

Cardiomyocytes Derived from Pluripotent Stem Cells Recapitulate Electrophysiological Characteristics of an Overlap Syndrome of Cardiac Sodium Channel Disease Running title: Davis et al.; Disease modelling using PSC cardiomyocytes

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Richard P. Davis, PhD1,6*; Simona Casini, PhD1*; Cathelijne W. van den Berg, MSc1*; Maaike Hoekstra, MSc2; Carol Ann Remme, MD, PhD2; Cheryl Dambrot, MSc1,4; Daniela Salvatori, PhD1,5; Dorien Ward-van Oostwaard, BSc1; Arthur A.M. Wilde, MD, PhD2; 1 Connie R. Bezzina, PhD2; Arie O. Verkerk, PhD3; Christian Freund, Ph PhD hD1* ; 1,6* Christine L. Mummery, PhD 1

Dept of Anatomy & Embryology, 4Dept of Cardiology, 5Proefdiercentrum, Leiden University Medical Center, Leiden; Cent Ce nter nt er,, Le er Leiden en n; 2De Dept D pt of Clinical & Experimental Experimenta tall Ca ta C Cardiology, rdiology, 3Dept off Anatomy, Anat An a omy, Embryology & Physiology, Heart Academic Center, University Phys Ph y io olo ogy gy, He Hear art Failure Research Center, Aca ademic Medical Ce ent n er,, Un Univ iversity y of Amsterdam, Amsterdam; Amst Am ster erda er dam da m; 6Ne Netherlands eth ther e la er land ndss Proteomics nd P ot Pr oteo e mi eo m css Institute, Instiitu tute te,, Ut te U Utrecht, reecht, cht,, T The he N Nethe Netherlands heerl rlan ands an ds equally **contributed con ntriibuteed ed equ ual ally ly

Correspondence: C orrespond nden dence: Richard Ri h d P. P Davis, D i PhD PhD or Christine Ch i tii L. L Mummery, M PhD PhD Department of Anatomy & Embryology Leiden University Medical Center Einthovenweg 20 2333 ZC Leiden The Netherlands Tel: 31-71-5269307 Fax: 31-30-2516464 E-mail: [email protected] or [email protected]

Journal Subject Codes: [106] Electrophysiology; [130] Animal models of human disease; [137] Cell biology/structural biology; [152] Ion channels/membrane transport 1


Abstract: Background - Pluripotent stem cells (PSCs) offer a new paradigm for modelling genetic cardiac diseases but it is unclear whether mouse and human PSCs can truly model both gain and loss of function genetic disorders affecting the Na+ current (INa) due to the immaturity of the PSCderived cardiomyocytes. To address this issue, we generated multiple PSC lines containing a Na+ channel mutation causing a cardiac Na+ channel overlap syndrome. Method and Results - Induced PSC (iPSC) lines were generated from mice carrying the Scn5a1798insD/+ (Scn5a-het) mutation. These mouse (m)iPSCs, along with wild-type miPSCs, were compared to the targeted mouse embryonic stem cell (mESC) line used to generate the mutant Downloaded from http://circ.ahajournals.org/ by guest on October 1, 2017

mice and to the wild-type mESC line. Patch-clamp experiments showed that the Scn5a-het y y had a significant g y and a larger g ppersistent INa when cardiomyocytes decrease in INa density compared to Scn5a-wt cardiomyocytes. Action potential (AP) measurements sho showed howe wedd a re we redu reduced duce du c d ce upstroke velocity and longer AP duration in Scn5a-het myocytes. These characteristics ecapi p tulated findings g from primary cardiomyocytes cardiomyocyt ytes isolated directly from adult Scn5a-het mice. recapitulated Finally, Fina Fi nall na lly, ll y, iPSCs iPS PS SCs were wer eree generated g ne ge nera ratedd from fr m a patient pati pa t ent with with h tthe he equ equivalent quiv i alen nt S SCN5A CN5 N5A A1795insD/+ mut mutation. utat atio on. Patch-clamp P atcchh clamp measurements meas me a ur as urem em ments ents on on the the derivative derivativve cardiomyocytes der carddioomy yocyt ocyttess revealed reveale ledd similar siimi mila larr changes la ch hange ange g s too tho tthose h se ho the mPSC-derived mPSCC-de Cd rived de ved ca card rdio iomy myoc my ocyt ytes yt es. inn the cardiomyocytes. Conclusions Conc nclu lusion ions - Here Herre we H w demonstrate dem emon onst stra rate t thatt bo both th ESCESC S - an andd iP iPSC iPSC-derived SC-d - er eriv ived ed car cardiomyocytes rdi d om omyo yocyte tess ca can an recapitulate ecapi p tulatee the the characteristics cha hara ract ra c er ct e issti tics c of cs of a combined comb co mbin mb i ed gain in ggai ainn and ai and loss loss of of function funccti fu tion on Na Na+ cchannel hann ha nnnel m mutation utation andd that the electrophysiological immaturity of PSC-derived cardiomyocytes does not preclude their use as an accurate model for cardiac Na+ channel disease.

