Effects of Wenxin Keli on Cardiac Hypertrophy and Arrhythmia via Regulation of the Calcium ...

13 Pages·2017·9.02 MB·English

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BioMed Research International
Volume 2017, Article ID 1569235, 12 pages
https://doi.org/10.1155/2017/1569235
Research Article
Effects of Wenxin Keli on Cardiac Hypertrophy and
Arrhythmia via Regulation of the Calcium/Calmodulin
Dependent Kinase II Signaling Pathway
XinyuYang,1,2YuChen,1,2,3YandaLi,1,2XiaomengRen,1,2
YanweiXing,1,2andHongcaiShang1,2
1TheKeyLaboratoryofChineseInternalMedicineoftheMinistryofEducation,
DongzhimenHospitalAffiliatedtoBeijingUniversityofChineseMedicine,Beijing100700,China
2Guang’anmenHospital,ChineseAcademyofChineseMedicalSciences,Beijing100053,China
3FujianHealthCollege,Fuzhou350101,China
CorrespondenceshouldbeaddressedtoYanweiXing;[email protected];[email protected]
Received 28 November 2016; Accepted 5 February 2017; Published 9 May 2017
AcademicEditor:AlexanderN.Orekhov
Copyright©2017XinyuYangetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
WeinvestigatedtheeffectsofWenxinKeli(WXKL)ontheCalcium/CalmodulindependentkinaseII(CaMKII)signaltransduction
pathwaywithtransverseaorticconstriction(TAC)rats.Echocardiographicmeasurementswereobtained3and9weeksafterthe
surgery.Meanwhile,theactionpotentials(APDs)wererecordedusingthewhole-cellpatchclamptechnique,andwesternblotting
wasusedtoassesscomponentsoftheCaMKIIsignaltransductionpathway.Atboth3and9weeksaftertreatment,thefractional
shortening(FS%)increasedintheWXKLgroupcomparedwiththeTACgroup.TheAPD90oftheTACgroupwaslongerthanthat
oftheShamgroupandwasmarkedlyshortenedbyWXKLtreatment.Westernblottingresultsshowedthattheproteinexpressions
ofCaMKII,phospholamban(PLB),andryanodinereceptor2(RYR2)werenotstatisticallysignificantamongthedifferentgroupsat
bothtreatmenttimepoints.However,WXKLtreatmentdecreasedtheproteinlevelandphosphorylationofCaMKII(Thr-286)and
increasedtheproteinlevelandphosphorylationofPLB(Thr-17)andthephosphorylationofRYR2(Ser-2814).WXKLalsodecreased
theaccumulationoftypeIIIcollagenfibers.Inconclusion,WXKLmayimprovecardiacfunctionandinhibitthearrhythmiaby
regulatingtheCaMKIIsignaltransductionpathway.
1.Introduction in the cardiomyocyte size. A previous study in a cohort of
690 athletes has found that 36% of the athletes died as a
Cardiovascular diseases are the most common threat to resultofcardiachypertrophy[4].Theseresultshighlightthe
humanhealthworldwideandaretheleadingcauseofmor- importance of finding suitable agents for treating cardiac
bidityamonghumans.Inmanycardiovasculardiseases,car- hypertrophy.
diachypertrophyisacommonpathologicalprocess,andthe
CaMK II belongs to the subfamily of multifunctional
cardiacarrhythmiainducedbyitisthemostcommoncause
Ser/Thrkinases,whichphosphorylateavarietyofsubstrates
of sudden cardiovascular death. Cardiac hypertrophy is a andregulatenumerouscellularfunctions[5–8]thatareinti-
maladaptive change in response to pressure overload and matelyinvolvedinheartdiseases[9–11].ActivationofCaMK
is also an important risk factor for developing heart failure II is an important step in the signaling of cardiac hyper-
[1]. Pathological hypertrophy is characterized by significant trophy.SeveralstudieshavedemonstratedthatCaMKIIplays
changes in the size, shape, wall thickness, and contractile important functions in the development of cardiac hyper-
functionofthecardiacchamber[2,3].Atthelevelofsingle trophybycausingimpairedgeneexpression[12].Increasing
cardiomyocytes,hypertrophyissimplydefinedasanincrease understandingofCaMKIIanditseffectsontheheartlends
2 BioMedResearchInternational
supporttoitspotentialasatherapeutictarget[13].Thecom- studyreceivedhumanecareincompliancewiththeNational
pound KN93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxyben- InstitutesofHealthGuidefortheCareandUseofLaboratory
zenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylben- Animals.
zylamine)hasbeenwidelyusedasapharmacologicaltoolto
inhibitCaMKIIinseveralstudies[14–16]. 2.3.EstablishmentoftheTACModelandSham-OperatedRats.
