Nicorandil Prevents Gaq-Induced Progressive Heart Failure and Ventricular Arrhythmias in ...

13 Pages·2012·2.59 MB·English

Nicorandil Prevents Ga -Induced Progressive Heart
q
Failure and Ventricular Arrhythmias in Transgenic Mice
Masamichi Hirose1*, Yasuchika Takeishi2, Tsutomu Nakada3, Hisashi Shimojo4, Toshihide Kashihara3,
Ayako Nishio5, Satoshi Suzuki2, Ulrike Mende6, Kiyoshi Matsumoto5, Naoko Matsushita7, Eiichi Taira7,
Fumika Sato1, Mitsuhiko Yamada3
1DepartmentofMolecularandCellularPharmacology,IwateMedicalUniversitySchoolofPharmaceuticalSciences,Shiwa,Iwate,Japan,2DepartmentofCardiologyand
Hematology,FukushimaMedicalUniversity,Fukushima,Fukushima,Japan,3DepartmentofMolecularPharmacology,ShinshuUniversitySchoolofMedicine,Matsumoto,
Nagano,Japan,4DepartmentofPathology,ShinshuUniversitySchoolofMedicine,Matsumoto,Nagano,Japan,5TheResearchCenterforHumanandEnvironmental
Science,ShinshuUniversity,Matsumoto,Nagano,Japan,6CardiovascularResearchCenter,DivisionofCardiology,RhodeIslandHospital&TheAlpertMedicalSchoolof
BrownUniversity,Providence,RhodeIsland,UnitedStatesofAmerica,7DepartmentofPharmacology,IwateMedicalUniversitySchoolofMedicine,Shiwa,Iwate,Japan
Abstract
Background:Beneficialeffectsofnicorandilonthetreatmentofhypertensiveheartfailure(HF)andischemicheartdisease
havebeensuggested.However,whethernicorandilhasinhibitoryeffectsonHFandventriculararrhythmiascausedbythe
activation of G protein alpha q (Ga ) -coupled receptor (GPCR) signaling still remains unknown. We investigated these
q
inhibitory effects ofnicorandil intransgenic micewith transient cardiac expression ofactivatedGa (Ga -TG).
q q
Methodology/PrincipalFindings:Nicorandil(6mg/kg/day)orvehiclewaschronicallyadministeredtoGa -TGfrom8to32
q
weeks of age, and all experiments were performed in mice at the age of 32 weeks. Chronic nicorandil administration
preventedtheseverereductionofleftventricularfractionalshorteningandinhibitedventricularinterstitialfibrosisinGa -
q
TG. SUR-2B and SERCA2 gene expression was decreased in vehicle-treated Ga -TG but not in nicorandil-treated Ga -TG.
q q
eNOS gene expression was also increased in nicorandil-treated Ga -TG compared with vehicle-treated Ga -TG.
q q
Electrocardiogram demonstrated that premature ventricular contraction (PVC) was frequently (more than 20 beats/min)
observed in7of10vehicle-treatedGa -TGbutinnone of10nicorandil-treatedGa -TG.TheQTintervalwassignificantly
q q
shorter in nicorandil-treated Ga -TG than vehicle-treated Ga -TG. Acute nicorandil administration shortened ventricular
q q
monophasic action potential duration and reduced the number of PVCs in Langendorff-perfused Ga -TG mouse hearts.
q
Moreover, HMR1098, a blocker of cardiac sarcolemmal K channels, significantly attenuated the shortening of MAP
ATP
durationinduced bynicorandil inthe Ga -TG heart.
q
Conclusions/Significance:These findings suggest that nicorandil can prevent the development of HF and ventricular
arrhythmiacausedbytheactivationofGPCRsignalingthroughtheshorteningoftheQTinterval,actionpotentialduration,
the normalizationofSERCA2gene expression.Nicorandil mayalso improvethe impairedcoronary circulationduring HF.
