camkii-based regulation of voltage-gated na...

DOI: 10.1161/CIRCULATIONAHA.112.105320

1

CaMKII-Based Regulation of Voltage-Gated Na+ Channel in Cardiac Disease

Running title: Koval et al.; CaMKII-based regulation of Nav in disease

Olha M. Koval, PhD1,2,*; Jedidiah S. Snyder, BS1,3,*; Roseanne M. Wolf, PhD1,4; Ryan E.

Pavlovicz, BS1,5; Patric Glynn, BS1,3; Jerry Curran, PhD1; Nicholas D. Leymaster, MS6; Wen

Dun, MD, PhD7; Patrick J. Wright, BS1; Natalia Cardona6; Lan Qian, MD2; Colleen C. Mitchell,

PhD4; Penelope A. Boyden, PhD7; Philip F. Binkley, MD1,8; Chenglong Li, PhD1,5; Mark E.

Anderson, MD, PhD2,6; Peter J. Mohler, PhD1,8,9; Thomas J. Hund, PhD1,3,8

1The Dorothy M. Davis Heart & Lung Research Inst, 8Dept of Internal Medicine, 9Dept of Physiology & Cell Biology, The Ohio State University Medical Center, Columbus, OH; 2Dept of Internal Medicine, 6Dept of Molecular Physiology & Biophysics, University of Iowa Carver College of Medicine, Iowa

City, IA; 3Dept of Biomedical Engineering, College of Engineering, 5Division of Medicinal Chemistry & Pharmocognosy, College of Pharmacy, The Ohio State University, Columbus, OH; 4Dept of Mathematics, University of Iowa, Iowa City, IA; 7Dept of Pharmacology, Center for Molecular

Therapeutics, Columbia University, New York, NY *contributed equally

Address correspondence to:

Thomas J. Hund, Ph.D.

The Dorothy M. Davis Heart and Lung Research Institute

The Ohio State University Medical Center

473 W. 12th Avenue

Columbus, OH 43210

Tel: 614 292-0755

Fax: 614 247-7799

E-mail: [email protected]

Journal Subject Codes: [109] Clinical genetics; [130] Animal models of human disease; [132] Arrhythmias-basic studies; [104] Structure; [157] Quantitative modeling

Anderson, MD, PhD2,6; Peter J. Mohler, PhD1,8,9; Thomas J. Hund, PhPhhDDD1,1,1 3,3,3 888

1The Dorothy M. Davis Heart & Lung Research Inst, 8Dept of Internal Medicine, 9Dept of Physiology &CeCeCellllll BBBioioiololologygg , ThThThee Ohio State University Medicaaal ll CeCeCenter, Columbus, OHHH;; 222DDeD pt of Internal Medicine,66DDeDept oof f f MMMolelecuculalar PhPhysysioi loogygy && BBioophhysysics,s, UnUnnivvversityty oof f Iowawa CCCarvever r CoC llegegee ofof Meddiccinine, Iowowa

CCiCity, IA; 33DeDeppptt t oofo BBBiioiomememedididicacaalll EnEnEngigigineneneeree inini g,g CCCololo lel ggge oof EnEnEngigiginenen erere ininng,g 555DiDiD vivivi isisioon oooff f MeMeMedididicicic nananalll ChChChemememisisi try& Pharmoccocoggnoossyy,, Colollleleegeg offf PPhPhaaarmmmacyyy, TTThe OOhhioo SSStatatatetete UUUniivvverrrsity, CCololuumumbubus, OOOHHH; 444DDeept t oofofMathememmatiiccss,, Unnnivvversitityy y ooof f Iowawawa, IoIoI wwaw CCCittty,y, IAA;A; 77DeDeDepppt t ofoff PPPharrmrmaacoloogogy,y, CCCenennteer ffofor r r MMolleecullaarr

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AdAdddress correspo dndence tto:

by guest on July 10, 2018http://circ.ahajournals.org/

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Abstract:

Background - Human gene variants affecting ion channel biophysical activity and/or membrane

localization are linked with potentially fatal cardiac arrhythmias. However, the mechanism for

many human arrhythmia variants remains undefined despite over a decade of investigation.

Post-translational modulation of membrane proteins is essential for normal cardiac function.

Importantly, aberrant myocyte signaling has been linked to defects in cardiac ion channel post-

translational modifications and disease. We recently identified a novel pathway for post-

translational regulation of the primary cardiac voltage-gated Na+ channel (Nav1.5) by CaMKII.

However, a role for this pathway in cardiac disease has not been evaluated.

Methods and Results - We evaluated the role of CaMKII-dependent phosphorylation in human

genetic and acquired disease. We report an unexpected link between a short motif in the Nav1.5

DI-DII loop, recently shown to be critical for CaMKII-dependent phosphorylation, and Nav1.5

function in monogenic arrhythmia and common heart disease. Experiments in heterologous cells

and primary ventricular cardiomyocytes demonstrate that human arrhythmia susceptibility

variants (A572D and Q573E) alter CaMKII-dependent regulation of Nav1.5 resulting in

abnormal channel activity and cell excitability. In silico analysis reveals that these variants

functionally mimic the phosphorylated channel resulting in increased susceptibility to

arrhythmia-triggering afterdepolarizations. Finally, we report that this same motif is aberrantly

regulated in a large animal model of acquired heart disease and in failing human myocardium.

Conclusions - We identify the mechanism for two human arrhythmia variants that affect Nav1.5

channel activity through direct effects on channel post-translational modification. We propose

that the CaMKII phosphorylation motif in the Nav1.5 DI-DII cytoplasmic loop is a critical nodal

point for pro-arrhythmic changes to Nav1.5 in congenital and acquired cardiac disease.

Keywords: arrhythmia (mechanisms); heart failure; long-QT syndrome; myocardial infarction; calmodulin dependent protein kinase II

g q p p vv

DI-DII loop, recently shown to be critical for CaMKII-dependent phosphorylatioionn, aaandndnd NNNaaavvv11.1.55

function in monogenic arrhythmia and common heart disease. Experiments in heterologous cells

and prp imary y ventricular cardiomyocytes demonstrate that human arrhyty hmia susceptibility

vavaariririaanantsts (AA5757572DD aandnd QQ57573E3 ) ala tet r CaCaMKII-depepenenndddent regegulu atioonn n oof NNaavv11.5 reesusulting g in

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egug lated inn aa a lalalargrgrgeee anananimimimalall mmmododdelelel ofoff acqcqcquiuiirereedd d hehehearartt t didid seeasaasee ananandd d inini fffaiaailililingngng hhhumumanaan mmmyoyoyocardium.


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Introduction

Since seminal studies of Keating and colleagues,1, 2 gene mutations in select ion channels have

been mechanistically linked to human arrhythmia through their effects on ion channel

biophysics.3 Discovery of these mutations has not only provided new insight into cellular

mechanisms for human disease, but has also advanced our fundamental understanding of ion

channel structure/function and cellular cardiology. Subsequently, a second class of human

arrhythmia mutations has been identified in genes encoding ion channel accessory proteins (e.g.

adapter and scaffolding proteins, channel subunits, chaperones) rather than ion channel alpha

subunits.4-11 These mutations cause disease by altering channel targeting and/or biophysical

activity and highlight the importance of proper channel localization within specific cellular

domains for normal heart function. Despite major inroads, the mechanistic link between specific

molecular defects, channel dysfunction and arrhythmias associated with many human arrhythmia

variants remains elusive. Protein phosphorylation has evolved as an essential mechanism for

regulating cell function in heart and other systems. In heart, tight spatial and temporal control of

local signaling domains is maintained to ensure proper regulation of key ion channels,

transporters and receptors. Importantly, alterations in post-translational modification of

membrane proteins are associated with increased susceptibility to congenital arrhythmia and

common causes of acquired arrhythmia, including heart failure.12-15 Here we identify the

mechanism for two human cardiac arrhythmia susceptibility variants in SCN5A (A572D and

Q573) based on direct defects in post-translational modification of a cardiac ion channel.

Voltage-gated Na+ channels (Nav) are critical for normal cell excitability and were

among the first ion channels to be linked to a specific congenital cardiac arrhythmia.2, 16 Almost

two decades of research has identified hundreds of human gene variants in SCN5A linked to

activity and highlight the importance of proper channel localization within specififficic cceelellulululalalarr r

domains for normal heart function. Despite major inroads, the mechanistic link between specific

momolelelecucucullalarrr dededefffectcttss,s, cchannel dysfunction and arrhhhyytythhmmias associatededed wwititthh h mmany human arrhythmia

vvariiiana ts remaininss ellususivivi ee.e. PPrProtototeiein n phphphososspphhoryyylaaationnn hhhas evevvolollveveed d aaas aan n esessseeentntiaiaialll memeechchchanana isii mm m fofofor

eegugugulalalatitit ngngg cccelelell l fufuuncncctitioonon iiin n heeearaa ttt ananand d d ototheheherr r sysysystttemememsss. InInn hheaeaeartrrt, tititighghht spspspatatiiiall l anannd dd tetetempmpmpoooralll conono trtrrolll oof

ocal signalinng g g dodod mamamaininns s s is mmmaiaa ntntntaiaiaineneed d tototo eeensnsnsururure ee prprp opopopere rrregegegulululatattioioion nn ofofof kkkeyeyey iiiononon ccchahahannnnnnelelels,s


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various forms of cardiac arrhythmia.17, 18 Nav1.5 dysfunction has also been identified in

common forms of acquired heart disease (e.g. heart failure and myocardial infarction) where

slow conduction and/or altered repolarization plays an important role in arrhythmia and sudden

death.19 We recently demonstrated that the multifunctional Ca2+/calmodulin-dependent protein

kinase II (CaMKII) directly phosphorylates Nav1.5 at residue S571 to decrease channel

availability and enhance persistent (late) current leading to increased susceptibility to

afterdepolarizations.20

A screen of identified human arrhythmia variants in SCN5A yielded two variants in the

Nav1.5 DI-DII loop whose mechanism was unsolved (A572D and Q573E). Here we

demonstrate that these variants are localized to the CaMKII phosphorylation motif of Nav1.5 and

alter functional regulation of Nav1.5. We also evaluate the role of the CaMKII phosphorylation

domain of Nav1.5 in a large animal model of acquired heart disease and in failing human hearts

and identify significant CaMKII-dependent changes in post-translational modification of Nav1.5

at S571 in diseased hearts. Based on these findings, we propose that S571 in the Nav1.5 DI-DII

cytoplasmic loop serves as a critical node for regulating channel function in diverse forms of

cardiac disease associated with arrhythmias and sudden death.