WenxinKeli(WXKL)isaChineseherbalextractdevel- The TAC surgery was performed in male Sprague-Dawley
opedattheGuang’anmenHospitaloftheChineseAcademy rats as described previously [19, 20]. Briefly, the rats were
ofChineseMedicalSciences,anditisthefirstChinesemed- anaesthetized by intraperitoneal injection of a 3% solution
icinetobeapprovedbytheChinesestateforuseinarrhyth- of chloral hydrate (300mg/kg). The preparation process
mia.ThemainingredientsofWXKLconsistofNardostachys included endotracheal intubation, positive pressure ventila-
chinensisBatal,Codonopsis,notoginseng,amber,andRhizoma tion,andpreoperativerecordingbytwelve-leadECG.Then,
Polygonati.Alargenumberofclinicaltrialshaveconfirmed thethoraxwasopened,anda4-0silksuturewaspassedunder
that WXKL can increase coronary blood flow, decrease theaortabetweentheoriginsoftherightinnominateandthe
myocardialoxygenconsumption,enhancemyocardialcom- leftcommoncarotidarteries.A6Gneedlewasplacedonthe
pliance,improvemyocardialhypoxiatolerance,relieveante- ascending aorta, and the suture was snugly tied around the
riorandposteriorcardiacloading,decreasemyocardialtissue needleandtheaorta.Theprobewasthenquicklyremoved.
damageinpatientswithhighbloodpressure,anddecreasethe Theskinwasclosed,andtheratsweremaintainedonaheat-
occurrence of arrhythmia [17]. This drug has been proven ingpaduntiltheyrecoveredfromtheanesthesia.TheSham-
to be beneficial in the treatment of various diseases, such operated animals underwent an identical procedure but
ascardiacarrhythmias,cardiacinflammation,cardiachyper- withouttheapplicationofligation.Aftersurgery,bothgroups
trophy, and chronic heart failure [18]. In the present study, were given tap water and normal chow in different cages.
wesoughttodeterminewhetherWXKLandKN93decrease Tocharacterizethemodel,echocardiographicmeasurements
cardiachypertrophyandarrhythmiabyregulatingtheCaMK wereobtained3and9weeksafterthesurgeryusingaVivid
IIsignaltransductionpathway. 7Dimensioncardiovascularultrasoundsystem(GEHealth-
care,Fairfield,Connecticut,UnitedStates)asdescribedpre-
2.MaterialsandMethods viously[21].Theindexesfortheleftventricularposteriorwall
thickness(LVPWS),leftventricularinternaldimensionsys-
2.1. WXKL. WXKL, consisting of Rhizoma Nardostachys, tole(LVIDs),andFS%wereshowninFigure2.
notoginseng,Codonopsis,amber,andRhizomaPolygonati,was
providedbytheBuChangGroup,Shandong,China.Accord- 2.4. Histological Examination. Rat heart samples were cut
ingtothenationalpharmacopoeia(NationalPharmacopoeia into transverse sections and stained with hematoxylin and
Committee, 2005), the total amount of Ginseng saponin eosin (H&E), Masson’s trichrome, and Sirius Red dye as
Rg1 (C42H72O14), notoginseng saponin R1 (C47H80O18), and described previously [22]. The stained sections were exam-
Ginseng saponin Rb1 (C54H92O23) should not be less than inedunderalightmicroscope(OLYMPUSBX51,Japan)and
17mg per bag (9g). The powdered WXKL compound was photographedat400xmagnificationformorphologicalanal-
dissolvedindistilledwaterbeforeuse. ysis.Meanwhile,theratmyocardialcellswereprocessedfor
transmissionelectronmicroscopy(H-600,Japan)according
2.2.AnimalGroupsandAdministrationofDrugs. Onehun- toroutineprocedures,aspreviouslydescribed[23].
dred male Sprague-Dawley rats (body weight: 140–160g),
purchasedfromtheVitalRiverExperimentalAnimalCenter 2.5.IsolationofCardiacVentricularMyocytes. Singlecardiac
(Beijing,China),wererandomlydividedintotwogroups:the ventricular myocytes were isolated from the hearts of the
TACgroup(𝑛=75)andtheShamgroup(𝑛=25)byusinga rats as previously described [24] with slight modifications.