Citation: Hirose M, Takeishi Y, Nakada T, Shimojo H, Kashihara T, et al. (2012) Nicorandil Prevents Gaq-Induced Progressive Heart Failure and Ventricular
ArrhythmiasinTransgenicMice.PLoSONE7(12):e52667.doi:10.1371/journal.pone.0052667
Editor:VladimirE.Bondarenko,GeorgiaStateUniversity,UnitedStatesofAmerica
ReceivedJune29,2011;AcceptedNovember19,2012;PublishedDecember20,2012
Copyright: (cid:2) 2012 Hirose etal. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisstudywassupportedinpartbyaGrant-in-AidforScientificResearchfromMinistryofEducation,Culture,Sports,ScienceandTechnology,Japan
(No.21590276)(toMH).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.Noadditional
externalfundingreceivedforthisstudy.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction demonstratedinacuteHF,suggestingapossibleeffectofthisdrug
inthetreatmentofHF[3].Infact,intravenousadministrationof
It is well known that the abnormalities in coronary hemody- nicorandilattenuatedexercise-inducedLVdiastolicdysfunctionin
namics in systolic heart failure (HF) are frequent. Myocardial
individuals with hypertrophic cardiomyopathy, probably as a
oxygen demand and consumption are increased and myocardial
result of its beneficial effect on abnormal coronary microcircula-
perfusion is also impaired in HF, which can result in myocardial
tion [4]. Moreover, chronic nicorandil administration prevented
ischemia, necrosis and apoptosis. This is potentially a factor
thedevelopmentofHFinDahlsalt-sensitivehypertensiveratsasa
contributing to progressive heart failure. Nicorandil is an ATP-
result of the promotion of myocardial capillary and arteriolar
sensitive K+ (K ) channel opener and a nitric oxide donor,
ATP growth[5].Thesefindingssupportthenotionthatnicorandilmay
whichdilatesepicardialandresistancecoronaryarteriesaswellas
ameliorate HF associated with defective coronary microcircula-
peripheral resistance arterioles and systemic veins. Thus, nicor-
tion.Ourpreviousstudydemonstratedadirecteffectofnicorandil
andil increases coronary blood flow, reduces preload and
on ventricular myocardium (i.e. shortening of ventricular action
afterload, and exerts an antianginal action [1,2]. In addition,
beneficial hemodynamic effects of nicorandil have also been
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Nicorandil-InducedPreventionofHeartFailure
potential), leading to the prevention of ventricular tachyarrhyth- Echocardiography wasalsoperformedinWT andGa -TGmice
q
mias during myocardialischemia[6]. at theageof 8 weeksbefore treatment withnicorandil.
It is known that the G protein aq-coupled receptor (GPCR)
signaling pathway plays a critical role in the development of Gross Anatomy and Histology
cardiac hypertrophy and HF [7–9]. Our previous study demon- WT, nicorandil-treated WT, vehicle-treated Gaq-TG, and
strated that a transgenic mouse with transient cardiac expression nicorandil-treated Gaq-TG female mice (n=10 each) were
ofactivatedGa (Ga -TG)developedchronicHFandventricular anesthetized and treated with sodium heparin (500 USP units/
q q
tachyarrhythmias[10–12].Whilenicorandilmayprovebeneficial kg i.v.). After a midline sternal incision, hearts were quickly
for the treatment of hypertensive heart failure as well as of excised.Theheartswerefixedwitha30%solutionofformalinin
ischemicheartdisease,itremainsunknownwhethernicorandilhas phosphate-buffered saline at roomtemperature for more than 24
inhibitory effects on the development of HF and ventricular hours, embedded in paraffin, andthencut serially fromtheapex
arrhythmiascausedbyactivationoftheG signalingpathway.We to the base. Six sections were stained with hematoxylin/eosin or
q
hypothesized that nicorandil can prevent the development of HF Masson’s trichrome for histopathological analysis. Transverse
andHF-inducedventriculararrhythmiasthroughimprovementof sections were captured digitally, and the cross-sectional diameter
coronary hemodynamics and ventricular electrophysiological ofatleast20cardiomyocytesineachsectionwasmeasuredusing
property. In the present study, we investigated the inhibitory theimageanalyzingsoftwareMacSCOPE(MITANICorporation,
effects of nicorandil on HF and ventricular arrhythmias in Ga - Tokyo) on a Macintosh computer. The measurements were
q
TG mice. performed on 3 sections in each preparation and averaged. To
assessthedegreeoffibrosis,digitalmicroscopicimagesweretaken
Materials and Methods fromthesectionsstainedwithMasson’strichromestainusinglight
microscopywithadigitalcamerasystem.Themeasurementswere
The experimental protocol was approved by the institutional performedon3imagesfromdifferentpartsoftheleftventriclein
animal experiments committee and complied with the Guide for each preparation as described previously [14]. The fibrosis
Care and Use of Laboratory Animals published by the US fraction was obtained by calculating the ratio of total connective
National Institutes of Health (NIH publication 85-23, revised areatototalmyocardialareafrom3imagesineachpreparation.
