Cardiac arrhythmia mechanisms in rats with heart failure induced by pulmonary hypertension

15 Pages·2012·2.26 MB·English

AmJPhysiolHeartCircPhysiol302:H2381–H2395,2012.
FirstpublishedMarch16,2012;doi:10.1152/ajpheart.01084.2011.
Cardiac arrhythmia mechanisms in rats with heart failure induced by
pulmonary hypertension
David Benoist,1,3,4 Rachel Stones,1,3 Mark J. Drinkhill,2,3 Alan P. Benson,1,3 Zhaokang Yang,1,3
Cecile Cassan,5 Stephen H. Gilbert,1,3,4 David A. Saint,6 Olivier Cazorla,5 Derek S. Steele,1,3
Olivier Bernus,1,3,4 and Ed White1,3
1InstituteofMembraneandSystemsBiology,2DivisionofCardiovascularandNeuronalRemodelling,and3Multidisciplinary
CardiovascularResearchCentre,UniversityofLeeds,Leeds,UnitedKingdom;4U-1045,InstitutNationaldelaSantéetdela
RechercheMédicale(INSERM),CentredeRechercheCardio-Thoracique,UniversitéBordeauxSegalen,Bordeaux;
5U-1046,INSERM,UniversitéMontpellier1and2,Montpellier,France;and6SchoolofMedicalSciences,Universityof
Adelaide,Adelaide,SouthAustralia,Australia
D
Submitted18November2011;acceptedinfinalform15March2012 o
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Benoist D, Stones R, Drinkhill MJ, Benson AP, Yang Z, majorcauseofdeathinpatients(6).Pulmonaryhypertensionis loa
CassanC,GilbertSH,SaintDA,CazorlaO,SteeleDS,Bernus associated with RV electrical remodeling (23, 26, 27) and a de
Ofa,iluWrehiitneduEc.edCabrydipaculmarorhnyatrhymhiyapmeretecnhsainoins.mAsminJraPthsywsioitlhHheeaarrtt h4i0g%heorfrsiuskffeorferasrr(h1y0t,h6m3)ia,san(d17le).thSauldadrrehnytdhematihasoacrceutrhsouingh3t0to– d from
CircPhysiol302:H2381–H2395,2012.FirstpublishedMarch16, beonecause,inadditiontoothermechanisms,suchaspulmo- h
2012; doi:10.1152/ajpheart.01084.2011.—Pulmonary hypertension nary embolism and pulmonary artery dissection. Interestingly, ttp
provokesrightheartfailureandarrhythmias.Betterunderstandingof the RV is not currently a target for therapeutic intervention in ://a
tnheewmtheecrhaapneiusmticsaupnpdreoralychinegsfthoerstehearhryhpyethrtmeniassivies,nfaeielidnegdrtioghftacvielnittartie- this disease (19). jph
e
cgleene(rRatVin)g. Tarhrehyathimmiaosfionuar mstouddeylwofasRVtofaidileunrteifiyndtuhceedmbeychpaunlimsmos- miTashecraenaarerisseev.eIrnaclreeastsaebdlieshleecdtrmiceaclhhaentiesrmogsebnyeiwtyhiacnhdasrrteheypther- art.p
nary hypertension. Rats were injected with monocrotaline to induce electrical restitution (1, 32, 53) increase the probability of hy
s
eitherRVhypertrophyorfailureorwithsaline(control).ECGswere alternans and arrhythmias. This is because heterogeneity in- io
measuredinconscious,unrestrainedanimalsbytelemetry.Inisolated creases the dispersion of refractoriness and steeper restitution log
hearts, electrical activity was measured by optical mapping and decreasesthestabilityofactionpotential(AP)duration(APD) y.o
myofiberorientationbydiffusiontensor-MRI.Sarcoplasmicreticular anddiastolicintervaladaptationtoachangeinheartrate,also rg
Ca2(cid:1) handling was studied in single myocytes. Compared with increasingthedispersionofrefractoriness.Thismakesconduc- b/
y
controlanimals,theT-waveoftheECGwasprolongedandinthree tion block and reentry more likely (9, 35, 49, 60). Alternans 1
ofsevenheartfailureanimals,prominentT-wavealternansoccurred. describetheeventswhereparameterssuchasAPDorintracel- 0.2
DheiasrctosrdaanndtaCcati2o(cid:1)ntproatnesnietinatl(aAltPer)naalntesrninanfsaoilcincugrrmedyoincyisteosl.atIendffaaiilliinngg lnualtaer iCna2s(cid:1)izecoonncenatrbateiaotn-to(-[bCeaa2t(cid:1)]bi)astirsa.nsTiheneyt aamreplietsutdaebliaslhteerd- 20.3
hearts,APdurationanddispersionwereincreased;conductionveloc- predictors of sudden cardiac death, and discordant alternans, 3.1
ityandAPrestitutionweresteeper.Thelatterwasintrinsictofailing o
single myocytes. Failing hearts had greater fiber angle disarray; this wrehgeiorensthoef tphaeravmenettreircliens,qaureestmioonstislikoeultyotof pghenaseeraitne rdeiefnfetrraenntt n N
correlatedwithAPduration.Failingmyocyteshadreducedsarco(en- o
do)plasmic reticular Ca2(cid:1)-ATPase activity, increased sarcoplasmic arrhythmias (9, 35, 49, 60). ve
reticular Ca2(cid:1)-release fraction, and increased Ca2(cid:1) spark leak. In Defective Ca2(cid:1) handling also leads to the generation of mb
hbyegpuernt,robpuhtieadltehrneaarntss dainddnmotyodceyvteelso,p.dyWsfeunccotniocnluadleatdhaapttaitniocrneahseadd SarRrhyrethlemasiaes.frIancctrieoans,edSRsarCcao2p(cid:1)lascmonicternett,icaunldumsl(oSwRe)dCSaR2(cid:1)Cleaa2k(cid:1), er 10
electricalandstructuralheterogeneityanddysfunctionalsarcoplasmic reuptake are thought to contribute to the generation of Ca2(cid:1) , 2
reticular Ca2(cid:1) handling increased the probability of alternans, a transientalternansandthustoAPDandT-wavealternans(via 01
proarrhythmic predictor of sudden cardiac death. These mechanisms modulation of Ca2(cid:1)-dependent currents) by facilitating beat- 7
are potential therapeutic targets for the correction of arrhythmias in to-beat fluctuations in SR Ca2(cid:1) release (38, 49). This is
hypertensive,failingRVs. because increased SR Ca2(cid:1) content favors SR Ca2(cid:1) leak and
electrocardiography; monocrotaline; calcium signaling; voltage-sen- placesactivatedryanodinereceptorsinarefractorystateforthe
sitivedyeimaging;alternans next beat, leading to fluctuations in SR Ca2(cid:1) release (48).