random number table. The TAC rats underwent transverse Briefly,5minutesaftertheratswereheparinized(100U/mL
aortic constriction surgery, and those in the Sham group 1mL/100gi.p.),theanimalswereanesthetizedwith3%chlo-
underwentanidenticalprocedurebutwithouttheapplication ralhydrate(0.5mL/100gi.p.).Theheartswererapidlyexcised
ofligation.The75TACratswererandomlyassignedtothree and mounted on the Langendorff apparatus and perfused
2+
treatment groups by using a random number table after viatheaortawithoxygenatedCa -freeTyrodesolutionfor
ultrasoniccardiogramevaluation:theTACgroup(𝑛 = 25), 5 minutes and then with Ca2+-free Tyrode solution con-
inwhichtheratsweretreatedwiththevehiclealone(distilled taining collagenase II (0.6mg/mL, Worthington, USA),
water,1mL/kg/day)byoraladministration;theWXKLgroup trypsin (0.24mg/mL, Amresco, USA), and proteinase E
(𝑛 = 25), in which the rats were treated with the WXKL (0.08mg/mL,Amresco,USA)for15–20minutesat37∘C.Sub-
compound(4g/kg/day)byoraladministration;andtheKN93 sequently, the ventricular tissue was excised, cut into small
group (𝑛 = 25), in which the rats were treated with KN93 piecesinadishcontainingKBsolution,andblowngentlyto
(14𝜇mol/kg/day)byintraperitonealinjection.After3weeks obtainsingleventricularmyocytes.Thecellsweremaintained
∘
oftreatment,10ratswererandomlyselectedfromeachgroup at4 CintheKBsolutionuntiluse.Allthesolutionswerecon-
∘
for the experiment. After 9 weeks of treatment, there were tinuouslygassedwith95%O2and5%CO2at37 C.Thesingle
15 rats in the Sham group, 10 rats in the TAC group (5 rats ventricular myocytes selected for electrophysiological mea-
died), 13 rats in the KN93 group (2 rats died), and 12 rats surementswererod-shaped,quiescent,andCa-tolerantand
in the WXKL group (3 rats died). All animals used in this hadclearcross-striationswithasmoothandglossysurface.
BioMedResearchInternational 3
2.6. Electrophysiological Recording. The whole-cell patch groupsweresignificantlylowerthanthoseoftheTACgroup
clamptechniquewasusedtorecordtheAPsusinganAxo- (Figure1(e)).Overall,thevaluesforPW/BWat9weekswere
patch700Bamplifier(AxonInstruments,USA)andthedata significantlyhigherthanthoseat3weeks.
were analyzed with pCLAMP 9.2 software (Axon Instru-
ments, USA). Borosilicate glass patch pipettes (resistance = 3.2.AssessmentofCardiacFunctionbyEchocardiography. We
3–5MΩ)werepulledusingaverticalpipettepuller(Narishige evaluatedcardiacfunctionbyacombinationofechocardio-
pp-830, Japan). The cells were maintained in an external graphy measurements that included the LVPWS, FS%, and
solutionfor5to10minutesafterperfusion,andthedatawere LVIDs(Figure2).Beforetreatment,comparedwiththeSham
recordedafterpiercingthecellfor5minutestostabilizethe group, the LVPWS of the TAC, WXKL, and KN93 groups
current.Datarecordingswereperformedatroomtempera- were significantly higher. After treatment for 3 weeks, the
∘
ture(22 C)within25minutestoavoidcurrentrundown.APs LVPWS of the KN93 and WXKL groups were not different
wereinitiatedinthecurrentclampmodeatarateof1.0Hz from that of the TAC group. Interestingly, after treatment
using30trainsofsuprathresholdcurrentpulses.Membrane for9weeks,theLVPWSbecamesignificantlythickerinthe
capacitance was calculated using the manual whole-cell KN93 and WXKL groups compared with the TAC group
capacitancecontrolsontheAxopatchamplifier. (Figure2(b)).Aftertreatmentfor3weeks,theFS%decreased
in the TAC group compared with the Sham group, but it
2.7.WesternBlotAnalysis. Theanimalswereeuthanizedafter increasedintheWXKLgroupcomparedwiththeTACgroup.
3weeksand9weeksofdrugadministration,andtheirhearts After9weeks,theFS%intheKN93andWXKLgroupswas
were immediately harvested and stored in liquid nitrogen significantlyhigherthanthatintheTACgroup(Figure2(c)).
until western blot analyses were performed. The antibodies However, the LVIDs of the TAC group had no significant
tothefollowingproteinswereused:CaMKIIdelta(1:1000, difference compared with those of the KN93 and WXKL
AbcamCorporation.),phospho-CaMKII(1:1000,CellSig- groupsafter3weeksoftreatment,buttheLVIDsdecreasedin
naling Technology Inc.), PLB (G-18) (1:200, Santa Cruz theKN93andWXKLgroupscomparedwiththeTACgroup
BiotechnologyInc.),p-phospholamban-R(1:200,SantaCruz aftertreatmentafter9weeksoftreatment(Figure2(d)).
