research article microrna-208a dysregulates apoptosis

Research ArticleMicroRNA-208a Dysregulates Apoptosis GenesExpression and Promotes Cardiomyocyte Apoptosis duringIschemia and Its Silencing Improves Cardiac Function afterMyocardial Infarction

Hasahya Tony1 Kai Meng2 Bangwei Wu3 Aijia Yu4 Qiutang Zeng1

Kunwu Yu1 and Yucheng Zhong1

1Laboratory of Cardiovascular Immunology Institute of Cardiology Union Hospital Tongji Medical CollegeHuazhong University of Science and Technology 1277 Jie-Fang Avenue Wuhan 430022 China2Department of Cardiology The Second Hospital of Shandong University Jinan Shandong 250033 China3Department of Cardiology Huashan Hospital Shanghai Medical College Fudan University Shanghai 200040 China4Department of Ultrasound Union Hospital Tongji Medical College Huazhong University of Science and Technology1277 Jie-Fang Avenue Wuhan 430022 China

Correspondence should be addressed to Qiutang Zeng zengqiutang007163com

Received 30 June 2015 Revised 11 September 2015 Accepted 4 October 2015

Academic Editor Hamid Boulares

Copyright copy 2015 Hasahya Tony et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Aims miR-208a is associated with adverse outcomes in several cardiac pathologies known to have increased apoptosis includingmyocardial infarction (MI) We investigated if miR-208a has proapoptotic effects on ischemic cardiomyocytes and if its silencinghas therapeutic benefits in MIMethods and Results The effect of miR-208a on apoptosis during ischemia was studied in culturedneonatal micemyocytes transfected with agomir or antagomir Differential gene expression was assessed usingmicroarraysMI wasinduced in male C57BL6 mice randomly assigned to antagomir (119899 = 6) or control group (119899 = 7) while sham group (119899 = 7) hadsham operation done Antagomir group received miR208a antagomir while control and sham group mice received vehicle onlyAt 7 and 28 days echocardiography was done and thereafter hearts were harvested for analysis of apoptosis by TUNEL methodfibrosis usingMassonrsquos trichrome and hypertrophy using hematoxylin and eosin miR-208a altered apoptosis genes expression andincreased apoptosis in ischemic cardiomyocytes Therapeutic inhibition of miR-208a decreased cardiac fibrosis hypertrophy andapoptosis and significantly improved cardiac function 28 days after MI Conclusion miR-208a alters apoptosis genes expressionand promotes apoptosis in ischemic cardiomyocytes and its silencing attenuates apoptosis fibrosis and hypertrophy afterMI withsignificant improvement in cardiac function

1 Introduction

Apoptosis a distinct form of cell death is at the heart ofboth mechanical and molecular mechanisms of cardiomy-ocyte loss during ischemia After MI there is heightenedapoptosis in the peri-infarct and noninfarcted myocardiumand this coincides with increased left ventricular diameterand decreased cardiac function [1] To this effect therapiesthat inhibit apoptosis in MI have been shown to improvecardiac function [2 3]

MicroRNAs (miR) are small noncoding ribonucleic acids(RNAs) measuring 18ndash25 nucleotides that are involved inposttranscription regulation of gene expression [4 5] miR-208a is a cardiac specific microRNA coded for in an intronof myosin heavy chain 6 (Myh6) gene and regulates cardiacconduction stress response and gene expression [6ndash9]Several researches have shown that its inhibition or deletionis associated with downregulation of cardiac fibrosis andhypertrophy in response to various stress stimuli [6ndash8] It hasalso been shown to be dysregulated in several cardiac diseases

Hindawi Publishing CorporationMediators of InflammationVolume 2015 Article ID 479123 11 pageshttpdxdoiorg1011552015479123

2 Mediators of Inflammation

including myocardial infarction and dilated cardiomyopathyin which it is associated with adverse outcomes [10 11]However no study as yet has been done to investigate its rolein cardiomyocyte apoptosis despite these cardiac pathologiesbeing associatedwith increasedmyocyte apoptosisWe there-fore investigated the effect of miR-208a on cardiomyocyteapoptosis and apoptosis related genes and if its silencinghas any therapeutic benefit in myocardial infarction Wereport that miR-208a upregulation alters apoptosis genesrsquoexpression and promotes cardiomyocyte apoptosis while itssilencing attenuates apoptosis and improves cardiac functionafter MI To the best of our knowledge our study is the firstto explore the antiapoptotic effects of miR-208a silencing andthe therapeutic benefits ofmiR-208a antagomir inmyocardialinfarction

2 Materials and Methods

21 Cardiomyocyte Cell Culture Transfection and SimulatedIschemia Neonatal mouse cardiomyocyte isolation and cul-ture were performed according to previously described pro-tocols [12] Cardiomyocytes were transfected with 02 nmolsmL of miR-208a agomir for gain of function or 04 nmolsmLof miR-208a antagomir for loss of function by directlyadding the agomir or antagomir formulation to the cellculture medium according to manufacturerrsquos instructions(RiboBio Co Ltd Guangzhou China) For Bax silenc-ing cardiomyocyte cells were transfected with Bax siRNA(51015840-AACAGAUCAUGAAGACAGGGG-31015840) using Ribofec-tamine reagent according to manufacturerrsquos instructions(RiboBio Co Ltd Guangzhou China) After 48 hours thedifferent treatment groups underwent simulated ischemia for12 hours by placing the cardiomyocytes in a hypoxia chamberwith 5 CO

2and 95 N

2at 37∘C in glucose-free DMEM

medium Sham group myocytes did not undergo simulatedischemia Myocytes were harvested fixed and assayed forapoptosis according to previously described protocol [13]Myocytes were also analyzed for miR-208a levels after cultur-ing

22 Microarray Profiling Microarray profiling was done oncustom-made array chips by a service provider (RiboBioCo Guangzhou China) RNA was isolated from culturedcontrol or miR-208a agomir transfected cardiomyocytes 72hours after transfection Differential gene expression betweencontrols and agomir transfected cardiomyocytes was ana-lyzed using permutation analysis of differential expressionGene clustering was performed with cluster 30 and heat mapimages generated in Java tree view Gene ontology analysiswas done using DAVID online tool [14 15] and apoptosispathway analysis was done using Ingenuity Pathway Analysis(httpwwwingenuitycom)

23 Animal Procedures All experiments were conductedaccording to the Guide For the Care and Use of LaboratoryAnimals and approved by the Animal Care and Utiliza-tion Committee of Union Hospital Tongji Medical CollegeHuazhong University of Science and Technology Wuhan

ChinaThe investigation conformed to theGuide for the CareandUse of LaboratoryAnimals published by theUSNationalInstitute of Health (2011)