Additionally,elevatedSRCa2(cid:1)contentandreleasefractionin
the presence of a slowed SR Ca2(cid:1) reuptake lead to intra-SR
ALTHOUGHRIGHTVENTRICULAR(RV)failuremostoftendevelops fluctuations in Ca2(cid:1) content (13).
as a consequence of left ventricular (LV) failure, RV failure Structuralchanges,suchasanalterationinconnexinexpres-
occursinmany,increasinglycommon,diseasesassociatedwith sion or increased fibrosis, which disrupts the normal myofiber
dysfunction of the pulmonary circulation (6, 18, 58). This organization, can slow conduction, create electrical heteroge-
includes pulmonary hypertension, where RV failure is the neity (31), and contribute to dysfunctional electrical activity
(16). Given the number of potential arrhythmic mechanisms,
thespecificoccurrenceandrelativeimportanceofthesemech-
Addressforreprintrequestsandothercorrespondence:E.White,Instituteof
anismsneedtobecharacterizedforanygivenpathology,ifthat
Membrane and Systems Biology, Garstang Bld., Univ. of Leeds, Leeds
LS29JT,UK(e-mail:[email protected]). pathology is to be fully understood and treated.
http://www.ajpheart.org 0363-6135/12Copyright©2012theAmericanPhysiologicalSociety H2381
H2382 ARRHYTHMIAMECHANISMSINRIGHTHEARTFAILURE
We have reported a proarrhythmic state in a model of RV measuredbyDopplerinthefour-chamberview.Dopplerimagingof
failure induced by pulmonary hypertension where changes in thepulmonaryoutflowwasrecordedinaparasternalviewatthelevel
ion channel gene expression and monophasic APD are pre- oftheaorticvalves.Pulmonaryarteryaccelerationtime(PAAT)was
dominantly found in the RV compared with the LV (3). The measuredfromtheonsetofsystolicflowtothemaximumpulmonary
outflow velocity and normalized to cardiac cycle length (CL). De-
purpose of this study was to investigate changes in RV elec-
creasing PAAT/CL can be used as a serial noninvasive indicator of
trophysiology and structure, at two stages of the disease, to
increasing pulmonary artery systolic pressure (20, 30). The velocity
identify the mechanisms responsible for the proarrhythmic
time integral of pulmonary artery flow was measured on the same
state.
images.Dopplerassessmentofthevelocitytimeintegralofaorticflow
was performed in a suprasternal view, with rats positioned on their
METHODS
back(54).
Experimentswereapprovedbythelocalethicalcommitteeandthe TelemetricmonitoringofECG.Surgicalimplantationoftelemetry
United Kingdom Home Office. Male Wistar rats (200 g) received a devices(TA10ETA-F20,DataSciences,St.Paul,MN)wasconducted
single intraperitoneal injection of monocrotaline (MCT) to induce under 2–3% isoflurane anesthesia using aseptic techniques. Devices
stable RV hypertrophy (HYP; 30 mg/kg in saline, N (cid:2) 11) or RV designed for mice, which are smaller and lighter than standard rat
D
failure(FAIL;60mg/kginsaline,N(cid:2)34)orwereinjectedwithan devices, were used to minimize the impact of implantation. ECGs o
equivalentvolumeofsaline[control(CON);N(cid:2)32].Animalswere were recorded in conscious, unrestrained rats. Two-hour sections of wn
weighed weekly for 3 wk postinjection and then daily. Ethical ap- data, acquired with Dataquest A.R.T. 4.1 Gold software (Data Sci- lo
a
proval required that FAIL animals were killed upon displaying clin- ences)midwaythroughthelightcycleofa12:12-hlight-darklighting d
e
icalsignsofheartfailure,e.g.,dyspnea,coldextremities,lethargy,or regime were analyzed with Ponemah software (P3 Plus version 4.4, d
any weight loss on consecutive days. CON and HYP animals were DataSciences).TheR-Rinterval(theinverseofheartrate),duration fro
killedonequivalentdayspostinjection. oftheQRScomplex(anindicatorofventricularactivationtime),QT m
aBnaexEstctehhre)otciwzaeirtddhioabguVrtaipvshpidyo7.nPtDaronoep(opGulEesrlyHeecbahrloetahctachraidrneigo)geraqanupimihpyaplewsda(ws2%iptheriafsoo1rflm0u-eMrdanHienz, ifAnroPtemDrv)Tawl-we(aarevnemipneedaaisckuarttoeodre.noEdfC(vTGepnet;trraaiccneuislnadwriecrraeetpooarllaosrofiztahanetiaoglnylozbetiadmlwed)ii,stphaenCrdshioatnirmto7ef http://a
transducer.Ratswerepositionedontheirleftside.Atwo-dimensional (AD Instruments), and a custom-written C(cid:1)(cid:1) program was used to jph
viewofboththeLVandRVwasobtainedatthelevelofthepapillary detectalternansinT-waveamplitudeandQTinterval.Analternating ea
muscles in a parasternal short-axis view (50). LV morphological 4%differenceinT-waveparametersforatleast10consecutivebeats rt.p
parametersweremeasuredfromM-modetracesrecordedthroughthe wascountedasanalternansevent,andthefrequencyoftheseevents h
y
anterior and posterior walls (11). LV fractional shortening (FS) was was recorded. These thresholds were chosen to allow manual verifi- s
calculatedasfollows:LVFS(cid:2)[(LVIDd(cid:3)LVIDs)/LVIDd(cid:4)100], cation and to accommodate signal-to-noise levels. Between 13,000 iolo
where LVIDd and LVIDs are the end-diastolic and end-systolic LV and22,000ECGwaveformswereanalyzedfromeachheart. g
y
internaldimensions,respectively. Optical mapping of electrical activity in isolated hearts. Isolated .o
RV wall thickness was assessed in two-dimensional parasternal hearts were perfused at 37°C with a modified Krebs-Henseleit solu- rg
short-axisviewindiastole.RVend-diastolicdiameterwasmeasured tioncontaining(inmmol/l)130NaCl,24NaHCO ,1.2NaH PO ,1 b/
3 2 4 y
afosutrh-cehmamaxbiemravlideiwst.anRcVeFfrSomwathsecRalVcuflareteedwaasllftoolltohwess:epRtuVmSFin(cid:2)a M95g%ClO2,25-5.6%glCuOco2s)e.,T4oKpCelr,foarnmd1o.p8tiCcaaClml2a(pppHinegquoiflibelreactetrdictaol7a.c4tiwviitthy 10.2
[(RVAd(cid:3)RVAs)/RVAd(cid:4)100],whereRVAdandRVAsaretheRV (29, 59), motion artifacts were prevented using 10 mmol/l 2,3- 2
0
areas in diastole and systole, respectively, measured in the four- butanedione monoxime (BDM) as an electromechanical uncoupler. .3
3
chamber view. The RV maximal value of E wave velocity was Thissolutionwasrecirculatedthroughoutthedurationoftheexperi- .1
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Fig.1.Echocardiographicimagesofcontrol , 2
(CON) and monocrotaline (MCT)-treated 0
1
[right ventricular (RV) failing (FAIL)] ani- 7
mals. A: two-dimensional (2-D) parasternal
short-axisviewinarepresentativeCONand
FAILanimal22daysaftersalineor60mg/kg
MCT treatment. RV, RV cavity; LV, left
ventricular(LV)cavity;IVS,interventricular
septum.InFAILanimals,theRVisenlarged
andhypertrophic,andtheLVmorphologyis
altered.B:pulmonaryarteryoutflow,obtained
by Doppler, was decreased in MCT-treated
animals.C:M-modetracesoftheLV(para-
sternal short-axis view). Images show evi-
dence of reduced LV diameters and de-
creasedcontractility.