Key words: differentiation; electrophysiology; stem cells; disease modelling; sodium ion channels

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Multiple cardiac arrhythmia syndromes including long-QT syndrome type 3 (LQT3), Brugada syndrome (BrS), progressive cardiac conduction disease and sinus node dysfunction have been linked to mutations in SCN5A, the gene encoding the Į-subunit of the cardiac sodium (Na+) channel.1,2 Most SCN5A mutations associated with LQT3 act by disrupting fast inactivation of the Na+ channel, resulting in a persistent inward Na+ current (INa) during the action potential (AP) plateau phase, and subsequently delaying ventricular repolarisation and prolonging the QT interval (gain of function mutations).3 In contrast, SCN5A mutations underlying BrS and Downloaded from http://circ.ahajournals.org/ by guest on October 1, 2017

conduction disease are loss of function mutations and are believed to reduce the total amount of available INa due to expression of non-functional channels, impaired intracellular trafficking and decreased membrane surface channel expression, or through altered channel gat gating tin ng pr prop properties. op per erti ties ti e .1,2 es Initially, it was believed that these arrhythmia syndromes constituted separate clinical en entities ntiiti ties es w wit with ithh iindividual it ndi diivi vidu d al SCN5A mutations leading ng to to one particular ar ele electrophysiological ect ctrrophysiological ro disorder. H However, ow wever, gen genotype-phenotype not otyp ype--ph yp p en not otyp ypee sstudies yp tud udie ud i s in ie n lar large rgee pe pedigrees edigreees ees ha have ave ve nnow ow w eestablished sttab bliishhed ed tthat hatt se ha sev several veral ver ral si ssingle ngle SCN5A with multiple manifestations consequence SC CN5 N5A A mutations mut m utat ut atio at ionns present pre rese sennt nt w wit ithh mu it mult ltip lt iplle le clinical cli linnica caal ma man niffest fest stat atiions at ns aass a co cons nseq equuen uence nce of the hee vvarious ario ar io ous us biophysical de defects efe fect ctts as aassociated sociat so ated at ed dw with ithh th it tthee mu muta mutations tati ta tion ti o s (t on (the h sso-called he o ca ocall l ed ooverlap ll veerl rlap ap ssyndromes). yndr yn drrom omes es). es ).4 T The he first reported was the in-frame insertion of an aspartic acid residue at position 1795 in the human protein (designated 1795insD), identified in a large Dutch family, with ECG features of bradycardia, ventricular and atrial conduction slowing, LQT3, and BrS.5,6 Transgenic mice carrying the mouse equivalent (1798insD) of the human SCN5A-1795insD mutation recapitulate the majority of the electrophysiological and ECG characteristics observed in patients, with Scn5a1798insD/+ (Scn5a-het) mice displaying signs of sinus node dysfunction in addition to prolonged PQ, QRS, and QTc intervals on surface ECGs.7,8 Patch-clamp analysis in isolated Scn5a-het cardiomyocytes demonstrated a prolonged AP duration (APD) and decreased upstroke

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velocity (Vmax) compared with wild-type (Scn5a+/+; Scn5a-wt) myocytes.7,8 Additionally, the mutation resulted in a reduction in INa density, underlying the observed conduction slowing in the Scn5a-het mice. Finally, a larger persistent inward INa was measured in cardiomyocytes from Scn5a-het mice, explaining the presence of the increased AP duration and QTc prolongation.9 Critically, in contrast to previous transfection studies using HEK cells,10 the majority of kinetic properties of the INa remained unchanged in the isolated Scn5a-het cardiomyocytes,7,8 underscoring the challenges in interpreting the electrophysiological properties of ion channel Downloaded from http://circ.ahajournals.org/ by guest on October 1, 2017

