olha m. koval, ph.d. nih public access jedidiah s. snyder ...€¦ · the dorothy m. davis heart...

CaMKII-Based Regulation of Voltage-Gated Na+ Channel inCardiac Disease

Olha M. Koval, Ph.D.*,1,2, Jedidiah S. Snyder, B.S.*,1,3, Roseanne M. Wolf, Ph.D.1,4, Ryan E.Pavlovicz, B.S.1,5, Patric Glynn, B.S.1,3, Jerry Curran, Ph.D.1, Nicholas D. Leymaster, M.S.6,Wen Dun, M.D., Ph.D.7, Patrick J. Wright, B.S.1, Natalia Cardona6, Lan Qian, M.D.2, ColleenC. Mitchell, Ph.D.4, Penelope A. Boyden, Ph.D.7, Philip F. Binkley, M.D.1,8, Chenglong Li,Ph.D.1,5, Mark E. Anderson, M.D.,Ph.D.2,6, Peter J. Mohler, Ph.D.1,8,9, and Thomas J. Hund,Ph.D.1,3,8

1The Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University MedicalCenter, Columbus, OH 43210, USA.2Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, IA52242, USA.3Department of Biomedical Engineering, College of Engineering, The Ohio State University,Columbus, OH 43210, USA.4Department of Mathematics, University of Iowa, Iowa City, IA 52242, USA.5Division of Medicinal Chemistry and Pharmocognosy, College of Pharmacy, The Ohio StateUniversity, Columbus, OH 43210, USA.6Department of Molecular Physiology and Biophysics, University of Iowa Carver College ofMedicine, Iowa City, IA 52242, USA.7Department of Pharmacology, Center for Molecular Therapeutics, Columbia University, NewYork, NY 10032, USA.8Department of Internal Medicine, The Ohio State University Medical Center, Columbus, OH43210, USA.9Department of Physiology & Cell Biology, The Ohio State University Medical Center, Columbus,OH 43210, USA.

AbstractBackground—Human gene variants affecting ion channel biophysical activity and/or membranelocalization are linked with potentially fatal cardiac arrhythmias. However, the mechanism formany 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-

Address correspondence to: Thomas J. Hund, Ph.D. The Dorothy M. Davis Heart and Lung Research Institute The Ohio StateUniversity Medical Center 473 W. 12th Avenue Columbus, OH 43210 [email protected] P: 614 292-0755; F: 614 247-7799.*These authors contributed equally.

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

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NIH Public AccessAuthor ManuscriptCirculation. Author manuscript; available in PMC 2013 October 29.

Published in final edited form as:Circulation. 2012 October 23; 126(17): . doi:10.1161/CIRCULATIONAHA.112.105320.

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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 inhuman genetic and acquired disease. We report an unexpected link between a short motif in theNav1.5 DI-DII loop, recently shown to be critical for CaMKII-dependent phosphorylation, andNav1.5 function in monogenic arrhythmia and common heart disease. Experiments in heterologouscells and primary ventricular cardiomyocytes demonstrate that human arrhythmia susceptibilityvariants (A572D and Q573E) alter CaMKII-dependent regulation of Nav1.5 resulting in abnormalchannel activity and cell excitability. In silico analysis reveals that these variants functionallymimic the phosphorylated channel resulting in increased susceptibility to arrhythmia-triggeringafterdepolarizations. Finally, we report that this same motif is aberrantly regulated in a largeanimal model of acquired heart disease and in failing human myocardium.

Conclusions—We identify the mechanism for two human arrhythmia variants that affect Nav1.5channel activity through direct effects on channel post-translational modification. We propose thatthe CaMKII phosphorylation motif in the Nav1.5 DI-DII cytoplasmic loop is a critical nodal pointfor pro-arrhythmic changes to Nav1.5 in congenital and acquired cardiac disease.