BiotechnologyInc.),andryanodinereceptor2(1:1000,Milli-
poreCorporation.).Theproteinswereseparatedby10%SDS- 3.3.EffectsofWXKLandKN93onMyocardialCellMorphol-
PAGEandtransferredontonitrocellulosemembranes,which ogyinaRatModelofCardiacHypertrophyInducedbyTAC.
werethenincubatedwithantibodiesat4∘C.Themembranes After 9 weeks of treatment, we isolated single ventricular
werefurtherincubatedfor2hoursatroomtemperature.ECL myocytes through enzymatic hydrolysis. After allowing the
visualizationwasperformed,andaGeneGnomeGelImaging cells to adhere to slides, the cells with neat edges and clear
System (Syngene Co.) was used to capture the resulting stripes were observed under a microscope. Compared with
images.ImageJ(Image-Proplusanalysissoftware)wasused thoseintheShamgroup,thesingleventricularmyocytesin
toanalyzethegelimages. the TAC group were larger (Figure 3). In addition, in the
KN93andWXKLgroups,thecellmorphologywasimproved,
and the degree of myocardial hypertrophy was decreased
2.8.StatisticalAnalyses. Allexperimentaldatawereexpressed
asthemeans±SD.Thedatawerestatisticallyevaluatedusing comparedwiththatintheTACgroup.
one-wayanalysisofvariance(ANOVA),andaposthocanaly-
3.4.ChangesintheMicrostructureoftheMyocardialTissuein
sis was performed using Fisher’s least significant difference
aRatModelofCardiacHypertrophyInducedbyTAC. Stained
(LSD)test.TheSPSSprogram(version20.0)wasusedforthe
analyses. A probability of 𝑃 < 0.05 was considered statis- sectionswereobservedunderalightmicroscope,andphotos
were taken under 400x magnification. Collagen deposition
ticallysignificant.ThepCLAMP9.2software(AxonInstru-
wasobservedunderordinarylightmicroscope.Theresultsof
ments, USA) and Origin 6.1 software (MicroCal Software,
Masson’strichromestainingshowedthatthecollagenfibers
USA)wereusedforthedataacquisitionandanalysis.
were stained green. The cytoplasm and muscle fibers were
stainedred.Theredbloodcellswerestainedorange,andthe
3.Results nuclei were stained blue-brown (Figure 4(a)). In the Sirius
Red staining, the collagen fibers were stained red, and the
3.1. Effects of Treatment with WXKL and KN93 for 3 and
nucleiwerestainedblue(Figure4(b)).
9 Weeks on Cardiac Morphology. The extent of myocardial
Aftertreatmentfor3weeks,Masson’strichromestaining
fibrosiswasimprovedintheKN93andWXKLgroupscom-
showedthattheTACgrouphadfibrosis,andcollagendepo-
paredwiththeTACgroupafter3and9weeksoftreatment
sitionwasapparent.Inaddition,aftertreatmentfor9weeks,
(Figure 1(c), HE staining 400x magnification). At 3 and 9
collagen deposition in the TAC group was even more pro-
weeks,theratiosofheartweighttobodyweight(HW/BW) nounced, and the degree of heart failure was significantly
of the TAC group were significantly higher than those of increased. In contrast, in the KN93 and WXKL groups, the
the Sham group, but those of the KN93 and WXKL groups extent of collagen deposition was greatly decreased (Fig-
werelessthanthevaluesfromtheTACgroup(Figure1(d)). ure4(a)).Asviewedunderalightmicroscope,theredstain-
Meanwhile, the ratios of pulmonary weight to body weight ingshowedthatthedegreeoffibrosiswasthemostintensein
(PW/BW)ofTACgroupwereonlyslightlyhigherthanthose the TAC group. However, fibrosis in the WXKL group was
oftheShamgroup,buttheratiosfromtheKN93andWXKL significantly decreased at 9 weeks (Figure 4(b)). Under a
4 BioMedResearchInternational
3 weeks 9 weeks
Sham TAC KN93 WXKL Sham TAC KN93 WXKL
(a)
(b)
(c)
TAC TAC
5 8 ∗
󳵳󳵳 󳵳󳵳 󳵳󳵳
mg/g) 34 󳵳# 󳵳# 󳵳# 󳵳# mg/g) 6 󳵳 󳵳# 󳵳# 󳵳# 󳵳#
( ( 4
W W
2
B B
W/ W/ 2
1
H P
0 0
Sham TAC93KN WXKL Sham TAC93KN WXKL Sham TAC93KN WXKL Sham TAC93KN WXKL
3 weeks 9 weeks 3 weeks 9 weeks
3 weeks 3 weeks
9 weeks 9 weeks
(d) (e)
Figure1:(a)RepresentativeimagesofheartsfromtheSham(𝑛=5),TAC(𝑛=5),KN93(𝑛=5),andWXKL(𝑛=5)groupsat3and9weeks
aftertreatment.Scale:1cm.(b)Representativeimagesofthelargestcross-sectionoftheheartfromeachgroupat3and9weeks(𝑛=5).(c)
Representativeimagesofleftventricularapicalbiopsyofeachgroupat3and9weeks(H&Estainingat400xmagnification;𝑛=5).(d)Ratios
ofheartweighttobodyweight(HW/BW)areshown(𝑛=5).(e)Ratiosofpulmonaryweighttobodyweight(PW/BW)areshownat3and
9weeks.(𝑛 = 5).󳵳𝑃 < 0.05and󳵳󳵳𝑃 < 0.01versustheShamgroup.#𝑃 < 0.05versustheTACgroup.∗𝑃 < 0.05versustheTACgroupat3
weeks.