24 Animals and Delivery of Antagomir Adult male C57BL6mice 8-week-old were purchased from Wuhan UniversityAnimal Research Center Mice were anesthetized by anintraperitoneal injection of pentobarbital sodium (50mgkg)orally intubated and connected to a rodent ventilator andECG monitoring was done using RM6240 data acquisitionsystem sampling at 1 kHz A left thoracotomy was performedby a horizontal incision at the third intercostal space andmyocardial infarction induced by ligation of the left anteriordescending coronary artery (LAD) according to previouslydescribed protocol [16] and then randomly assigned toAntagomir group (119899 = 6) or control group (119899 = 7) Shamgroupmice (119899 = 7) had the same procedure done but withoutligation of the LAD Starting within 2 hours after MI eachantagomir groupmouse received a total of 300 nmols of miR-208a antagomir given over 3 consecutive days (days 0 1 and2) using 02mLs of normal saline as vehicle by low pressuretail vein injection while control and sham group mice eachreceived 02mLs of vehicle only by the same method overthe same period of time Echocardiography was performedat 7 and 28 days and thereafter animals were euthanized andhearts harvested for further analysis

25 Histology At 28 days animals were euthanized andhearts harvested cut transversely into blocks and fixed in4 paraformaldehyde These were then embedded in OCTcompound (BHD UK) and then transversely cut into 5120583mthick cryosections for histological analysis Two cryosectionsfrom each of the blocks were stained withMassonrsquos trichromefor assessment of fibrosis and infarct sizeThe infarct size wasmeasured as the sum of the epicardial and endocardial scarlength divided by the sum of LV epicardial and endocardialcircumferences

For assessment of fibrosis we randomly selected 4 fieldsper slide in the peri-infarct zone (PIZ) or 5 fields per slidein the noninfarct zone (NIZ) and calculated collagen volumefraction (CVF) as the ratio of blue stained (collagen) area tothe total tissue area The collagen-rich border zone of vesselsand the scar were excluded from the analysis Image analysiswas done using Image-Pro Plus software version 6

Cardiomyocyte hypertrophy was assessed by capturingcardiomyocyte images over 5 different fields per slide (40xobjective) Using Image-Pro Plus software version 6 weobtained mean cardiomyocyte cross-sectional areas whichwere compared between groups

26 Apoptosis Assay At 28 days after MI or after 12 hoursof simulated cardiomyocyte ischemia tissues or cells respec-tively were fixed with 4 paraformaldehyde and apoptosisassay was done by TUNEL method according to previouslydescribed protocol [13]

27 Quantitative Real-Time PCR Total RNA was extractedfromcells and tissue samples usingTrizol reagent (Invitrogen

Mediators of Inflammation 3

00

05

10

15

20

25

miR

-208

a fol

d ch

ange

AntagomirControl

lowast

(a)

0

500

1000

1500

2000

2500

miR

-208

a fol

d ch

ange

AgomirControl

lowastlowast

(b)

00

05

10

15

miR

-208

a fol

d ch

ange

AntagomirControl

lowast

(c)

Figure 1 miR-208a agomir and antagomir transfection significantly upregulated or downregulated miR-208a respectively (lowast119901 lt 005lowastlowast119901 lt 0001) (a) miR-208a antagomir transfection significantly downregulated miR-208a in cultured neonatal cardiomyocytes 48 hours

after transfection (119899 = 4 samples per group) (b) Agomir transfection into neonatal cardiomyocytes significantly upregulated miR-208a(119899 = 4 samples per group) (c) miR-208a antagomir significantly downregulated miR-208a at 28 days after MI (119899 = 4 per group)

USA) according to the manufacturerrsquos protocol cDNA wassynthesized from2120583g of total RNAwithTakararsquos reverse tran-scription kit (Takara China) according to manufacturerrsquosinstructions in a 25 120583L volume including 2 120583g total RNA andreverse transcription primer for mRNA genes or randomprimers for U6 rRNA and miR-208a specific primers (Bulge-Loop miRNA qPCR Primers from RiboBio China) Real-time PCR was carried out with the reagents of a Sybr greenI mix (Takara China) in a 20120583L reaction volume (10 120583LSybr green I mix 200mM forward and reverse primer and2 120583L cDNA template) on an MJ Opticon Monitor chromo4

instrument (Bio-Rad USA) using the following protocol95∘C for 20 s 40 cycles of 95∘C for 10 s 60∘C for 20 s and70∘C for 1 s Normalization for miR-208a was done usingU6 while Bax expression was normalized using GAPDH U6and Bulge-Loop miRNA qPCR Primers were from RiboBioChina The following primer sets were used for BAX andGAPDH

BAX

Forward TGTTTGCTGATGGCAACTTCReverse GATGGTTCTGATCAGCTCGG


Table 1 MicroRNA-208a upregulated or downregulated genes involved in apoptosis

Downregulated UpregulatedGene log 2 ratio Ratio (fold change) 119901 value Gene log 2 ratio Ratio (fold change) 119901 valueSTEAP3 minus07095433 16352864 00011179 BAX 0836016276 17851141 00084057CRADD minus0823218 17693482 00112386 BOK 1294252566 2452499 00005049CYLD minus06113516 15276898 00183018 C9 074852223 1680071 00019129CASP8 minus08671301 18240308 00016388 ERN1 2265447191 48080343 391E minus 05COL4A3 minus08231795 17693011 00129512 HIPK2 0593421677 1508821 187E minus 02CIDEB minus06968248 16209334 0001492 HMOX1 0644592878 15632981 00483578IRF1 minus06296009 15471369 00230882 HRAS1 1335399535 25234536 00043852PPP1R13B minus1029947 20419492 00006539 WNT7B 1931190212 3813697 295E minus 04DMRT2 minus10451381 20635638 00004388ERCC2 minus08137374 17577592 744E minus 05GSDMA minus06040422 15199694 00236305NUDT2 minus07902432 17293659 00099716

GAPDH

Forward 51015840-GCTGGCGCTGAGTACGTCGTGGAGT-31015840

Reverse 51015840-CACAGTCTTCTGGGTGGCAGTGATGG-31015840

Data analyses were performed using the 2minusΔΔCt method

28 Echocardiography To assess if therapeutic silencing ofmiR-208a improves cardiac function in MI 2-dimensionaltransthoracic echocardiography studies were performed on4-5 mice from each group lightly anesthetized with pento-barbital sodium (25mgkg) on days 7 and 28 using a GEVoluson ultrasound system equipped with an 115MHZ highfrequency probe Cardiac imaging was done in a parasternalshort-axis view at the level of the papillary muscles torecord M-mode and determine fractional shortening (FS)and ejection fraction (EF) which are measures of cardiacfunction

29 Statistical Analysis Statistical analysis was done usingSPSS version 20 Groups were compared using indepen-dent Studentrsquos 119905-test or one-way ANOVA with the Tukeyor Games-Howell post hoc tests as appropriate Statisticalsignificance was defined as a 119901 value lt005 and values arepresented as mean plusmn SEM

3 Results

31 miR-208a Antagomir and Agomir Are Effective in Silencingor Upregulating miR-208a Respectively Expression of miR-208a in vivo and in vitro was analyzed using quantitative RT-PCR We found that 04 nmolmL of miR-208a antagomirsignificantly downregulated miR-208a expression in culturedcardiomyocytes (Figure 1(a)) Similarly miR-208a agomir

upregulated miR-208a expression in neonatal cardiomy-ocytes 48 hours after transfection (Figure 1(b)) To silencemiR-208a 300 nmols of miR-208a antagomir per mouse wasadministered after MI and this significantly downregulatedmiR-208a expression at 28 days after MI (Figure 1(c))