Keywordsarrhythmia (mechanisms); calmodulin dependent protein kinase II; heart failure; ion channels;long-QT syndrome; myocardial infarction

IntroductionSince seminal studies of Keating and colleagues,1, 2 gene mutations in select ion channelshave been mechanistically linked to human arrhythmia through their effects on ion channelbiophysics.3 Discovery of these mutations has not only provided new insight into cellularmechanisms for human disease, but has also advanced our fundamental understanding of ionchannel structure/function and cellular cardiology. Subsequently, a second class of humanarrhythmia mutations has been identified in genes encoding ion channel accessory proteins(e.g. adapter and scaffolding proteins, channel subunits, chaperones) rather than ion channelalpha subunits.4-11 These mutations cause disease by altering channel targeting and/orbiophysical activity and highlight the importance of proper channel localization withinspecific cellular domains for normal heart function. Despite major inroads, the mechanisticlink between specific molecular defects, channel dysfunction and arrhythmias associatedwith many human arrhythmia variants remains elusive. Protein phosphorylation has evolvedas 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 properregulation of key ion channels, transporters and receptors. Importantly, alterations in post-translational modification of membrane proteins are associated with increased susceptibilityto congenital arrhythmia and common causes of acquired arrhythmia, including heartfailure.12-15 Here we identify the mechanism for two human cardiac arrhythmiasusceptibility 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 amongthe first ion channels to be linked to a specific congenital cardiac arrhythmia.2, 16 Almosttwo decades of research has identified hundreds of human gene variants in SCN5A linked tovarious forms of cardiac arrhythmia.17, 18 Nav1.5 dysfunction has also been identified incommon forms of acquired heart disease (e.g. heart failure and myocardial infarction) whereslow conduction and/or altered repolarization plays an important role in arrhythmia andsudden death.19 We recently demonstrated that the multifunctional Ca2+/calmodulin-

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dependent protein kinase II (CaMKII) directly phosphorylates Nav1.5 at residue S571 todecrease channel availability and enhance persistent (late) current leading to increasedsusceptibility to afterdepolarizations.20

A screen of identified human arrhythmia variants in SCN5A yielded two variants in theNav1.5 DI-DII loop whose mechanism was unsolved (A572D and Q573E). Here wedemonstrate that these variants are localized to the CaMKII phosphorylation motif of Nav1.5and alter functional regulation of Nav1.5. We also evaluate the role of the CaMKIIphosphorylation domain of Nav1.5 in a large animal model of acquired heart disease and infailing human hearts and identify significant CaMKII-dependent changes in post-translational modification of Nav1.5 at S571 in diseased hearts. Based on these findings, wepropose that S571 in the Nav1.5 DI-DII cytoplasmic loop serves as a critical node forregulating channel function in diverse forms of cardiac disease associated with arrhythmiasand sudden death.

MethodsMolecular biology

Nav1.5 α-subunit cDNA was engineered in-frame into pIRES2-EGFP (Clontech). Nav1.5arrhythmia variant and TTX-sensitive (C373Y) constructs were generated by Quikchangemethod using WT Nav1.5 as template. Vectors were completely sequenced. Nav1.5constructs were co-transfected with murine pcDNA3.1 T287D constitutively activeCaMKIIδ20 or empty vector into HEK and primary myocytes using X-tremeGENE 9(Roche). For HEK experiments, Nav1.5 β-subunit (pcDNA3.1 hβ1) was co-transfected withthe Nav1.5 α-subunit. To verify successful co-transfection of CaMKII with channelconstructs, Na+ current was measured with and without competitive CaMKII inhibitor,autocamtide-2-related inhibitory peptide (AIP, AnaSpec), in the pipette.

ElectrophysiologyElectrophysiological recordings were obtained from GFP-positive cells. Whole cell sodiumcurrents were measured using standard protocols as described in detail.20-22 Actionpotentials (APs) were recorded using perforated (amphotericin B) patch-clamp technique atphysiological temperature with pacing frequency of 1 Hz. Detailed electrophysiologicalprotocols and conditions are provided in online-only Supplemental Material.

Computational modelTransmembrane currents and ion concentration changes are described by a well-validatedmodel 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 variantNav1.5 may be found in online-only Supplemental Material (Supplemental Tables I, II, andIII).