AJP-HeartCircPhysiol•doi:10.1152/ajpheart.01084.2011•www.ajpheart.org
ARRHYTHMIAMECHANISMSINRIGHTHEARTFAILURE H2383
Table 1. Echocardiographic parameters for CON and monocrotaline-treated animals
Day0 Day16 Day(cid:7)21
CON FAIL CON FAIL CON FAIL
Bodyweight,g 226(cid:6)4 219(cid:6)3 325(cid:6)3 299(cid:6)7† 353(cid:6)5 303(cid:6)6‡
Heartrate,beats/min 408(cid:6)6 414(cid:6)7 383(cid:6)10 391(cid:6)9 376(cid:6)13 318(cid:6)10†
Cardiacoutput,ml/min 91.4(cid:6)2.1 96.5(cid:6)3.8 102.1(cid:6)4.9 100.4(cid:6)4.4 92.4(cid:6)4.0 35.0(cid:6)3.3‡
LV
Posteriorwallthicknessatdiastole,101cm 15(cid:6)0 15(cid:6)0 15(cid:6)0 16(cid:6)0 16(cid:6)0 18(cid:6)1*
Internaldimensionatdiastole,mm 7.8(cid:6)0.2 7.5(cid:6)0.1 8.5(cid:6)0.2 8.1(cid:6)0.2 8.6(cid:6)0.2 7.1(cid:6)0.1‡
Relativewallthickness 0.35(cid:6)0.01 0.37(cid:6)0.01 0.34(cid:6)0.01 0.36(cid:6)0.01 0.34(cid:6)0.01 0.43(cid:6)0.01‡
FS,% 52(cid:6)1 50(cid:6)1 49(cid:6)2 50(cid:6)2 50(cid:6)1 43(cid:6)2*
Velocitytimeintegralofaorticflow,mm 39.9(cid:6)0.6 41.9(cid:6)1.5 43.4(cid:6)2.0 42.8(cid:6)1.7 40.8(cid:6)1.1 18.6(cid:6)1.3‡
RV
Wallthicknessatdiastole,mm 0.64(cid:6)0.02 0.62(cid:6)0.01 0.62(cid:6)0.01 0.66(cid:6)0.02 0.63(cid:6)0.01 1.40(cid:6)0.06‡
End-diastolicdiameter,cm 0.48(cid:6)0.01 0.48(cid:6)0.01 0.50(cid:6)0.01 0.52(cid:6)0.02 0.52(cid:6)0.02 0.76(cid:6)0.04‡ D
FS,% 29(cid:6)3 31(cid:6)2 27(cid:6)2 23(cid:6)1 30(cid:6)2 12(cid:6)2‡ o
w
Ewave,m/s 0.90(cid:6)0.02 0.88(cid:6)0.02 0.87(cid:6)0.03 0.88(cid:6)0.04 0.88(cid:6)0.02 0.69(cid:6)0.04‡ n
LVFS/RVFS 2.6(cid:6)0.2 2.3(cid:6)0.2 2.5(cid:6)0.3 2.8(cid:6)0.2 2.4(cid:6)0.1 5.4(cid:6)1.0* lo
a
Pulmonaryartery d
PAAT/CL 0.17(cid:6)0.01 0.18(cid:6)0.01 0.16(cid:6)0.00 0.11(cid:6)0.01† 0.15(cid:6)0.01 0.07(cid:6)0.00‡ ed
Velocitytimeintegral,cm 5.69(cid:6)0.22 5.40(cid:6)0.16 6.04(cid:6)0.17 4.89(cid:6)0.14‡ 6.16(cid:6)0.21 2.47(cid:6)0.22‡ fro
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Valuesaremeans(cid:6)SE;N(cid:2)8animalsinthecontrol(CON)groupand8animalsinthemonocrotaline-treated[failing(FAIL)]group.Shownareparameters h
iEnwCaOvNevaenldocFitAyI;LPAanAimT/aClsLa,tp0u,lm16o,naanryda(cid:7)rt2e1rydaacycselpeorsatttiroenattmimenet.noLrVm,alleizftedvetnotrciacrled;iaFcSc,yfcrlaectlieonngatlhs.h*oPrte(cid:9)nin0g.0;5R;V†P,r(cid:9)igh0t.0v1en;t‡riPcl(cid:9)e;E0.0w0a1v.e,maximalvalueof ttp://a
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ment.Heartswerestimulatedwiththepacingelectrodeplacedatthe calculatedfromthediffusionmeasurements,andtissuestructurewas e
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RwVereeptihceanrdilaoladsuedrfawceithinathe5-p(cid:5)ogs/tmerliobroilnutserv(1entmrilc)uloafrrdeig-4io-An.NHEePaPrtSs quaAnntiafilyedsisfroofmDtThe-MseRvIecdtaotrassuestisnghaisn-bheoeunseprseovfitowuasrley.described (4). rt.ph
(Biotium)dissolvedinDMSO.Thedyewasexcitedat530nmusing Fiber inclination (helix) angles were extracted from 2-mm-thick and ys
monochromatic light-emitting devices. The emission light was sepa- 15°-wide sectors of the equatorial LV and RV free walls. Any io
rated with a 700 (cid:6) 50-nm filter. Excitation light and emission filter papillary muscle data were digitally removed. Wall thickness, range log
y
wavelengths were selected to provide the optimal voltage-sensitive ofhelixangles,andtransmuralrateofchangeofthehelixanglewere .o
fluorescent signal. The fluorescent signal was acquired with a calculatedfromlinearfits.TheR2valueofthelinearfitwasusedto rg
0.25-mmspatialresolutionat1kHzthroughacharge-coupleddevice assessheterogeneityinfiberangle. b/
camera(SciMeasureAnalyticalSystems)mountedwithalens(focal SimultaneousmeasurementofshorteningandintracellularCa2(cid:1)in y 1
length:12mm,apertureratio:1:0.8,Computar).TheRVwasorien- singleventricularmyocytes.Singlecardiacmyocyteswereisolatedas 0.2
tatedsothatitfacedtheexcitationlightsourceandthecamera. previously described (40). After perfusion with a collagenase- and 2
Anepicardialmid-RVfreewallregionof2.5(cid:4)2.5mmwasused protease-containingsolution,theRVfreewallwasdissectedandRV 0.3
forconstructingdynamicAPandconductionvelocity(CV)restitution myocyteswereisolated.Myocyteswerestoredat20–23°Candused 3.1
curves.APsrecordedovera5-ssequenceunderwentensembleaver- within10h. o
aging followed by temporal (1.5 ms kernel) and spatial (1.0 mm n N
kernel)filtering.ParametersanalyzedincludedAPDat80%repolar- o
ization(APD80)andCV,whichwasobtainedfromtheactivationtime Table 2. Animal and organ characteristics of CON and vem
across the sampled section of the ventricular free wall. Dynamic monocrotaline-treated animals
b
restitution characteristics were assessed by increasing stimulation e
frequency and measuring parameters at each steady state. APD dis- CON HYP FAIL r 1
0
persion was calculated as the difference between the maximum and Animals/group 15 12 17 , 2
minimumAPD80overthewholeimagedportionoftheRVsurfacein Bodyweight,g 315(cid:6)4 300(cid:6)7e 264(cid:6)5c 01
