Stretch-Sensitive KCNQ1 Mutation

9
Stretch-Sensitive KCNQ1 Mutation A Link Between Genetic and Environmental Factors in the Pathogenesis of Atrial Fibrillation? Robyn Otway, PHD,* Jamie I. Vandenberg, MB, BS, PHD,†‡ Guanglan Guo, PHD,* Anthony Varghese, PHD,§ M. Leticia Castro, BMEDSC(HONS),* Jian Liu, PHD,* JingTing Zhao, PHD,† Jane A. Bursill, BTC,† Ken R. Wyse, BSC,† Haley Crotty, B BIOMED SC,* Olivia Baddeley, MSC,* Bruce Walker, MB, BS, PHD, Dennis Kuchar, MD, FACC, Charles Thorburn, MB, CHB, Diane Fatkin, MD*‡ Darlinghurst and Kensington, Australia; and River Falls, Wisconsin Objectives This study sought to evaluate mutations in genes encoding the slow component of the cardiac delayed rectifier K current (I Ks ) channel in familial atrial fibrillation (AF). Background Although AF can have a genetic etiology, links between inherited gene defects and acquired factors such as atrial stretch have not been explored. Methods Mutation screening of the KCNQ1, KCNE1, KCNE2, and KCNE3 genes was performed in 50 families with AF. The effects of mutant protein on cardiac I Ks activation were evaluated using electrophysiological studies and human atrial action potential modeling. Results One missense KCNQ1 mutation, R14C, was identified in 1 family with a high prevalence of hypertension. Atrial fibrillation was present only in older individuals who had developed atrial dilation and who were genotype posi- tive. Patch-clamp studies of wild-type or R14C KCNQ1 expressed with KCNE1 in CHO cells showed no statistically significant differences between wild-type and mutant channel kinetics at baseline, or after activation of adenyl- ate cyclase with forskolin. After exposure to hypotonic solution to elicit cell swelling/stretch, mutant channels showed a marked increase in current, a leftward shift in the voltage dependence of activation, altered channel kinetics, and shortening of the modeled atrial action potential duration. Conclusions These data suggest that the R14C KCNQ1 mutation alone is insufficient to cause AF. Rather, we suggest a model in which a “second hit”, such as an environmental factor like hypertension, which promotes atrial stretch and thereby unmasks an inherited defect in ion channel kinetics (the “first hit”), is required for AF to be mani- fested. Such a model would also account for the age-related increase in AF development. (J Am Coll Cardiol 2007;49:578–86) © 2007 by the American College of Cardiology Foundation Atrial fibrillation (AF) is a cardiac arrhythmia characterized by rapid and irregular activation of the atrium. Loss of coordinated atrial contraction results in blood stasis and reduced ventricular filling. Consequently, AF is a major risk factor for thromboembolic stroke and heart failure. The prevalence of AF increases with age, ranging from 1% in young adults to nearly 10% of those older than 80 years of age (1). Although considerable advances have been made in pharmacologic and nonpharmacologic approaches to ther- apy, morbidity, and mortality associated with AF remain high (2). Despite the medical and economic importance of this condition, very little is known about its underlying etiology. Atrial fibrillation occurs generally as a complication of cardiac and systemic diseases, such as hypertension, coro- nary artery disease, valvular heart disease, and cardiomyop- athies. Increased atrial pressure and/or volume with subse- quent chamber dilation and activation of stretch-sensitive signaling pathways has been thought to be integral to AF development. Once AF is established, continued electrical From the *Sr. Bernice Research Program in Inherited Heart Diseases and the †Mark Cowley Lidwill Program in Cardiac Electrophysiology and Biophysics, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia; ‡Faculties of Medicine and Life Sciences, University of New South Wales, Kensington, New South Wales, Australia; §Department of Computer Science, University of Wisconsin at River Falls, River Falls, Wisconsin; and the Cardiology Department, St. Vincent’s Hospital, Darlinghurst, New South Wales, Australia. Supported by the Sylvia and Charles Viertel Charitable Foundation, Melbourne, Victoria, Australia; the National Health and Medical Research Council, Canberra, Australian Capital Territory, Australia; and St. Vincent’s Clinic Foundation, Sydney, New South Wales, Australia. Drs. Otway and Vandenberg contributed equally to this work. Manuscript received July 14, 2006; revised manuscript received September 5, 2006, accepted September 27, 2006. Journal of the American College of Cardiology Vol. 49, No. 5, 2007 © 2007 by the American College of Cardiology Foundation ISSN 0735-1097/07/$32.00 Published by Elsevier Inc. doi:10.1016/j.jacc.2006.09.044

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Journal of the American College of Cardiology Vol. 49, No. 5, 2007© 2007 by the American College of Cardiology Foundation ISSN 0735-1097/07/$32.00P

Stretch-Sensitive KCNQ1 MutationA Link Between Genetic and EnvironmentalFactors in the Pathogenesis of Atrial Fibrillation?

Robyn Otway, PHD,* Jamie I. Vandenberg, MB, BS, PHD,†‡ Guanglan Guo, PHD,*Anthony Varghese, PHD,§ M. Leticia Castro, BMEDSC(HONS),* Jian Liu, PHD,* JingTing Zhao, PHD,†Jane A. Bursill, BTC,† Ken R. Wyse, BSC,† Haley Crotty, B BIOMED SC,* Olivia Baddeley, MSC,*Bruce Walker, MB, BS, PHD,� Dennis Kuchar, MD, FACC,� Charles Thorburn, MB, CHB,�Diane Fatkin, MD*‡�

Darlinghurst and Kensington, Australia; and River Falls, Wisconsin

Objectives This study sought to evaluate mutations in genes encoding the slow component of the cardiac delayed rectifierK� current (IKs) channel in familial atrial fibrillation (AF).