Electrostatic potential molecular modelingNav1.5 loop motif (W565-S577) structures were built and energy minimized using Amber.27

Electrostatic potentials were computed by the Delphi finite-difference Poisson-Boltzmannsolver28 and mapped to molecular surfaces of W565-S577 containing the CaMKIIphosphorylation site.

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Experimental model of myocardial infarction and immunoblottingMyocardial infarction (MI) was produced in canines by total coronary artery occlusion, asdescribed previously.29, 30 Ventricular lysates were prepared and analyzed by SDS-PAGE asdescribed.20, 30 Immunoblotting was performed using validated affinity-purified antibodiesto phospho-Nav1.5(S571) or total Nav1.5.20 Slight differences in protein loading werecorrected using an internal control standard (rabbit polyclonal antibody to actin (SantaCruz)). This investigation conforms to the Guide for the Care and Use of LaboratoryAnimals published by the National Institutes of Health (Pub. No. 85-23,1996).

Human tissue samplesLeft ventricular tissue was obtained from explanted hearts of patients undergoing orthotopicheart transplantation through The Cooperative Human Tissue Network: MidwesternDivision at The Ohio State University. Approval for use of human subjects was obtainedfrom the Institutional Review Board of The Ohio State University. Left ventricular tissuefrom healthy donor hearts not suitable for transplantation (subclinical atherosclerosis, age,no matching recipients) was obtained through the Iowa Donors Network and the NationalDisease Research Interchange. The investigation conforms with the principles outlined inthe Declaration of Helsinki. Age and sex were the only identifying information acquiredfrom tissue providers and the Iowa Human Subjects Committee deemed that informedconsent was not required.

Adult cell isolation and pacingIsolated ventricular myocytes from WT and AC3-I adult mice (2-3 mos) were plated onto a6-well tray precoated with laminin (Invitrogen). Cells were pretreated for 30 min with thephosphatase inhibitor okadaic acid (2 μM) and paced for 10 min at 2 Hz using the C-pacemultichannel stimulator (Ionoptix). 100 nM isoproterenol was added immediately prior toonset of pacing. A subset of cells was pretreated for 30 min before onset of pacing with theCaMKII inhibitor KN-93 (10 μM). Cell lysates were analyzed by SDS-PAGE andimmunoblotting. Slight differences in protein loading were corrected using monoclonal anti-GAPDH (Fitzgerald) as an internal control standard.

StatisticsP 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 was usedfor post-hoc testing (SigmaPlot 12.0). If the data distribution failed normality tests using theShapiro-Wilk test, rank-based ANOVA and Dunn’s multiple-comparisons test wereperformed. The null hypothesis was rejected for P<0.05.

Additional methods are provided in online only Data Supplement.

ResultsHuman Nav1.5 arrhythmia variants proximate to CaMKII phosphorylation site disruptchannel regulation

We recently identified Nav1.5 S571 in the DI-DII loop as a target for CaMKII-mediatedphosphorylation and key regulatory point for multiple Nav properties, including channelavailability, recovery from inactivation, and late current.20 Our findings suggested thatvariation in this regulatory region could potentially result in significant cardiac dysfunctionin vivo. As a first step in evaluating a link between the CaMKII regulatory motif anddisease, we analyzed this region for potential human cardiovascular disease variants andidentified two variants proximate to the CaMKII regulatory motif: A572D and Q573E

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(Figure 1).31-35 Q573E was originally identified in a genetic screen of LQTS probands withautosomal dominant Romano Ward syndrome.33 The A572D variant was initiallycharacterized in a proband with Romano-Ward LQTS, later found in larger arrhythmiacohorts, 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 (neutralto negative) at residues immediately adjacent to the CaMKII phosphorylation site S571.Thus, while not involving direct changes to S571, we hypothesized that these proximatevariants alter CaMKII-dependent modulation of Nav1.5 resulting in altered susceptibility topro-arrhythmic phenotypes.