polarizedlightmicroscope,SiriusRedstainedthetypeIcol- ridgeswerestillbroken.Aftertreatmentfor9weeks,inthe
lageninredoryellowandthetypeIIIcollageningreen.Sirius Sham group, the myocardial cells were basically complete,
Red-stainedsectionsclearlydisplayedthetypeIandtypeIII andthearrangementofthefilamentswasmoreregular.The
collagen fibers (Figure 4(c)). The results showed that the structureofthemitochondriawasalsonormal.Onceagain,in
typeIcollagenfiberswerearrangedclosely,showingstrong theTACgroup,themyocardialcellsweredissolvedtoamuch
refraction, and they appeared bright orange red or bright greaterextent,andtheirarrangementwasmoredisordered.
yellow.Incontrast,thestainingfortypeIIIcollagenfiberswas Themorphologyofthemitochondriawasalsosubstantially
veryweakandnotasapparent. altered, and the crest had been fractured. However, after
KN93 and WXKL drug treatment, the myocardial cells
3.5. Ultrastructural Changes in Cardiac Myocytes in a Rat showedslightrecovery,buttherewasstillasignificantchange
ModelofCardiacHypertrophyInducedbyTAC. Aftertreat- inthemorphologyofmitochondria(Figure5).
ment for 3 weeks, analysis of the ultrastructure of cardiac
myocytes showed that the myocardial cells of the Sham 3.6. Effects of WXKL and KN93 on the APs after Treatment
group were arranged in a compact manner, and the muscle for3and9Weeks. TheAPswererecordedbyapplicationofa
sections were clear and complete. The mitochondrialstruc- 900-pAcurrentpulsewithadurationof3msat1Hzinthe
turewasnormal,andtherewereabundantglycogengranules. currentclampmode.Aftertreatmentfor3weeks,theAPD90
However,intheTACgroup,manyofthecardiacmusclecells wasslightlyprolongedintheTACgroupcomparedwiththat
hadbeendissolved,andsomegapsappeared.Themitochon- in the Sham group (Figure 6(a)). The APD90 values of the
dria were swollen, and the ridges were broken. After KN93 ShamandTACgroupswere139.0±9.3msand150.3±11.6ms,
andWXKLdrugtreatment,somemyocardialcellshadbeen respectively (Figure 6(c)). After treatment with KN93 and
dissolved, the mitochondria still appeared swollen, and the WXKL, the APD90 changed slightly to 146.4 ± 6.5ms and
BioMedResearchInternational 5
Vehicle Vehicle KN93 WXKL
m
3 weeks
c
1
m
9 weeks
c
1
Sham TAC
(a)
0.45 ∗󳵳
#
󳵳 # 70 󳵳
LVPWS (cm) 000...334050 ∗󳵳󳵳󳵳 󳵳󳵳# ∗## FS (%) 60 󳵳 # 󳵳#∗#
󳵳∗
0.25 50
0 3 9 0 3 9
Weeks Weeks
Sham KN93 Sham KN93
TAC WXKL TAC WXKL
(b) (c)
0.7 󳵳∗
m)
S(c 0.6 󳵳󳵳##∗∗
VID #∗
L
0.5
0 3 9
Weeks
Sham KN93
TAC WXKL
(d)
Figure2:(a)TypicalechocardiographyimagesfromtheSham(𝑛=10),TAC(𝑛=10),KN93(𝑛=10),andWXKL(𝑛=10)groupsat3and
9weeks.Scale:0.5cm.(b)LVPWSofeachgroup(𝑛 = 10).(c)FS%ofeachgroup(𝑛 = 10).(d)LVIDsofeachgroup(𝑛 = 10).󳵳𝑃 < 0.05
versustheShamgroup.#𝑃<0.05versustheTACgroup.∗𝑃<0.05versuscorrespondingvaluesat3weeks.