Background Although AF can have a genetic etiology, links between inherited gene defects and acquired factors such asatrial stretch have not been explored.

Methods Mutation screening of the KCNQ1, KCNE1, KCNE2, and KCNE3 genes was performed in 50 families with AF. Theeffects of mutant protein on cardiac IKs activation were evaluated using electrophysiological studies and humanatrial action potential modeling.

Results One missense KCNQ1 mutation, R14C, was identified in 1 family with a high prevalence of hypertension. Atrialfibrillation was present only in older individuals who had developed atrial dilation and who were genotype posi-tive. Patch-clamp studies of wild-type or R14C KCNQ1 expressed with KCNE1 in CHO cells showed no statisticallysignificant differences between wild-type and mutant channel kinetics at baseline, or after activation of adenyl-ate cyclase with forskolin. After exposure to hypotonic solution to elicit cell swelling/stretch, mutant channelsshowed a marked increase in current, a leftward shift in the voltage dependence of activation, altered channelkinetics, and shortening of the modeled atrial action potential duration.

Conclusions These data suggest that the R14C KCNQ1 mutation alone is insufficient to cause AF. Rather, we suggest amodel in which a “second hit”, such as an environmental factor like hypertension, which promotes atrial stretchand thereby unmasks an inherited defect in ion channel kinetics (the “first hit”), is required for AF to be mani-fested. Such a model would also account for the age-related increase in AF development. (J Am Coll Cardiol2007;49:578–86) © 2007 by the American College of Cardiology Foundation

ublished by Elsevier Inc. doi:10.1016/j.jacc.2006.09.044

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trial fibrillation (AF) is a cardiac arrhythmia characterizedy rapid and irregular activation of the atrium. Loss ofoordinated atrial contraction results in blood stasis andeduced ventricular filling. Consequently, AF is a major risk

rom the *Sr. Bernice Research Program in Inherited Heart Diseases and the †Markowley Lidwill Program in Cardiac Electrophysiology and Biophysics, Victor Changardiac Research Institute, Darlinghurst, New South Wales, Australia; ‡Faculties ofedicine and Life Sciences, University of New South Wales, Kensington, New Southales, Australia; §Department of Computer Science, University of Wisconsin at

iver Falls, River Falls, Wisconsin; and the �Cardiology Department, St. Vincent’sospital, Darlinghurst, New South Wales, Australia. Supported by the Sylvia andharles Viertel Charitable Foundation, Melbourne, Victoria, Australia; the Nationalealth and Medical Research Council, Canberra, Australian Capital Territory,ustralia; and St. Vincent’s Clinic Foundation, Sydney, New South Wales, Australia.rs. Otway and Vandenberg contributed equally to this work.

dManuscript received July 14, 2006; revised manuscript received September 5, 2006,

ccepted September 27, 2006.

actor for thromboembolic stroke and heart failure. Therevalence of AF increases with age, ranging from �1% inoung adults to nearly 10% of those older than 80 years ofge (1). Although considerable advances have been made inharmacologic and nonpharmacologic approaches to ther-py, morbidity, and mortality associated with AF remainigh (2).Despite the medical and economic importance of this

ondition, very little is known about its underlying etiology.trial fibrillation occurs generally as a complication of

ardiac and systemic diseases, such as hypertension, coro-ary artery disease, valvular heart disease, and cardiomyop-thies. Increased atrial pressure and/or volume with subse-uent chamber dilation and activation of stretch-sensitiveignaling pathways has been thought to be integral to AF

evelopment. Once AF is established, continued electrical

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579JACC Vol. 49, No. 5, 2007 Otway et al.February 6, 2007:578–86 Stretch-Sensitive Mutations in AF

nd structural remodeling within the atria promote arrhyth-ia maintenance (3,4). Atrial fibrillation can also occur in

he absence of precipitating disorders or cardiac structuralhanges (“lone” AF), which suggests that primary electro-hysiological defects could also be an important risk factor.The role of inherited gene defects in the pathogenesis of

F has only recently begun to be appreciated (5,6). KCNQ1as the first disease gene identified for familial AF, with a

ingle missense mutation, S140G, found in 1 family (7).CNQ1 encodes the pore-forming alpha subunit of the

hannel that conducts the slow component of the delayedectifier K� current (IKs), which contributes to repolariza-ion of the cardiac action potential. KCNQ1 can interactith alternative beta subunits, KCNE1, KCNE2, andCNE3. Mutations in KCNQ1 and its binding partnersave previously been associated with the long QT syn-rome, a disorder complicated by ventricular tachyarrhyth-ias and sudden death (8). In contrast to the long-QT–

ausing KCNQ1 mutations that have loss-of-functionffects, the S140G KCNQ1 mutation was shown to induce

gain of function of cardiac IKs channels. The S140Gariant is the only KCNQ1 mutation reported to date indult-onset familial AF. Recently, a de novo KCNQ1utation was shown to cause AF in utero (9). A gain-of-

unction mutation, R27C, in the KCNE2 gene has beendentified in 2 unrelated kindreds with AF (10).