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 alterNav function and/or CaMKII regulation, we measured Nav current (INa) from human full-length GFP-WT and variant channels (expressed with the hβ1 subunit) in HEK cells +/−constitutively active CaMKIIδ-T287D (Figure 2). A572D and Q573E variants showedincreased late current (measured as current amplitude 50 ms after peak) at baselinecompared to WT, similar to S571E (phospho-mimetic) and WT+CaMKIIδ (Figure 2A-B).The Nav blocker ranolazine (10μM, specific for late current44,45) normalized late current forWT, A572D and Q573E variants with CaMKIIδ(T287D) (Figure 2A, P=NS vs. WT, n=5,not shown).37, 38 A572D and Q573E variants also showed a leftward (hyperpolarizing) shiftin Nav1.5 steady-state inactivation and slowing of recovery from inactivation at baselinecompared to WT, similar to S571E and WT+CaMKIIδ(T287D) (Figure 2C-H). In contrast,S571A (phospho-resistant) INa properties were not different from WT at baseline (Figure2B,E,H). Immunoblot analysis showed similar expression of WT and human arrhythmiavariants expressed in HEK cells (Supplemental Figure 1 in online-only SupplementalMaterial). Changes in Nav properties observed in WT+CaMKIIδ(T287D) were prevented bythe CaMKII peptide inhibitor AIP (10 μM) (Supplemental Figure 2 in online-onlySupplemental Material). These data indicate that A572D and Q573E human variantsresulting in charge substitution immediately juxtaposed to the CaMKII phosphorylation sitemimic CaMKII phosphorylation (A572D>Q573E) and suggest a pro-arrhythmic mechanismassociated with these human variants.

Human variants alter cardiomyocyte membrane excitabilityWe next determined whether phospho-mimetic A572D and Q573E mutant channelsproduced abnormal cell excitability in primary cardiomyocytes. To express WT and mutantchannels in cardiomyocytes and distinguish from endogenous channels, we createdtetrodotoxin (TTX) sensitive constructs containing a point mutation (C373Y) at a criticalaromatic residue where TTX binds. The C373Y mutation increases TTX sensitivity of Navby almost three orders of magnitude.39 Ventricular myocytes were transfected with GFP-expressing TTX-sensitive WT and mutant constructs (Figure 3). INa was measured fromGFP-positive cells 24 hours following Nav channel expression (Figure 3). Consistent withfindings in HEK cells, A572- and Q573-expressing cardiomyocytes showed increased latecurrent compared to WT, which was blocked by 10 μM ranolazine (Figure 3A-B).Furthermore, a significant leftward shift in steady-state inactivation was measured in A572Dand Q573E variants compared to WT (Figure 3C-D). Differences in endogenous Navfunction were eliminated by low dose (10 nM) TTX sufficient to block TTX-sensitiveNav1.5 channels (Figure 3A,B,D-F). Peak current was comparable in transfected cells atbaseline 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 (Figure3E-F). These data support findings in HEK cells that A572D and Q573E variants act in aphospho-mimetic manner.

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Having validated successful expression of TTX-sensitive Nav channels in primarycardiomyocytes, we measured APs in cardiomyocytes 24 hours following expression of WTand variant constructs (Figure 4). A572D- and Q573-expressing cardiomyocytes showedsignificant prolongation of APD90 compared to WT or vehicle, consistent with the clinicalphenotype of QT prolongation32, 33 (Figure 4A and B). Furthermore, afterdepolarizationswere observed in A572D-expressing myocytes (2 out of 9 cells, Figure 4A, red arrow).Ranolazine (10μM) or low dose (10 nM) TTX eliminated differences in late current andAPD90 between Q573E, A572D and WT-expressing myocytes and eliminatedafterdepolarizations in A572D-expressing cells (Figure 4A-B). These data indicate thatA572D and Q573E variants mimic channel phosphorylation and delay AP repolarization byincreasing late INa in cardiomyocytes.