143.4 ± 4.1ms, respectively. The WXKL group thus showed 3.7. Effects of WXKL and KN93 on the Expression of CaMK
ashortenedAPD90.Aftertreatmentfor9weeks,theAPD90 II and Related Proteins. Western blotting analysis was per-
wassignificantlyprolongedintheTACgroupcomparedwith formedtoexaminetheexpressionofCaMKII,Thr-286,PLB,
theShamgroup(Figure6(b)).APD90 oftheShamandTAC Thr-17,RYR2,andSer-2814intheleftventricularapexamong
groupswere142.7±3.9msand175.4±4.2s,respectively(Fig- thefourexperimentalgroups(Figure7(a)).
ure6(c)).AftertreatmentwithKN93andWXKL,theAPD90 Aftertreatmentfor3andfor9weeks,thedifferenceinthe
changed significantly to 160.4 ± 5.0ms, and 150.5 ± 4.6ms. expressionofCaMKIIwasnotstatisticallysignificantamong
TheresultsthusshowedthatWXKLandKN93shortenedthe anyofthegroups(Figure7(b)).However,thelevelofThr-286
APD90. was increased in the TAC group compared with that in the
6 BioMedResearchInternational
Sham TAC
(a) (b)
TAC + KN93 TAC + WXKL
(c) (d)
Figure3:SingleventricularmyocytesfromtheSham(a),TAC(b),KN93(c),andWXKL(d)group(400x).
Shamgroup.ThelevelsofThr-286intheWXKLandKN93 remodeling and tissue remodeling in hypertrophic cardiac
groupsweresignificantlydecreasedcomparedtointheTAC myocytes.
group(Figure7(c)).Therewasasimilartrendinthechange Cardiac hypertrophy is a common pathological change
aftertreatmentbothfor3weeksandfor9weeksonthelevels thatincreasestheincidenceandmortalityinmanycardiovas-
ofThr-286. culardiseases.Thesechangesarefrequentlyinducedbyelec-
The protein expression of PLB was not statistically sig- tricalremodelingandgenesisofarrhythmia.Theresultsfrom
nificant after treatment for 9 weeks (Figure 7(d)). At both histology and immunohistochemistry analyses showed that
treatmenttimepoints,theproteinexpressionofThr-17was WXKLandKN93hadasignificanteffectonthehypertrophic
significantlydecreasedintheTACgroupcomparedwiththe myocardium.H&EandMasson’strichromestainingshowed
Shamgroup.However,thelevelsofThr-17intheWXKLand thatthecardiomyocytesintheTACgroupweresignificantly
KN93groupswereincreasedaftertreatment(Figure7(e)). larger and more loosely arranged. Cardiac fibrosis is a
At both 3 and 9 weeks after treatment, the difference commonresponseofthehearttomanyformsofinjuryand
in the level of RyR2 protein expression was not statistically isthekeypathologicalprocessinvariouscardiovasculardis-
significantamongthedifferentgroups(Figure7(f)).Atboth eases [25]. In cardiac fibrosis, excessive collagen deposition
treatment time points, however, the levels of Ser-2814 were and extracellular matrix accumulation result in myocardial
decreasedintheTACgroupcomparedwiththeShamgroup, hypertrophy,cardiacdysfunction,andarrhythmias[26–28].
whereasitwashigherthanintheTACgroupintheWXKL Theexperimentalresultsrevealedthat,intheTACgroup,the
andKN93groups(Figure7(g)). gap between cells had widened and the residual number of
myocardialcellsdecreased,showingoverallfibrosis.Micro-
4.Discussion graphsindicatedthatWXKLandKN93improvedthetissue
morphology.Theenhancementofcollagendepositionplays
This study revealed that WXKL and KN93 significantly animportantroleinadverseremodelingofthecardiactissue
decreasedthedevelopmentofcardiachypertrophy,thussug- [29]. Hence, stained sections were observed under a light
gestingthatWXKLandKN93preservecardiacfunctionafter microscope, and the results showed that KN93 and WXKL
TACinrats.Ourresultsalsoshowedthatthebeneficialeffects greatlydecreasedthedegreeofcollagendeposition.Theultra-
of WXKL and KN93 might be attributed to the shortening structure of cardiac myocytes showed that the myocardial
of APD90 and regulation of the CaMK II signal transduc- cells in rats also showed slight recovery after KN93 and
tion pathway, thus eventually improving cardiac electrical WXKL drug treatments. Thus, WXKL and KN93 improved
BioMedResearchInternational 7
Vehicle Vehicle KN93 WXKL
3 weeks
9 weeks
Sham TAC
(a)
Vehicle Vehicle KN93 WXKL
3 weeks
200𝜇M
9 weeks
Sham TAC
(b)
Vehicle Vehicle KN93 WXKL
3 weeks
9 weeks
Sham TAC
(c)
Figure4:Representativeimagesof(a)Masson’strichromestainingmethodtoevaluatecardiacfibrosisofeachgroupat3and9weeks(𝑛=5),
(b)SiriusRedstainingtoevaluatecardiacfibrosisofeachgroupat3and9weeks(𝑛=5),and(c)SiriusRedstainingtoevaluatecardiacfibrosis
ofeachgroupat3and9weeksunderpolarizedlight(𝑛=5).