To further define the relative importance of perturbationf cardiac IKs channel function in the pathogenesis ofamilial AF, we performed genetic analyses of the KCNQ1,CNE1, KCNE2, and KCNE3 genes in a cohort of 50indreds. Functional and computational modeling studiesere performed to evaluate the consequences of mutantrotein on IKs channel activity.

ethods

linical evaluation. Informed, written consent was ob-ained for participation in a protocol approved by thenstitutional Human Research Ethics Committee. Studyubjects were evaluated by history and physical examination,2-lead electrocardiography, and transthoracic echocardiog-aphy. Familial AF was suspected when 2 or more first-egree family members presented with documented nonval-ular AF. The deoxyribonucleic acid (DNA) was isolatedrom peripheral blood samples using conventionalechniques.

utation screening. Protein-coding sequences of theCNQ1, KCNE1, KCNE2, and KCNE3 genes were ampli-ed by polymerase chain reaction (PCR) from genomicNA. Amplimers were purified and sequenced using theig Dye terminator (version 3.1, Applied Biosystems, Fos-

er City, California) and were analyzed on an ABI PRISM700 DNA Analyzer (UNSW DNA Analysis Facility,ydney, New South Wales, Australia). DNA sequenceariants were confirmed by restriction-enzyme digestion in

amily members and in 100 normal control subjects. w

ell culture. Chinese hamstervary (CHO) cells were co-ransfected with KCNQ1 (kindlyonated by Prof. Jentsch, Uni-ersity of Hamburg, Hamburg,ermany) and KCNE1 in a ratio

f 1:4 using lipofectamine (In-itrogen, Carlsbad, California).n experiments in which the-kinase adaptor protein yotiao

kindly donated by Prof. Scott,regon Health Sciences Univer-

ity, Portland, Oregon) was in-luded, the ratio of constructsas 1:1:4 (yotiao:KCNQ1:CNE1). The KCNE1 was sub-cloned into the pTSV40

ector (Invitrogen), which contains eGFP under a separateromoter, and transfected cells were identified by detectionf eGFP fluorescence (11). The R14C mutation was intro-uced into the KCNQ1 coding sequence using the mega-rimer PCR method (12).atch clamping. Cells were superfused with normal Ty-

ode solution (in mM): 129 NaCl, 5 Na pyruvate, 5 Nacetate, 4 KCl, 1 MgCl2, 1.8 CaCl2, 11.1 glucose, 5-(2-hydroxyethyl)piperazine-1-ethanesulfonic acidHEPES) (titrated to pH 7.4 with NaOH) at 37°C.orskolin was added to the superfusate at a concentration of�M. Cell swelling was induced by reducing the NaCl in

he superfusate from 135 mM to 100 mM (osmolalityeduced from �280 mOsm to �215 mOsm). The patchipette solution contained (in mM): 30 KCl, 110 potassiumspartate, 1 MgCl2, 5 magnesium adenosine triphosphate,0 ethylene glycol tetraacetic acid, 5 HEPES (pH 7.4 withotassium hydroxide). The liquid:liquid junction potential,15 mV, was corrected for in all experiments. Recordingsere made using an Axopatch 200A or Axopatch 200B

mplifier (Axon Instruments, Foster City, California). Ca-acitance current transients were electronically subtracted,nd series resistance compensation was at least 80%. Resid-al uncompensated series resistance was typically �1 M�.xperiments in which currents resulted in voltage errors of5 mV because of residual series resistance were excluded

rom the analysis. Current signals were filtered at 2 kHz andampled at 5 kHz. Acquisition and analysis of data wereerformed using pClamp9 software (Axon Instruments,nion City, California). All summary data are expressed asean � SEM. Statistical comparisons were performed

sing analysis of variance or Student t test.trial cardiac modeling. Two human atrial cardiomyocyteodels (13,14) were modified to include IKs (15). In

ddition, the model of Courtemanche et al. (14) wasodified to stabilize the long-term behavior (16). The

arameters of the IKs current were determined using exper-mental data obtained in CHO cells transfected with WTnd mutant KCNQ1-KCNE1 (Appendix). Each cell model

Abbreviationsand Acronyms

AF � atrial fibrillation

CHO � Chinese hamsterovary

DNA � deoxyribonucleicacid

IKs � slow component ofthe delayed rectifier K�

current

PCR � polymerase chainreaction

WT � wild-type

as paced at 1, 2, and 4 Hz using

1-ms current pulses for

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580 Otway et al. JACC Vol. 49, No. 5, 2007Stretch-Sensitive Mutations in AF February 6, 2007:578–86

,602 s to reach a steady state. A differential-algebraicumerical integration scheme (17) was implemented using��, Windows (Microsoft, Redmond, Washington) andac OS X (Apple, Cupertino, California) software.

esults

utation screening. Mutation screening of the codingequences of the KCNQ1, KCNE1, KCNE2, and KCNE3enes was performed in 50 familial AF probands usingNA sequence analysis. One missense mutation in theCNQ1 gene was identified in the proband of Family D.R.