Computational model of phospho-mimetic Nav1.5 variantsTo determine whether measured differences in Nav function are responsible for APprolongation in A572D- and Q573E-expressing myocytes and to predictelectrophysiological consequences in human, we performed computational modeling usingdetailed Markov models of WT and variants integrated into a computer model of the humanventricular AP23 (Figure 5). State rate transitions in a Markov model of Nav1.524-26 weredetermined for WT, A572D and Q573E channels based on our electrophysiologicalmeasurements (parameters provided in online-only Supplemental Material, SupplementalTable 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 Navfunction and cell excitability (Figure 5D). Consistent with experiments (Figure 2-4),phospho-mimetic A572D and Q573E human variants were associated with increased late INaand prolonged APD (Figure 5E-F). Furthermore, slow pacing (cycle length = 4,000 ms)unmasked afterdepolarizations in variant-expressing cells (Figure 5G-H). These resultsindicate that measured changes in Nav function are sufficient to produce APD prolongationand afterdepolarizations in A572D- and Q573E-expressing cells.

Finally, we simulated effects of ranolazine (10μM) on APs from WT and A572-expressingcardiomyocytes. Ranolazine was assumed to preferentially block the Nav1.5 open state withon- and off-rates of 8.2 μM−1s−1 and 22 s−1, respectively (Figure 5A).40, 41 Consistent withexperimental observations37, ranolazine preferentially blocked late INa (Figure 5H).Moreover, in agreement with our myocyte experiments, ranolazine normalized APD invariant-expressing cells and eliminated afterdepolarizations at slow pacing (Figure 5G).These data support our hypothesis that A572D and Q573E variants prolong APD byincreasing late INa and indicate that late INa blockers (ranolazine) may be an effectivetherapy for human patients with Nav variants that mimic CaMKII phosphorylation (A572Dand Q573E).

CaMKII-dependent Nav1.5 phosphorylation is dysregulated in acquired heart diseaseOur findings suggest that the CaMKII phosphorylation motif plays an important role inregulating channel activity and cell membrane excitability. Similar defects in Nav channelfunction (e.g. increased late current) are observed in heart failure.42 Therefore, wehypothesized that this same site contributes to cellular phenotypes associated with commondisease. We evaluated levels of Nav1.5 phosphorylation at S571 in a large animal model ofcardiac arrhythmias following myocardial infarction (MI), where abnormalities in CaMKIIand Nav1.5 activities have previously been identified.29, 43-45 Immunoblot analysis revealedsignificantly elevated levels of phospho-Nav1.5 (S571) (but not total Nav1.5) in the infarctborder zone 5 days post-occlusion (Figure 6A-B) without any change in remote regions fromnormal noninfarcted or five-day post-occlusion hearts (Figure 6A-B). These data areconsistent with previous reports of activated CaMKII and abnormal Nav activity in the

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canine infarct border zone.29, 43-45 Furthermore, these data identify a potential mechanisticlink between increased CaMKII activity and Nav dysfunction following MI.

Finally, to determine whether CaMKII phosphorylation of Nav1.5 at S571 is involved inhuman disease, we analyzed left ventricular (LV) samples from human patients with non-ischemic heart failure (HF) (Figure 6C-E). We observed significantly increased levels ofphospho-CaMKII(T287) in HF samples (Figure 6C,D), consistent with previous reports ofincreased CaMKII activity.46 In parallel, we measured a small but significant increase inphospho-Nav1.5(S571) in HF samples compared to normals (Figure 6C,E) without anysignificant difference in total Nav1.5 (Figure 6C,E). Thus, dysregulation of Nav1.5 at S571is found in a large animal model of MI and in human HF, supporting the notion of S571 as anodal point for electrical dysfunction in common disease.

While our data are consistent with a role for CaMKII in increased phospho-Nav1.5(S571) indisease, multiple signaling cascades are activated in the diseased heart. Therefore, toestablish a causal link between CaMKII and dysregulation of Nav1.5 at S571 in disease, wedetermined phospho-Nav1.5(S571) levels in isolated WT ventricular murine myocytessubjected to pacing (2 Hz) in the presence of isoproterenol and okadaic acid to simulatestress conditions where CaMKII activity is elevated47 (Figure 7). Myocytes from transgenicmice expressing a CaMKII inhibitory peptide (AC3-I)48 were subjected to the same protocolto determine the role of CaMKII in Nav1.5 phosphorylation during stress. Increased levels ofphospho-CaMKII(T287) and phospho-Nav1.5(S571) were observed in WT paced cellstreated 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 inhibitorKN-93 (10 μM) 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 animportant molecular link between CaMKII overactivity and abnormal Nav function in heartdisease, including human HF.