each heart. The threshold criterion for APD and AP amplitude Heartweight,g 1.30(cid:6)0.04 1.60(cid:6)0.08c 1.53(cid:6)0.03b 7
alternans was a beat-to-beat difference of (cid:7)2SD of the mean mea- Lungweight,g 1.71(cid:6)0.07 2.27(cid:6)0.11c 2.81(cid:6)0.16c
suredparameter. Heartweight/bodyweight,
Diffusion tensor-MRI. After the optical mapping experiments, mg/g 4.14(cid:6)0.13 5.37(cid:6)0.30c 5.84(cid:6)0.17c
some hearts were preserved by perfusion with 4% formalin. Fixed Lungweight/Bodyweight,
hearts were immersed in the perfluoropolyether fomblin. Diffusion mg/g 5.42(cid:6)0.21 7.62(cid:6)0.39c,d 10.70(cid:6)0.70c
Ventricularweights
tensor (DT)-MRI imaging was performed with a NMR spectrometer
Hearts/group 10 6 11
witha9.4-Tmagnet.Thespectrometerhadanimaginganddiffusion RVweight,g 0.26(cid:6)0.02 0.44(cid:6)0.03c 0.43(cid:6)0.02c
probeheadtoenableDTstobemeasuredateachvoxelwithinatissue, LVweight,g 0.60(cid:6)0.02 0.56(cid:6)0.03 0.51(cid:6)0.02a
the eigenvectors of which quantified the fiber orientation and sheet RVweight/LVweight,
structure throughout the tissue. High-resolution (200 (cid:5)m isotropic) mg/mg 0.44(cid:6)0.03 0.81(cid:6)0.07b 0.85(cid:6)0.04c
imaging of the orthotropic fiber and sheet structure was performed
withareasonablyshortscantimeof(cid:8)8h.Diffusionofprotonswas Valuesaremeans(cid:6)SE.Shownarewholeanimal,organ,andventricular
weights from animals used in optical mapping/diffusion tensor (DT)-MRI
measuredthroughoutthetissueinasetof12optimizeddirections(45)
experiments and single myocyte experiments from CON, hypertrophied
using a three-dimensional diffusion-weighted spin-echo sequence
(HYP), and FAIL groups. RV and LV weights are RV and LV free wall
withreducedencodingat20°C(repetitiontime:500ms,echotime:15 weights after an exposure to collagenase from animals used in the single
ms,diffusiongradientswith2-msdurationand7-msseparation,b(cid:2) myocytesexperiments.aP(cid:9)0.05,bP(cid:9)0.01,andcP(cid:9)0.001vs.theCON
1,000 s/mm2). DTs, and the eigenvectors of these tensors, were group;dP(cid:9)0.01andeP(cid:9)0.001,HYPgroupvs.FAILgroup.
AJP-HeartCircPhysiol•doi:10.1152/ajpheart.01084.2011•www.ajpheart.org
H2384 ARRHYTHMIAMECHANISMSINRIGHTHEARTFAILURE
Fig. 2. ECGs in conscious, unrestrained CON
and FAIL rats. A: representative ECG traces
from a CON and FAIL rat 22 days after treat-
ment.B:R-Rinterval.C:QRSduration.D:QT
interval.E:timefromT-wavepeaktoend(T )
pe
at 1, 15, and (cid:7)21 days after treatment. Open
bars, CON animals; solid bars, FAIL animals.
Dataaremeans(cid:6)SE;N(cid:2)9CONanimalsand
9FAILanimals.**P(cid:9)0.01and***P(cid:9)0.001
vs.CON.TheincreasedQTintervalisconsistent D
withaprolongedactionpotential(AP)duration ow
(dAisPpDer)siaonndoifnAcrPeaDs.edTpewithincreasedglobal nlo
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Cells were placed in a chamber on the stage of an inverted at340and380nmusingamonochromatorspectrophotometersystem a
microscope (Diaphot, Nikon) and continuously superfused at (cid:8)1 (CairnResearch).Emittedlightat510nmwascollectedbyaphoto- rt.p
ml/minwithaHEPES-basedTyrodesolutionat37°Ccontaining(in multiplier, and the ratio of emitted light in response to 340- and h
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mmol/l) 137 NaCl, 5.4 KCl, 0.33 NaH PO , 0.5 MgCl ·6H O, 5 380-nm illumination (340-to-380-nm ratio) used as an index of s
HEPES, 5.6 glucose, and 1 CaCl2 (pH ad2just4ed to 7.4 wit2h Na2OH). [Ca2(cid:1)]i.The[Ca2(cid:1)]itransientamplitudewasthedifferencebetween iolo
Cellswerefieldstimulatedviaplatinumbathelectrodesusinga5-ms thediastolicandpeaksystolic340-to-380-nmratio.Sarco(endo)plas- gy
pulse at stimulation frequencies between 1 and 9 Hz to investigate mic reticular Ca2(cid:1)-ATPase (SERCA) is the Ca2(cid:1) pump responsible .o
[Ca[2C(cid:1)a]2i(cid:1)t]rianwsiaesntmsoannidtofrreadctiuosninaglsthhoertflenuionrge.scent Ca2(cid:1) probe fura-4 fcoerllsthweeurpetasktiemouflaCtae2d(cid:1)toinstoteathdeySsRta.teToataassfersesqtuheenaccytiovfity5oHfzS.ESRtiCmAu-, byrg/
AM (Molecular probes) (34). Isolated cells were loaded with fura-4 lation was stopped, and Tyrode solution containing 20 mmol/l caf- 1
0
AM (2 (cid:5)mol/l) for 20 min at 20–23°C, resuspended in Tyrode feine was applied via a rapid solution changer in close proximity to .2
solution,andthenstoredforatleast30mintoallowdeesterification thecell.CaffeinereleasesCa2(cid:1)fromtheSR,andtheamplitudeofthe 20
ofthedye.Myocyteswerealternatelyilluminatedwithexcitationlight caffeine-inducedCa2(cid:1)-transientisanindexofSRCa2(cid:1)content.The .3
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ECGrecordingshowninFig.2fromaFAIL 1
7
rat 22 days after treatment. T-waves alter-
nated between taller and longer (L) and
shorter (S) forms. B: successive alternating
T-wave amplitudes and QT intervals from
another FAIL animal (cid:7)21 days after treat-
ment.CandD:percentagesofbeatsina1-h
recordingperiodassociatedwithalternansof
T-waveamplitude(C)orQTinterval(D)in7
CON animals (Œ) and 7 FAIL animals ((cid:1)).