Fig. 1). This sequence variant, 40C to T, in exon 1A of theCNQ1 gene, alters the amino acid at codon 14 from arginine

o cysteine (R14C). The R14C variant results in loss of an Afe1ite and was independently confirmed in the proband andamily members by restriction enzyme digestion. The sequencehange was not found in 200 control chromosomes and hadot been reported in a compendium of KCNQ1 variants in 744ealthy individuals (18). No mutations were identified in theCNE1, KCNE2, or KCNE3 genes.amily phenotype evaluation. The proband in Family.R., II-4, had been diagnosed with AF at the age of 59

Figure 1 Pedigree of Family D.R.

Solid symbols denote presence of hypertension (left) and/or atrial fibrillation (AF)jects are shown as squares and female subjects as circles. The family proband (aindicated.

linical Phenotype of Living Genotype-Positive Individuals in Family

Table 1 Clinical Phenotype of Living Genotype-Positive Individu

FamilyIdentification AF

Age at Dx*(yrs)

Age at Study(yrs) HT/Ant

II-1 Chronic† 50 90 Yes/

II-4 Chronic‡ 59 83 Yes/

II-5 Chronic§ 77 80 Yes/

III-1 No N/A 56 Yes/

III-2 No N/A 49 Yes/

III-4 No N/A 48 No

III-8 No N/A 44 No

III-10 No N/A 45 No

III-13 No N/A 51 Yes/

III-14 No N/A 49 No

Age at first electrocardiographically documented AF. †Intermittent palpitations for �20 years behronic AF. �Reported as moderately dilated for patient size.

AF � atrial fibrillation; AntiHT Rx � antihypertensive drug therapy (angiotensin-converting enzyme inhibi

VDD � left ventricular end-diastolic diameter; LVFS � left ventricular fractional shortening; LVWT � left

ears (Table 1). A detailed family history showed a highrevalence of hypertension (Fig. 1, Table 1, supplementaryable 1 [Appendix]). Of the 10 individuals with hyperten-

ion, 6 were positive and 4 were negative for the R14Cariant. Atrial fibrillation was present only in those individ-als who were genotype positive and who had atrial dilation.valuation of electrocardiographic recordings showed that

orrected QT intervals were normal for all family memberstudied, with the exception of 1 individual who had a leftundle branch block.unctional studies of the R14C KCNQ1 variant. Ex-ression of wild-type (WT) IKs (WT KCNQ1 � KCNE1)r R14C IKs (R14C KCNQ1 � KCNE1) channels inHO cells resulted in slowly activating currents character-

stic of cardiac IKs channels (Fig. 2A). The peak currentensity measured at the end of the 4-s pulse was similar forhe 2 channels (e.g., 165 � 17 pA/pF at �60 mV for WTKs compared with 172 � 21 pA/pF for R14C IKs). Theoltage for half-activation for WT IKs channels, 28 � 1.4V (n � 13), also was not statistically different from that

or R14C IKs channels, 28 � 1.9 mV (n � 12) (Fig. 2A).imilar results were obtained in Xenopus oocytes (data not

, and striped symbols denote individuals with uncertain phenotype. Male sub-and the presence (�) or absence (�) of the R14C KCNQ1 mutation are

Family D.R.

LVDD(mm)

LVFS(%)

LVWT(mm)

LA(mm)

PRInterval

(ms)QT/QTc

(ms)

53 42 12 52 N/A 410/426

50 41 10 59 N/A 434/434

38 37 7 42� N/A 374/395

45 35 9 31 132 410/433

55 30 8 40 146 412/421

53 37 8 39 148 386/448

46 35 6 35 158 428/413

51 33 9 35 144 376/406

46 30 12 27 120 338/402

57 38 6 36 140 466/461

agnosis of chronic AF. ‡Asymptomatic at time of diagnosis. §Paroxysmal AF documented before

(right)rrow)

D.R.

als in

iHT Rx

Yes

Yes

Yes

Yes

No

No

fore di

tors/beta-blockers); Dx � AF diagnosis; HT � hypertension; LA � left atrium; LV � left ventricular;ventricular wall thickness.

581JACC Vol. 49, No. 5, 2007 Otway et al.February 6, 2007:578–86 Stretch-Sensitive Mutations in AF

Figure 2 Baseline Characteristics of WT and R14C IKs Channels

(A) Typical examples of currents recorded from wild-type (WT) (KCNQ1 � KCNE1) and R14C (KCNQ1-R14C � KCNE1) IKs channels in Chinese hamster ovary (CHO) cells during4-s depolarization steps from a holding potential of �80 mV to test potentials in the range �50 to �70 mV followed by repolarization to �60 mV (voltage protocol shown ininset). Scale bars are 0.5 s and 50 pA/pF. Horizontal dashed line indicates zero current level. Solid and dashed arrows indicate points where currents were measured for cur-rent-voltage relationships (upper panel to right) and tail currents for isochronal activation curves (lower panel on right), respectively. Tail current data were normalized to themaximum current value (Imax) and fitted with a Boltzmann function: I/Imax � [1 � exp((V0.5 � Vt)/k)]�1, where V0.5 is the half-activation voltage, Vt is the test potential, and k isthe slope factor (values for V0.5 and k are summarized in supplementary Table 2 [Appendix]). (B) Typical examples of currents recorded during 4-s depolarization steps to volt-ages in the range 0 to �60 mV (voltage protocol shown in inset). Scale bars as for panel A. IKs channels undergo transitions through multiple pre-open closed states beforefinally opening, as indicated by the sigmoidal shape of activation time courses. Rates of activation were estimated by fitting an exponential function (dashed lines) to the sec-ond half of the activation time course. Lower panel summarizes the time constants for activation at voltages in the range �20 to �60 mV for WT (red) and R14C (blue) IKs

channels. (C) Typical examples of tail currents recorded at voltages in the range �60 mV to �120 mV (voltage protocol shown in inset). Rates of deactivation were obtained byfitting a single exponential to tail current recordings. Lower panel summarizes time constants for deactivation in the range �120 mV to �60 mV for WT (red) and R14C (blue)