DiscussionThe 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 beenassociated with abnormal cell excitability and arrhythmia in heart disease including humanHF. Here we use a variety of novel reagents and computer models to demonstrate a linkbetween defects in CaMKII-dependent phosphorylation of Nav1.5 and both congenital andacquired human disease. We show that two previously identified human SCN5A arrhythmiavariants are localized to the CaMKII phosphorylation motif of the Nav1.5 DI-DII loop.Experimental studies in heterologous cells demonstrate that these variants, resulting innegative charge substitution adjacent to the phosphorylation site, partially recapitulateCaMKII phosphorylation effects on channel availability, recovery and late current.Furthermore, these defects in the CaMKII phosphorylation motif alter the channel’sresponse to active CaMKII. Expression of these variants in myocytes and computersimulations reveal delayed AP repolarization and afterdepolarizations due to increased lateINa. Computational modeling reveals that the isolated (13-mer) DI-DII domainscorresponding to A572D and Q573E variants possess very similar electrostatic andstructural features to the S571-phosphorylated channel, suggesting that they may havesimilar binding interactions with downstream partners including regions of the channel itself(e.g. channel pore) (Supplemental Figure 3 in online-only Supplemental Material). Whilethese simulations do not account for the true three-dimensional structure of the intact DI-DIIloop, they support our experimental data indicating that human variants A572D and Q573E

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confer arrhythmia susceptibility by structurally and functionally mimicking thephosphorylated channel. Finally, we report dysregulation of Nav1.5 at S571 in commonforms of heart disease, including human HF, suggesting a common molecular link betweenknown defects in CaMKII activity, Nav dysfunction and arrhythmias. Future studies areneeded to determine whether targeting this motif (genetically or pharmacologically) will beeffective in preventing arrhythmia and/or progression of disease.

Remarkable progress has been made in understanding links between specific congenitalmolecular defects and human disease. Structure-function studies on ion channels coupledwith expression studies in heterologous cells have greatly advanced our understanding of notonly monogenic disease but also more common acquired disease. A classic example of ourability to link defects at the molecular level to a clinical phenotype comes from LQTSmutations found in the DIII-DIV linker or C-terminal region that increase late current byinterfering with rapid channel inactivation (e.g. ΔKPQ). Here, we propose that A572D andQ573E human variants belong to an emerging class of atypical ion channel mutations thatproduce abnormal cell excitability and arrhythmia by affecting post-translationalmodification.5, 13, 49 Previous studies have yielded conflicting results regarding thefunctional status of the A572D variant.35, 36 Expression studies in oocytes report fasterrecovery from inactivation with no change in steady-state availability (late current was notassessed).35 Other studies have found decreased availability, altered recovery and increasedlate 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 andtherefore 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 inisolation and may depend on environmental cues/cofactors. Also, it is interesting to considerthe possibility that A572D may be linked with diseases other than inherited arrhythmiasyndromes (cardiomyopathy, heart failure). Similarly, while our data establish a role forS571 in CaMKII-dependent regulation of Nav1.5, the precise mechanism is likely morecomplicated with potential contributions from multiple nearby sites.50 It will be importantgoing forward to determine exactly how phosphorylation/mutation events in this DI-DII “hotspot” conspire to regulate channel activity.