32miR-208aAlters Apoptosis Genes Expression and PromotesCardiomyocyte Apoptosis during Ischemia Apoptosis a dis-tinct form of cell death is a contributor to cardiac dys-function remodeling and heart failure following MI [17 18]We investigated the role of miR-208a in cardiomyocyte apop-tosis in an ischemic setting Simulated ischemia of neonatalcardiomyocytes following transfection with miR-208a agom-ir or antagomir showed that miR-208a upregulation signif-icantly increases apoptosis while its silencing blunts apopto-sis during ischemia (Figures 2(a) and 2(b)) To gain insightinto the effect of miR-208a on apoptosis related genes weemployed custom-builtmicroarrays (RiboBioCoGuangzhouChina) Hierarchical clustering and heat map visualization ofthe differentially expressed genes are shown in Figure 3(a)Cluster analysis of the differentially expressed genes revealedthat eight apoptosis related genes were upregulated whiletwelve apoptosis related genes were downregulated (Table 1)A closer inspection of the dysregulated genes showed thatmiR-208a tended to downregulate genes favoring the extrin-sic apoptosis pathway while upregulating those that favorthe intrinsic pathway of apoptosis Analysis of differen-tially expressed apoptosis genes highlighted Bax Casp8 andHRAS1 as involved in the canonical apoptosis pathways(Figure 3(b)) Bax is an essential member of the intrinsicpathway of apoptosis To decipher if Bax was essential formiR-208a induced apoptosis we used siRNA to silence Baxgene Bax siRNA attenuated the proapoptotic effect of miR-208a agomir (Figure 2(c)) suggesting that miR-208a func-tions to promote apoptosis at least in part by upregulatingBax

Mediators of Inflammation 5TU

NEL

DA

PI

Antagomir ControlSham Agomir

(a)

Perc

enta

ge o

f apo

ptot

ic

Ant

agom

ir

Con

trol

Sham

Ago

mir

lowast

lowast

lowast

0

5

10

15

nucle

i per

fiel

d

(b)

BAX

siRN

A

Scra

mbl

edsiR

NA-

cont

rol

Scra

mbl

edsiR

NA-

agom

ir

BAX

siRN

A-ag

omir

lowast lowast

lowast

0

5

10

15

20

nucle

i per

fiel

dPe

rcen

tage

of a

popt

otic

(c)

Figure 2 miR-208a upregulation promotes cardiomyocyte apoptosis during ischemia and its silencing attenuates myocyte apoptosis (lowast119901 lt005) (a) Representative TUNEL stained images of neonatal cardiomyocytes showing increased apoptosis following agomir treatment anddecreased apoptosis following antagomir treatment and simulated myocyte ischemia (times20 objective) (b) Graph of percentage apoptosisfollowing simulated ischemia of neonatal cardiomyocytes shows that miR-208a agomir significantly upregulated myocyte apoptosis whilemiR-208a antagomir transfection attenuated ischemia induced apoptosis (119899 = 3 per group) (c) Bax silencing using siRNA attenuated miR-208a agomir induced cardiomyocyte apoptosis during ischemia (119899 = 3 per group)

33 miR-208a Silencing Attenuates Apoptosis in MI Apop-tosis has been observed to occur in the peri-infarct andnoninfarctedmyocardiumand is a contributor to cardiac dys-function remodeling and heart failure following MI [17 18]To investigate the antiapoptotic effects of miR-208a silencingin an MI setting we treated mice with 300 nmols of miR-208a antagomir Antagomir attenuated peri-infarct apoptosisat 28 days a finding that is novel and may have therapeuticsignificance (Figures 4(a) and 4(b)) In the noninfarct area

however the difference in apoptosis between antagomir andcontrol group hearts only showed a trend towards reductionbut did not reach statistical significance (Figure 4(c)) qPCRshowed that miR-208a silencing in MI also decreased Baxlevels at 28 days (Figure 4(d))

A comparison of infarct area sizes between control andantagomir groups showed a trend towards reduction ininfarct size by antagomir but this did not reach statisticalsignificance (Figure 4(e))


Con

trol 2

Con

trol 1

Ago

mir

1

Ago

mir

2

0 1minus1

Row

Color key

Z-score

(a)

Extracellular spaceCytoplasm

TNFFasL

TNFR

DNA damage

FAS

CASP

CASP

810NIK

IKK

RTK

ASK1 RASc-RAF

BIM

CDC2

pSORSK

BAD

BCL-XL

CYCSHTRA2

BAXBCL-B

cIAP

DIABLO

APAF1

AIF ENDO G

zVAD-FMK

MCL1

HTRA2

BIRC6

BID

tBID

P BFL-12

CASP9

CASP3

CASP

CAD

6CASP

7

DNA

PARP ACINUS

fragmentationDNAfragmentation

ERstress Calpain

CASP12

p53 JNK1

tBD

DIABLO AIF

MEK12

12ERK

Cellshrinkage

Membraneblebbing

Chromatin condensation

Apoptosis

LaminFodrinA

GAS2ROCK1

PLC120574

PKC

I120581B

I120581B

NF-120581B

NF-120581B

Mitochondria

Growthfactor

MKK47

CAD

DNArepair

BAK BCL-2

(120572 120576 120579)

CYCS

ENDO G

(b)

Figure 3 Heatmap visualization and pathway analysis of differentially expressed genes (a)Hierarchical clustering and heatmap visualizationof the differentially expressed genes after agomir based upregulation of miR-208a (b) Apoptosis pathway analysis highlighted Bax Caspase8and RAS as the miR-208a regulated genes involved in the canonical apoptosis pathways


Antagomir ControlSham

TUN

ELD

API

(a)

00

05

10

15

20

25

Apop

totic

nuc

lei (

)

AntagomirControl Sham

lowast

lowast

(b)


lowast

00

02

04

06

08

10

Apop

totic

nuc

lei (

)

(c)


lowast

lowast

00

05

10

15

20

BAX

fold

chan

ge

(d)

AntagomirControl0

10

20

30

40

50

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rct a

rea (

)

(e)

Figure 4 miR-208a silencing attenuates Bax expression and cardiomyocyte apoptosis at 28 days after MI (lowast119901 lt 005) (a) Representativetunnel stained images of the peri-infarct area showing that antagomir treatment decreased apoptosis in the peri-infarct area at day 28 (times20objective) (b) Antagomir treatment significantly attenuated percentage apoptosis in the peri-infarct area (119899 = 3 per group) (c) Bar graphof percentage apoptotic nuclei in the noninfarcted area showing no significant difference between antagomir group and control (119899 = 3) (d)miR-208a antagomir downregulated Bax expression at 28 days after MI (119899 = 4 per group) (e) Although there was a trend towards reductionin infarct size in miR-208a antagomir treated mice compared to controls this did not reach statistical significance (119899 = 3 per group)