1996).TheanimalexperimentswerealsoapprovedbytheShinshu
University School of Medicine Animal Studies Committee Western Blot Analysis
(approval ID200044). Totalproteinwaspreparedfromtheventricularmyocardiumof
anesthetized WT, vehicle-treated Ga -TG and nicorandil-treated
q
Experimental Animals Ga -TG mice (n=6 each) using a lysis buffer (Cell Signaling
q
A transgenic mouse (Ga -TG mouse) with transient cardiac Technology,Inc.Danvers,MA)toexaminetheproteinexpression
q
expression of activated Ga was used [12]. The wild-type (WT) of TRPC isoforms. Protein concentrations assayed, and equal
q
mice used in this study are littermates from the non-transgenic amounts of the proteins were subjected to 10% SDS-PAGE and
mice.ThegenotypesoftheWTandGa -TGmicewereidentified transferred to PVDF membranes. To ensure equivalent protein
q
by polymerase chain reaction (PCR) with the use of tail genomic loading and to verify efficient protein transfer, membranes were
DNA as a template as previously reported. Our previous studies stained with Ponceau S before incubating with primary isoform-
demonstrated that Ga -TG mice developed HF but not ventric- specific antibodies against TRPC isoforms (TRPC 3 and 6;
q
SIGMA,SaintLouis,MO)andactin[15].Immunoreactivebands
ular arrhythmias at the age of 16 weeks, whereas they developed
were detected with an ECL kit (Amersham Biosciences Corp.,
both by 32 weeks. Therefore, to examine effects of chronic
Piscataway,NJ).Thedensitometricintensityofbandsrepresenting
nicorandil administration on HF and ventricular arrhythmias,
TRPC isoforms was normalized to that of actin. In addition, to
nicorandil (6 mg/kg/day) or vehicle was orally administered to
examine potential basal effects of long-term nicorandil treatment
Ga -TGmicefrom8to32weeksofage.Inaddition,toexamine
q in WT mice, a western blot analysis was performed in vehicle-
potential basal effects of long-term nicorandil treatment in WT
treated andnicorandil-treated WT mice (n=5 each).
mice, nicorandil (6mg/kg/day) or vehicle was also administered
to WT mice from 8 to 32 weeks of age. All experiments were
Quantification of mRNA by Real-time PCR
performed in 32-week-old mice. All mice were anesthetized with
Total RNA was prepared from the ventricular myocardium of
sodium pentobarbital (30mg/kg) applied intraperitoneally. The
anesthetized WT, vehicle-treated Ga -TG and nicorandil-treated
adequacyofanesthesiawasmonitoredbywatchingheartrateand q
Ga -TGmice (n=6 each)withIsogen (NipponGene Co.LTD.,
the frequency and the degree of motion of the sternum and of q
Tokyo, Japan) according to the manufacturer’s instructions. One
movement oftheextremities.
microgram of total RNA was used as a template for reverse
transcriptionwiththeSuperScriptFirst-Strandsynthesissystemfor
Echocardiography
qRT-PCR (Invitrogen, Carlsbad, CA). To generate a standard
WT, nicorandil-treated WT, vehicle-treated Ga -TG, and
q curveformRNAquantification,partialcDNAfragmentsofatrial
nicorandil-treated Ga -TG female mice (n=6 each) were
q natriuretic factor (ANF), B-type natriuretic peptide (BNP), b-
anesthetized, and cardiac function was assessed with echocardi-
myosin heavy chain (b-MHC), Kir6.1, Kir6.2, sulfonylurea
ography (GE Yokogawa Medical System, Tokyo, Japan) as
receptor (SUR) 1, SUR2A, SUR2B, endothelial nitric oxide
previously described [13]. Hearts were viewed at the level of the
synthese(eNOS),inducibleNOS(iNOS),connectivetissuegrowth
papillary muscles along the short axis. In M-mode tracings, the
factor(CTGF),collagentype1,phspholamban(PLB),sarco(endo)-
average of three consecutive beats was used to measure the plasmic reticulum Ca2+-ATPase 2 (SERCA2), sodium/calcium
following parameters: interventricular septum thickness, left exchanger 1 (NCX1) and acidic ribosomal protein P0 (ARPP0)
ventricularend-diastolicdimension(LVEDd),end-systolicdimen- were amplified from the heart cDNA by PCR with DNA
sion (LVESd) and fractional shortening (LVFS), which was polymeraseExTaqHS(TakaraBio,Shiga,Japan)andsubcloned
calculated as follows: (LVEDd - LVESd)/LVEDd 6100%. intothepGEM-TEasyvector(Promega,Madison,WI).Real-time
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Nicorandil-InducedPreventionofHeartFailure
PCR was performed with an ABI Prism 7900HT Sequence PVC)wasdefinedasmorethan20beats/min.InMAPsignalsof
Detection System (Applied Biosystems, Foster City, CA). The all Langendorff hearts, automated algorithms were used to
PCR mixture (20ml) contained FastStart Universal SYBR Green determine depolarization time relative to a single fiducial point
Master(Rox)(RocheDiagnostics),standardcDNA(102to106ng (i.e.,thestimulus).Depolarizationtimewasdefinedasthepointof
per reaction) or 0.2 ml of reverse-transcribed cDNA samples and maximal positive derivative in the action potential upstroke (dV/
100nMofforwardandreverseprimers.Allprimersusedarelisted dt ). Repolarization time was defined as the time when
max
in Table S1. To choose a suitable internal control for this study, repolarizationreachedalevelof50%.MAPdurationwasdefined
glyceraldehyde 3-phosphate dehydrogenase, b-actin, b-glucuron- as the difference between repolarization time and depolarization
idase, and ARPP0 were tested, and it was found that the time.
expression of ARPP0 mRNA was most constant between the
groups.Thus,theexpressionofeachgenewasnormalizedtothat Data Analysis
ofARPP0mRNA.Thespecificityofthemethodwasconfirmedby Alldataareshownasthemean6SE.Ananalysisofvariance
a dissociation analysis according to the instructions supplied by
with Bonferroni’s test was used for the statistical analysis of
AppliedBiosystems.Inaddition,toexaminepotentialbasaleffects
multiple comparisons of data. Fisher’s exact test was used to
oflong-termnicorandiltreatmentinWTmice,thereal-timePCR
compare the incidence of VT between different conditions.
was performed in WT and nicorandil-treated WT mice (n=5
P,0.05was consideredstatistically significant.
each).