Alternans were quantified as at least a 4%
beat-to-beat alternans of magnitude for at
least 10 beats. Box plots in C and D show
means,SEs,and25–75%ranges.
AJP-HeartCircPhysiol•doi:10.1152/ajpheart.01084.2011•www.ajpheart.org
ARRHYTHMIAMECHANISMSINRIGHTHEARTFAILURE H2385
decayoftheelectricallyandcaffeine-stimulatedCa2(cid:1)transientswere edlyalongtheirlongaxisatarateof166lines/s.ACoherentsapphire
fittedtosingleexponentials,andthedifference(K )wasusedas laserwasusedtoexcitethedyeat488nm,andemittedfluorescence
SERCA
anindexofSERCAactivity,becauseCa2(cid:1)cannotaccumulateinthe wasmeasuredat(cid:7)515nm.Ca2(cid:1)sparkswereidentifiedandanalyzed
SRinthepresenceofcaffeineandcytosolicCa2(cid:1)declinesprincipally withImageJsoftware(NationalInstitutesofHealth)usingtheSpark-
duetoextrusionfromthecellviatheNa(cid:1)/Ca2(cid:1)exchanger(seeRefs. masterplugin.Parametersanalyzedweresparkfrequency,amplitude,
12and14fordetails). durationtohalf-decay,andwidthathalf-decay(61,62).Ca2(cid:1)spark
Cell shortening was measured using a video-edge detector (Cres- mass and spark leak were calculated as previously described (25) as
cent Electronics) and NTSC TM640 camera (JAI Pulnix) and ex- follows:mass(cid:2)sparkamplitude(cid:4)sparkwidth3(cid:4)1.206[changein
pressed as a percentage of the diastolic cell length. Cell length and fluorescence/initial fluorescence ((cid:10)F/F ·(cid:5)m3)] and SR Ca2(cid:1) spark
0
[Ca2(cid:1)] weremonitoredsimultaneouslyinthesamecell.Signalswere leak(cid:2)sparkmass(cid:4)sparkfrequency((cid:10)F/F ·(cid:5)m3·s(cid:3)1).
i 0
recorded by an Axon Digidata at a sample frequency of 1 kHz and Single myocyte electrophysiology. Single myocytes were impaled
analyzed with pCLAMP 9.2 (Axon Instruments). The threshold cri- with sharp microelectrodes containing 0.6 mol/l KCl (resistance:
terion for Ca2(cid:1) transient alternans was a 5% beat-to-beat change in 20–30M(cid:11)).CellsweresuperfusedwithTyrodesolutionat37°C,and
amplitudeovera30-beatperiod. APs were stimulated via an AxoClamp 2B amplifier (Axon Instru-
Measurement of Ca2(cid:1) sparks by confocal microscopy. Intact, ments) in bridge mode by the injection of 2-ms current pulses just
quiescentmyocyteswereloadedfor15minat20–22°Cwith6(cid:5)mol/l abovethresholdamplitudeatstimulationfrequenciesfrom1to9Hz. D
o
fluo-4AM.Thereafter,cellsweresuperfusedwithTyrodesolutionat L-typeCa2(cid:1)current(I )wasrecordedinvoltage-clampedmyocytes w
20–22°C. Ca2(cid:1) sparks were assessed with a confocal microscope usingtheswitch-clampteCcahLniquewithaswitchingfrequencyof3kHz. nlo
(NikonDiaphotEclipseTE2000invertedmicroscopeequippedwitha Myocytes were held at (cid:3)40 mV to inactivate Na(cid:1) current, and 0.1 ad
confocalscanhead,Bio-RadMicroRadiance2000,anda(cid:4)60water- mmol/l BaCl2 and 5 mmol/l 4-aminopyridine were used to block K(cid:1) ed
immersion objective) in line-scan mode. Cells were scanned repeat- currents (inward rectifier and transient outward K(cid:1) currents, respec- fro
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Fig.4.ElectricalandCa2(cid:1)alternansinheartsofMCT-treatedrats.A:differenceinAPamplitude(left)andAPD(right)betweentwosuccessivebeats(beats
1and2)fromtworegions(aandb)ofaRVfromaFAILheart.APsareshownbetweenthetwomaps.Eachregiondisplaysamplitudeanddurationalternans
outofphase(discordant)witheachother.B:proportionofheartsshowingAPDalternansfromN(cid:2)5CON,6RVhypertrophied(HYP),and6FAILhearts.
*P(cid:9)0.05vs.CON;#P(cid:9)0.05,HYPvs.FAIL.AstheseverityoftheMCTtreatmentincreased,theprobabilityofinducingelectricalalternansincreased.
C:cellshorteningandintracellularCa2(cid:1)concentration([Ca2(cid:1)])transientsmeasuredsimultaneouslyinasingleRVmyocytefromaFAILheartstimulatedat
i
afrequencyof9Hz.Themyocytedisplayed[Ca2(cid:1)] transientandshorteningalternans.D:proportionofmyocytesshowingalternansfromn(cid:2)18CON,14HYP,
i
and14FAILmyocytes,N(cid:2)3hearts/group.***P(cid:9)0.001vs.CON.AlternanswereprovokedinFAILmyocytesbutnotinCONmyocytes.
AJP-HeartCircPhysiol•doi:10.1152/ajpheart.01084.2011•www.ajpheart.org
H2386 ARRHYTHMIAMECHANISMSINRIGHTHEARTFAILURE
tively).Steady-stateI waselicitedbya100-msdepolarizingstepto0 weight-to-bodyweightratio,andRV-to-LVweightratio(CON(cid:9)
CaL
mVandwasmeasuredasthedifferencebetweenthepeakinwardcurrent HYPandFAILanimals;Table2).
andthecurrentattheendofthedepolarizingstep.Thetimeconstantof ChangesintheECGwithRVfailure.ECGswererecordedby
inactivationwasmeasuredbyfittingasingleexponentialtothedecaying
radiotelemetryinconscious,unrestrainedanimals(Fig.2A).After
I .ThevoltagedependenceofI wasassessedbydepolarizingthe
CaL CaL 3wkoftreatment,theR-RintervalofFAILanimalswassignif-
cell from (cid:3)40 to (cid:1)60 mV in 10-mV increments of 300-ms duration.