IKs channels.

sed�WnRa2R

RpK

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582 Otway et al. JACC Vol. 49, No. 5, 2007Stretch-Sensitive Mutations in AF February 6, 2007:578–86

hown). Rates of activation, estimated by fitting a singlexponential function to the second half of the currents recordeduring depolarization steps to voltages in the range �20 to60 mV, were slightly slower in R14C IKs compared withT IKs channels (Fig. 2B), however, these differences were

ot statistically significant. Similarly, rates of deactivation of14C IKs channels were slightly slower than WT IKs channels,

lthough the differences were not statistically significant (Fig.C). Overall, very little difference was found between WT and14C IKs channels under baseline conditions.To determine whether there might be differences in

14C IKs channel responses to protein kinase A signalingathways, CHO cells were transfected with WT or R14C

Figure 3 Effects of Fsk on WT and R14C IKs Channels

(A) Forskolin (Fsk), 1 �M, resulted in an increase in current amplitude (green) forin current recorded at the end of a 1-s depolarization pulse to �20 mV in both WTdensity measured after 4-s depolarization steps (protocol as shown in Figure 2A) fthe voltage-dependence of activation for WT (red dashed line � control, red boxesBoth shifts were statistically significant (p � 0.05). The Fsk caused an acceleratioboth WT and R14C IKs channels. Symbols in panels E and F are the same as for p

CNQ1, KCNE1, and yotiao. Activation of adenylate t

yclase with 1 �M forskolin resulted in a gradual increase inurrent amplitude over a period of �5 min, which wasimilar for WT and R14C IKs channels (Figs. 3A and 3B).orskolin caused a small increase in maximum currentensity for both WT and R14C IKs channels (Fig. 3C) andsmall but significant leftward shift in the voltage depen-

ence of activation (Fig. 3D). There was, however, notatistically significant difference between forskolin-timulated R14C and forskolin-stimulated WT IKs chan-els. Forskolin resulted in similar changes in the rates ofctivation and deactivation for both WT and R14C IKs

hannels (Figs. 3E and 3F).To evaluate R14C IKs channel responses to stretch,

ild-type (WT) and R14C IKs channels. (B) The Fsk resulted in an �60% increaseand R14C (blue) IKs channels. (C) The Fsk caused a small increase in currentWT and R14C IKs channels. (D) The Fsk caused a leftward shift in the V0.5 of

Fsk) and R14C (blue dashed line � control, blue boxes � � Fsk) IKs channels.e rates of activation (E) and a deceleration of the rates of deactivation (F) for.

both w(red)

or both� �

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ransfected CHO cells were exposed to hypotonic solution

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583JACC Vol. 49, No. 5, 2007 Otway et al.February 6, 2007:578–86 Stretch-Sensitive Mutations in AF

o elicit cell swelling (19). An increase in current (Figs. 4A,B, and 4C), leftward shift in the voltage dependence ofctivation (Fig. 4D), acceleration of activation, and slowingf deactivation (Figs. 4E and 4F) were observed. Theseffects were all more marked in R14C IKs channels than in

T IKs channels. For example, the V0.5 of the voltageependence of activation was shifted to a greater extent in14C IKs channels (23 � 2.1 mV, n � 6) than in WT IKs

hannels (13 � 1.9 mV, n � 6, p � 0.05). Values for V0.5 andfor all experiments are summarized in supplementary Table(Appendix). The slowing of deactivation at �60 mV was alsoore marked for R14C IKs channels (e.g., �deact,�60 mV

Figure 4 Effects of Cell Swelling on WT and R14C IKs Channels

(A) Hypotonic solution resulted in an increase in current amplitude (green) for bottion, currents recorded at the end of a 1-s depolarization pulse to �20 mV increasIKs channels (WT vs. R14C, p � 0.05). (C) Hypotonic solution caused a greater in(D) Hypotonic solution caused a greater leftward shift in the V0.5 of the voltage deobtained from Boltzmann fits to the steady-state activation curves are summarizedactivation (E) and deceleration of the rates of deactivation (F) for both WT and R1R14C than for WT IKs channels. Symbols in panels D to F are the same as in Figu

ncrease from 531 � 41 ms to 937 � 65 ms, n � 6) p

ompared with WT (�deact,�60 mV increase from 453 � 42s to 640 � 60 ms, n � 6) (Fig. 4F). The latter finding is

ikely to be of particular significance at faster heart ratesecause slower deactivation will result in accumulation ofurrent during subsequent action potentials.ffects of R14C KCNQ1 on atrial electrophysiology. Toetermine whether the kinetic differences and changes inhole cell conductance observed with cell swelling for WT and14C IKs channels would alter atrial electrophysiology, we

valuated 2 human atrial cardiomyocyte models (13,14) thatere modified to incorporate an IKs model (15). The R14CCNQ1 mutation alone had a minimal effect on atrial action

-type (WT) and R14C IKs channels. (B) After 5-min exposure to hypotonic solu-a greater extent in R14C (blue, 215 � 34%) compared with WT (red, 96 � 34%)in current density at all voltages for R14C compared with WT IKs channels.

nce of activation for R14C than for WT IKs channels (values for V0.5 and kpplementary Table 2). Hypotonic solution caused an acceleration of the rates ofchannels. However, the changes in values for �deact were more marked for

h wilded to

creasependein Su

4C IKs

re 3.

otential characteristics (data not shown). Cell swelling caused

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584 Otway et al. JACC Vol. 49, No. 5, 2007Stretch-Sensitive Mutations in AF February 6, 2007:578–86

greater reduction in the action potential duration for R14CKs channels compared with WT IKs channels with bothodels (Fig. 5, Table 2), although the difference between WT

nd mutant channels was more marked in the Nygren model.