Interestingly, despite the potentially lethal cellular phenotype associated with delayed APrepolarization observed in our myocyte studies and computer simulations, the allelefrequency for at least one of the variants (A572D) is relatively high (~0.5%). This raises thequestion, why are these variants not associated with a more severe clinical phenotype? Onepossibility suggested by our data is that although the variants mimic the phosphorylatedchannel, they also make the channel resistant to further regulation by endogenous CaMKII(see Figure 2) thereby preventing exacerbation of the phenotype by multiple factors knownto increase CaMKII activity (e.g. β-adrenergic stimulation). Furthermore, the inherent natureof 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 thiscompound regulation may be a cellular phenotype less problematic than either one alone.Alternatively, allelic imbalance and variable expression of variant Nav1.5 channels mayproduce heterogeneity in Nav function and clinical phenotype.51 Finally, and perhaps mostimportantly, our findings predict that individuals harboring these variants will in partresemble a much larger population of HF patients (display S571 hyper-phosphorylation,Figure 6), suggesting that while this modification may increase susceptibility to arrhythmiait is not 100% penetrant.

While our data support a critical role for CaMKII in dysregulation of Nav1.5, we cannotexclude contributions of other kinase pathways to this regulatory site in disease. In fact, weexpect that while CaMKII is the primary regulatory kinase for this site, other kinases also

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contribute to phosphorylation at S571. Furthermore, phosphorylation of other sites, inaddition to S571, likely contributes to the disease phenotype, as Nav1.5 is directly regulatedby both PKC and PKA, whose activities are altered in disease.19 This may be true in humanHF, in particular, where we measured a significant but relatively modest change inphosphorylation of S571. While our data demonstrate elevated Nav1.5 pS571 in forms ofcanine and human disease, it will be important to establish the relative contribution of thesechanges to dysfunction across disease pathologies. Finally, oxidation and other post-translational modifications may regulate channel activity through direct (e.g. lipidperoxidation) or indirect (e.g. PKC-dependent phosphorylation) pathways. Thus, futurestudies will be important to determine the relative contribution of phosphorylation of Nav1.5at S571 to dysfunction in diseased heart, as well as upstream signals (e.g. β-adrenergicreceptor stimulation, angiotensin II, reactive oxygen species, Ca2+).

In summary, we identified the molecular mechanism for two human Nav1.5 variantslocalized to the CaMKII regulatory motif in the Nav1.5 DI-DII loop and provide data tosupport that a similar defect is present in a large animal model of ischemic heart disease andin human HF. These studies add to mounting evidence that defects in local signaling andprotein post-translational modification help define the disease phenotype associated with abroad range of cardiac arrhythmia syndromes. It will be interesting in the future to determinewhether therapies targeting CaMKII-dependent regulation of Nav1.5 at S571 will beeffective in reducing arrhythmia burden in these patients.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsThis 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 ScienceFoundation Grant DMS-1022466 (CCM), the Gilead Sciences Research Scholars Program in CardiovascularDisease (TJH), Saving Tiny Hearts Society (PJM), and the Fondation Leducq Transatlantic Alliance for CaMKIISignaling (08CVD01, MEA, PJM).

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Figure 1. Human arrhythmia variants proximate to Nav1.5 CaMKII-phosphorylation node(A) Schematic illustrating the spectrin-based signaling complex at the cardiomyocyteintercalated disc for regulation of Nav1.5 and cell membrane excitability. The actin-associated polypeptide βIV-spectrin complexes CaMKII with Nav1.5 (via ankyrin-G) tofacilitate direct phosphorylation at S571 in the Nav DI-DII linker. (B) Human variantsadjacent to the Nav1.5 phosphorylation site (A572D and Q573E) have been linked to cardiacarrhythmia (long-QT syndrome).

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Figure 2. Human arrhythmia Nav1.5 variants adjacent to CaMKII phosphorylation domainmimic channel phosphorylation(A) Nav current (INa) traces measured from HEK cells expressing WT, A572D or Q573Echannels (co-expressed with hβ1 beta subunit) +/− 10 μM ranolazine to block late INa. Navfunction was also measured in S571A- and S571E-expressing cells as negative (phospho-resistant) and positive (phospho-mimetic) controls, respectively. Dashed line indicatesmeasurement time of late current (50 ms after peak). (B) Summary data for late currentmeasured at 50 ms after peak and expressed as percentage of peak (**P<0.005, ***P<0.001vs. WT at baseline; n=5 except Q573E+CaMKII (n=6)), (C-D) steady-state inactivationcurves (*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) Navrecovery from inactivation curves (*P<0.05 vs. A572D and Q573E, n=5), and (H) recovery

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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 increasedlate 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, theS571A phospho-resistant mutant (+/− CaMKII) resembles WT at baseline (withoutCaMKII).