Ca2(cid:1)currentavailabilitywasassessedbyclampingbackto(cid:3)40mVfor icantly shorter that of than CON animals (Fig. 2B), and hence
heart rate was faster, in contrast to measurements made in anes-
10msbeforeatestpulseto0mVwasapplied.Currentwasnormalized
tocellcapacitanceandexpressedaspA/pF.Alldatawereacquiredwith thetized animals (see DISCUSSION). There were no significant dif-
anAxonDigidataata5-kHzsamplingrateandanalyzedwithpCLAMP ferencesinQRSdurationsbetweenCONandFAILanimals(Fig.
9.2software(AxonInstruments). 2C).TheQTintervalofFAILanimalswasprolongedatboth15
Statistical analysis. Data are expressed as means (cid:6) SE. Statistical and (cid:7)21 days (Fig. 2D). At (cid:7)21 days, T was significantly
pe
differences between groups were tested with unpaired t-tests, one- or prolonged(Fig.2E).LengtheningoftheQTintervalisconsistent
two-wayANOVA,orlinearregressionasappropriate.Statisticallysig-
with a prolongation of the mean APD and increased T with
nificant difference was assumed when P (cid:9) 0.05. Animal, heart, and pe
increasedglobaldispersionofAPD(seebelow).
myocytenumbersforeachexperimentaregivenintherelevantfigures/tables. D
T-wave alternans were recorded in some conscious, unre- o
w
strained FAIL animals (Fig. 3A). Alternans of both T-wave n
RESULTS lo
amplitude and QT interval were observed (Fig. 3B). When a
d
Cardiac function in RV failure. Echocardiographic record- alternans were quantified as a 4% difference in successive e
d
ingsfromanesthetizedanimalsshowedasignificantdepression amplitude for at least 10 consecutive beats, alternans of T- fro
in heart rate and function after 3 wk of failure treatment. wave height (Fig. 3C) and QT interval (Fig. 3D) comprised m
PAAT, normalized to cardiac CL [a noninvasive index of (cid:7)9%ofmeasuredbeatsintwo(QTinterval)orthree(T-wave h
preudlumcoendariynaFrtAerIyLsyasntiomliaclsp.reIsnsutrhee(2R0,V3,0)w],alwlatshiscigknniefiscsanatnldy ianmteprlvitaul)deo)rosfesveevnen(TF-AwIaLveanaimmpallistubduet)(cid:9)o1f%seovfenbeCatOsNinasinxim(QalTs ttp://a
jp
end-diastolic diameter were increased, whereas RVFS was de- (13,000–22,000 beats were analyzed from each heart). Ven- h
e
creased. The LV showed a reduction in diameter, contractility, tricular ectopic beats, measured in the same recordings, were a
and aortic outflow. Thus, FAIL rats displayed both RV and LV rare: four events in total from three of seven FAIL animals and rt.p
h
insufficiencyafter3wkoftreatment(Fig.1andTable1).There twoeventsintotalfromoneofsevenCONanimals.Theoccur- y
s
was an increase in the heart weight-to-body weight ratio, lung rence of T-wave alternans could not be correlated with other io
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Fig.5.OpticalmappingofAPsinRVsofCONandMCT-treatedrats.A:opticalAPsrecordedfromtheRVmidlineinarepresentativeCON,HYP,andFAIL
heart.B:color-codedAPDat80%repolarization(APD )mapoverthesurfaceoftheRVataresolutionof0.25mm2fromarepresentativeCONandFAIL
80
heart.C:APD measuredattheRVmidline(*inB).D:APD dispersion(maximumAPD(cid:3)minimumAPD)ineachheart.E:dynamicAPD restitution.
80 80 80
F:maximumslopeoftheAPD restitutioncurve.Dataaremeans(cid:6)SEforN(cid:2)5CON,6HYP,and6FAILhearts.**P(cid:9)0.01and***P(cid:9)0.001vs.CON;
80
#P(cid:9)0.05,HYPvs.FAIL.AstheseverityoftheMCTtreatmentincreased,APDincreasedandAPDrestitutionsteepened.
AJP-HeartCircPhysiol•doi:10.1152/ajpheart.01084.2011•www.ajpheart.org
ARRHYTHMIAMECHANISMSINRIGHTHEARTFAILURE H2387
parametersoftheECG,suchasR-Rinterval.However,thethree and were always discordant. One FAIL heart showed a
animals showing T-wave alternans appeared to be the most transition from alternans to a 30-s period of nonsustained
severelyaffectedbyMCT;theyhadthethreelargestheartweight- fibrillation during pacing at 12 Hz. Alternans were not
to-body weight ratios and the three lowest body temperatures of observed in HYP or CON hearts; thus, there was a signifi-
thesevenFAILanimalsattheexperimentalendpoint. cant difference in the occurrence of AP alternans (CON and
Alternans in isolated hearts and single myocytes. We next HYP (cid:9) FAIL hearts; Fig. 4B).
established whether correlates of in vivo T-wave alternans APD alternans have been associated with dysfunctional
could be identified in isolated hearts and single myocytes. Ca2(cid:1) homeostasis, resulting in [Ca2(cid:1)] and contractile altern-
i
Electricalactivitywasmeasuredbyopticalmappinginisolated ans. When single RV myocytes were stimulated to contract at
hearts.APalternanswereobservedinFAILheartspacedabove 7–9 Hz, CON myocytes did not display alternans, but RV
10Hz.Figure4Ashows alternans of both AP amplitude and myocytesfromFAILheartsdid(Fig.4C).OnlyoneinsixLV
APD in two regions of the same RV in a FAIL heart. FAIL myocytes developed alternans. The proportion of RV
Alternansinthetworegionswereoutofphaseandtherefore FAIL myocytes displaying alternans was significantly greater
discordant. Alternans occurred in four of six FAIL hearts thanCONmyocytes(Fig.4D).Thus,theincreasedpresenceof
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Fig. 6. APs and L-type Ca2(cid:1) currents (I ) in single RV myocytes from CON and MCT-treated rats. A and B: intracellular membrane potential from a
CaL
representativeCON(A)andFAIL(B)RVmyocyteatstimulationfrequenciesbetween1and9Hz.C:APDat90%repolarization(APD )restitutioncurvefor
90
CONandFAILmyocytes.Dataaremeans(cid:6)SE;n(cid:2)3CONand8FAILmyocytes.ConsistentwithopticallyrecordedAPsinwholehearts,APsinsingleFAIL
RVmyocytesdisplayedalongerAPD(P(cid:9)0.001atallfrequencies)andasteeperrestitutioncurvethanCONmyocytes(P(cid:9)0.001).BCL,basiccyclelength.