Methods

Molecular biology: Nav1.5 -subunit cDNA was engineered in-frame into pIRES2-EGFP

(Clontech). Nav1.5 arrhythmia variant and TTX-sensitive (C373Y) constructs were generated by

Quikchange method using WT Nav1.5 as template. Vectors were completely sequenced. Nav1.5

constructs were co-transfected with murine pcDNA3.1 T287D constitutively active CaMKII 20

or empty vector into HEK and primary myocytes using X-tremeGENE 9 (Roche). For HEK

experiments, Nav1.5 -subunit (pcDNA3.1 h 1) was co-transfected with the Nav1.5 -subunit.

demonstrate that these variants are localized to the CaMKII phosphorylation mootttiifif ooof f NaNaNavv1.1.1.55 5 ana d

alter functional regulation of Nav1.5. We also evaluate the role of the CaMKII phosphorylation

doomamamaininin oofff NaNaNav1..55 5 inin a large animal model of acqcqquiuiu rred heart diseasasse ananndd d inin failing human hearts

annd d d idi entify siigngnniffficicanannt CaCaCaMKMKMKIIII-deded ppependndndent chhhanggegess innn ppposostt-t ttrtrananslslatatioionnnall momooddidifificacaatitiionono of f f NaNaNav11.1 5

att SSS57577111 inin dddisisiseaeasssedd d heheeararrtsts. . BBBasasededed ooonn ththhesesesee e fffindndndininngsgss,,, wwwe ppprororopopooseses thahahat t t SSS577171 iiin nn thththee NaNaNavv11.5 5 DIDI---DDIDII I

cytoplasmic lolooopopop ssserererveveesss asss aaa cccriiitititicacac ll l nonoodedede fffororor rrregegegulululatatatining g g chchchananannenenelll fufuuncncnctitit ononon iiin nn didiiveveversrsrseee fofof rms of


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To verify successful co-transfection of CaMKII with channel constructs, Na+ current was

measured with and without competitive CaMKII inhibitor, autocamtide-2-related inhibitory

peptide (AIP, AnaSpec), in the pipette.

Electrophysiology: Electrophysiological recordings were obtained from GFP-positive cells.

Whole cell sodium currents were measured using standard protocols as described in detail.20-22

Action potentials (APs) were recorded using perforated (amphotericin B) patch-clamp technique

at physiological temperature with pacing frequency of 1 Hz. Detailed electrophysiological

protocols and conditions are provided in online-only Supplemental Material.

Computational model: Transmembrane currents and ion concentration changes are described by

a well-validated model of the human ventricular myocyte23 with a Markov model of voltage-

gated Na+ current (INa) assuming one normal and one variant allele (50% variant channels).24-26

Transition rate expressions, model parameters, and initial conditions for WT and variant Nav1.5

may be found in online-only Supplemental Material (Supplemental Tables I, II, and III).

Electrostatic potential molecular modeling: Nav1.5 loop motif (W565-S577) structures were

built and energy minimized using Amber.27 Electrostatic potentials were computed by the

Delphi finite-difference Poisson-Boltzmann solver28 and mapped to molecular surfaces of W565-

S577 containing the CaMKII phosphorylation site.

Experimental model of myocardial infarction and immunoblotting: Myocardial infarction (MI)

was produced in canines by total coronary artery occlusion, as described previously.29, 30

Ventricular lysates were prepared and analyzed by SDS-PAGE as described.20, 30

Immunoblotting was performed using validated affinity-purified antibodies to phospho-

Nav1.5(S571) or total Nav1.5.20 Slight differences in protein loading were corrected using an

a well-validated model of the human ventricular myocyte23 with a Markov modelell ooff f vovooltltltagagage-e-e-

gated Na+ current (I( NaI ) assuming one normal and one variant allele (50% variant channels).24-26

Trrananansisisitititiononon rrratatate exexxpprpressions, model parameters, ananand d initial conditioioonsn ffororor WWT and variant Nav1.5

mmayy y be foundd iin n onnnliliinnene--onononlylyly SSSupupplplpleememenenntal MaMaM terriiaall (SuSuSuppppllel mmemennntaalal TTTababa lleess III, IIIII, ananand d d IIIIIII)II .

ElEllececectrtrtrososo tatatititic cc popottetenntntiaiall mmomoleleccuculalalarrr mommoddedelililingngng:: NaNaN vvv1..55 lolol opopop mmotototififf (W5W5W56665--S5S577777 )) ) ststrururuccctururresss wweeeree e

built and enererrgygygy mmmininnimimmizii ededd uuusis ngngng AAAmbmbmbererer.272727 ElElElececectrtrrososstat titiic cc popopotetetentntntiaiai lsss wwwererere e e cococompmpmputututededed bbby the


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internal control standard (rabbit polyclonal antibody to actin (Santa Cruz)). This investigation

conforms to the Guide for the Care and Use of Laboratory Animals published by the National

Institutes of Health (Pub. No. 85-23,1996).

Human tissue samples: Left ventricular tissue was obtained from explanted hearts of patients

undergoing orthotopic heart transplantation through The Cooperative Human Tissue Network:

Midwestern Division at The Ohio State University. Approval for use of human subjects was

obtained from the Institutional Review Board of The Ohio State University. Left ventricular

tissue from healthy donor hearts not suitable for transplantation (subclinical atherosclerosis, age,

no matching recipients) was obtained through the Iowa Donors Network and the National

Disease Research Interchange. The investigation conforms with the principles outlined in the

Declaration of Helsinki. Age and sex were the only identifying information acquired from tissue

providers and the Iowa Human Subjects Committee deemed that informed consent was not

required.

Adult cell isolation and pacing: Isolated ventricular myocytes from WT and AC3-I adult mice

(2-3 mos) were plated onto a 6-well tray precoated with laminin (Invitrogen). Cells were

pretreated for 30 min with the phosphatase inhibitor okadaic acid (2 M) and paced for 10 min at

2 Hz using the C-pace multichannel stimulator (Ionoptix). 100 nM isoproterenol was added

immediately prior to onset of pacing. A subset of cells was pretreated for 30 min before onset of

pacing with the CaMKII inhibitor KN-93 (10 M). Cell lysates were analyzed by SDS-PAGE

and immunoblotting. Slight differences in protein loading were corrected using monoclonal anti-

GAPDH (Fitzgerald) as an internal control standard.

Statistics: P values were determined with unpaired Student’s t test (2-tailed) for single

comparisons. Multiple comparisons were analyzed using one-way ANOVA. The Bonferroni test

Disease Research Interchange. The investigation conforms with the principles ououutllinini ededed iiinn n thththee

Declaration of Helsinki. Age and sex were the only identifying information acquired from tissue

prrovovvidididerererss ananand dd thheee IIoIowa Human Subjects Commitititttet eee deemed that inini formrmmeeded consent was not

eequuuired.

AdAddulululttt cecec llll iiisososolalatitiionnn aandndnd pppacacinining:g:g: IsIsIsololo atattededd vvveneentrtrricici uuulaarar mmmyoyoyocycycyteeesss frfrfromomm WTWTWT aandndnd AAAC3C33--III adddultlt mmmicicee e

2-3 mos) weererere ppplalalateteted d d onono tooo aaa 66-wewewelllll ttraaay yy prprp ecececoaoaoateteed d d wiwiw thhh lllamamaminininininin ((Innnvivivitrtrtrogogogenenen).). CeCeCelllllls ss weww re


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was used for post-hoc testing (SigmaPlot 12.0). If the data distribution failed normality tests

using the Shapiro-Wilk test, rank-based ANOVA and Dunn’s multiple-comparisons test were

performed. The null hypothesis was rejected for P<0.05.

Additional methods are provided in online only Data Supplement.

Results

Human Nav1.5 arrhythmia variants proximate to CaMKII phosphorylation site disrupt

channel regulation.