34 miR-208a Silencing Attenuates Myocyte Hypertrophy andCardiac Fibrosis in MI At one month after MI antagomirtreatment significantly decreased cardiomyocyte hypertro-phy (Figures 5(a) and 5(b)) a known pathological responseassociated with heart failure and sudden death [19 20]Moreover miR-208a antagomir also decreased percentagefibrosis at day 28 (Figures 5(c) 5(d) and 5(e)) Increasedfibrosis is known to reduce cardiac contractility and tocontribute to reduction of cardiac function after MI [21] andthus its reduction by antagomir is potentially beneficial Theabove results were consistent with previous reports whichshowed that miR-208a silencing or knockout attenuatescardiac fibrosis and hypertrophy in response to cardiac stress[6 7]

35 Therapeutic Silencing of miR-208a Improves CardiacFunction after MI Aiming to see any beneficial effects oflong term silencing ofmiR-208a on cardiac function afterMImice received a total of 300 nmols of miR-208a antagomirand echo was done at 7 and 28 days Antagomir treatmentsignificantly attenuated cardiac dysfunction after MI (Table 2and Figures 5(f) and 5(g))

4 Discussion

The current study investigated the role of miR-208a in apop-tosis during cardiomyocyte ischemia and the therapeuticpotential of miR-208a antagomir in myocardial infarctionHere we reveal for the first time that miR-208a promotes


Sham Antagomir Control

(a)

Control Antagomir Sham

lowast

lowastlowast

0

100

200

300

400

Card

iom

yocy

te ar

ea(120583

M2 )

(b)


lowastlowast

0

5

10

15

Fibr

osis

()

(c)


(d)


lowastlowast

lowastlowast

0

10

20

30

40

50

Fibr

osis

()

(e)


lowast

lowastlowast

0

20

40

60

Mea

n fr

actio

nal s

hort

enin

g

(f)

Figure 5 Continued



(g)

Figure 5miR-208a silencing attenuates cardiomyocyte hypertrophy andfibrosis and improves cardiac function at 28 days afterMI (lowast119901 lt 005lowastlowast119901 lt 0001) (a) Representative images of hematoxylin and eosin staining indicate a decrease in cardiomyocyte size in antagomir treated

mice at 28 days after MI Scale bar = 40 120583m (b) Graph comparing mean cardiomyocyte sizes at 28 days after MI Antagomir treatmentdecreased the mean cardiomyocyte size compared to controls (119899 = 3 per group) (c) Graph showing percentage fibrosis in the noninfarctedarea Antagomir treatment decreased fibrosis in the noninfarcted area compared to controls at 28 days after MI (119899 = 4 per group) (d)Representative images showing miR-208a antagomir decreased fibrosis in the noninfarcted area at 28 days after MI (e) Bar graph of peri-infarct fibrosis quantification shows reduction in peri-infarct fibrosis in antagomir treatment group at 28 days after MI (119899 = 4 per group) (f)miR-208a silencing improved cardiac function as measured by fractional shortening at 28 days after MI (119899 = 4 per group) (g) Representativeechocardiography images showing improved wall motion in antagomir treated group compared to control at day 28 after MI

Table 2 Mean echocardiographic parameters at 7 and 28 days after MI

LVIDd LVIDd LVIDs LVIDs EF EF FS FS7 days 28 days 7 days 28 days 7 days 28 days 7 days 28 days

Sham 232 plusmn 032 282 plusmn 026 115 plusmn 015 154 plusmn 011 866 plusmn 114lowast 826 plusmn 23lowast 496 plusmn 114lowast 45 plusmn 245lowast

Antagomir 368 plusmn 033 396 plusmn 041 236 plusmn 037 259 plusmn 042 725 plusmn 619 710 plusmn 560lowast 360 plusmn 455 350 plusmn 42lowast

Control 36 plusmn 02 407 plusmn 056 25 plusmn 026 304 plusmn 069 648 plusmn 676 534 plusmn 879 308 plusmn 444 236 plusmn 472LVIDd left ventricular internal diameter diastole LVIDs left ventricular internal diameter systole EF ejection fraction FS fractional shortening lowast119901 lt 005compared to controls

myocyte apoptosis during ischemia and further show thattherapeutic administration ofmiR-208a antagomir attenuatescardiomyocyte apoptosis hypertrophy and fibrosis coupledwith improvement in cardiac function after MI

Following myocardial infarction nonischemic myocyteapoptosis ensues resulting in further myocardial loss andventricular dysfunction [2 19 20] Because apoptosis plays acritical role in both mechanical and molecular mechanismsof cardiac dysfunction and remodeling after MI [19 20]blunting it is an attractive therapeutic target Our finding thatmiR-208a antagomir mitigates ischemia induced apoptosisopens a window for its possible therapeutic application inMI Despite noting no difference in infarct size between thegroups the observed decrease in apoptosis levels could still bea contributor to improved contractile performance as otherstudies have shown [2] Interestingly some researchers haveshown that cardiac miR-208a in human MI is persistentlyupregulated with the highest levels observed in patients whodied within 24 hours [10] Thus targeted silencing of miR-208a in human MI may be a potentially viable and beneficialtherapeutic option

Although miR-208a has been shown to be proprolif-erative under certain conditions we demonstrated that itis proapoptotic in the ischemic setting [22 23] It is notuncommon for microRNAs or other genes to have oppositeeffects under different conditions For example miR-494 hasbeen shown to have both antiapoptosis and proapoptosiseffects under different circumstances [24] Similarly p21 (Waf1) a target of miR-208a has also been reported to be both

anti- and proapoptosis depending on prevailing conditions[25]

To investigate the genetic basis of miR-208a inducedapoptosis we employed custom-built microarrays whichprovided an insight into the miR-208a regulated genes asso-ciated with apoptosis There was a noted trend towardsdownregulation of genes involved in the extrinsic apoptosispathway and upregulation of those in the intrinsic apoptosispathway Among those upregulated was Bax an essentialmember in the intrinsic pathway which was also highlightedby pathway analysis of the differentially expressed apoptosisgenes (Figure 3(a)) In MI Bax was also shown to be down-regulated by miR-208a silencing and may thus be playinga role in miR-208a mediated myocyte apoptosis duringischemia Bax functions by controlling access of upstreamapoptotic signals to the mitochondria and together with Bakis essential for intrinsic pathway mediated apoptosis [1726] Given that microRNAs can upregulate gene expressionby binding to the promoter regions and target sites in themRNA or indirectly by downregulating repressors [27ndash29]we scanned the Bax promoter regions and mRNA for anymiR-208a binding sites Results revealed no predicted miR-208a binding sites both in the gene promoter regions and inmRNA suggesting that miR-208a may be acting indirectlyto alter Bax expression Our findings only open a windowand more studies will be needed to further decipher themechanisms involved in miR-208a mediated apoptosis

Upon histological examination we observed significantlyreduced myocyte hypertrophy in the noninfarcted area of


the LV following antagomir treatment Given that heightenedcardiomyocyte hypertrophy in the noninfarcted region isassociated with remodeling and cardiac dysfunction [19] thenoted decrease in myocyte hypertrophymay be a contributorto the observed improvement in cardiac function Moreoverantagomir therapy attenuated fibrosis a known contributorto post-MI cardiac dysfunction [30] Studies have shown thatmiR-208a functions via upregulation of endoglin to increasemyocardial fibrosis and its silencing downregulates endoglinand reduces type 1 collagen synthesis [31 32] The observeddecrease in fibrosis provides yet another possible mechanismby which miR-208a antagomir may contribute to improvingcardiac function after MI