D:representativeI evokedbyadepolarizationfrom(cid:3)40to0mV.Theredcurveshowstheexponentialfitusedtocalculatethetimeconstantofinactivation.
CaL
EandF:current-voltage(E )relationshipofI (E)andI availability(F)inCONandFAILmyocytesstimulatedat1Hz.Schematicvoltageprotocolsare
m CaL CaL
shown above the data. G: representative trace showing the negative effect of increasing stimulation frequency on the amplitude of I . H and
CaL
I:meandatafortheeffectofstimulationfrequencyonI density(H)andtheI timeconstantofinactivation(tau;I).Dataaremeans(cid:6)SE.*P(cid:9)0.05,
CaL CaL
**P(cid:9)0.01,and***P(cid:9)0.001vs.CONinEandvs.1HzinHandI;n(cid:2)12CONand15FAILmyocytesfromN(cid:2)3CONand5FAILhearts.Theeffect
ofincreasedstimulationfrequencywasnotstatisticallydifferentbetweenCONandFAILmyocytes.
AJP-HeartCircPhysiol•doi:10.1152/ajpheart.01084.2011•www.ajpheart.org
H2388 ARRHYTHMIAMECHANISMSINRIGHTHEARTFAILURE
Fig.7.Opticalmappingofconductionvelocity(CV)
in RVs of CON and MCT-treated rats. A: color-
codedactivationtimesacrossarepresentativeRV
surface.B:CVatastimulationfrequencyof5Hz
in CON, HYP, and FAIL hearts measured at the
RV midline (* in A). C: CV restitution curves in
CON,HYP,andFAILhearts.D:slopeoftheCV
restitution curve from CON, HYP, and FAIL
hearts. Data are means (cid:6) SE for N (cid:2) 5 CON, 6
D
HYP,and6FAILhearts.*P(cid:9)0.05and***P(cid:9) o
0.001vs.CON;#P(cid:9)0.05,HYPvsFAIL.Asthe w
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severityoftheMCTtreatmentincreased,CVres- lo
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alternans in FAIL animals could be traced from single myo- was significantly increased in FAIL hearts with no difference rt.p
cytes to isolated whole hearts and to the ECG in vivo. in the range of fiber angles across the ventricle (Table 3). hy
s
Electrical restitution as a mechanism of alternans. Optical Therefore, the gradient of the fiber angle change was signifi- io
mappingwasusedtomeasureAPDinisolatedhearts(Fig.5A). cantly shallower in the RV of FAIL hearts than the other two lo
g
APD80 was mapped across the whole RV epicardial surface groups or FAIL LV (Fig. 8B and Table 3). In addition, there y.o
m(Fiidgr.e5gBio)n. A(atstaerpisakciinngFfirge.q5uBe)ncinycroefa5seHdz(w, mitheaCnOANP(cid:9)D8H0YatPth(cid:9)e wasaisndsiegxneidficbayntthlyegRr2eaotferthdeisfiabrrearyhoelfixfibaenrgslefraogmaiRnsVtpFoAsiItLionhe(aFritgs., brg/
y
FAIL hearts; Fig. 5C). We observed a significant correlation 8B and Table 3). The level of fiber disarray was significantly 1
(R2 (cid:2) 0.83, P (cid:9) 0.001) between the degree of cardiac hyper- correlatedwiththeheartweight-to-bodyweightratioandAPD80 0.2
2
ntrootpshhyow(hne)a.rAt PwDeigdhistp-teor-sbioondywawseaiglshotirnactrioea)saendd(CAOPND8(cid:9)0 (HdYatPa at5DyHszfu(nFcitgi.on8aCl)SbRutCnao2t(cid:1)writehleAasPeDasdiaspmeresciohnanoisrmCVof.alternans. 0.33
and FAIL; Fig. 5D). Pacing frequency was increased at inter- The amplitude of Ca2(cid:1) transients evoked by electrical stimu- .1
o
vals to 13 Hz to measure dynamic APD restitution (Fig. 5E); lation at 5 Hz was compared with that evoked by rapid n
theslopeoftheAPDrestitutioncurvewassignificantlysteeper exposure to 20 mmol/l caffeine (Fig. 9A) to assess SERCA N
o
intheFAILgroupthanintheCONandHYPgroups(Fig.5F). function and SR Ca2(cid:1) release. In FAIL cells compared with ve
APrecordingsfromsinglemyocytesrevealedthatsteeperAPD CON cells, there was a significant increase in SR Ca2(cid:1) load m
b
rAe–sCtit)u.tBioonthwianscarenaisnetdrinAsPicDprdoipspeertrysioofnFaAndILinmcyreoacsyetdess(loFpige.o6f, b(Foitgh.N9aB(cid:1))/aCnad2(cid:1)SRexcrehlaenagseerf(rpalcutisoonth(eFrigC.a92C(cid:1))reamndovaadlepcartehawsaeyisn, er 10
APD restitution have been reported to be associated with an excluding SERCA; Fig. 9D) and SERCA activity (Fig. 9E). , 2
increased occurrence of alternans (see DISCUSSION). Ca2(cid:1)sparkparametersweremeasured(Fig.10AandTable4). 01
7
WehypothesizedthatmodificationofICaLmightexplainthe Ca2(cid:1)sparkmasswassignificantlyincreasedinbothHYPand
steeper APD restitution and altered Ca2(cid:1) handling of FAIL FAILmyocytescomparedwithCONmyocytes(Fig.10B),and
heartsandmyocytes.However,weobservednodifferencesin SR Ca2(cid:1) spark leak was significantly greater in FAIL myo-
eitherthecurrentdensityorrateofinactivationofICaLbetween cytes (CON (cid:9) HYP (cid:9) FAIL myocytes; Fig. 10C) due to
CON and FAIL myocytes as stimulation frequency was in- increasedsparkwidthandfrequency(Table4).Theamplitude
creased (Fig. 6, D–I). of[Ca2(cid:1)] transientsandcellshorteningatstimulationfrequen-
i
OpticalmappingwasalsousedtomeasureCV(Fig.7A).At cies from 1 to 9 Hz were measured (Fig. 11). Compared with
apacingfrequencyof5Hz,CVwasnotsignificantlydifferent CON myocytes, FAIL myocytes displayed significantly larger
between the three groups of hearts (Fig. 7B). However, as amplitudes at 1 Hz, but these fell steeply as stimulation
pacing frequencyincreased,weobservedchangesinCVrestitu- frequency was increased.
tion(Fig.7C).Theslopesoftheserelationshipsweresignificantly
different between the three groups of hearts (CON (cid:9) HYP (cid:9)
DISCUSSION
FAILhearts;Fig.7D).