We recently identified Nav1.5 S571 in the DI-DII loop as a target for CaMKII-mediated

phosphorylation and key regulatory point for multiple Nav properties, including channel

availability, recovery from inactivation, and late current.20 Our findings suggested that variation

in this regulatory region could potentially result in significant cardiac dysfunction in vivo. As a

first step in evaluating a link between the CaMKII regulatory motif and disease, we analyzed this

region for potential human cardiovascular disease variants and identified two variants proximate

to the CaMKII regulatory motif: A572D and Q573E (Figure 1).31-35 Q573E was originally

identified in a genetic screen of LQTS probands with autosomal dominant Romano Ward

syndrome.33 The A572D variant was initially characterized in a proband with Romano-Ward

LQTS, later found in larger arrhythmia cohorts, and likely has an allele frequency of ~0.5% in

the general population.31, 32, 34-36 Both variants result in charge substitution consistent with

effects of phosphorylation (neutral to negative) at residues immediately adjacent to the CaMKII

phosphorylation site S571. Thus, while not involving direct changes to S571, we hypothesized

that these proximate variants alter CaMKII-dependent modulation of Nav1.5 resulting in altered

susceptibility to pro-arrhythmic phenotypes.

phosphorylation and key regulatory point for multiple Nav properties, including g chchchananannenenell l

availability, recovery from inactivation, and late current.20 Our findings suggested that variation

n tthihiss s rereregugugulalaatott ryyy rrregegion could potentially resultt iiinnn ssignificant cardiacc dddysysy function in vivo. As a

fifiirstt t sts ep in eevavaalululuatata ninngg g a a lililinknknk bbbetetetweweweenenen ttheheh CCCaMaMaMKIKIII rreguguulalalatototoryryy mmmototo ififif aandndnd dddiseaeaasesese,, wwwe e e anananalalyzyzyzededed tthis

eegigig oonon for ppotototenenttiiaaal hhumumuman cccarardidid ovovasasa cucuullaar r ddiiseeeasaseee vvavaririiananntsts aannnd iidedenntifiieedd d twtwwoo vavarir ananantts prprroxoximimmaaate

ooo ttthehehe CaCaCaMKMKMKIIIIII rrregegegulululatatatorororyyy momomotititifff::: A5A5A5727272DDD anananddd Q5Q5Q5737373EEE (F(F(Figigigururureee 1)1)1).fffff 311-3535 Q5Q5Q5737373EEE wawawasss orororigigiginininalalallylyly


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In previous studies, variants in this region were analyzed to assess basic gain- or loss-of-

function status with mixed results.35, 36 Therefore, to determine whether these variants alter Nav

function and/or CaMKII regulation, we measured Nav current (INa) from human full-length GFP-

WT and variant channels (expressed with the h 1 subunit) in HEK cells +/- constitutively active

CaMKII -T287D (Figure 2). A572D and Q573E variants showed increased late current

(measured as current amplitude 50 ms after peak) at baseline compared to WT, similar to S571E

(phospho-mimetic) and WT+CaMKII (Figure 2A-B). The Nav blocker ranolazine (10 M,

specific for late current44,45) normalized late current for WT, A572D and Q573E variants with

CaMKII (T287D) (Figure 2A, P=NS vs. WT, n=5, not shown).37, 38 A572D and Q573E variants

also showed a leftward (hyperpolarizing) shift in Nav1.5 steady-state inactivation and slowing of

recovery from inactivation at baseline compared to WT, similar to S571E and

WT+CaMKII (T287D) (Figure 2C-H). In contrast, S571A (phospho-resistant) INa properties

were not different from WT at baseline (Figure 2B,E,H). Immunoblot analysis showed similar

expression of WT and human arrhythmia variants expressed in HEK cells (Supplemental Figure

1 in online-only Supplemental Material). Changes in Nav properties observed in

WT+CaMKII (T287D) were prevented by the CaMKII peptide inhibitor AIP (10 M)

(Supplemental Figure 2 in online-only Supplemental Material). These data indicate that A572D

and Q573E human variants resulting in charge substitution immediately juxtaposed to the

CaMKII phosphorylation site mimic CaMKII phosphorylation (A572D>Q573E) and suggest a

pro-arrhythmic mechanism associated with these human variants.

Human variants alter cardiomyocyte membrane excitability

We next determined whether phospho-mimetic A572D and Q573E mutant channels produced

abnormal cell excitability in primary cardiomyocytes. To express WT and mutant channels in

( ) ( g , , , ) QQ

also showed a leftward (hyperpolarizing) shift in Nav1.5 steady-state inactivationonn anand d d slslslowowowininingg g ofof

ecovery from inactivation at baseline compared to WT, similar to S571E and

WTWTWT+++CaCaMKMKKIIIIII (TT28287D7D) ) (F(Figigurree 2C-H)H). IIn contn rarastt,, S57171A A (phohospspsphoo--reresis stannt)t) INaNaI proropeperties

wwew rrere not differennnt frommm WWT T aatt basselelininee ((Figgururre 2BB,B,EE,H)H)H). IIImmmmmuunnoooblott annnalallysyssis shhohowwweddd sssimiiilaaarr

exprpresesessisisiononon of f WTWTWT aaandndnd hhhumumumann aaarrrrrrhyhyhyththmimimiaa a vavavariririanantststs exexexprpresesessssed dd ininin HHHEKEKEK cccelelellslsls ((SuSuupppppplelelemementntntalalal FFFigigigurururee

1 in online-ononlylyl SSSupupplpllememenenttatal l MaMateteririalall).)). ChChChanangeges s inini NaNavv pproropepertieiess obobbseservrvededd iin n


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cardiomyocytes and distinguish from endogenous channels, we created tetrodotoxin (TTX)

sensitive constructs containing a point mutation (C373Y) at a critical aromatic residue where

TTX binds. The C373Y mutation increases TTX sensitivity of Nav by almost three orders of

magnitude.39 Ventricular myocytes were transfected with GFP-expressing TTX-sensitive WT

and mutant constructs (Figure 3). INa was measured from GFP-positive cells 24 hours following

Nav channel expression (Figure 3). Consistent with findings in HEK cells, A572- and Q573-

expressing cardiomyocytes showed increased late current compared to WT, which was blocked

by 10 M ranolazine (Figure 3A-B). Furthermore, a significant leftward shift in steady-state

inactivation was measured in A572D and Q573E variants compared to WT (Figure 3C-D).

Differences in endogenous Nav function were eliminated by low dose (10 nM) TTX sufficient to

block TTX-sensitive Nav1.5 channels (Figure 3A,B,D-F). Peak current was comparable in

transfected cells at baseline and showed a similar decrease with 10 nM TTX (57.3±4.6%,

63.2±6.2%, 61.7±4.6%, in WT, A572D, Q573E, respectively, P=NS for WT vs. A572D or

Q573E), indicating significant and comparable expression levels between WT and variants

(Figure 3E-F). These data support findings in HEK cells that A572D and Q573E variants act in

a phospho-mimetic manner.

Having validated successful expression of TTX-sensitive Nav channels in primary

cardiomyocytes, we measured APs in cardiomyocytes 24 hours following expression of WT and

variant constructs (Figure 4). A572D- and Q573-expressing cardiomyocytes showed significant

prolongation of APD90 compared to WT or vehicle, consistent with the clinical phenotype of QT

prolongation32, 33 (Figure 4A and B). Furthermore, afterdepolarizations were observed in

A572D-expressing myocytes (2 out of 9 cells, Figure 4A, red arrow). Ranolazine (10 M) or

low dose (10 nM) TTX eliminated differences in late current and APD90 between Q573E,

Differences in endogenous Nav function were eliminated by low dose (10 nM) TTTTTXXX sufufuffifificiicieneent t toto

block TTX-sensitive Nav1.5 channels (Figure 3A,B,D-F). Peak current was comparable in

rranannsfsfsfeecectetedd d cececellss atatat bbaseline and showed a similaar r ddeecrease with 10100 nM M M TTTTTX (57.3±4.6%,

63.222±6± .2%, 61.1 7±77±444.6%6%%, iinin WWWTT,T, AAA5757572D2DD,, Q573733E, rrreesspeectctctiviveeelyyy, PP=N=N=NSS fofoor WTWTT vvs.s AAA5757572D2DD oor

Q5Q55737373E)E)E),, ininindididicacatitiingngg sisiiggngnififi icicannnt tt ananand d d cocompmpmparararaabablelee eexxpxprreresssssioioon n n lllevevevelslss bebebetwtwtweeeeen nn WTWTWT aaandndnd vvavarririannntsts

Figure 3E-F)F)F).. TTTheheheseses ddatata a a sususuppppppororort t fififindnddininingsgsgs iin n n HEHEHEKKK cecec lllllsss thththatatat AAA5755 2D2D2D aaandndnd QQQ575773E3E3E vavavariririants act in


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A572D and WT-expressing myocytes and eliminated afterdepolarizations in A572D-expressing

cells (Figure 4A-B). These data indicate that A572D and Q573E variants mimic channel

phosphorylation and delay AP repolarization by increasing late INa in cardiomyocytes.

Computational model of phospho-mimetic Nav1.5 variants

To determine whether measured differences in Nav function are responsible for AP prolongation

in A572D- and Q573E-expressing myocytes and to predict electrophysiological consequences in

human, we performed computational modeling using detailed Markov models of WT and

variants integrated into a computer model of the human ventricular AP23 (Figure 5). State rate

transitions in a Markov model of Nav1.524-26 were determined for WT, A572D and Q573E

channels based on our electrophysiological measurements (parameters provided in online-only

Supplemental Material, Supplemental Table II) (Figure 5A-C). The Nav1.5 Markov model was

then incorporated into a well-validated model of the human ventricular AP23 to determine the

effect of variants on Nav function and cell excitability (Figure 5D). Consistent with experiments

(Figure 2-4), phospho-mimetic A572D and Q573E human variants were associated with

increased late INa and prolonged APD (Figure 5E-F). Furthermore, slow pacing (cycle length =

4,000 ms) unmasked afterdepolarizations in variant-expressing cells (Figure 5G-H). These

results indicate that measured changes in Nav function are sufficient to produce APD

prolongation and afterdepolarizations in A572D- and Q573E-expressing cells.

Finally, we simulated effects of ranolazine (10 M) on APs from WT and A572-

expressing cardiomyocytes. Ranolazine was assumed to preferentially block the Nav1.5 open

state with on- and off-rates of 8.2 M-1s-1 and 22 s-1, respectively (Figure 5A).40, 41 Consistent

with experimental observations37, ranolazine preferentially blocked late INa (Figure 5H).