Using 2D echocardiography we demonstrated improvedcardiac function at 28 days afterMI in animals therapeuticallytreated with miR-208a antagomir The observed improve-ment in cardiac function is likely due to a combination offactors including decrease in apoptosis hypertrophy andfibrosis

To the best of our knowledge our study is the firstto demonstrate that antagomir based miR-208a silencingcan attenuate cardiomyocyte apoptosis during ischemia andimprove cardiac function after MI We believe that this willprovide a basis for development of therapies targeting miR-208a in MI

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the institution where the work wasperformed This work was performed at the Laboratory ofCardiovascular Immunology Institute of Cardiology UnionHospital Tongji Medical College Huazhong University ofScience and Technology This work was supported by theNational Natural Science Foundation of China (81070237 and81270354 to Qiutang Zeng)

References

[1] E Palojoki A Saraste A Eriksson et al ldquoCardiomyocyteapoptosis and ventricular remodeling after myocardial infarc-tion in ratsrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 280 no 6 pp H2726ndashH2731 2001

[2] A Diwan M Krenz F M Syed et al ldquoInhibition of ischemiccardiomyocyte apoptosis through targeted ablation of Bnip3restrains postinfarction remodeling in micerdquo The Journal ofClinical Investigation vol 117 no 10 pp 2825ndash2833 2007

[3] D Engel R Peshock R C Armstong N Sivasubramanianand D L Mann ldquoCardiac myocyte apoptosis provokes adversecardiac remodeling in transgenic mice with targeted TNFoverexpressionrdquo American Journal of PhysiologymdashHeart andCirculatory Physiology vol 287 no 3 pp H1303ndashH1311 2004

[4] L-A Macfarlane and P R Murphy ldquoMicroRNA biogenesisfunction and role in cancerrdquo Current Genomics vol 11 no 7pp 537ndash561 2010

[5] M Ha and V N Kim ldquoRegulation of microRNA biogenesisrdquoNature Reviews Molecular Cell Biology vol 15 no 8 pp 509ndash524 2014

[6] T E Callis K Pandya H Y Seok et al ldquoMicroRNA-208a is aregulator of cardiac hypertrophy and conduction in micerdquo TheJournal of Clinical Investigation vol 119 no 9 pp 2772ndash27862009

[7] R L Montgomery T G Hullinger H M Semus et al ldquoTher-apeutic inhibition of miR-208a improves cardiac function andsurvival during heart failurerdquo Circulation vol 124 no 14 pp1537ndash1547 2011

[8] E van Rooij D Quiat B A Johnson et al ldquoA family of microR-NAs encoded by myosin genes governs myosin expression andmuscle performancerdquoDevelopmental Cell vol 17 no 5 pp 662ndash673 2009

[9] E Van Rooij L B Sutherland X Qi J A Richardson J Hilland E N Olson ldquoControl of stress-dependent cardiac growthand gene expression by amicroRNArdquo Science vol 316 no 5824pp 575ndash579 2007

[10] E Bostjancic N Zidar D Stajer and D Glavac ldquoMicroRNAsmiR-1 miR-133a miR-133b and miR-208 are dysregulated inhuman myocardial infarctionrdquo Cardiology vol 115 no 3 pp163ndash169 2010

[11] M Satoh Y Minami Y Takahashi T Tabuchi and M Naka-mura ldquoExpression of microRNA-208 is associated with adverseclinical outcomes in human dilated cardiomyopathyrdquo Journal ofCardiac Failure vol 16 no 5 pp 404ndash410 2010

[12] M Ieda T Tsuchihashi K N Ivey et al ldquoCardiac fibroblastsregulate myocardial proliferation through 1205731 integrin signal-ingrdquo Developmental Cell vol 16 no 2 pp 233ndash244 2009

[13] A Aries P Paradis C Lefebvre R J Schwartz and M NemerldquoEssential role of GATA-4 in cell survival and drug-inducedcardiotoxicityrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 101 no 18 pp 6975ndash69802004

[14] D W Huang B T Sherman and R A Lempicki ldquoSystematicand integrative analysis of large gene lists using DAVID bioin-formatics resourcesrdquo Nature Protocols vol 4 no 1 pp 44ndash572009

[15] D W Huang B T Sherman and R A Lempicki ldquoBioin-formatics enrichment tools paths toward the comprehensivefunctional analysis of large gene listsrdquo Nucleic Acids Researchvol 37 no 1 pp 1ndash13 2009

[16] A Nagashima R Watanabe M Ogawa et al ldquoDifferent rolesof PPAR-120574 activity on physiological and pathological alterationafter myocardial ischemiardquo Journal of Cardiovascular Pharma-cology vol 60 no 2 pp 158ndash164 2012

[17] M T Crow K Mani Y-J Nam and R N Kitsis ldquoThe mito-chondrial death pathway and cardiac myocyte apoptosisrdquo Cir-culation Research vol 95 no 10 pp 957ndash970 2004

[18] L Qian LW Van Laake Y Huang S Liu M FWendland andD Srivastava ldquomiR-24 inhibits apoptosis and represses Bim inmouse cardiomyocytesrdquo Journal of Experimental Medicine vol208 no 3 pp 549ndash560 2011

[19] B A French and C M Kramer ldquoMechanisms of postinfarctleft ventricular remodelingrdquo Drug Discovery Today DiseaseMechanisms vol 4 no 3 pp 185ndash196 2007

[20] M G Sutton and N Sharpe ldquoLeft ventricular remodeling aftermyocardial infarction pathophysiology and therapyrdquo Circula-tion vol 101 no 25 pp 2981ndash2988 2000


[21] J Wang W Huang R Xu et al ldquoMicroRNA-24 regulates car-diac fibrosis aftermyocardial infarctionrdquo Journal of Cellular andMolecular Medicine vol 16 no 9 pp 2150ndash2160 2012

[22] Y Zhang Y Wang X Wang et al ldquoInsulin promotes vascularsmooth muscle cell proliferation via microRNA-208-mediateddownregulation of p21rdquo Journal of Hypertension vol 29 no 8pp 1560ndash1568 2011

[23] E Bostjancic M Jerse D Glavac and N Zidar ldquomiR-1 miR-133ab and miR-208a in human fetal hearts correlate to theapoptotic and proliferation markersrdquo Experimental Biology andMedicine vol 240 no 2 pp 211ndash219 2015

[24] X Wang X Zhang X-P Ren et al ldquoMicroRNA-494 targetingboth proapoptotic and antiapoptotic proteins protects againstischemiareperfusion-induced cardiac injuryrdquo Circulation vol122 no 13 pp 1308ndash1318 2010

[25] A L Gartel and A L Tyner ldquoThe role of the cyclin-dependentkinase inhibitor p21 in apoptosisrdquoMolecular Cancer Therapeu-tics vol 1 no 8 pp 639ndash649 2002

[26] M F van Delft and D C S Huang ldquoHow the Bcl-2 family ofproteins interact to regulate apoptosisrdquoCell Research vol 16 no2 pp 203ndash213 2006

[27] R F Place L-C Li D Pookot E J Noonan and R DahiyaldquoMicroRNA-373 induces expression of genes with complemen-tary promoter sequencesrdquo Proceedings of the National Academyof Sciences of the United States of America vol 105 no 5 pp1608ndash1613 2008