Drug
Electrocardiography (ECG)
NicorandilwaskindlyprovidedbyChugaiPharmaceuticalCo.
WT, nicorandil-treated WT, vehicle-treated Ga -TG, and
q (Tokyo, Japan).
nicorandil-treated Ga -TG female mice (n=10 each) were
q
anesthetized. Electrocardiography (ECG) lead II was recorded
Results
for10mininallmice.ECGleadIIwasrecordedandfiltered(0.1
to 300Hz), digitized with 12-bit precision at a sampling rate of Nicorandil Prevented the Progression of Cardiomegaly
1000Hzperchannel(MicrostarLaboratoriesInc.,Bellevue,WA,
and Contractile Dysfunction in Ga -TG Mice
USA),transmittedintoamicrocomputorandsavedonCD-ROM. q
Toevaluatewhetherchronicadministrationofnicorandilcould
prevent the progression of HF in Ga -TG mice, cardiac
Monophasic Action Potential (MAP) q
morphology was examined in WT, nicorandil-treated WT,
WT and vehicle-treated Ga -TG mice were anesthetized and
q vehicle-treated Ga -TG and nicorandil-treated Ga -TG mice at
treated with sodium heparin (500 USP units/kg i.v.). After a q q
the age of 32 weeks. Gross examination of the four-chamber
midlinesternalincision,heartswerequicklyexcisedandconnected
section of the heart revealed all chambers to be dilated in the
to a modified Langendorff apparatus. A polyterafluoroethylene-
vehicle-treatedGa -TGheartcomparedwithWTandnicorandil-
coated silver unipolar electrode was used to stimulate the q
treated Gaq-TG hearts (Fig. 1A). The vehicle-treated Ga -TG
epicardialsurfaceoftheanteriorleftventricleattwicethediastolic q
mouse exhibited marked cardiomegaly. The heart/body weight
threshold current with a duration of 1 ms. A monophasic action
ratio increased in vehicle-treated Ga -TG mice compared with
potential(MAP)electrodewasplacedontheepicardialsurfaceof q
WT mice, but nicorandil significantly reduced the ratio in Ga -
theposteriorleftventricle,andMAPwasrecordedfor10secata q
TGmice(Table1).Theleftatrialsize/tibiallengthratiowasalso
basic cycle length of 200ms to measure MAP duration. Each
increased in vehicle-treated Ga -TG compared with WT hearts.
preparation was perfused under constant flow conditions with q
Nicorandil decreased the ratio in Ga -TG hearts (Table 1).
oxygenated (95%oxygen, 5%CO ) Tyrode’s solutioncontaining q
2 Echocardiography was performed, and representative M-mode
in mM: NaCl, 141.0; KCl, 5.0; CaCl , 1.8; NaHCO , 25.0;
2 3 echocardiogramsareshowninFigure1B.ComparedwiththeWT
MgSO ,1.0;NaH PO ,1.2;HEPES,5;anddextrose,5.0(pHof
4 2 4 mice, vehicle-treated Ga -TG mice exhibited impaired left
7.4at3661uC).Perfusionpressurewasmeasuredwithapressure q
ventricular contractility and chamber dilation as demonstrated
transducer (Nihon Kohden Co, Tokyo, Japan) and maintained
bythemarkedlyreducedLVFSandtheincreasedLVEDd(Fig.1B
within a pressure range (50–60mmHg) by adjusting flow. The
and Table 2). However echocardiographic parameters such as
MAPsignalswerefiltered(0.3to300Hz),amplified(10006)and
reducedLVFSandincreasedLVEDdweresignificantlyimproved
recorded. Perfusion pressure and flow were continuously moni-
innicorandil-treatedGa -TGcomparedwithvehicle-treatedGa -
tored during each experiment. q q
TG mice (Fig. 1B and Table 2). Moreover, LVEDd and LVFS
First, to examine direct effects of nicorandil on ventricular
age-dependentlychangedinGa -TGmice,whereasthevaluesin
action potential in Ga -TG mouse hearts, MAP was recorded q
q nicorandil-treatedGa -TGmiceat32weeksweresimilartothose
fromtheepicardialsurfaceoftheposteriorleftventricleinvehicle- q
at8weeks,indicatingthatnicorandilpreventedtheprogressionof
treated WT and Ga -TG mouse hearts (n=8 each) before and
q cardiomegaly and contractile dysfunction in Ga -TG mice
after the application of nicorandil (1 and 10mM). Next, to q
(Fig.1C).