Moreover, in agreement with our myocyte experiments, ranolazine normalized APD in variant-

channels based on our electrophysiological measurements (parameters providedd iiin onoo lililineene-o-o-onlnnly y

Supplemental Material, Supplemental Table II) (Figure 5A-C). The Nav1.5 Markov model was

hhenenn iiincncncorororpopoporrrateed dd iininto a well-validated model of f f ththt ee human ventririicuc laarrr AAAP23 to determine the

efffeeectc of varianantststs ooon NaNaN vvv fffunununctctctioion n anannd d ceeell eexcxccitabbbilliity (F(F(Figiggururure e 5D5DD)).). CoCoConnssisistetetenntnt wwwititi h h h exexxpepeeririmememennts

FFigigigururureee 22-4-44),),), pphohoosppphoho--mmimimemetitit c A5A5A572727 DD D ananandd d Q5Q5573737 EE huhuumamaann n vavaariririananntstss wewewerere aasssssococociaiateteed d d wiwiithth

ncreased latete IIINaNaNI aaandndn prprprololoongngngeddd AAAPDPDPD (F(F(Figigigururu ee e 5E5E5E-F-FF).).). FuFuFurtrtthehehermrmmororo e,e,, slsllowowow pppacaca ininng g g (c(c(cycycyclel length =


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expressing cells and eliminated afterdepolarizations at slow pacing (Figure 5G). These data

support our hypothesis that A572D and Q573E variants prolong APD by increasing late INa and

indicate that late INa blockers (ranolazine) may be an effective therapy for human patients with

Nav variants that mimic CaMKII phosphorylation (A572D and Q573E).

CaMKII-dependent Nav1.5 phosphorylation is dysregulated in acquired heart disease

Our findings suggest that the CaMKII phosphorylation motif plays an important role in

regulating channel activity and cell membrane excitability. Similar defects in Nav channel

function (e.g. increased late current) are observed in heart failure.42 Therefore, we hypothesized

that this same site contributes to cellular phenotypes associated with common disease. We

evaluated levels of Nav1.5 phosphorylation at S571 in a large animal model of cardiac

arrhythmias following myocardial infarction (MI), where abnormalities in CaMKII and Nav1.5

activities have previously been identified.29, 43-45 Immunoblot analysis revealed significantly

elevated levels of phospho-Nav1.5 (S571) (but not total Nav1.5) in the infarct border zone 5 days

post-occlusion (Figure 6A-B) without any change in remote regions from normal noninfarcted or

five-day post-occlusion hearts (Figure 6A-B). These data are consistent with previous reports of

activated CaMKII and abnormal Nav activity in the canine infarct border zone.29, 43-45

Furthermore, these data identify a potential mechanistic link between increased CaMKII activity

and Nav dysfunction following MI.

Finally, to determine whether CaMKII phosphorylation of Nav1.5 at S571 is involved in

human disease, we analyzed left ventricular (LV) samples from human patients with non-

ischemic heart failure (HF) (Figure 6C-E). We observed significantly increased levels of

phospho-CaMKII(T287) in HF samples (Figure 6C,D), consistent with previous reports of

increased CaMKII activity.46 In parallel, we measured a small but significant increase in

evaluated levels of Nav1.5 phosphorylation at S571 in a large animal model of ccaaardididiaccc

arrhythmias following myocardial infarction (MI), where abnormalities in CaMKII and Nav1.5

acctitiivivivitititieseses hhhavavave e prprrevevevioi usly been identified.29, 43-4555 Immmmunoblot annalaa yssisiss rrreevealed significantly

lellevvvata ed levells s ofoo ppphohospsphohoho--d NaNaNavv1.1.555 (S(S( 5575711) (bububut nooot ttotaaal NaNav11.1.5)5) innn ttheheh innnfararrctctct bobordrdrdererer zzonnnee 5 55 ddadays

popoststst-o-ooccccc lulusisisioonon ((FFFiggugureree 66A6A-B-BB) ) ) wiwiwithththouoout t anannyy y cchchananangegee ininn rrremememotototee rereregigiiononnss s frfromomom nnoorormmamal l nonononiinfnfnfararctctteddd oro

five-day post-t-ocococclcllusususioioon n heearara tststs (((FiFiFigugugurerr 666A-A-A B)B)B .. TTThehehesesee datatata a a ararare ee cococonsnsn isisistetetentntnt wwwititith h prprprevevevioioiousuu reports offf


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phospho-Nav1.5(S571) in HF samples compared to normals (Figure 6C,E) without any

significant difference in total Nav1.5 (Figure 6C,E). Thus, dysregulation of Nav1.5 at S571 is

found in a large animal model of MI and in human HF, supporting the notion of S571 as a nodal

point for electrical dysfunction in common disease.

While our data are consistent with a role for CaMKII in increased phospho-Nav1.5(S571)

in disease, multiple signaling cascades are activated in the diseased heart. Therefore, to establish

a causal link between CaMKII and dysregulation of Nav1.5 at S571 in disease, we determined

phospho-Nav1.5(S571) levels in isolated WT ventricular murine myocytes subjected to pacing (2

Hz) in the presence of isoproterenol and okadaic acid to simulate stress conditions where

CaMKII activity is elevated47 (Figure 7). Myocytes from transgenic mice expressing a CaMKII

inhibitory peptide (AC3-I)48 were subjected to the same protocol to determine the role of

CaMKII in Nav1.5 phosphorylation during stress. Increased levels of phospho-CaMKII(T287)

and phospho-Nav1.5(S571) were observed in WT paced cells treated with isoproterenol and

okadaic acid compared to control (untreated without pacing). In contrast, stressed AC3-I

myocytes showed no change in phospho-CaMKII or phospho-Nav1.5 compared to control. Pre-

treatment of WT myocytes with the CaMKII inhibitor KN-93 (10 M) also prevented stress-

induced changes in phospho-CaMKII and phospho-Nav1.5(S571). Together these data support

that S571 in the Nav1.5 DI-DII serves as an important molecular link between CaMKII

overactivity and abnormal Nav function in heart disease, including human HF.

Discussion

The vertebrate heart has evolved highly specialized pathways for regulating post-translational

modifications of ion channels, transporters and membrane receptors. Importantly, defects in

local signaling and regulation of specific ion channels have been associated with abnormal cell

CaMKII activity is elevated47 (Figure 7). Myocytes from transgenic mice expresessisingnn aa CCCaMaMaMKKKII

nhibitory peptide (AC3-I)48 were subjected to the same protocol to determine the role of

CaCaMKMKMKIIIIII iiinnn NaNN v1.1.555 php osphorylation during stresss.s.s IInncreased levelslss of f phphphooospho-CaMKII(T287)

annd d d php ospho-NaNavv11.5(5((S5S5S5717171))) wewewerere ooobbsbseerrvved iinn WTWTT pppaceeedd d cecelll sss trtreeaeatteted d wiww tthh iisososoprprototterere enene oolo aaandndnd

okkkadadadaiaiaiccc acacididid ccomommpapapareredd ttoto ccononntrtrololol (((unuuntrtrreaeaateteteddd wiwiwiththououout t papapacicicingngng).. InInn ccononontrtraasastt,t, sstrtrtresesssesedd d ACACAC33--II

myocytes shohowewewed d d nonono ccchahah ngnggee e inin ppphohohospsps hohoho-C-CCaMaMaMKIKIKII II ororor ppphohohospspsphohoho--NaNaNav1.1.1.5 5 5 cococompmpmpararededed ttto o o cococontrol. Pre--


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excitability and arrhythmia in heart disease including human HF. Here we use a variety of novel

reagents and computer models to demonstrate a link between defects in CaMKII-dependent

phosphorylation of Nav1.5 and both congenital and acquired human disease. We show that two

previously identified human SCN5A arrhythmia variants are localized to the CaMKII

phosphorylation motif of the Nav1.5 DI-DII loop. Experimental studies in heterologous cells

demonstrate that these variants, resulting in negative charge substitution adjacent to the

phosphorylation site, partially recapitulate CaMKII phosphorylation effects on channel

availability, recovery and late current. Furthermore, these defects in the CaMKII

phosphorylation motif alter the channel’s response to active CaMKII. Expression of these

variants in myocytes and computer simulations reveal delayed AP repolarization and

afterdepolarizations due to increased late INa. Computational modeling reveals that the isolated

(13-mer) DI-DII domains corresponding to A572D and Q573E variants possess very similar

electrostatic and structural features to the S571-phosphorylated channel, suggesting that they

may have similar binding interactions with downstream partners including regions of the channel

itself (e.g. channel pore) (Supplemental Figure 3 in online-only Supplemental Material). While

these simulations do not account for the true three-dimensional structure of the intact DI-DII

loop, they support our experimental data indicating that human variants A572D and Q573E

confer arrhythmia susceptibility by structurally and functionally mimicking the phosphorylated

channel. Finally, we report dysregulation of Nav1.5 at S571 in common forms of heart disease,

including human HF, suggesting a common molecular link between known defects in CaMKII

activity, Nav dysfunction and arrhythmias. Future studies are needed to determine whether

targeting this motif (genetically or pharmacologically) will be effective in preventing arrhythmia

and/or progression of disease.

variants in myocytes and computer simulations reveal delayed AP repolarizationn andndn

afterdepolarizations due to increased late INaI . Computational modeling reveals that the isolated

1133--mememe )r)r) DDDIII-D-D-DIII dddoomomains corresponding to A572722D D and Q573E vavaarir anntststs pppossess very similar

lelleccctrt ostatic anand dd stttruuctctc urrralalal fefefeatataturureseses ttto o ththee S555771-phohohosphohohoryrylalal tteed d chchananneneel, suguggggegeststinining g ththhataa ttthheheyyy

mamaay y y hahahaveve sssimimimililaarr bbbinindddingngng iintntteere acacactititionoonss wiwiwiththth dddowowownsnsstrrreaeam m m papaparrrtnenenersrss incncnclulul dddinngng rrregegegioioi nsnsns ooof tththeee chchanannnel

tself (e.g. chhananannenen ll l popoporerer ) ) ) (SSSupupupplpllemememenenentaaall l FiFiFigugugurerere 33 iin nn onono liliinenene--onononlylyly SSSupuppplplplemememenenentatat l l MaMaMateteeriririala ). While