Increased fiber disarray in FAIL hearts. Cardiac structure A whole animal to single myocyte approach was used to
wasinvestigatedbyDT-MRIinrandomlyselectedheartsfrom investigate which established arrhythmic mechanisms were
eachgroupafteropticalmapping(Fig.8A).RVwallthickness present in the MCT-induced model of pulmonary artery
AJP-HeartCircPhysiol•doi:10.1152/ajpheart.01084.2011•www.ajpheart.org
ARRHYTHMIAMECHANISMSINRIGHTHEARTFAILURE H2389
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Fig.8.Diffusiontensor(DT)-MRIofmyocardialfiberorientationinCONandMCT-treatedrats.A:equatorialshort-axisslicesfromrepresentativeCON,HYP,
and FAIL hearts showing color-coded fiber inclination (helix) angle. Angles of (cid:3)90 to (cid:3)60° and from (cid:1)60 to (cid:1)90° are represented as (cid:3)60 and (cid:1)60,
respectively,forclarity.B:fiberinclination(helix)angleasafunctionoftransmuraldistancefromthemidwallforrepresentativeCON,HYP,andFAILhearts.
Dataarefrom2-mm-thickand15°-widesectorsfromtheequatorialRVfreewall.FiberanglerotationwaslesssteepandfiberdisarrayincreasedinFAILhearts
(seeTable3).C:correlationsbetweenmyocardialfiberdisarray,asindexedbytheR2ofthefiberhelixanglederivedfromDT-MRIimagingandtheheart
weight-to-bodyweightratio(HW/BW;left)andAPD at5Hz(right)TherespectiveRandPvaluesareshown.DatainCarefromN(cid:2)4CON,4HYP,and
80
3FAILhearts.FiberdisarraywassignificantlycorrelatedwithHW:BWandAPD .
80
hypertension at two stages of the disease. In animals with the ECG of some conscious, unrestrained animals were
RV failure, steep electrical restitution, myofiber disarray, seen. We suggest these mechanisms are the cause of the
and dysfunction of SR Ca2(cid:1) handling were identified. Con- proarrhythmic substrate of this model (3).
sistent with the presence of these mechanisms, [Ca2(cid:1)] Becausewe(3)havepreviouslyreportedthatAPremodeling
i
transient alternans and discordant AP alternans, established in response to MCT is primarily RV rather than LV, and
precursors of serious arrhythmias, could be provoked in because we did not see significantly elevated levels of Ca2(cid:1)
isolated cells and hearts. Furthermore, T-wave alternans in alternans in LV myocytes from FAIL hearts, for brevity and
AJP-HeartCircPhysiol•doi:10.1152/ajpheart.01084.2011•www.ajpheart.org
H2390 ARRHYTHMIAMECHANISMSINRIGHTHEARTFAILURE
Table 3. Characteristics of myocardial fiber orientation from DT-MRI of CON and monocrotaline-treated hearts
WallThickness,mm
mm RV/LV HelixRange,° HelixSlope,°/mm R2
LV
CON 1.98(cid:6)0.12 164(cid:6)12 (cid:3)50(cid:6)5.0 0.90(cid:6)0.04
HYP 2.13(cid:6)0.21 139(cid:6)11 (cid:3)60(cid:6)11.0 0.84(cid:6)0.10
FAIL 2.20(cid:6)0.18 135(cid:6)14 (cid:3)45(cid:6)2.7 0.84(cid:6)0.10
RV
CON 1.20(cid:6)0.04 0.62(cid:6)0.05 148(cid:6)9 (cid:3)73(cid:6)7.8 0.72(cid:6)0.05
HYP 1.63(cid:6)0.14 0.78(cid:6)0.07 141(cid:6)5 (cid:3)70(cid:6)7.0 0.75(cid:6)0.04
FAIL 1.97(cid:6)0.12* 0.91(cid:6)0.10 128(cid:6)12 (cid:3)37(cid:6)3.6* 0.46(cid:6)0.07*
Valuesaremeans(cid:6)SE;N(cid:2)4CONhearts,4HYPhearts,and3FAILhearts.ShownareLVandRVwallthicknesses,fiberinclination(helix)angleranges
andslopes,andR2valuesfordatafrom2-mm-thickand15°-widesectorsfromtheequatorialLVandRVfreewallsinCON,HYP,andFAILhearts.Decreased
R2isameasureofincreasedfiberdisarray.*P(cid:9)0.05versustheCONgroup.
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twostagesofthedisease.MCTtreatmentproducespulmonary 21, 24) and increased heart rates when conscious and unre- d
e
hypertension, as we (and other groups) have previously dem- strained (see Fig. 2 and Ref. 51). We conclude that FAIL d
onstrated (e.g., Ref. 3). Here, we used PAAT normalized to animals are more sensitive to anesthesia (see Ref. 21), like fro
m
cardiacCLasaserial,noninvasiveindexofincreasingpulmo- human sufferers of heart failure (7). h
nary artery systolic pressure (20, 30). Our indexes of dose- TheincreasedQTintervalinFAILanimalswasmirroredby ttp
dependentchangesincardiacsize,function,andsymptomsare anincreaseinRVAPDinbothisolatedwholeheartsandsingle ://a
consistentwithpreviousstudiesusingthismodel(20,21,24). myocytes. The longer APD is thought to be due to decreased jp
Compared with CON animals, FAIL animals displayed de- K(cid:1)currents,principallytransientoutwardandinwardrectifier he
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Fig.9.Dysfunctionalsarcoplasmicreticulum(SR)Ca2(cid:1)regulationinRVmyocytesofMCT-treatedrats.A:[Ca2(cid:1)] transientsinamyocyteelectricallystimulated
i
at5Hz.Stimulationwasthenstopped,and20mmol/lcaffeinewasrapidlyappliedtoreleaseCa2(cid:1)fromtheSR.B:SRCa2(cid:1)contentestimatedfromtheamplitude
of the caffeine-induced Ca2(cid:1) transient. C: SR Ca2(cid:1)-release fraction calculated from the relative amplitude of the electrically and caffeine-induced Ca2(cid:1)
transients.D:estimateoffunctionoftheNa(cid:1)/Ca2(cid:1)exchanger,basedonthedecayofthecaffeine-inducedCa2(cid:1)transient(K ).E:sarco(endo)plasmic
caffeine
Ca2(cid:1)-ATPase(SERCA)function,calculatedfromthedifferencebetweenthetimeconstantofdecayoftheelectricallyandcaffeine-stimulatedCa2(cid:1)transients
(K ).n(cid:2)11CON,13HYP,and10FAILmyocytesfromN(cid:2)4CON,3HYP,and3FAILhearts.**P(cid:9)0.01and***P(cid:9)0.001vs.CON;##P(cid:9)0.01
SERCA
and###P(cid:9)0.001,HYPvs.FAIL.EvidenceispresentedfordysfunctionalSRCa2(cid:1)uptakeandSRCa2(cid:1)-releasefractioninFAILmyocytes.
AJP-HeartCircPhysiol•doi:10.1152/ajpheart.01084.2011•www.ajpheart.org