Figure 5 Modeled Effects of R14C IKs on Atrial Action Potentia

Modeled atrial action potentials using the (i) Nygren (13) and (ii) Courtemanche (1tion for 6 min at 1 Hz (A), 2 Hz (B), and 4 Hz (C). Control data for WT and R14Cin models with R14C (blue) compared with WT IKs channels. The heterozygous phetypes but is only shown for the Nygren model, panel (i), where the differences can

odel-Derived Values for APD90 for the Nygren andourtemanche Human Atrial Action Potential Models

Table 2 Model-Derived Values for APD90 for the Nygren andCourtemanche Human Atrial Action Potential Models

Nygren Courtemanche

1 Hz 2 Hz 4 Hz 1 Hz 2 Hz 4 Hz

Control 228 150 115 293 202 132

WT � swelling 200 133 89 250 176 107

R14C � swelling 142 99 63 237 168 101

mPD90 � action potential duration measured at 90% of repolarization (measured in ms); WT �

ild-type.

iscussion

ere we report a novel stretch-responsive KCNQ1 mutationn a kindred with late-onset familial AF. Functional studiesndicate that the R14C variant does not alter baseline IKs

urrent but does have a gain-of-function effect with cellwelling. Our findings suggest a “two-hit” model in whichenetically predisposed individuals (the first “hit”) require aecond key factor, such as atrial dilation (the second “hit”)or AF to be manifested.

Although the IKs channel is thought to have a major rolen repolarization of the human ventricle, its physiologicalignificance in the atrium is unclear because it provides onlysmall contribution to atrial repolarization under resting

onditions (20). The KCNQ1 protein comprises 6

an atrial action potential models. Results shown were obtained after stimula-ndistinguishable (black). Cell swelling caused more action potential shorteninge (WT/R14C, dashed blue) was intermediate between the WT and R14C pheno-en clearly.

ls

4) humwere inotypbe se

embrane-spanning motifs (S1-S6) with cytoplasmic N-

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585JACC Vol. 49, No. 5, 2007 Otway et al.February 6, 2007:578–86 Stretch-Sensitive Mutations in AF

nd C-terminal domains. The majority (�90%) of long-T–causing KCNQ1 mutations reported to date have been

ocated in the transmembrane and C-terminal domains21). The R14C variant is located within the short cyto-lasmic N-terminus of the KCNQ1 protein. This regionas previously been implicated in cardiomyocyte responseso sympathetic stimulation. Specifically, cyclic adenosineonophosphate–mediated regulation of cardiac IKs channel

unction, which is dependent on a macromolecular complex,omprised of protein kinase A, protein phosphatase 1, andhe A-kinase adaptor protein yotiao (22,23), results inhosphorylation of Ser27 in the N-terminal domain ofCNQ1. We found, however, that activation of adenylate

yclase with forskolin had similar effects on WT and R14CKs channels. The N-terminal domain of KCNQ1 is alsonvolved in cellular responses to mechanical stretch. Cell

embrane stretch can activate mechanosensitive ion chan-els by direct effects on membrane curvature or thickness,r by changes in cytoskeletal-membrane protein interac-ions. The KCNQ1 N-terminal domain interacts withhe actin cytoskeleton and is thought to act as a sensor ofellular stretch (24). The IKs channel activation in cardiacyocytes has been shown to be stretch responsive,hether stretch is elicited by positive pressure or cell

welling (25,26). Our data confirm the stretch sensitivityf normal IKs channels and show enhanced effects in14C mutant channels.Epidemiologic studies have shown that age, hyperten-

ion, and left atrial dilation (a marker of atrial stretch) arendependent risk factors for AF (1,3). Hypertension canause left atrial dilation as a result of elevated left ventricularlling pressures and impaired diastolic function. Progressivetrial dilation predisposes to AF. Once AF is established,urther increases in atrial size may occur as a consequence oftructural remodeling of the atrial wall. Therefore, oneossible interpretation of the findings in Family D.R. is that14C KCNQ1 predisposes to hypertension rather than AF,

nd that the occurrence of AF in affected family members isredominantly related to patient age and the sequelae ofypertension. It is notable, however, that 4 of the 10 familyembers with hypertension did not carry the R14C variant,

nd that 2 of the 5 family members with hypertension whoere age �80 years did not carry the R14C variant and didot have AF. None of the genotype-positive individualsith hypertension who were age �80 years had either atrialilation or AF. A parsimonious explanation for these data ishat the combination of R14C KCNQ1, together with atrialilation, associated with hypertension and increasing age,romotes AF development in this family.KCNQ1 is not only present in the main body of the left