examine the mechanisms underlying the effect of nicorandil on
ventricularactionpotential,MAPwasrecordedinvehicle-treated
Nicorandil Inhibited Myocardial Fibrosis but not the
Ga -TGmousehearts(n=4)inthepresenceofnicorandilalone,
q
and in the presence of nicorandil plus HMR1098 (30 mM), a mRNA Expression of Profibrotic Genes and
blocker of cardiacsarcolemmal K channels. Cardiomyocyte Hypertrophy in Ga -TG Mice
ATP q
We examined the effects of chronic nicorandil administration
Electrophysiological Measurement on left ventricular myocardial fibrosis and cardiomyocyte hyper-
In all mice examined, P, PR, QRS complex, QT, and RR trophy in WT, vehicle-treated Ga -TG and nicorandil-treated
q
intervals were measured from ECG lead II. The number of Ga -TGmiceattheageof32weeks.Extensiveinterstitialfibrosis
q
premature ventricular contractions (PVCs) per minute was intheleftventriclewasobservedinvehicle-treatedGa -TGhearts
q
calculated from ECG lead II. A high incidence of PVCs (High compared with WT and nicorandil-treated Ga -TG hearts
q
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Nicorandil-InducedPreventionofHeartFailure
Figure1.Effectsofnicorandiloncardiacmorphologyandontheleftventricularcontractilefunction.PanelA:Grossexaminationofthe
four-chambersectionofaheartanditshistologystainedwithhematoxylin/eosininaWT(wild-type),WT+nicorandil,Ga -TG,andGa TG+nicorandil
q q
mouse heart. Original magnification: 1.256. Panel B: Representative M-mode echocardiograms of a WT, WT+nicorandil, Ga -TG, and
q
Ga TG+nicorandil mouse at the age of 32 weeks. Panel C: Age-dependent changes in left ventricular fractional shortening (LVFS) and left
q
ventricularend-diastolicdimension(LVEDd)inWT,WT+nicorandil,Ga -TG,andGa TG+nicorandilmice.WT+nicorandil,nicorandil-treatedWT;Ga -
q q q
TG,vehicle-treatedGa -TG;Ga TG+nicorandil,nicorandil-treatedGa TG.Miceatageofthe32weekswereused.
q q q
doi:10.1371/journal.pone.0052667.g001
(Fig.2A).Thedegreeofmyocardialfibrosisintheleftventriclewas gene expression relevant to fibrotic changes. The expression of
significantly greater in vehicle-treated Ga -TG mice than in WT CTGF and collagen type 1 was significantly upregulated in Ga -
q q
mice (Fig. 2B). Nicorandil significantly reduced the increased TG hearts compared with WT mouse hearts (Fig. 2C). In
interstitial fibrosis in the left ventricle of Ga -TG mice (Fig. 2B). nicorandil-treated Ga -TG hearts, the gene expression of CTGF
q q
Wenextexaminedtheexpressionofprofibroticgenes,suchasthe andcollagentype1wassimilartothatinvehicle-treatedGa -TG
q
genesforCTGFandcollagentype1,toinvestigatewhetherthese hearts (Fig.2C).
morphological observations were accompanied by alterations in
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Nicorandil-InducedPreventionofHeartFailure
Table1.Generalparametersandtheincidenceofprematureventricularcontraction(PVC)inWT,WT+nicorandil,Ga -TG,andGa -
q q
TG+nicorandil mice.
Parameters WT WT+nicorandil Gaq-TG Gaq-TG+nicorandil
BW(g) 29.861.5 24.260.3b 29.861.4 29.761.2
HW(mg) 13062.4 12564.0 17068.8b 14869.5+
HW/BW(mg/g) 4.460.2 5.160.2 5.863a 5.060.5
LA/TL(mm/mm) 0.1460.01 0.1760.01 0.2760.02b 0.2160.02b,+
PVC 1/10 0/10 9/10b 3/10+
PVC(.20beats/min) 0/10 0/10 7/10b 0/10$
Dataarethemean6SEobtainedfrom10miceforeachgroup.ap,0.05,bp,0.01vs.WT,+p,0.05,$p,0.01vs.valuesincorrespondingparametersofGaq-TG.