8 BioMedResearchInternational
Sham TAC KN93 WXKL
3 weeks
9 weeks
Sham TAC
Figure5:Theultrastructureofmyocardialcellsineachgroupat3and9weeks(𝑛=5)(Magnification10,000x).
cardiac fibrosis and decreased the degree of cardiac hyper- that WXKL and KN93 contributed to the improvement of
trophy. cardiacfunctionandinhibitedtheprocessofcardiachyper-
Cardiacfibrosiscannotonlycausecardiachypertrophy trophy.
but also affect cardiac function. It can also cause substitu- Proper functioning of the heart depends on normal
tion of the myocardium with nonfunctional fibrotic tissue, actionpotential,whichinturndependsonthenormalfunc-
thus leading to impaired ventricular systolic and diastolic tioningofionchannels.Theabnormalitythatismostcom-
functions, as well as atrial and ventricular arrhythmias and monlyfoundinanimalmodelsofcardiachypertrophyisthe
heart failure [30, 31]. Here, we evaluated cardiac function prolongation of the APD [33–35]. It has been shown that
through a combination of echocardiography measurements CaMK II inhibition has little effect on APD in a transgenic
thatincludedtheparametersLVPWS,FS%,andLVIDs.After mouse with increased endogenous CaMK II activity [36].
treatmentfor3weeks,theLVPWSoftheKN93andWXKL However,treatmentwithKN93hasbeenshowntocontribute
groups were not any thinner than that in the TAC group. totheshorteningofAPD[37].Inourmodel,wefoundthat
Interestingly,aftertreatmentfor9weeks,theLVPWSwassig- theAPDoftheTACgroupwassignificantlyprolongedcom-
nificantlythickerintheKN93andWXKLgroupscompared paredtothatoftheShamgroupafter3weeks,aresultcon-
with the TAC group. These results showed that myocardial sistentwithresultsfrompreviousstudies[38,39].Aftertreat-
hypertrophyhadformedafter3weeks,andWXKLandKN93 mentwithKN93andWXKL,theAPD90changedslightly,but
hadaneffectofdecreasinghypertrophy.However,thecardiac thischangewasnotstatisticallysignificant.After9weeksof
chamberswereenlarged,andthemyocardiumbecamethin- treatment,theresultsshowedthatWXKLandKN93further
nerintheTACgroupafter9weeks.Afterboth3and9weeks shortenedtheAPD90.Becausesomestudieshavereportedthe
oftreatment,theratiosofHW/BWandPW/BWoftheTAC useofWXKLforthetreatmentofpatientswitharrhythmia,
group were higher than those of the Sham group, but the testing this parameter may be a reasonable and effective
ratiosoftheKN93andWXKLgroupswerelessthanthatof choice[39,40].Thus,thispaperinvestigatedtheantiarrhyth-
theTACgroup.Theenlargementoftheventricleandincrease mic effects of WXKL and KN93 in the TAC model using
incardiacweightresultinadecreaseincardiacfunction,thus an electrophysiological technology. Other studies have also
resulting in cardiac decompensation [32]. After 3 weeks of demonstrated that increased CaMK II activity is linked not
treatment, the FS% decreased in the TAC group compared only to cardiomyopathy [41] but also to fatal arrhythmias,
with the Sham group, but it increased in the WXKL group owing to the prolongation of APDs and development of
compared with the TAC group. After 9 weeks of treatment, prematuredepolarization.
the FS% of the KN93 and WXKL groups was significantly CaMKIIoverexpressioncontributestothedevelopment
higherthanthatoftheTACgroup.However,theLVIDsofthe ofheartfailure[42],isassociatedwitharrhythmias[43–46],
TAC group were significantly different from those of the and has detrimental consequences in irreversible ischemia
KN93 and WXKL groups after 3 weeks, but, after 9 weeks and perfusion injury [47]. CaMK II is one of the most
2+
oftreatment,theLVIDsdecreasedintheKN93andWXKL important regulators of intracellular Ca -cycling and acts
groupscomparedwiththeTACgroup.Theseresultsshowed through phosphorylation of various proteins such as RyR2
BioMedResearchInternational 9
20mV
50ms 3ms
900pA
Sham
Sham
TAC
TAC
KN93
KN93
WXKL
WXKL
−75mV 1HZ −75mV 1HZ
3 weeks 9 weeks
(a) (b)
200 󳵳
∗∗
#
∗∗
##
150
ms)
(
D90 100
P
A
50
0
m C 3 L m C 3 L
Sha TA KN9 WXK Sha TA KN9 WXK
3 weeks 9 weeks
3 weeks
9 weeks
(c)
Figure6:RepresentativeAPtracesrecordedfromeachgroup.(a)APsofeachgrouptreatedfor3weeks(𝑛=5).(b)APsofeachgrouptreated
for9weeks(𝑛=5).(c)APD90ofeachgroupaftertreatmentwithKN93and5g/LWXKLfor3weeksand9weeks(𝑛=5).∗∗𝑃<0.01versus
theShamgroup.#𝑃<0.05and##𝑃<0.01versustheTACgroup.󳵳𝑃<0.05versustheTACgroupafter3weeks.