trium, but is also highly expressed in the pulmonary veins27). Recent studies have shown that AF is frequentlynitiated by focal ectopic activity in the vicinity of theulmonary veins (28). Although the mechanisms by whichulmonary veins contribute to arrhythmia development are

ot well understood, dilation of the pulmonary venous ostia m

as been implicated. Hypertension and persistent AF haveoth been associated with increased ostial pulmonary veiniameter (29). It is intriguing to speculate that stretch-nduced augmentation of pulmonary venous IKs channelctivation could be involved specifically in the initiationnd/or maintenance of AF in the family investigated here.on channel defects that shorten action potential durationight be expected to be associated not only with AF, but

lso with a short QT interval. The presence of normal QTntervals in genotype-positive individuals in Family D.R. isntirely consistent with our finding that R14C KCNQ1 doesot alter baseline IKs channel function, and can most readilye explained by a differential extent of atrial and ventricularhamber stretch.

The low prevalence of KCNQ1 mutations in the presenttudy (1 of 50 families) is similar to findings in 2 otherohorts of individuals with AF. Chen et al. (7) identified aCNQ1 mutation in 1 of 7 families with no mutations

dentified in 19 sporadic cases, and Ellinor et al. (30)eported no mutations in a series of 141 unrelated subjectsith lone AF. Although KCNQ1 gene mutations are not a

ommon cause of familial AF, identification of these mu-ations is useful because there are potentially major impli-ations for the management of genotype-positive familyembers. Although drug therapy with selective IKs-

locking agents (31) may prove to be useful in some familiesith gain-of-function KCNQ1 mutations, our findings

uggest that in others, aggressive treatment of hypertensionnd maintenance of normal left atrial size may be relativelyore important, particularly in young individuals, in whomF may be potentially preventable.There are 2 main limitations of this study. The small

umber of affected individuals limits the definitive conclu-ions that can be drawn, and further studies in larger patientopulations are required. Cell swelling is frequently used assurrogate experimental model for cell stretch because theasic physical principle involved in both perturbations isistortion of the surface membrane and its intrinsic integralembrane proteins. The effects of acute cell swelling

nduced by hypotonic solution do not, however, fully repro-uce those of chronic atrial dilation. Despite these limita-ions, we suggest that our data provide proof of principle forignificant interactions between genetic and environmentalactors in the pathogenesis of AF. Our findings have aumber of potentially important implications for bothxperimental and clinical studies. For example, a modifyingffect of environmental factors needs to be taken intoccount when performing in vitro studies of putative AF-ausing mutations, and might explain the apparently nega-ive functional assays of a recently described KCNE3 se-uence variant (32). More generally, in populations ofndividuals with conditions such as hypertension that pre-ispose to atrial dilation, identification of genetic riskactors may select a subgroup with an increased propensityo develop AF, in whom close medical surveillance and

onitoring of atrial size are warranted. Further investiga-

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586 Otway et al. JACC Vol. 49, No. 5, 2007Stretch-Sensitive Mutations in AF February 6, 2007:578–86

ion of the roles of inherited gene defects and environmentalactors in the development of AF may have major ramifi-ations for the clinical management not only of the relativelyare familial syndrome, but also of the more commonlyccurring acquired disorder.

cknowledgmentshe authors thank all of the participating family members,

asmine Hessell and Louise Lynagh for assistance with clinicalata collection, Robert Graham for critical review of theanuscript, and the following physicians for referring study

robands: Ruth Arnold, Richard Cranswick, Deborah Hayes,eter Hayes, Christopher Hayward, Anne Keogh, Jim Leitch,lex Levendel, Peter Macdonald, Matthew Pincus, Davidoss, Jonathan Silberberg, Paul West, and Thomas Yeoh.

eprint requests and correspondence: Dr. Diane Fatkin, Victorhang Cardiac Research Institute, Level 6, 384 Victoria Street, Dar-

inghurst NSW 2010, Australia. E-mail: [email protected].

EFERENCES

1. Kannel WB, Wolf PA, Benjamin EJ, Levy D. Prevalence, incidence,prognosis and predisposing conditions for atrial fibrillation:population-based estimates. Am J Cardiol 1998;82:2N–9N.

2. Benjamin EJ, Wolf PA, D’Agostino RB, Silbershatz H, Kannel WB,Levy D. Impact of atrial fibrillation on the risk of death. TheFramingham Heart Study. Circulation 1998;98:946–52.

3. Wijffels MC, Kirchhof CJ, Dorland R, Allessie MA. Atrial fibrillationbegets atrial fibrillation. A study in awake chronically instrumentedgoats. Circulation 1995;92:1954–68.

4. Nattel S. New ideas about atrial fibrillation 50 years on. Nature2002;415:219–26.

5. Darbar D, Herron KJ, Ballew JD, et al. Familial atrial fibrillation isa genetically heterogeneous disorder. J Am Coll Cardiol 2003;41:2185–92.

6. Fox CS, Parise H, D’Agostino RB, et al. Parental atrial fibrillation asa risk factor for atrial fibrillation in offspring. JAMA 2004;291:2851–5.

7. Chen YH, Xu SJ, Bendahhou S, et al. KCNQ1 gain-of-functionmutation in familial atrial fibrillation. Science 2003;299:251–4.