doi:10.1371/journal.pone.0052667.t001
Microscopic observation revealed that the cross-sectional vehicle-treatedGa -TGmouseheartscomparedwiththatinWT
q
diameter of cardiomyocytes was profoundly increased in vehicle- mouse hearts (Fig. 4). Interestingly in nicorandil-treated Ga -TG
q
treated Ga -TG mice compared with WT mice (Fig. 2D). The hearts,thegeneexpressionofSUR2BbutnotKir6.2orSUR2A
q
increasecross-sectionaldiameterwasnotattenuatedinnicorandil- wassimilartothatinWThearts(Fig.4).Previousstudyhasshown
treated Ga -TGmice (Fig.2D). that nicorandil upregulates eNOS expression in rat hearts with
q
myocardial infarction [17]. We also examined the mRNA
Nicorandil did not Inhibit Fetal Gene Expression and expression of eNOS and iNOS in WT, vehicle-treated Gaq-TG
and nicorandil-treated Ga -TG mice at 32 weeks of age. The
TRPC Channel Protein Levels in Ga -TG Mice q
q mRNA expression of eNOS was significantly increased in
We next examined the mRNA expression of fetal type genes
nicorandil-treated Ga -TG hearts compared with vehicle-treated
suchas ANF,b-MHCandBNPinWT, vehicle-treated Ga -TG q
q Ga -TGhearts(Fig.4).Next,weexaminedthemRNAexpression
and nicorandil-treated Ga -TG mice at the age of 32 weeks. q
q of PLB, SERCA2, and NCX1 in WT, vehicle-treated Ga -TG
Expression of ANF, BNP and b-MHC was significantly upregu- q
and nicorandil-treated Ga -TG mouse hearts at the age of 32
latedinGa -TGheartscomparedwithWTmousehearts(Fig.3A). q
q weeks. The expression of PLB and SERCA2 was significantly
In nicorandil-treated Ga -TG hearts, the gene expression of
q downregulated in vehicle-treated Ga -TG hearts compared with
ANF, BNP, and b-MHC was similar to that in vehicle-treated q
that in WT mouse hearts (Fig. 5). Interestingly in nicorandil-
Ga -TG hearts (Fig. 3A). A recent study suggested that the
q treated Ga -TG hearts, the gene expression of SERCA2 but not
activation of TRPC channels participated in the generation of q
PLB was similar to that in WT hearts (Fig. 5). In contrast to the
cardiachypertrophy[16].Moreover,theproteinlevelsofTRPC3
expression of PLB and SERCA2, NCX1 expression was similar
and 6 were increased in Ga -TG mouse hearts [11]. Therefore,
q amongWT,vehicle-treatedGa -TGandnicorandil-treatedGa -
q q
we examined effects of nicorandil on the protein expression of
TG mouse hearts.
TRPCchannelsubtypesinGa -TGhearts.ThelevelsofTRPC3
q
and 6 channels were significantly increased in Ga -TG hearts
q NicorandilReducestheNumberofPrematureVentricular
compared with WT hearts (Fig. 3B). The expression of TRPC 3
Contractions (PVCs) in Ga -TG Mice
and6wasnotsignificantlydifferentbetweenvehicle-treatedGa - q
q ECG lead II was recorded for 10min in anesthetized WT,
TG andnicorandil-treated Ga -TGmouse hearts (Fig.3B).
q vehicle-treated Ga -TG and nicorandil-treated Ga -TG mice.
q q
Shown in Figure 6 are representative ECGs. The upper 2 cases
Gene Expression of ATP-sensitive K+ (K ) Channel
ATP showventriculararrhythmiasrecordedfromvehicle-treated Ga -
Subunit, Nitric Oxide Synthase (NOS), and Ca2+-handling q
TG mice:incase 1,PVCwas frequently observed; incase2,the
Proteins ECG demonstrated consecutive ventricular beats, which are
We examined the mRNA expression of ATP-sensitive K+ (K- features of VT. In contrast, the lower 3 cases recorded from a
ATP) channel subunits such as Kir 6.1, Kir 6.2, SUR1, SUR2A WT, nicorandil-treated WT, Ga -TG, and nicorandil-treated
q
and SUR2B in WT, vehicle-treated Ga -TG and nicorandil- Ga -TGmouseshowedPwavesandQRScomplexeswithregular
q q
treated Ga -TG mice at the age of 32 weeks. The expression of RR intervals without any arrhythmia, indicating a sinus rhythm.
q
Kir6.2,SUR2A,andSUR2Bwassignificantlydownregulatedin Table 1 shows the overall data for ventricular arrhythmias.
Table2. Echocardiographic parameters inWT, WT+nicorandil, Ga -TG, and Ga -TG+nicorandil mice.
q q
Parameters WT WT+nicorandil Gaq-TG Gaq-TG+nicorandil
IVS(mm) 0.7660.05 0.6860.03 0.5560.04b 0.6260.07a
LVEDd(mm) 2.860.12 2.860.07 3.460.12b 2.860.16$
LVFS(%) 49.862.9 47.061.4 26.761.6 37.060.9b,$
Dataarethemean6SEobtainedfrom6miceforeachgroup.ap,0.05,bp,0.01vs.WT,+p,0.05,$p,0.01vs.valuesincorrespondingparametersofGaq-TG.LVEDd,
leftventricularend-diastolicdimension;IVS,intraventricularseptum.