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Remarkable progress has been made in understanding links between specific congenital

molecular defects and human disease. Structure-function studies on ion channels coupled with

expression studies in heterologous cells have greatly advanced our understanding of not only

monogenic disease but also more common acquired disease. A classic example of our ability to

link defects at the molecular level to a clinical phenotype comes from LQTS mutations found in

the DIII-DIV linker or C-terminal region that increase late current by interfering with rapid

channel inactivation (e.g. KPQ). Here, we propose that A572D and Q573E human variants

belong to an emerging class of atypical ion channel mutations that produce abnormal cell

excitability and arrhythmia by affecting post-translational modification.5, 13, 49 Previous studies

have yielded conflicting results regarding the functional status of the A572D variant.35, 36

Expression studies in oocytes report faster recovery from inactivation with no change in steady-

state availability (late current was not assessed).35 Other studies have found decreased

availability, altered recovery and increased late current in A572D but only with the common

polymorphism H558R.36 Importantly, previous studies did not analyze A572D function in the

setting of CaMKII signaling and therefore lack controls that would facilitate a more thorough

comparison (e.g. CaMKII co-transfection/inhibition). It is very likely that the A572D may have

limited pathogenicity in isolation and may depend on environmental cues/cofactors. Also, it is

interesting to consider the possibility that A572D may be linked with diseases other than

inherited arrhythmia syndromes (cardiomyopathy, heart failure). Similarly, while our data

establish a role for S571 in CaMKII-dependent regulation of Nav1.5, the precise mechanism is

likely more complicated with potential contributions from multiple nearby sites.50 It will be

important going forward to determine exactly how phosphorylation/mutation events in this DI-

DII “hot spot” conspire to regulate channel activity.

have yielded conflicting results regarding the functional status of the A572D variriianannt.tt 3355,, 36336

Expression studies in oocytes report faster recovery from inactivation with no change in steady-

ttatatteee aavav iiailalalabbibillityy (((llalatte current was not assessed).33535 OOther studies hahh vee fffoouound decreased

avvaiaiillability, allteterrreddd rerecocooveveryryy aaandnd iiincncncrereeasssed lllattte cuuurrrrennt tt iinin AAA557572D2DD bbutut ooonnlly y wiwiwithth ttheheh cccomommmmomonnn

popolylylymomomorprprphihihismssm HHH55558R8RR.3366 ImImpopoportrtrtanananttltly,y, ppprerereviviviouuus ss stststududdieiees s dididid d d nonoott t aananalalalyzyzyzeee AA5A5727272D DD fufufuncnnctitioonon iin n thtthe ee

etting of CCaMaMaMKIKIK II I sisisigngngnalala innng g g anana d d d ththt ererereffforororeee lalalackckck ccononontrtrroloo sss thththatatat wwwouououlddd fafafacicicilililitatatatetete aaa mmmororore ee thththorough


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Interestingly, despite the potentially lethal cellular phenotype associated with delayed AP

repolarization observed in our myocyte studies and computer simulations, the allele frequency

for at least one of the variants (A572D) is relatively high (~0.5%). This raises the question, why

are these variants not associated with a more severe clinical phenotype? One possibility

suggested by our data is that although the variants mimic the phosphorylated channel, they also

make the channel resistant to further regulation by endogenous CaMKII (see Figure 2) thereby

preventing exacerbation of the phenotype by multiple factors known to increase CaMKII activity

(e.g. -adrenergic stimulation). Furthermore, the inherent nature of Nav1.5 regulation by

CaMKII is complex with loss-of-function (decreased availability) coupled with gain-of-function

(increased late current) effects. Thus, the net result of this compound regulation may be a

cellular phenotype less problematic than either one alone. Alternatively, allelic imbalance and

variable expression of variant Nav1.5 channels may produce heterogeneity in Nav function and

clinical phenotype.51 Finally, and perhaps most importantly, our findings predict that individuals

harboring these variants will in part resemble a much larger population of HF patients (display

S571 hyper-phosphorylation, Figure 6), suggesting that while this modification may increase

susceptibility to arrhythmia it is not 100% penetrant.

While our data support a critical role for CaMKII in dysregulation of Nav1.5, we cannot

exclude contributions of other kinase pathways to this regulatory site in disease. In fact, we

expect that while CaMKII is the primary regulatory kinase for this site, other kinases also

contribute to phosphorylation at S571. Furthermore, phosphorylation of other sites, in addition

to S571, likely contributes to the disease phenotype, as Nav1.5 is directly regulated by both PKC

and PKA, whose activities are altered in disease.19 This may be true in human HF, in particular,

where we measured a significant but relatively modest change in phosphorylation of S571. While

increased late current) effects. Thus, the net result of this compound regulationn mamamay y bebebe aa

cellular phenotype less problematic than either one alone. Alternatively, allelic imbalance and

vavariririabababllele expxpxpreesssioioionn n of variant Nav1.5 channels mmamayyy produce heteeroror geeneneneiitity in Nav function and

cllinnnicical phenototypyppee.511 FiFinanallllllyyy, aandndd pppeerhhahapps mmmooost immmpportrtrtaanantltlyyy, ooururr fffinindidinngngss pprpredededicictt ththhatata inini ddidiviviiduduuals

haharbrbrbororrininingg g thththeesesee vavavaririr ananntss wwill l ininin ppparararttt rereseses mbmbmbllle aaa mmmucucchh lalal rgrgrgererer popopopupuulalaatititiononn oooff HFHFHF papapatititienenentsts ((ddidispspplllay y y

S571 hyper-p-pphohohospspsphohohoryryylalaationonon, , , Fiiigugugureree 6))),,, sususuggggggesesestititingngng thththatt wwwhihihilelele ttthihihis s momomodididififificacacatitit ononon mmmayayay iiincn rease


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our data demonstrate elevated Nav1.5 pS571 in forms of canine and human disease, it will be

important to establish the relative contribution of these changes to dysfunction across disease

pathologies. Finally, oxidation and other post-translational modifications may regulate channel

activity through direct (e.g. lipid peroxidation) or indirect (e.g. PKC-dependent phosphorylation)

pathways. Thus, future studies will be important to determine the relative contribution of

phosphorylation of Nav1.5 at S571 to dysfunction in diseased heart, as well as upstream signals

(e.g. -adrenergic receptor stimulation, angiotensin II, reactive oxygen species, Ca2+).

In summary, we identified the molecular mechanism for two human Nav1.5 variants

localized to the CaMKII regulatory motif in the Nav1.5 DI-DII loop and provide data to support

that a similar defect is present in a large animal model of ischemic heart disease and in human

HF. These studies add to mounting evidence that defects in local signaling and protein post-

translational modification help define the disease phenotype associated with a broad range of

cardiac arrhythmia syndromes. It will be interesting in the future to determine whether therapies

targeting CaMKII-dependent regulation of Nav1.5 at S571 will be effective in reducing

arrhythmia burden in these patients.

Funding Sources: This work was funded by National Institutes of Health (NIH) Grants HL096805, HL114893 (TJH), HL084583, HL083422 (PJM), HL079031, HL096652, HL113001, and HL070250 (MEA), HL066140 (PAB), National Science Foundation Grant DMS-1022466 (CCM), the Gilead Sciences Research Scholars Program in Cardiovascular Disease (TJH), Saving Tiny Hearts Society (PJM), and the Fondation Leducq Transatlantic Alliance for CaMKII Signaling (08CVD01, MEA, PJM).

Conflict of Interest Disclosures: Dr. Anderson is cofounder of Allosteros Therapeutics and inventor on patents claiming to treat heart failure and arrhythmias by CaMKII inhibition.

References:

hat a similar defect is present in a large animal model of ischemic heart disease aaandndd innn hhuhumamaman n

HF. These studies add to mounting evidence that defects in local signaling and protein post-

rranannslslslatatatiiionananall l mmomodididiffificac tion help define the diseasese phhenotype assococciai teed d d wwwith a broad range of

caardddiiac arrhytthmhmmiaaa ssynynndrdrdromomeseses. ItItt wwwili ll bbee inntterrrestiiinngg inn n tththee fuuututurree toto ddeeeteerrmimiinnene wwheheh ththt erer ttthehheraraappipies

aargrggetetetinininggg CaCaCaMKMKMKIII-d-d-depepepennndedentntt rregeggulululaatatioioon nn ofofof NNNaaavvv1.1..555 aatat SSS57577111 wiwiwilllll bbbe e efefeffefeectctivivi ee ininin rereredududuccicingngng

arrhythmia bburururdededen n n ininin thththesee e e e papapatienenentstst ..


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4411. FFrF edj S, SSammmpspsson KKJJ, LLiiuiu HH, KaKassss RRRS. MMMoooleccululara bbbasasasiisis ooff raaanooolaziinenene bblllocckck of f LQLQQTTT-333 muuuttaantoodididiumumum cchahaannnnn elelsss: eeevividddenncncee fofofor sisisitetete ooof f aacactititiononon. BrBrBr JJJ PPPhahaarmrmmacacacololl. . 2020006066;1;1; 4448::1:16-6-242424..

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51. Leoni AL, Gavillet B, Rougier JS, Marionneau C, Probst V, Le Scouarnec S, Schott JJ, Demolombe S, Bruneval P, Huang CL, Colledge WH, Grace AA, Le Marec H, Wilde AA, Mohler PJ, Escande D, Abriel H, Charpentier F. Variable Nav1.5 protein expression from the wild-type allele correlates with the penetrance of cardiac conduction disease in the Scn5a(+/-)mouse model. PLoS One. 2010;5:e9298.

Figure Legends:

Figure 1 – Human arrhythmia variants proximate to Nav1.5 CaMKII-phosphorylation node. (A)

Schematic illustrating the spectrin-based signaling complex at the cardiomyocyte intercalated

disc for regulation of Nav1.5 and cell membrane excitability. The actin-associated polypeptide

IV-spectrin complexes CaMKII with Nav1.5 (via ankyrin-G) to facilitate direct phosphorylation

at S571 in the Nav DI-DII linker. (B) Human variants adjacent to the Nav1.5 phosphorylation

site (A572D and Q573E) have been linked to cardiac arrhythmia (long-QT syndrome).