[28] S Vasudevan Y Tong and J A Steitz ldquoSwitching from repres-sion to activation microRNAs can up-regulate translationrdquoScience vol 318 no 5858 pp 1931ndash1934 2007

[29] Y Shan J Zheng R W Lambrecht and H L BonkovskyldquoReciprocal effects of micro-RNA-122 on expression of hemeoxygenase-1 and hepatitis C virus genes in human hepatocytesrdquoGastroenterology vol 133 no 4 pp 1166ndash1174 2007

[30] F See A Kompa J Martin D A Lewis andH Krum ldquoFibrosisas a therapeutic target post-myocardial infarctionrdquo CurrentPharmaceutical Design vol 11 no 4 pp 477ndash487 2005

[31] B-W Wang G-J Wu W-P Cheng and K-G Shyu ldquoMic-roRNA-208a increases myocardial fibrosis via endoglin involume overloading heartrdquo PLoS ONE vol 9 no 1 Article IDe84188 2014

[32] K-G Shyu B-W Wang G-J Wu C-M Lin and H ChangldquoMechanical stretch via transforming growth factor-beta1 acti-vates microRNA208a to regulate endoglin expression in cul-tured rat cardiac myoblastsrdquo European Journal of Heart Failurevol 15 no 1 pp 36ndash45 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


including myocardial infarction and dilated cardiomyopathyin which it is associated with adverse outcomes [10 11]However no study as yet has been done to investigate its rolein cardiomyocyte apoptosis despite these cardiac pathologiesbeing associatedwith increasedmyocyte apoptosisWe there-fore investigated the effect of miR-208a on cardiomyocyteapoptosis and apoptosis related genes and if its silencinghas any therapeutic benefit in myocardial infarction Wereport that miR-208a upregulation alters apoptosis genesrsquoexpression and promotes cardiomyocyte apoptosis while itssilencing attenuates apoptosis and improves cardiac functionafter MI To the best of our knowledge our study is the firstto explore the antiapoptotic effects of miR-208a silencing andthe therapeutic benefits ofmiR-208a antagomir inmyocardialinfarction

2 Materials and Methods

21 Cardiomyocyte Cell Culture Transfection and SimulatedIschemia Neonatal mouse cardiomyocyte isolation and cul-ture were performed according to previously described pro-tocols [12] Cardiomyocytes were transfected with 02 nmolsmL of miR-208a agomir for gain of function or 04 nmolsmLof miR-208a antagomir for loss of function by directlyadding the agomir or antagomir formulation to the cellculture medium according to manufacturerrsquos instructions(RiboBio Co Ltd Guangzhou China) For Bax silenc-ing cardiomyocyte cells were transfected with Bax siRNA(51015840-AACAGAUCAUGAAGACAGGGG-31015840) using Ribofec-tamine reagent according to manufacturerrsquos instructions(RiboBio Co Ltd Guangzhou China) After 48 hours thedifferent treatment groups underwent simulated ischemia for12 hours by placing the cardiomyocytes in a hypoxia chamberwith 5 CO

2and 95 N

2at 37∘C in glucose-free DMEM

medium Sham group myocytes did not undergo simulatedischemia Myocytes were harvested fixed and assayed forapoptosis according to previously described protocol [13]Myocytes were also analyzed for miR-208a levels after cultur-ing

22 Microarray Profiling Microarray profiling was done oncustom-made array chips by a service provider (RiboBioCo Guangzhou China) RNA was isolated from culturedcontrol or miR-208a agomir transfected cardiomyocytes 72hours after transfection Differential gene expression betweencontrols and agomir transfected cardiomyocytes was ana-lyzed using permutation analysis of differential expressionGene clustering was performed with cluster 30 and heat mapimages generated in Java tree view Gene ontology analysiswas done using DAVID online tool [14 15] and apoptosispathway analysis was done using Ingenuity Pathway Analysis(httpwwwingenuitycom)

23 Animal Procedures All experiments were conductedaccording to the Guide For the Care and Use of LaboratoryAnimals and approved by the Animal Care and Utiliza-tion Committee of Union Hospital Tongji Medical CollegeHuazhong University of Science and Technology Wuhan

ChinaThe investigation conformed to theGuide for the CareandUse of LaboratoryAnimals published by theUSNationalInstitute of Health (2011)

24 Animals and Delivery of Antagomir Adult male C57BL6mice 8-week-old were purchased from Wuhan UniversityAnimal Research Center Mice were anesthetized by anintraperitoneal injection of pentobarbital sodium (50mgkg)orally intubated and connected to a rodent ventilator andECG monitoring was done using RM6240 data acquisitionsystem sampling at 1 kHz A left thoracotomy was performedby a horizontal incision at the third intercostal space andmyocardial infarction induced by ligation of the left anteriordescending coronary artery (LAD) according to previouslydescribed protocol [16] and then randomly assigned toAntagomir group (119899 = 6) or control group (119899 = 7) Shamgroupmice (119899 = 7) had the same procedure done but withoutligation of the LAD Starting within 2 hours after MI eachantagomir groupmouse received a total of 300 nmols of miR-208a antagomir given over 3 consecutive days (days 0 1 and2) using 02mLs of normal saline as vehicle by low pressuretail vein injection while control and sham group mice eachreceived 02mLs of vehicle only by the same method overthe same period of time Echocardiography was performedat 7 and 28 days and thereafter animals were euthanized andhearts harvested for further analysis

25 Histology At 28 days animals were euthanized andhearts harvested cut transversely into blocks and fixed in4 paraformaldehyde These were then embedded in OCTcompound (BHD UK) and then transversely cut into 5120583mthick cryosections for histological analysis Two cryosectionsfrom each of the blocks were stained withMassonrsquos trichromefor assessment of fibrosis and infarct sizeThe infarct size wasmeasured as the sum of the epicardial and endocardial scarlength divided by the sum of LV epicardial and endocardialcircumferences

For assessment of fibrosis we randomly selected 4 fieldsper slide in the peri-infarct zone (PIZ) or 5 fields per slidein the noninfarct zone (NIZ) and calculated collagen volumefraction (CVF) as the ratio of blue stained (collagen) area tothe total tissue area The collagen-rich border zone of vesselsand the scar were excluded from the analysis Image analysiswas done using Image-Pro Plus software version 6

Cardiomyocyte hypertrophy was assessed by capturingcardiomyocyte images over 5 different fields per slide (40xobjective) Using Image-Pro Plus software version 6 weobtained mean cardiomyocyte cross-sectional areas whichwere compared between groups

26 Apoptosis Assay At 28 days after MI or after 12 hoursof simulated cardiomyocyte ischemia tissues or cells respec-tively were fixed with 4 paraformaldehyde and apoptosisassay was done by TUNEL method according to previouslydescribed protocol [13]

27 Quantitative Real-Time PCR Total RNA was extractedfromcells and tissue samples usingTrizol reagent (Invitrogen


00

05

10

15

20

25

miR

-208

a fol

d ch

ange

AntagomirControl

lowast

(a)