doi:10.1371/journal.pone.0052667.t002
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Nicorandil-InducedPreventionofHeartFailure
Figure2.Effectsofnicorandilontheleftventricularfibrosisandonconnectivetissuegrowthfactor(CTGF)andcollagentype1
geneexpression.PanelA:HistologyoftheleftventriclestainedwithMasson’strichromeinaWT,WT+nicorandil,Ga -TG,andGa TG+nicorandil
q q
mouse. Original magnification: 406. Panel B: Comparison of the fibrosis fraction in the left ventricle in WT, WT+nicorandil, Ga -TG, and
q
Ga TG+nicorandilmice.PanelC:QuantitativeanalysesofCTGFandcollagentype1geneexpressionbyreal-timereal-timereversetranscriptase-
q
polymerasechainreaction(RT-PCR)inWT,Ga -TGandGa -TG+nicorandilhearts.DataforCTGFandcollagentype1werenormalizedtothosefor
q q
ARPP0. Data are the mean 6 SE obtained from 6 mice for each group. Panel D: Comparison of cardiomyocyte size in the left ventricle in WT,
WT+nicorandil,Ga -TG,andGa TG+nicorandilmice.
q q
doi:10.1371/journal.pone.0052667.g002
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Nicorandil-InducedPreventionofHeartFailure
Figure3.EffectsofnicorandilonANP,BNP,andb-MHCgeneexpressionsandonproteinexpressionofcanonicaltransientreceptor
potential(TRPC)channelisoforms.PanelA:QuantitativeanalysesofANP,BNP,andb-MHCgeneexpressionbyreal-timeRT-PCRinWT,Ga -TG
q
andGa -TG+nicorandilhearts.DataforANP,BNP,andb-MHCwerenormalizedtothoseforARPP0.Dataarethemean6SEobtainedfrom6micefor
q
eachgroup.PanelB:ExpressionofTRPCchannelisoformsinWT,Ga -TGandGa -TG+nicorandilhearts.TRPCisoformexpressionwasnormalizedto
q q
actinexpressionandexpressedrelativetowt(setat1).Dataarethemean6SEobtainedfrom6miceforeachgroup.ANF,atrialnatriureticfactor;
BNP,B-typenatriureticpeptide;b-MHC,b-myosinheavychain;ARPP0,acidicribosomalproteinP0.Miceattheageof32weekswereused.
doi:10.1371/journal.pone.0052667.g003
VentriculararrhythmiassuchasahighPVCcount(morethan20 Nicorandil Restores the Baseline Values of P and QT
beats/min) were not observedinWT andnicorandil-treated WT Intervals in Ga -TG Mice
q
mice.Incontrast,ahighnumberofPVCswasobservedin7of10
Table 3 shows overall data for the electrophysiological
vehicle-treatedGaq-TGmice(Table1).Interestingly,ahighPVC parameters in WT, nicorandil-treated WT, vehicle-treated Ga -
q
count was not observed in any nicorandil-treated Gaq-TG mice TG and nicorandil-treated Ga -TG mice at 32 weeks of age. All
q
tested,indicatingasignificantreductionofventriculararrhythmias ECG parameters were longer in vehicle-treated Ga -TG mice
q
innicorandil-treatedGa -TGmicecomparedwithvehicle-treated
q than WT mice. Interestingly, while the PR and RR interval was
Gaq-TG mice. stillprolongedinnicorandil-treatedGa -TGmicecomparedwith
q
WT mice, the P interval and QT interval were restored to the
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Nicorandil-InducedPreventionofHeartFailure
Figure4.QuantitativeanalysesofKir6.1(A),Kir6.2(A),SUR1(B),SUR2A(B),SUR2B(B),eNOS(C),andiNOS(C)geneexpressionby
real-timeRT-PCRinWT,Ga -TGandGa -TG+nicorandilhearts.DataforKir6.1,Kir6.2,SUR1,SUR2A,SUR2B,eNOS,andiNOSwerenormalized
q q
tothoseforARPP0.Dataarethemean6SEobtainedfrom6miceforeachgroup.SUR1,sulfonylureareceptor1;SUR2A,sulfonylureareceptor2A;
SUR2B,sulfonylureareceptor2B;eNOS,endothelialnitricoxidesynthase;iNOS,induciblenitricoxidesynthase;ARPP0,acidicribosomalproteinP0.
Miceattheageof32weekswereused.
doi:10.1371/journal.pone.0052667.g004
normal level in nicorandil-treated Ga -TG compared with steady state pacing at a cycle length of 200msec. Ventricular
q
vehicle-treated Ga -TGmice. MAP duration was prolonged in the Ga -TG heart compared
q q
with the WT heart before acute nicorandil administration. After
Acute Application of Nicorandil Shortens the Prolonged thenicorandiltreatment,MAPdurationshortenedintheGa -TG
q
Left Ventricular MAP Duration and Prevents Ventricular heart but not WT heart (Fig. 7A). In addition, from the pooled
data (Fig. 7B), nicorandil (1 and 10mM) significantly shortened
Tachyarrhythmias in Ga -TG Mice
q ventricular MAP duration in Ga -TG hearts but not in WT
ShowninFigure7Aarerepresentativeexamplesofventricular q
hearts. Moreover, HMR1098 significantly attenuated the short-
MAPs recorded from the posterior left ventricle in Langendorff-
ening of MAP duration induced by nicorandil in the Ga -TG
perfused WT and Ga -TG hearts at the age of 32 weeks during q
q heart (Fig. 7C). Shown in Figure 7D are representative examples
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Nicorandil-InducedPreventionofHeartFailure
Figure 5. Quantitative analyses of PLB, SERCA2, and NCX1 gene expression by real-time RT-PCR in WT, Ga -TG and Ga -
q q
TG+nicorandilhearts.DataforPLB,SERCA2,andNCX1werenormalizedtothoseforARPP0.Dataarethemean6SEobtainedfrom6micefor
eachgroup.PLB,phospholamban;NCX1,sodium/calciumexchanger1;ARPP0,acidicribosomalproteinP0.Miceattheageof32weekswereused.