Hudmon A. Ca2 /calmodulin-dependent protein kinase II (CaMKII) regulates carddiaiaaccc sosodidiumum channel NaV1.5 gating by multiple phosphorylation sites. J Biol Chem. 2012;287877 1:19989 565656-1-119898986969.

51. Leoni AL, Gavillet B, Rougier JS, Marionneau C, Probst V, Le Scouarnec S, Schott JJ, Demolombe S, Bruneval P, Huang CL, Colledge WH, Grace AA, Le Marec H, Wilde AA, MoMoohlhlhlererer PPPJ,J,J EEEsscanannddede D, Abriel H, Charpentier F. VVVaaariable Nav1.55 pproteteeininin expression from the wiwiwildldd-ttype allllleeele e cococorrrrelelatatatesess wwwitith h thththe e e pepenenetrtranancece oof f caaardiaaccc cococondn ucucucttitioon n didiseseseasee ininn tttheheh SScncn5a5a5 (+(+( /-/-/ ))mmouususe model. PLPLPLooSoS OOOneee. 202020101010;5;5:e:ee9929299898..

FiFigugurere LLegegenendsds::


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Figure 2 – Human arrhythmia Nav1.5 variants adjacent to CaMKII phosphorylation domain

mimic channel phosphorylation. (A) Nav current (INa) traces measured from HEK cells

expressing WT, A572D or Q573E channels (co-expressed with h 1 beta subunit) +/- 10 M

ranolazine to block late INa. Nav function was also measured in S571A- and S571E-expressing

cells as negative (phospho-resistant) and positive (phospho-mimetic) controls, respectively.

Dashed line indicates measurement time of late current (50 ms after peak). (B) Summary data for

late current measured at 50 ms after peak and expressed as percentage of peak (**P<0.005,

***P<0.001 vs. WT at baseline; n=5 except Q573E+CaMKII (n=6)), (C-D) steady-state

inactivation curves (*P<0.05 vs. S571E, A572D, Q573E; n=5 except Q573E and S571E (n=6)),

(E) steady-state inactivation V1/2 (**P<0.005, ***P<0.001 vs. WT at baseline), (F-G) Nav

recovery from inactivation curves (*P<0.05 vs. A572D and Q573E, n=5), and (H) recovery time

constants (*P<0.05, **P<0.005, ***P<0.001 vs. WT at baseline) in WT and variant-expressing

cells +/- CaMKII. S571E, A572D and Q573E-expressing cells show increased late INa and

slowed recovery from inactivation similar to WT+CaMKII. Neither S571E, A572D nor Q573E

show further changes in Nav function with CaMKII. In contrast, the S571A phospho-resistant

mutant (+/- CaMKII) resembles WT at baseline (without CaMKII).

Figure 3 – Expression of human arrhythmia variants in cardiomyocytes. Nav current (INa) was

measured in 3-day old neonatal mouse cardiomyocytes transfected with vehicle, WT, A572D or

Q573E channels engineered to have increased sensitivity to TTX (C373Y).39 (A) INa traces, (B)

late INa amplitude +/-10 M ranolazine (**P<0.005 and ***P<0.001 vs. control (no

TTX/ranolazine); n=8 for control, n=5 for ranolazine), (C) steady-state inactivation curves

(*P<0.05 vehicle vs. A572D or Q573E; n=5 except for WT (n=4) and A572D (n=6)), and (D)

E) steady-state inactivation V1/2V (**P<0.005, ***P<0.001 vs. WT at baseline), (F(FF-G)G)G) NaNaNavvv

ecovery from inactivation curves (*P<0.05 vs. A572D and Q573E, n=5), and (H) recovery time

coonsnsnstatatantnts (*(*(*P<P<P 0.0.050505, , **P<0.005, ***P<0.001 vs. WWTWT at baseline)) inini WWWTTT aand variant-expressing

ceellls s +/- CaMKMKIIII. SS57571E1EE, A5A5A577272D DD aananddd QQQ57333EEE-exxprprressisisingngng ccceelllsls ssshhoow iinnccrreaeaaseeed d lalatetet IIINaNaaI aaandnd

llowowwededed rrrecececovovovereryy y frrromomom inananacctivvvatata ioioionn n ssisimmimilalaarrr tototo WWWT+T+T+CCaCaMKMKMKIIIIII.. NNNeieiithththererer SSS5757571E1E, A5A5A5727272DDD nononor r Q5Q5Q5737373E EE

how furtherer ccchhhananangegeges s ininin NNaaavvv fffununnctctc ioioon n wiwiw ththth CCCaMaMaMKIKIKII.I.I. Innn cccononontrtrtrasasast,t,t thehehe SSS5757571A1A1A ppphohohospspsphohoho--resistant


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steady-state inactivation V1/2 (*P<0.05, ***P<0.001 vs. vehicle control (no drug)) were

measured +/- low dose (10 nM) TTX to block exogenous current. (E) Current-voltage

relationships were also measured +/- low dose TTX to verify similar expression levels for WT

and human arrhythmia variants. (*P<0.0.05 control vs. 10 nM TTX; n=7 for control, n=5 for

TTX). (F) Summary data comparing peak TTX-sensitive (exogenous) current-voltage

relationship in WT and variant-expressing myocytes (P=N.S. vs. WT)

Figure 4 – Human arrhythmia variants adjacent to CaMKII phosphorylation site delay AP

repolarization when expressed in cardiomyocytes. WT, A572D and Q573E channels engineered

to have increased sensitivity to TTX (C373Y)39 were expressed in neonatal mouse

cardiomyocytes. (A) APs and (B) APD90 from vehicle, WT, A572D and Q573E-expressing

cardiomyocytes +/- 10 nM TTX (to block exogenous Na+ current) (*P<0.05, **P<0.005 and

***P<0.001 vs. control (no TTX); n=12 for control, n=5 for ranolazine and TTX). A572D and

Q573E significantly increase APD90 compared to WT and increase the likelihood of

afterdepolarizations (red arrow in A). Low dose TTX or 10 M ranolazine eliminates

differences in APD90. Myocytes were paced at 1Hz.

Figure 5 – Computational model to determine role of altered Nav function in AP prolongation

for human arrhythmia variants. (A) Markov model to simulate Nav function including

transitions between multiple inactivated (red), closed (blue), open (green), and ranolazine-bound

(gray) states. Parameter estimation results comparing experimentally measured (black) and

simulated (red) values for (B) steady-state inactivation and (C) late current. (D) Schematic of

mathematical model of human ventricular AP used to determine effects of mutant Nav function

o have increased sensitivity to TTX (C373Y)39 were expressed in neonatal mouussse

cardiomyocytes. (A) APs and (B) APD90 from vehicle, WT, A572D and Q573E-expressing

caardrddioioiomymymyocococytytytes +++///-- 10 nM TTX (to block exogenenenouuus Na+ current)t)) (*PP<0<0<0.0. 5, **P<0.005 and

******PP<0.001 vvs.s cccooontrtroloo (((nonono TTTTXTTX););; nnn==1122 for cooontrrooll, n=5=5=5 ffooor rrrananololazazinineee aanand d d TTTTT X)X)). AAA5575 2D2D2D aaannnd

Q5Q55737373EE E sisigngngnififificicananntltlly y y ininncrcrreaeaseee AAAPDPDPD99090 ccomomompapapareredd d totoo WWWTT T ananndd d inincrcrcreaeaasesee ttthehee liikikelellihihihooooo d d d oofof

afterdepolarrizizzatatatioioi nsnsns ((rerer d dd ararrrororoww iiin n n AAA).) LLLowowow dddososose e TTTTTTX XX ororor 111000 M M M raranononolalalazizizinenene eeliliimimiminananatetetesss


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on cardiac repolarization. Simulated (E) APs and (F) INa from WT (black), A572D (red), and

Q573 (gray) expressing cells. Results shown at steady-state at pacing cycle length = 1,000 ms.

Simulated (G) APs and (H) INa in WT- (black) and A572D- (red) expressing cells at steady-state

during slow pacing (cycle length = 4,000 ms). Afterdepolarizations were observed in A572D

and Q573E (not shown) variants. Simulating block of late INa with 10 M ranolazine (C)

reduced late INa and afterdepolarizations (gray lines in A and B) consistent with experiments.

Figure 6 – Increased phosphorylation of Nav1.5 at S571 in canine model of myocardial

infarction and human heart failure. (A) Representative immunoblots and (B) densitometric

measurements (normalized to actin and expressed relative to normal levels) of phospho-

Nav1.5(S571) (left) and total Nav1.5 (right) from remote and border zone regions of normal

(noninfarcted) and infarcted hearts (**P<0.005 compared to remote; n=4 for remote and n=5 for

BZ). (C) Representative immunoblots and (D) densitometric measurements of total CaMKII

and phospho-CaMKII(T287) and (E) total Nav1.5, phospho-Nav1.5 from left ventricular samples

of normal and failing human hearts (*P<0.05 vs. normal, ***P<0.001 vs. remote; n=7). For

densitometric measurements, all samples were analyzed on the same gel and normalized to

corresponding actin levels from same blot. Equal protein loading was ensured by BCA assay

and verified by analysis of Coomassie stains of gel.