0

500

1000

1500

2000

2500

miR

-208

a fol

d ch

ange

AgomirControl

lowastlowast

(b)

00

05

10

15

miR

-208

a fol

d ch

ange

AntagomirControl

lowast

(c)





BAX





GAPDH






3 Results





NEL

DA

PI


(a)

Perc

enta

ge o

f apo

ptot

ic

Ant

agom

ir

Con

trol

Sham

Ago

mir

lowast

lowast

lowast

0

5

10

15

nucle

i per

fiel

d

(b)

BAX

siRN

A

Scra

mbl

edsiR

NA-

cont

rol

Scra

mbl

edsiR

NA-

agom

ir

BAX

siRN

A-ag

omir

lowast lowast

lowast

0

5

10

15

20

nucle

i per

fiel

dPe

rcen

tage

of a

popt

otic

(c)






Con

trol 2

Con

trol 1

Ago

mir

1

Ago

mir

2

0 1minus1

Row

Color key

Z-score

(a)


TNFFasL

TNFR

DNA damage

FAS

CASP

CASP

810NIK

IKK

RTK

ASK1 RASc-RAF

BIM

CDC2

pSORSK

BAD

BCL-XL

CYCSHTRA2

BAXBCL-B

cIAP

DIABLO

APAF1

AIF ENDO G

zVAD-FMK

MCL1

HTRA2

BIRC6

BID

tBID

P BFL-12

CASP9

CASP3

CASP

CAD

6CASP

7

DNA

PARP ACINUS


ERstress Calpain

CASP12

p53 JNK1

tBD

DIABLO AIF

MEK12

12ERK

Cellshrinkage

Membraneblebbing


Apoptosis

LaminFodrinA

GAS2ROCK1

PLC120574

PKC

I120581B

I120581B

NF-120581B

NF-120581B

Mitochondria

Growthfactor

MKK47

CAD

DNArepair

BAK BCL-2

(120572 120576 120579)

CYCS

ENDO G

(b)




TUN

ELD

API

(a)

00

05

10

15

20

25

Apop

totic

nuc

lei (

)


lowast

lowast

(b)


lowast

00

02

04

06

08

10

Apop

totic

nuc

lei (

)

(c)


lowast

lowast

00

05

10

15

20

BAX

fold

chan

ge

(d)

AntagomirControl0

10

20

30

40

50

Infa

rct a

rea (

)

(e)




4 Discussion




(a)


lowast

lowastlowast

0

100

200

300

400

Card

iom

yocy

te ar

ea(120583

M2 )

(b)


lowastlowast

0

5

10

15

Fibr

osis

()

(c)


(d)


lowastlowast

lowastlowast

0

10

20

30

40

50

Fibr

osis

()

(e)


lowast

lowastlowast

0

20

40

60

Mea

n fr

actio

nal s

hort

enin

g

(f)

Figure 5 Continued



(g)




















Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

05

10

15

20

25

miR

-208

a fol

d ch

ange

AntagomirControl

lowast

(a)

0

500

1000

1500

2000

2500

miR

-208

a fol

d ch

ange

AgomirControl

lowastlowast

(b)

00

05

10

15

miR

-208

a fol

d ch

ange

AntagomirControl

lowast

(c)





BAX





GAPDH






3 Results





NEL

DA

PI


(a)

Perc

enta

ge o

f apo

ptot

ic

Ant

agom

ir

Con

trol

Sham

Ago

mir

lowast

lowast

lowast

0

5

10

15

nucle

i per

fiel

d

(b)

BAX

siRN

A

Scra

mbl

edsiR

NA-

cont

rol

Scra

mbl

edsiR

NA-

agom

ir

BAX

siRN

A-ag

omir

lowast lowast

lowast

0

5

10

15

20

nucle

i per

fiel

dPe

rcen

tage

of a

popt

otic

(c)






Con

trol 2

Con

trol 1

Ago

mir

1

Ago

mir

2

0 1minus1

Row

Color key

Z-score

(a)


TNFFasL

TNFR

DNA damage

FAS

CASP

CASP

810NIK

IKK

RTK

ASK1 RASc-RAF

BIM

CDC2

pSORSK

BAD

BCL-XL

CYCSHTRA2

BAXBCL-B

cIAP

DIABLO

APAF1

AIF ENDO G

zVAD-FMK

MCL1

HTRA2

BIRC6

BID

tBID

P BFL-12

CASP9

CASP3

CASP

CAD

6CASP

7

DNA

PARP ACINUS


ERstress Calpain

CASP12

p53 JNK1

tBD

DIABLO AIF

MEK12

12ERK

Cellshrinkage

Membraneblebbing


Apoptosis

LaminFodrinA

GAS2ROCK1

PLC120574

PKC

I120581B

I120581B

NF-120581B

NF-120581B

Mitochondria

Growthfactor

MKK47

CAD

DNArepair

BAK BCL-2

(120572 120576 120579)

CYCS

ENDO G

(b)




TUN

ELD

API

(a)

00

05

10

15

20

25

Apop

totic

nuc

lei (

)


lowast

lowast

(b)


lowast

00

02

04

06

08

10

Apop

totic

nuc

lei (

)

(c)


lowast

lowast

00

05

10

15

20

BAX

fold

chan

ge

(d)

AntagomirControl0

10

20

30

40

50

Infa

rct a

rea (

)

(e)




4 Discussion




(a)


lowast

lowastlowast

0

100

200

300

400

Card

iom

yocy

te ar

ea(120583

M2 )

(b)


lowastlowast

0

5

10

15

Fibr

osis

()

(c)


(d)


lowastlowast

lowastlowast

0

10

20

30

40

50

Fibr

osis

()

(e)


lowast

lowastlowast

0

20

40

60

Mea

n fr

actio

nal s

hort

enin

g

(f)

Figure 5 Continued



(g)




















Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















GAPDH






3 Results





NEL

DA

PI


(a)

Perc

enta

ge o

f apo

ptot

ic

Ant

agom

ir

Con

trol

Sham

Ago

mir

lowast

lowast

lowast

0

5

10

15

nucle

i per

fiel

d

(b)

BAX

siRN

A

Scra

mbl

edsiR

NA-

cont

rol

Scra

mbl

edsiR

NA-

agom

ir

BAX

siRN

A-ag

omir

lowast lowast

lowast

0

5

10

15

20

nucle

i per

fiel

dPe

rcen

tage

of a

popt

otic

(c)






Con

trol 2

Con

trol 1

Ago

mir

1

Ago

mir

2

0 1minus1

Row

Color key

Z-score

(a)


TNFFasL

TNFR

DNA damage

FAS

CASP

CASP

810NIK

IKK

RTK

ASK1 RASc-RAF

BIM

CDC2

pSORSK

BAD

BCL-XL

CYCSHTRA2

BAXBCL-B

cIAP

DIABLO

APAF1

AIF ENDO G

zVAD-FMK

MCL1

HTRA2

BIRC6

BID

tBID

P BFL-12

CASP9

CASP3

CASP

CAD

6CASP

7

DNA

PARP ACINUS


ERstress Calpain

CASP12

p53 JNK1

tBD

DIABLO AIF

MEK12

12ERK

Cellshrinkage

Membraneblebbing


Apoptosis

LaminFodrinA

GAS2ROCK1

PLC120574

PKC

I120581B

I120581B

NF-120581B

NF-120581B

Mitochondria

Growthfactor

MKK47

CAD

DNArepair

BAK BCL-2

(120572 120576 120579)