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Figure 3. Expression of human arrhythmia variants in cardiomyocytesNav current (INa) was measured in 3-day old neonatal mouse cardiomyocytes transfectedwith vehicle, WT, A572D or Q573E channels engineered to have increased sensitivity toTTX (C373Y).39 (A) INa traces, (B) late INa amplitude +/−10 μM ranolazine (**P<0.005and ***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) 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 exogenouscurrent. (E) Current-voltage relationships were also measured +/− low dose TTX to verify

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similar expression levels for WT and human arrhythmia variants. (*P<0.0.05 control vs. 10nM 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)

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Figure 4. Human arrhythmia variants adjacent to CaMKII phosphorylation site delay APrepolarization when expressed in cardiomyocytesWT, A572D and Q573E channels engineered to have increased sensitivity to TTX(C373Y)39 were expressed in neonatal mouse cardiomyocytes. (A) APs and (B) APD90 fromvehicle, WT, A572D and Q573E-expressing cardiomyocytes +/− 10 nM TTX (to blockexogenous Na+ current) (*P<0.05, **P<0.005 and ***P<0.001 vs. control (no TTX); n=12for control, n=5 for ranolazine and TTX). A572D and Q573E significantly increase APD90compared to WT and increase the likelihood of afterdepolarizations (red arrow in A). Lowdose TTX or 10 μM ranolazine eliminates differences in APD90. Myocytes were paced at1Hz.

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Figure 5. Computational model to determine role of altered Nav function in AP prolongation forhuman arrhythmia variants(A) Markov model to simulate Nav function including transitions between multipleinactivated (red), closed (blue), open (green), and ranolazine-bound (gray) states. Parameterestimation results comparing experimentally measured (black) and simulated (red) valuesfor (B) steady-state inactivation and (C) late current. (D) Schematic of mathematical modelof human ventricular AP used to determine effects of mutant Nav function on cardiacrepolarization. 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

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A572D and Q573E (not shown) variants. Simulating block of late INa with 10 μMranolazine (C) reduced late INa and afterdepolarizations (gray lines in A and B) consistentwith experiments.

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Figure 6. Increased phosphorylation of Nav1.5 at S571 in canine model of myocardial infarctionand human heart failure(A) Representative immunoblots and (B) densitometric measurements (normalized to actinand 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) Representativeimmunoblots and (D) densitometric measurements of total CaMKII and phospho-CaMKII(T287) and (E) total Nav1.5, phospho-Nav1.5 from left ventricular samples ofnormal and failing human hearts (*P<0.05 vs. normal, ***P<0.001 vs. remote; n=7). Fordensitometric measurements, all samples were analyzed on the same gel and normalized to

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corresponding actin levels from same blot. Equal protein loading was ensured by BCA assayand verified by analysis of Coomassie stains of gel.

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Figure 7. CaMKII contributes to increased phosphorylation of Nav1.5 at S571 in stressconditions(A-B) Representative immunoblots from isolated adult (A) WT and (B) AC3-I myocytessubjected to pacing (10 min, 2 Hz) in the presence of isoproterenol (100 nM) and okadaicacid (2 μM) to simulate stress conditions and enhance CaMKII-dependent regulation. Asubset of WT cells were pretreated with CaMKII inhibitors KN-93 (10μM) for 30 min priorto pacing. (C-D) Densitometric measurements (normalized to GAPDH and expressedrelative 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 andKN-93) prevents increased phosphorylation of Nav1.5 at S571 observed in stressed WTmyocytes.

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olha m. koval, ph.d. nih public access jedidiah s. snyder ...€¦ · the dorothy m. davis heart...

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