Figure 7 – CaMKII contributes to increased phosphorylation of Nav1.5 at S571 in stress

conditions. (A-B) Representative immunoblots from isolated adult (A) WT and (B) AC3-I

myocytes subjected to pacing (10 min, 2 Hz) in the presence of isoproterenol (100 nM) and

okadaic acid (2 M) to simulate stress conditions and enhance CaMKII-dependent regulation. A

measurements (normalized to actin and expressed relative to normal levels) of phphhosossphphpho-o-o-

Nav1.5(S571) (left) and total Nav1.5 (right) from remote and border zone regions of normal

nnonononinininffafarccteteted)dd aandndnd ini farcted hearts (**P<0.005 coocommmpared to remoootet ; n=n==44 4 for remote and n=5 for

BBZ)).). (C) Repeprereeseentntaaativvvee imimimmmumunonoobblbloottss anddd ((DD) ddedennsitittoomomeetrriic c mmemeaasururememmenenntstss oof f totototataal l CaCaCaMKMKMKIIIII

ananddd phphphosososphphpho-o-o CaCaaMKMKMKIIIII(T(TT28282 7))) aandndnd (E(E(E)) totototatatalll NNaNavvv1.1.1 55,5, ppphohoospspphohoho-NNaNavv1.555 frfromomom llefefft t t veveventntntririricuculllar r r sasampmpmpleles

of normal annd d d fafafailillinining g g huhuhumamaan n n heheheararartstst (((*P*PP<0<0<0.0.00555 vsvsvs. . nononormrmrmalalal,, *******P*P*P<0<0<0.000010101 vvvs.s.s. rrrememe ototote;e;e; nnn=7=7=7). For


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subset of WT cells were pretreated with CaMKII inhibitors KN-93 (10 M) for 30 min prior to

pacing. (C-D) Densitometric measurements (normalized to GAPDH and expressed relative to

normal levels) of (C) phospho-CaMKII(T287), and (D) phospho-Nav1.5(S571) (**P<0.005 and

***P<0.001 vs. control; n=4). Note that CaMKII inhibition (AC3-I and KN-93) prevents

increased phosphorylation of Nav1.5 at S571 observed in stressed WT myocytes.


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and Thomas J. HundMitchell, Penelope A. Boyden, Philip F. Binkley, Chenglong Li, Mark E. Anderson, Peter J. MohlerCurran, Nicholas D. Leymaster, Wen Dun, Patrick J. Wright, Natalia Cardona, Lan Qian, Colleen C.

Olha M. Koval, Jedidiah S. Snyder, Roseanne M. Wolf, Ryan E. Pavlovicz, Patric Glynn, Jerry Channel in Cardiac Disease+CaMKII-Based Regulation of Voltage-Gated Na

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2012 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation published online September 24, 2012;Circulation.

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Koval et al. CaMKII-based regulation of Nav in disease - CIRCULATIONAHA/2012/105320

1

SUPPLEMENTAL MATERIAL

Supplemental Methods

Electrophysiology: Current recordings were measured by conventional whole-cell patch-clamp

technique with an Axon 200B patch-clamp amplifier controlled by a personal computer using a

Digidata 1320A acquisition board driven by pClamp 8.0 software (Axon Instruments, Foster City,

CA). Electrophysiological recordings were obtained from GFP-positive cells. Whole cell sodium

currents were measured using standard protocols as described in detail1-3. Briefly, a whole-cell

bath solution containing 10 mM NaCl, 130 mM choline chloride, 4.5 mM KCl, 1.8 mM CaCl2, 2.0

mM MgCl2, 10.0 mM Hepes, and 5.5 mM glucose, pH 7.35, titrated with KOH. The pipette

solution contained 130 mM CsCl, 0.5 mM CaCl2, 2 mM MgCl2, 5 mM Na2ATP, 0.5 mM GTP, 5

mM EGTA, and 10 mM Hepes, pH 7.3, titrated with CsOH. Current recordings were low-pass

filtered at 5 kHz and digitized at a sampling rate of 20 kHz. Standard protocols were used to

measure current-voltage relationship, steady-state inactivation, recovery from inactivation and

late current (provided as insets in corresponding Figure panels).1-3 Briefly, IV curves were

measured by step voltage pulses from a holding potential of -120 mV. Late current was

measured as current amplitude 50 ms after peak and expressed as percentage of peak.

Steady-state inactivation was measured as a step pulse from -120 for 500 ms followed by test

pulse to 0 mV. Recovery from inactivation was measured using a prepulse to 0 mV for 500 ms

(P1), followed by return to holding potential of -120 mV for variable duration (interpulse interval),

then a test pulse to 0 mV for 10 ms (P2). Current recording experiments were conducted at

room (21-23ºC) and physiological temperature (37ºC) for HEK cells and cardiomyocytes,

respectively. Recording pipettes, fabricated from borosilicate glass, had resistance of 2-4 MΩ,

when filled with recording solution. All solutions were adjusted to 275-295 mOsm.

Action potentials (APs) were evoked by brief current pulses 1.5–4 pA, 0.5–1 ms at a

pacing frequency of 1 Hz. AP duration (APD) was assessed as the time from the AP upstroke


2

to 90% repolarization to baseline (APD90).4 APs were recorded using the perforated

(amphotericin B) patch-clamp technique in Tyrode’s solution (bath) with the pipette filled with

(mmol/L): 130 potassium aspartate, 10 NaCl, 10 HEPES, 0.04 CaCl2, 2.0 MgATP, 7.0

phosphocreatine, 0.1 NaGTP, and amphotericin B 240 μg/mL, with the pH adjusted to 7.2 with

KOH.5 APs were measured at physiological temperature.

Neonatal mouse cardiomyocytes: Hearts were isolated from postnatal day 1 mice and

cultured as previously described.1, 6

Computational model of Nav1.5 and ventricular action potential: Parameter estimation to

determine rate constants for models of WT and mutant INa was performed using the Levenberg-

Marquardt method in COPASI (v.4.8.35).7 Nav1.5 models were incorporated into a human

ventricular cell model8 which was paced from rest (initial conditions provided in Supplemental

Table III) to steady state using a conservative current stimulus9 (cycle length = 1,000 unless

otherwise stated, stimulus amplitude = -60 A/F, stimulus duration = 1.0 ms).

Experimental model of myocardial infarction and immunoblotting: Myocardial infarction

(MI) was produced in canines by total coronary artery occlusion, as described previously. 10, 11

A cardiectomy was performed five days after surgery and thin tissue slices from visible

epicardial border zone and from remote area away from the infarct (left ventricular base) were

flash frozen for immunoblot analysis. Ventricular lysates were prepared for immunoblot analysis

as described.3, 11 Equal quantities of protein were analyzed by SDS-PAGE (3-8% Tris acetate

gels). Immunoblotting was performed using validated affinity-purified antibodies to phospho-

Nav1.5(S571) or total Nav1.5 created against a 20 amino acid sequence

(ESESHHTSLLVPWPLRRT-phosphoS-A) that includes the CaMKII phosphorylation site at

S571.3 Resulting antibodies produce a single dominant band around 220 kDa corresponding to

Nav1.5. Slight differences in protein loading were corrected using an internal control standard


3

(rabbit polyclonal antibody to actin (Santa Cruz)). This investigation conforms to the Guide for

the Care and Use of Laboratory Animals published by the National Institutes of Health (Pub. No.

85-23,1996).

Adult cell isolation and pacing: Ventricular myocytes from WT and AC3-I adult mice (2-3

mos) were isolated as described,12 washed in modified Tyrode’s solution containing (mM): 137

NaCl, 5.4 KCl, 0.5 MgCl2, 0.16 NaH2PO4, 3 NaHCO3, 5 HEPES-NaOH and 5 glucose, pH

adjusted to 7.4 with NaOH, and resuspended in modified Tyrode’s supplemented with (mM) 1.2

CaCl2, 2 BSA, 2 L-carnitine, 5 creatin, and 5 taurine. Cells were plated onto a 6-well tray

precoated with laminin (Invitrogen). After 90 min at 37°, media was aspirated and replaced with

fresh supplemented Tyrode’s. Cells were pretreated for 30 min with the phosphatase inhibitor

okadaic acid (2 M) and paced (amplitude=35 V, duration=3ms) for 10 min at 2 Hz using the C-

pace multichannel stimulator (Ionoptix). 100 nM isoproterenol was added immediately prior to

onset of pacing. A subset of cells were pretreated for 30 min before onset of pacing with the

CaMKII inhibitor KN-93 (10 M). Cell lysates were analyzed by SDS-PAGE and

immunoblotting. Slight differences in protein loading were corrected using monoclonal anti-

GAPDH (Fitzgerald) as an internal control standard.


4

Supplemental Table I. Transition rate expressions for mathematical model of Nav1.5

Transition rate (ms-1) Reference

a1 = P1a1/(P2a1 exp(-(Vm+2.5)/17)) + 0.20 exp(-(Vm+2.5)/150) Bondarenko et al.13

a2 = P1a1/(P2a1 exp(-(Vm+2.5)/15)) + 0.23 exp(-(Vm+2.5)/150) “

a3 = P1a1/(P2a1 exp(-(Vm+2.5)/12)) + 0.25 exp(-(Vm+2.5)/150) “

a4 = 1.0/(P1a4 exp(-(Vm+7.0)/16.6) + 0.393956) “

a5 = P1a5 exp(-Vm/P2a5) “

a6 = a4/P1a6 “

a7 = P1a7 a4 “

a8 = P1a8 Grandi et al.14

b1 = P1b1 exp(-(Vm+2.5)/P2b1) Bondarenko et al.13

b2 = P1b2 exp(-(Vm-P2b2)/P2b1) “

b3 = P1b3 exp(-(Vm-P2b3)/P2b1) “

b4 = (a3 a4 a5)/(b3 b5) “

b5 = P1b5 + P2b5(Vm+7.0) “

b6 = P1b6 a5 “

b7 = P1b7 a5 “

b8 = P1b8 Grandi et al.14

a9 = [Ranolazine] P1a9

b9 = P1b9


5

Supplemental Table II. Parameters for mathematical models of wildtype and variant Nav1.5

Parameters Wildtype A572D Q573E Source

P1a1 7.5207 6.8920 4.6305 Fit to IV curve.

P2a1 0.1027 Bondarenko et al.13

P1a4 0.188495 “

P1a5 7.0e-7 “

P2a5 7.7 “

P1b1 0.1917 “

P2b1 20.3 “

P1b2 0.2 “

P2b2 2.5 “

P1b3 0.22 “

P2b3 7.5 “

P1b5 0.0108469 0.0604095 0.0453576 Fit to inactivation and IV curves.