doi:10.1371/journal.pone.0052667.g005
ofventricularrepetitivebeatsintheLangendorff-perfuedGa -TG
q
heart during spontaneous sinus rhythm before and after acute
nicorandil administration. PVC and ventricular repetitive beats
were frequently observed in the Ga -TG heart before nicorandil
q
was administered (Fig. 7D, top). After the nicorandil treatment,
MAPdurationwasshortened,andPVCandrepetitiveventricular
beatswerenotinducedintheGa -TGheart(Fig.7D,middleand
q
bottom).HighPVCwasobservedin6of8Langendorff-perfused
Ga -TG hearts before nicorandil treatment but only 1 of 8 Ga -
q q
TG hearts after(p,0.05).
Effects of Long-term Treatment with Nicorandil on WT
Mouse Hearts
Chronicnicorandiltreatmentfrom8to32weeksofagehadno
effectonelectricalandcontractilefunction,hypertrophy,fibrosis,
(Figs.1,2,6,andTables1,2,and3).Figure8showsoveralldata
for several genes and TRPC protein expression in WT and
nicorandil-treated WT hearts. The chronic nicorandil treatment
hadnoeffectongeneandproteinexpressioninWTmousehearts
(Fig.8).
Discussion
Inthisstudy,wefoundthatchronicadministrationofnicorandil
for 24 weeks prevented the progression of heart failure and
ventricular arrhythmia in Ga -TG mice. We also found that
q
nicorandil inhibited ventricular interstitial fibrosis, attenuated the
Figure6.ECGleadIIrecordingsfromWT,WT+nicorandil,Ga -
q
TG, and Ga +nicorandil mice. The upper 2 ECGs show premature decreased SUR2B gene expression, and attenuated or prevented
q
ventricular contraction (PVC) and ventricular repetitive beats in thedecreaseofeNOSandSERCA2inGa -TGmousehearts.It
q
anesthetized Gaq-TG mice. The lower 3 ECGs recorded from a WT, hasbeensuggestedthatabnormalitiesincoronaryhemodynamics
WT+nicorandil, and Gaq-TG+nicorandil mouse show P wave and QRS in systolic heart failure contribute to ventricular remodeling,
complexwitharegularRRintervalwithoutanyarrhythmia,indicating
myocardial dysfunction and progressive heart failure. Both
sinus rhythm. Mice at the age of 32 weeks were used. See text for
details. endothelium-dependent and -independent mechanisms of coro-
doi:10.1371/journal.pone.0052667.g006 nary vasodilatation are impaired in heart failure [18–20]. For
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Nicorandil-InducedPreventionofHeartFailure
Table3. Electrocardiographic parameters inWT,WT+nicorandil, Ga -TG, and Ga -TG+nicorandil mice.
q q
Parameters WT WT+nicorandil Gaq-TG Gaq-TG+nicorandil
P(msec) 1861 1661 2561b 2261+
RR(msec) 14265 166610 18669b 193616b
PR(msec) 3963 4362 6263b 5565b
QRS(msec) 1660.5 1560.5 1961a 1762
QT(msec) 3462 3262 4362b 3463+
Dataarethemean6SEobtainedfrom6miceforeachgroup.ap,0.05,bp,0.01vs.WT,+p,0.05vs.valuesincorrespondingparametersofvehicle-treatedGaq-TG.
doi:10.1371/journal.pone.0052667.t003
Figure7.Effectsofnicorandilontheventricularactionpotentialdurationandspontaneousprematureventricularbeats.PanelA:
Representativeexamplesofmonophasicactionpotentials(MAPs)recordedfromtheposteriorleftventricleinaLangendorff-perfusedWTandGa -TG
q
heartduringsteadystatepacingatacyclelengthof200msec.PanelB:OveralldataforMAPdurationinWTandGa -TGheartsbeforeandafteracute
q
nicorandiladministration.PanelC:OveralldataforMAPdurationinGaq-TGheartsduringthecontrol,inthepresenceofnicorandil(10mM)alone,andin
thepresenceofnicorandilplusHMR1098(30mM),ablockerofcardiacsarcolemmalKATPchannels.PanelD:Spontaneousprematureventricularbeatsin
aLangendorff-perfusedGa -TGheartbeforeandafteracutenicorandiladministration.Miceattheageof32weekswereused.Seetextfordetails.
q
doi:10.1371/journal.pone.0052667.g007
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