8. Keating MT, Sanguinetti MC. Molecular and cellular mechanisms ofcardiac arrhythmias. Cell 2001;104:569–80.

9. Hong K, Piper DR, Diaz-Valdecantos A, et al. De novo KCNQ1mutation responsible for atrial fibrillation and short QT syndrome inutero. Cardiovasc Res 2005;68:433–40.

0. Yang Y, Xia M, Jin Q, et al. Identification of a KCNE2 gain-of-function mutation in patients with familial atrial fibrillation. Am JHum Genet 2004;75:899–905.

1. Lu Y, Mahaut-Smith MP, Varghese A, Huang CL, Kemp PR,Vandenberg JI. Effects of premature stimulation on HERG K�

channels. J Physiol 2001;537:843–51.2. Clarke CE, Hill AP, Zhao J, et al. Effect of S5P �-helix charge

mutants on inactivation of hERG K� channels. J Physiol 2006;573:291–304.

3. Nygren A, Fiset C, Firek L, et al. Mathematical model of an adulthuman atrial cell: the role of K� currents in repolarization. Circ Res1998;82:63–81.

4. Courtemanche M, Ramirez RJ, Nattel S. Ionic mechanisms underly-

ing human atrial action potential properties: insights from a mathe-matical model. Am J Physiol 1998;275:H301–21. p

5. Terrenoire C, Clancy CE, Cormier JW, Sampson KJ, Kass RS.Autonomic control of cardiac action potentials: role of potassiumchannel kinetics in response to sympathetic stimulation. Circ Res2005;96:e25–34.

6. Kneller J, Ramirez RJ, Chartier D, Courtemanche M, Nattel S.Time-dependent transients in an ionically based mathematical modelof the canine atrial action potential. Am J Physiol Heart Circ Physiol2002;282:H1437–51.

7. Varghese A, Sell G. A conservation principle and its effect on theformulation of Na-Ca exchanger current in cardiac cells. J Theor Biol1997;189:33–40.

8. Ackerman MJ, Tester DJ, Jones GS, Will ML, Burrow CR, CurranME. Ethnic differences in cardiac potassium channel variants: impli-cations for genetic susceptibility to sudden cardiac death and genetictesting for congenital long QT syndrome. Mayo Clin Proc 2003;78:1479–87.

9. Rees SA, Vandenberg JI, Wright AR, Yoshida A, Powell T. Cellswelling has differential effects on the rapid and slow components ofdelayed rectifier potassium current in guinea pig cardiac myocytes.J Gen Physiol 1995;106:1151–70.

0. Wang Z, Fermini B, Nattel S. Rapid and slow components of delayedrectifier current in human atrial myocytes. Cardiovasc Res 1994;28:1540–6.

1. Tester DJ, Will ML, Haglund CM, Ackerman MJ. Compendium ofcardiac channel mutations in 541 consecutive unrelated patientsreferred for long QT syndrome genetic testing. Heart Rhythm2005;2:507–17.

2. Marx SO, Kurokawa J, Reiken S, et al. Requirement of a macromo-lecular signaling complex for � adrenergic receptor modulation of theKCNQ1-KCNE1 potassium channel. Science 2002;295:496–9.

3. Kurokawa J, Motoike HK, Rao J, Kass RS. Regulatory actions of theA-kinase anchoring protein yotiao on a heart potassium channeldownstream of PKA phosphorylation. Proc Natl Acad Sci U S A2004;101:16374–8.

4. Grunnet M, Jespersen T, MacAulay N, et al. KCNQ1 channels sensesmall changes in cell volume. J Physiol 2003;549:419–27.

5. Sasaki N, Mitsuiye T, Wang Z, Noma A. Increase of the delayedrectifier K� and Na�-K� pump currents by hypotonic solutions inguinea pig cardiac myocytes. Circ Res 1994;75:887–95.

6. Wang Z, Mitsuiye T, Noma A. Cell distension-induced increase ofthe delayed rectifier K� current in guinea pig ventricular myocytes.Circ Res 1995;78:466–74.

7. Melnyk P, Ehrlich JR, Pourrier M, Villeneuve L, Cha TJ, Nattel S.Comparison of ion channel distribution and expression in cardiomy-ocytes of canine pulmonary veins versus left atrium. Cardiovasc Res2005;65:104–16.

8. Yamane T, Shah DC, Jais P, et al. Dilation as a marker of pulmonaryveins initiating atrial fibrillation. J Interv Card Electrophysiol 2002;6:245–9.

9. Herweg B, Sichrovsly T, Polosajian L, Rozenshtein A, Steinberg JS.Hypertension and hypertensive heart disease are associated withincreased ostial pulmonary vein diameter. J Cardiovasc Electrophysiol2005;16:2–5.

0. Ellinor PT, Moore RK, Patton KK, Ruskin JN, Mollak MR, MacRaeCA. Mutations in the long QT gene, KCNQ1, are an uncommoncause of atrial fibrillation. Heart 2004;90:1487–8.

1. Bauer A, Koch M, Kraft P, et al. The new selective IKs-blockingagent HMR 1556 restores sinus rhythm and prevents heart failurein pigs with persistent atrial fibrillation. Basic Res Cardiol 2005;100:270 – 8.

2. Zhang D, Liang B, Lin J, Liu B, Zhou Q, Yang Y. KCNE3 R53Hsubstitution in familial atrial fibrillation. Chin Med J 2005;118:1735–8.

APPENDIX

or supplementary Tables 1 and 2, and the equations and constants for I ,

Ks

lease see the online version of this article.