CYCS

ENDO G

(b)




TUN

ELD

API

(a)

00

05

10

15

20

25

Apop

totic

nuc

lei (

)


lowast

lowast

(b)


lowast

00

02

04

06

08

10

Apop

totic

nuc

lei (

)

(c)


lowast

lowast

00

05

10

15

20

BAX

fold

chan

ge

(d)

AntagomirControl0

10

20

30

40

50

Infa

rct a

rea (

)

(e)




4 Discussion




(a)


lowast

lowastlowast

0

100

200

300

400

Card

iom

yocy

te ar

ea(120583

M2 )

(b)


lowastlowast

0

5

10

15

Fibr

osis

()

(c)


(d)


lowastlowast

lowastlowast

0

10

20

30

40

50

Fibr

osis

()

(e)


lowast

lowastlowast

0

20

40

60

Mea

n fr

actio

nal s

hort

enin

g

(f)

Figure 5 Continued



(g)




















Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NEL

DA

PI


(a)

Perc

enta

ge o

f apo

ptot

ic

Ant

agom

ir

Con

trol

Sham

Ago

mir

lowast

lowast

lowast

0

5

10

15

nucle

i per

fiel

d

(b)

BAX

siRN

A

Scra

mbl

edsiR

NA-

cont

rol

Scra

mbl

edsiR

NA-

agom

ir

BAX

siRN

A-ag

omir

lowast lowast

lowast

0

5

10

15

20

nucle

i per

fiel

dPe

rcen

tage

of a

popt

otic

(c)






Con

trol 2

Con

trol 1

Ago

mir

1

Ago

mir

2

0 1minus1

Row

Color key

Z-score

(a)


TNFFasL

TNFR

DNA damage

FAS

CASP

CASP

810NIK

IKK

RTK

ASK1 RASc-RAF

BIM

CDC2

pSORSK

BAD

BCL-XL

CYCSHTRA2

BAXBCL-B

cIAP

DIABLO

APAF1

AIF ENDO G

zVAD-FMK

MCL1

HTRA2

BIRC6

BID

tBID

P BFL-12

CASP9

CASP3

CASP

CAD

6CASP

7

DNA

PARP ACINUS


ERstress Calpain

CASP12

p53 JNK1

tBD

DIABLO AIF

MEK12

12ERK

Cellshrinkage

Membraneblebbing


Apoptosis

LaminFodrinA

GAS2ROCK1

PLC120574

PKC

I120581B

I120581B

NF-120581B

NF-120581B

Mitochondria

Growthfactor

MKK47

CAD

DNArepair

BAK BCL-2

(120572 120576 120579)

CYCS

ENDO G

(b)




TUN

ELD

API

(a)

00

05

10

15

20

25

Apop

totic

nuc

lei (

)


lowast

lowast

(b)


lowast

00

02

04

06

08

10

Apop

totic

nuc

lei (

)

(c)


lowast

lowast

00

05

10

15

20

BAX

fold

chan

ge

(d)

AntagomirControl0

10

20

30

40

50

Infa

rct a

rea (

)

(e)




4 Discussion




(a)


lowast

lowastlowast

0

100

200

300

400

Card

iom

yocy

te ar

ea(120583

M2 )

(b)


lowastlowast

0

5

10

15

Fibr

osis

()

(c)


(d)


lowastlowast

lowastlowast

0

10

20

30

40

50

Fibr

osis

()

(e)


lowast

lowastlowast

0

20

40

60

Mea

n fr

actio

nal s

hort

enin

g

(f)

Figure 5 Continued



(g)




















Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con

trol 2

Con

trol 1

Ago

mir

1

Ago

mir

2

0 1minus1

Row

Color key

Z-score

(a)


TNFFasL

TNFR

DNA damage

FAS

CASP

CASP

810NIK

IKK

RTK

ASK1 RASc-RAF

BIM

CDC2

pSORSK

BAD

BCL-XL

CYCSHTRA2

BAXBCL-B

cIAP

DIABLO

APAF1

AIF ENDO G

zVAD-FMK

MCL1

HTRA2

BIRC6

BID

tBID

P BFL-12

CASP9

CASP3

CASP

CAD

6CASP

7

DNA

PARP ACINUS


ERstress Calpain

CASP12

p53 JNK1

tBD

DIABLO AIF

MEK12

12ERK

Cellshrinkage

Membraneblebbing


Apoptosis

LaminFodrinA

GAS2ROCK1

PLC120574

PKC

I120581B

I120581B

NF-120581B

NF-120581B

Mitochondria

Growthfactor

MKK47

CAD

DNArepair

BAK BCL-2

(120572 120576 120579)

CYCS

ENDO G

(b)




TUN

ELD

API

(a)

00

05

10

15

20

25

Apop

totic

nuc

lei (

)


lowast

lowast

(b)


lowast

00

02

04

06

08

10

Apop

totic

nuc

lei (

)

(c)


lowast

lowast

00

05

10

15

20

BAX

fold

chan

ge

(d)

AntagomirControl0

10

20

30

40

50

Infa

rct a

rea (

)

(e)




4 Discussion




(a)


lowast

lowastlowast

0

100

200

300

400

Card

iom

yocy

te ar

ea(120583

M2 )

(b)


lowastlowast

0

5

10

15

Fibr

osis

()

(c)


(d)


lowastlowast

lowastlowast

0

10

20

30

40

50

Fibr

osis

()

(e)


lowast

lowastlowast

0

20

40

60

Mea

n fr

actio

nal s

hort

enin

g

(f)

Figure 5 Continued



(g)




















Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















TUN

ELD

API

(a)

00

05

10

15

20

25

Apop

totic

nuc

lei (

)


lowast

lowast

(b)


lowast

00

02

04

06

08

10

Apop

totic

nuc

lei (

)

(c)


lowast

lowast

00

05

10

15

20

BAX

fold

chan

ge

(d)

AntagomirControl0

10

20

30

40

50

Infa

rct a

rea (

)

(e)




4 Discussion




(a)


lowast

lowastlowast

0

100

200

300

400

Card

iom

yocy

te ar

ea(120583

M2 )

(b)


lowastlowast

0

5

10

15

Fibr

osis

()

(c)


(d)


lowastlowast

lowastlowast

0

10

20

30

40

50

Fibr

osis

()

(e)


lowast

lowastlowast

0

20

40

60

Mea

n fr

actio

nal s

hort

enin

g

(f)

Figure 5 Continued



(g)




















Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)


lowast

lowastlowast

0

100

200

300

400

Card

iom

yocy

te ar

ea(120583

M2 )

(b)


lowastlowast

0

5

10

15

Fibr

osis

()

(c)


(d)


lowastlowast

lowastlowast

0

10

20

30

40

50

Fibr

osis

()

(e)


lowast

lowastlowast

0

20

40

60

Mea

n fr

actio

nal s

hort

enin

g

(f)

Figure 5 Continued



(g)




















Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(g)




















Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article microrna-208a dysregulates apoptosis

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