P2b5 2e-5 Bondarenko et al.13

P1a6 1000.0 “

P1b6 6.0448e-3 2.5121e-3 2.6199e-3 Fit to recovery.

P1a7 1.05263e-5 Bondarenko et al. 13

P1b7 0.02 “

P1a8 4.0933e-13 1.4458e-4 6.5226e-5 Fit to late current.

P1b8 9.5e-4 Grandi et al.14

P1a9 8.2 mM-1ms-1 Wang et al.15

P1b9 0.022 “ Maximum INa conductance

7.35 9.75 11.60 Fit to peak IV curve.


6

Supplemental Table III. Initial conditions for state variables in mathematical model of human ventricular action potential*

State variable Definition WT A572D Q573E C1 INa closed state 0.0003850597267 0.0003849053676 0.0003848309613C2 “ 0.02639207662 0.02638818278 0.02638630536 C3 “ 0.7015088787 0.7015704583 0.7016001465 IC2 INa inactive state 0.009845083654 0.009840770794 0.009838691924 IC3 “ 0.2616851145 0.2616320393 0.2616064508 IF “ 0.0001436395221 0.0001435402196 0.0001434923636IM1 INa intermediate

inactivation state 3.913769904e-05 3.909441247e-05 3.907357776e-05

IM2 “ 3.381242427e-08 3.376098643e-08 3.373623669e-08 LC1 INa burst mode closed

state 1.659002962e-13 1.658337887e-13 1.65801729e-13

LC2 “ 1.137084204e-11 1.136916421e-11 1.136835517e-11 LC3 “ 3.02240205e-10 3.022667309e-10 3.022795176e-10 O INa open state 9.754706096e-07 9.747956198e-07 9.744703275e-07 LO INa burst mode open

state 4.202747013e-16 4.199838793e-16 4.198437236e-16

C1,mut Mutant INa closed state - 0.000126208303 7.166307322e-05 C2,mut “ - 0.009441839154 0.007980529607 C3,mut “ - 0.2739249316 0.3446438288 IC2,mut Mutant INa inactive state - 0.02242017495 0.01409702075 IC3,mut “ - 0.6504500644 0.6087880685 IFmut “ - 0.0002996886807 0.0001265875713IM1,mut Mutant INa intermediate

inactivation state - 0.0001977778539 8.00620291e-05

IM2,mut “ - 1.707703298e-07 6.911707407e-08 LC1,mut Mutant INa burst mode

closed - 1.920497509e-05 4.919562139e-06

LC2,mut “ - 0.001436754012 0.0005478513486LC3,mut “ - 0.04168284782 0.02365928023 Omut Mutant INa open state - 2.929101154e-07 1.117291082e-07 LOmut Mutant INa burst mode

open state - 4.457180191e-08 7.670035154e-09

d L-type Ca2+ current activation gate

1.638090607e-05 1.637609944e-05 1.637378239e-05

f L-type Ca2+ voltage-dependent inactivation gate

0.9999364487 0.9999364687 0.9999364783

fca L-type Ca2+ calcium-dependent inactivation gate

1.0 1.0 1.0

Xr1 Rapidly activating K+ current activation gate

0.0001497389543 0.0001496918853 0.000149669196

Xr2 Rapidly activating K+ current activation gate

0.4963056182 0.4963285448 0.4963395991

Xs Slowly activating K+ current activation gate

0.002723153193 0.00272272626 0.002722520434

r Transient outward K+ current activation gate

1.61573943e-08 1.615146813e-08 1.614861155e-08

s Transient outward K+ current inactivation gate

0.9999986683 0.9999986689 0.9999986692


7

g Ryanodine receptor Ca2+ release activation gate

0.9999961213 0.9999961354 0.9999961421

[Ca2+]i Ca2+ concentration in myoplasm (mM)

4.387145251e-05 4.384476863e-05 4.383218716e-05

[Ca2+]SR Ca2+ concentration in sarcoplasmic reticulum (mM)

0.1969056818 0.1967445959 0.196669525

[Na+]i Na+ concentration in myoplasm (mM)

8.355040031 8.349674873 8.3471155

[K+]i K+ concentration in myoplasm (mM)

145.439625 145.4478242 145.451705

Vm Transmembrane potential (mV)

-87.64533404 -87.64753511 -87.64859638

* Model equations for human ventricular cell model and Nav1.5 model are found in original publications.8,

13, 14, 16


8

Supplemental Figure 1 – Expression of wildtype (WT) and human arrhythmiavariants in HEK cells. Immunoblots of total Nav1.5 and GAPDH (loading control) onlysates from HEK cells expressing WT, A572D, Q573E channels or untransfected cells.Equal protein loading was ensured by BCA assay and verified by Coomassie stain ofgel.

Supplemental Figure 2 – CaMKII inhibitor AIP reverses effects of CaMKII(T287D) on Nav1.5 expressed in HEK cells. (A) Steady-state inactivation and (B) late current in HEK cells expressing WT Nav1.5 and CaMKII(T287D) at baseline (black) and with 10 M AIP (red). AIP prevents the depolarizing shift in steady-state availability and increased late current observed with CaMKII(T287D) expression (*P<0.05, n=5).


9

Supplemental Figure 3 – Electrostatic potential maps of wildtype and humanvariant Nav1.5 DI-DII loop motif conformations. Conformations of isolated (13-mer)DI-DII domains (left) and electrostatic potential maps (right) shown for (A) basalwildtype (S571 in green), (B) phosphorylated wildtype (phospho-S571 modification ingold), (C) A572D human variant (D572 in green), and (D) Q573E human variant (573Ein green). Note that human variants have very similar electrostatic features to thephosphorylated wildtype channel (conserved negative charges marked by asterisk attop of panels B-D). Electrostatic potentials were computed by the Delphi finite-difference Poisson-Boltzmann solver and mapped to molecular surfaces of W565-S577 from hNav1.5 (units in kT/e).


10

Supplemental References

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2. Wagner S, Dybkova N, Rasenack EC, Jacobshagen C, Fabritz L, Kirchhof P, Maier SK, Zhang T, Hasenfuss G, Brown JH, Bers DM, Maier LS. Ca/calmodulin-dependent protein kinase II regulates cardiac Na channels. J Clin Invest. 2006;116:3127-3138.

3. Hund TJ, Koval OM, Li J, Wright PJ, Qian L, Snyder JS, Gudmundsson H, Kline CF, Davidson NP, Cardona N, Rasband MN, Anderson ME, Mohler PJ. A betaIV spectrin/CaMKII signaling complex is essential for membrane excitability in mice. J Clin Invest. 2010;120:3508-3519.

4. Koval OM, Guan X, Wu Y, Joiner ML, Gao Z, Chen B, Grumbach IM, Luczak ED, Colbran RJ, Song LS, Hund TJ, Mohler PJ, Anderson ME. CaV1.2 beta-subunit coordinates CaMKII-triggered cardiomyocyte death and afterdepolarizations. Proc Natl Acad Sci U S A. 2010;107:4996-5000.

5. Wu Y, Gao Z, Chen B, Koval OM, Singh MV, Guan X, Hund TJ, Kutschke W, Sarma S, Grumbach IM, Wehrens XH, Mohler PJ, Song LS, Anderson ME. Calmodulin kinase II is required for fight or flight sinoatrial node physiology. Proc Natl Acad Sci U S A. 2009;106:5972-5977.

6. Bhasin N, Cunha SR, Mduannayake M, Gigena MS, Rogers TB, Mohler PJ. Molecular basis for PP2A regulatory subunit B56 alpha targeting in cardiomyocytes. Am J Physiol Heart Circ Physiol. 2007;293:H109-119.

7. Hoops S, Sahle S, Gauges R, Lee C, Pahle J, Simus N, Singhal M, Xu L, Mendes P, Kummer U. COPASI--a COmplex PAthway SImulator. Bioinformatics. 2006;22:3067-3074.

8. Ten Tusscher KH, Noble D, Noble PJ, Panfilov AV. A model for human ventricular tissue. Am J Physiol Heart Circ Physiol. 2004;286:H1573-1589.

9. Hund TJ, Kucera JP, Otani NF, Rudy Y. Ionic charge conservation and long-term steady state in the Luo-Rudy dynamic model of the cardiac cell. Biophys J. 2001;81:3324-3331.

10. Pu J, Boyden PA. Alterations of Na+ currents in myocytes from epicardial border zone of the infarcted heart. A possible ionic mechanism for reduced excitability and postrepolarization refractoriness. Circ Res. 1997;81:110-119.

11. Hund TJ, Wright PJ, Dun W, Snyder JS, Boyden PA, Mohler PJ. Regulation of the ankyrin-B-based targeting pathway following myocardial infarction. Cardiovasc Res. 2009;81:742-749.

12. Li J, Kline CF, Hund TJ, Anderson ME, Mohler PJ. Ankyrin-B regulates Kir6.2 membrane expression and function in heart. J Biol Chem. 2010;285:28723-28730.

13. Bondarenko VE, Szigeti GP, Bett GC, Kim SJ, Rasmusson RL. Computer model of action potential of mouse ventricular myocytes. Am J Physiol Heart Circ Physiol. 2004;287:H1378-1403.

14. Grandi E, Puglisi JL, Wagner S, Maier LS, Severi S, Bers DM. Simulation of Ca-Calmodulin-Dependent Protein Kinase II on Rabbit Ventricular Myocyte Ion Currents and Action Potentials. Biophys J. 2007;93:3835-3847.

15. Wang GK, Calderon J, Wang SY. State- and use-dependent block of muscle Nav1.4 and neuronal Nav1.7 voltage-gated Na+ channel isoforms by ranolazine. Mol Pharmacol. 2008;73:940-948.

16. Clancy CE, Tateyama M, Kass RS. Insights into the molecular mechanisms of bradycardia-triggered arrhythmias in long QT-3 syndrome. J Clin Invest. 2002;110:1251-1262.

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