mutations in heterologous expression systems. The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) and cardiomyocyttes, of ooffers fers fe rs a nnew ew differentiate these cells into many cell types, including functional cardiomyocytes, paradigm for modelling human disease.11,12 However, the electrophysiological immaturity of pl lurrip ipot oten ot en nt st stem tem cce el ell-derived cardiomyocytes (P PSCSC-CMs) might in nflue ueenc ncee whether they are able pluripotent cell-derived (PSC-CMs) influence too recapitulate recapitulate ec eN Naa+ cchannel h nn ha nel llos loss osss off ffunction os unct un ctiion m mutations utattioons th that hat affect affe af fect ctt the the rrapid apiid uupstroke pstr ps trok okke of the he A AP. P.. In huma hu human mann embryonic ma embr em bryyon br yonic stem stem m cell-derived cel ell-de d ri de rive vedd cardiomyocytes ve caard dio iomy myoc my o ytees oc es (hESC-CMs), (hE hESC SC-C SC CMs), ), the thee relative reelat elattiv i e contribution co ont ntri ribbut bution tion off INa to the phase phas asse 0 of the the h AP AP is a matter mattte t r of debate, ddeb ebaate eb t , wi with t uupstroke th pstr ps trok tr okee ve ok vvelocities loci lo citi ci tiies aaro around r un ro undd 8 V/ V V/ss typicallyy detected in hESC-CMs13-16 compared with ~200V/s in adult cardiomyocytes.17 In hiPSC-derived CMs (hiPSC-CMs) similar low upstroke velocities have also been reported.18-20 While mouse (m)PSC-CMs show an increase in INa and upstroke velocity during prolonged in vitro differentiation,21-23 the latter is still smaller than that observed in isolated primary adult mouse cardiomyocytes.8 Therefore it remains uncertain whether PSC-CMs can recapitulate the electrophysiological features of Na+ channel loss of function mutations. In this study, we generated iPSC lines from Scn5a-het mice and from wild-type littermates, and derived functional cardiomyocytes from these. In addition, the mESC line used

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to introduce the Scn5a-1798insD mutation into the mice, and the parental (Scn5a-wt) control mESC line were also differentiated into cardiomyocytes. The biophysical properties of the Na+ channel and AP characteristics in cardiomyocytes derived from these two PSC sources were examined and compared to those observed in cardiomyocytes isolated from Scn5a-wt and -het mice that were described previously.8,9 Both in vitro Scn5a-het models recapitulated the electrophysiological phenotype observed in the corresponding primary adult cardiomyocytes. Finally to ascertain whether some of the electrophysiological features underlying LQT3 and BrS Downloaded from http://circ.ahajournals.org/ by guest on October 1, 2017

could also be detected in human cardiomyocytes, we generated a hiPSC line from a patient carrying the SCN5A-1795insD variant. Cardiomyocytes derived from these cells also displayed he INa and AP properties of this cardiac Na+ channelopathy. Collectively, these re rresults esuult ltss the demonstrate the utility of PSCs in modelling even complex ion channel disorders.

Me Met th thods Methods Methods Supplemental Ann eexpanded xpan xp ande dedd Me de eth hod odss sect ssection ec io on is ava aavailable vail ilab able ab le iinn th thee Su Supp upp pleeme ment ntal nt a Material. al Mat a erria al. 24-26 26 Mouse Mous se and a d human an h ma hu man iPSCs iPSC iP SCss were SC wer generated gen ener erat er ated at e essentially ed essse sent ntia iall ia llly ass described ddes e cr crib ibed ib ed previously, ppre r viiou re ousl sly, sl y,24 either

by retroviral or lentiviral transduction of vectors encoding Oct4, Sox2, Klf4 and c-Myc. The undifferentiated PSC lines were maintained using standard procedures.27,28 Teratomas were generated by injecting undifferentiated iPSCs into NOD-SCID mice. Mouse PSCs were differentiated in vitro as embryoid bodies (EBs) using a “hanging drop” protocol,27 while human iPSCs were induced to differentiate to cardiomyocytes by co-culture on visceral endoderm-like (END-2) cells as described previously.28,29 Immunofluorescence and gene expression analyses were performed as described previously.28,30 PSC-CMs were isolated from differentiated EBs by enzymatic dissociation and the electrophysiological recordings obtained by using either ruptured

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or perforated patch-clamp techniques. Results are expressed as mean±SEM. Comparisons were made using either unpaired Student’s t-test, one-way ANOVA or two-way repeated measure ANOVA (rmANOVA), followed by a Holm-Sidak test for post-hoc analysis. P