andPLB[48].WhenCaMKIIexpressionandactivationare Nevertheless, WXKL and KN93 also significantly increased
2+
increased, RyR2 phosphorylation and the diastolic SR Ca theexpressionofSer-2814.Thesepossiblyenhancetheability
2+
leakagearealsoincreased[49];thisdiastolicSRCa leakcan tomodulatetheCaMKIIsignaltransductionpathway.This
initiateDADsinwhichthedepolarizingcurrentconsistsofan maybethemostimportantmechanismbywhichWXKLand
inwardNa+/Ca2+exchange[50].However,dephosphorylated KN93inhibitcardiachypertrophy.
PLBinhibitsSERCA2aactivity,whereasthephosphorylation
of PLB by cAMP-dependent protein kinase or CaMK II 5.Conclusions
2+
reverses SERCA2a inhibition, thus increasing Ca uptake
into the luminal SR [51]. In the present study, the protein Insummary,thepresentstudyevaluatedtheeffectsofWXKL
expressionofThr-286wassignificantlyincreasedintheTAC and KN93 on the expression of CaMK II and on the elec-
group.WXKLandKN93significantlydecreasedthelevelof trophysiologicalparametersinTACrats.Theresultsdemon-
Thr-286inratswithTAC.WXKLandKN93alsoincreased stratedthattheprolongationofAPD90resultingfromcardiac
the level of Thr-17 in rats with cardiac hypertrophy. The hypertrophy was the electrophysiological mechanism caus-
protein levels of RyR2 slightly recovered after WXKL and ing cardiac arrhythmias, and KN93 and WXKL shortened
KN93 treatment, but there were no significant differences. the process and improved electrical remodeling. We also
10 BioMedResearchInternational
3 weeks 9 weeks
VehicleVehicleKN93WXKLVehicleVehicleKN93WXKL
CaMKII 50
p-CaMKII 50
PLB 47
p-PLB 27 Da)
k
(
RYR2 233
p-RYR2 233
GAPDH 37
Sham TAC Sham TAC
(a)
ADPH (Folds) 0011....6802 ∗ ADPH (Folds) 0011....6802 ∗∗ # # ∗∗ # # DPH (Folds) 0011....6802 ∗
MKII/G 00..24 MKII/G 00..24 LB/GA 00..24
Ca 0.0 Ca 0.0 P 0.0
Sham TAC KN93 WXKL Sham TAC KN93 WXKL p- Sham TAC KN93 WXKL Sham TAC KN93 WXKL Sham TAC KN93 WXKL Sham TAC KN93 WXKL
3 weeks 9 weeks 3 weeks 9 weeks 3 weeks 9 weeks
3 weeks
3 weeks
9 weeks 3 weeks
9 weeks
9 weeks
(b) (c) (d)
DPH (Folds) 0011....6802 ∗∗ ## ## ∗∗ ## ## ADPH (Folds) 0011....6802 ADPH (Folds) 0011....6802 ∗ # ## ∗∗ ## ##
LB/GA 00..24 YR2/G 00..24 YR2/G 00..24
p-P 0.0 R 0.0 p-R 0.0
Sham TAC KN93 WXKL Sham TAC KN93 WXKL Sham TAC KN93 WXKL Sham TAC KN93 WXKL Sham TAC KN93 WXKL Sham TAC KN93 WXKL
3 weeks 9 weeks 3 weeks 9 weeks 3 weeks 9 weeks
3 weeks 3 weeks 3 weeks
9 weeks 9 weeks 9 weeks
(e) (f) (g)
Figure7:(a)TheexpressionofCaMKIIandrelatedproteinsintheCaMKIIsignaltransductionpathwayintheleftventricularareasafter
treatmentfor3and9weekswereanalyzedbywesternblotting.(b)TheexpressionofCaMKII(𝑛 = 5).(c)Theexpressionofp-CaMKII
(Thr-286)(𝑛=5).(d)TheexpressionofPLB(𝑛=5).(e)Theexpressionofp-PLB(Thr-17)(𝑛=5).(f)TheexpressionofRyR2(𝑛=5).(g)
Theexpressionofp-RyR2(Ser-2814)(𝑛 = 5).∗𝑃 < 0.05and∗∗𝑃 < 0.01versustheShamgroup.#𝑃 < 0.05and##𝑃 < 0.01versustheTAC
group.