Articulo bioquimica

The International Journal of Biochemistry & Cell Biology 69 (2015) 5261

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The International Journal of Biochemistry& Cell Biology

jo u r n al homep ag e: www.elsev ier .com/ locate /b ioce l

Inhibition of MEF2A prevents hyperglycemia-indmatrix accumulation by blocking Akt and TGF-1cardiac broblasts

Xueying Chena, Guoliang Liub, Wei Zhanga, Jianing Zhangc, YuWenqian Donga, Ershun Lianga, Yun Zhanga, Mingxiang Zhanga The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education anQilu Hospital ob Henan Provin ity, Kac School of Fored Department o

a r t i c l e i n f o

Article history:Received 15 June 2015Received in re19 SeptemberAccepted 13 O

Keywords:HyperglycemiDiabetesCardiac brobMyocyte enhaCardiac remod

a b s t r a c t

Myocyte enhancer factor 2A (MEF2A) functions in muscle-specic and/or growth factor-related tran-

1. Introdu

Diabetesease that ca

Abbreviatinormal glucosprotein kinasetozotocin; MOtissue inhibitoJun NH2-term8, Cell Countininterventriculend-systolic dshortening; E/matrix.

CorresponXi Road, Jinan,

E-mail add

http://dx.doi.o1357-2725/ vised form 2015ctober 2015

c

lastsncer factor 2Aeling

scription and is involved in cell growth, survival, and apoptosis. To evaluate the role of this transcriptionfactor in cardiac broblasts (CFs) in diabetes mellitus, we performed a series of in vitro and in vivo experi-ments. We used short hairpin RNA (shRNA) to inhibit the expression of MEF2A in CFs in vitro. Inhibition ofMEF2A signicantly reduced hyperglycemia-induced CF proliferation and migration, myobroblast dif-ferentiation, matrix metalloproteinase (MMP) activities, and collagen production. Furthermore, MEF2Ainhibition attenuated HG-induced activation of the mitogen-activated protein kinase (MAPK), Akt, andTGF-1/Smad signaling pathways. For in vivo analysis in a mouse model, type-1 diabetes was inducedby streptozotocinand MEF2A expression was knocked down by myocardial injection with lentiviruscarrying shRNA-MEF2A. Cardiac function was assessed by echocardiography. Total collagen depositionwas assessed by Massons trichrome and Picrosirius red staining. Knockdown of MEF2A ameliorateddiabetes-induced cardiac dysfunction and collagen deposition. Our study suggests that inhibition ofMEF2A could alleviate HG-induced extracellular matrix accumulation by blocking the activation of Aktand TGF-1/Smad signaling pathway in CFs. Thus, inhibition of MEF2A has therapeutic potential in thetreatment of diabetic-induced cardiac remodeling.

2015 Elsevier Ltd. All rights reserved.

ction

is an independent risk factor for cardiovascular dis-n cause cardiac remodeling and heart failure (Ban and

ons: MEF2A, myocyte enhancer factor 2A; CFs, cardiac broblasts; NG,e; HG, high glucose; OC, osmotic control; MAPK, mitogen-activateds; shRNAs, short-hairpin RNAs; RNAi, RNA interference; STZ, strep-I, multiplicity of infection; MMP, matrix metalloproteinase; TIMP1,r of metalloproteinase; TGF-1, transforming growth factor-1; JNK,inal kinase; ERK1/2, extracellular-regulated protein kinase 1/2; CCK-g Kit-8; PE, phycoerythrin; 7-AAD, 7-amino-actinomycin D; IVS, thear septum; LVEDD, left ventricular end-diastolic dimension; LVESD, LVimension; LVPW, LV posterior wall; EF, ejection fraction; FS, fractionalA, the ratio of early to late mitral inow velocity; ECM, extracellular

ding author at: Shandong University, Qilu Hospital, No. 107, Wen Hua Shandong 250012, China.ress: [email protected] (M. Zhang).

Twigg, 2008). Cardiac remodeling is one of the major pathophysi-ological processes contributing to increased myocardial stiffnessand hampered systolic ejection, which eventually lead to heartfailure (Kong et al., 2014). However, no curative treatment for car-diac remodeling has been developed to date. Cardiac broblasts(CFs) are the critical mediators of physiological and pathologicalcardiac remodeling. CFs respond to stimuli in various ways, includ-ing proliferation, migration, myobroblast differentiation, matrixgeneration and degradation, secretion of cytokines etc. (Souderset al., 2009). Many studies (Bugyei-Twum et al., 2014; Shamhartet al., 2014; Tang et al., 2007) have shown that high glucose (HG)stimulates cardiac remodeling by inducing CF proliferation andactivation; however, the molecular mechanisms underlying CF-mediated cardiac remodeling are not clear.

Myocyte enhancer factor 2 (MEF2) is a widely distributed DNA-binding transcription factor that activates various muscle-specicand growth factor- and stress-induced genes (McKinsey et al.,2002). MEF2 DNA-binding activity depends on four separate gene

rg/10.1016/j.biocel.2015.10.0122015 Elsevier Ltd. All rights reserved.f Shandong University, Jinan, Shandong, Chinacial Key Engineering Laboratory of Antibody Drugs, School of Medicine, Henan Universign Languages and Literature, Shandong University, Chinaf Medicinal Chemistry, School of Pharmacy, Shandong University, Chinauced extracellular/Smad activation in

gang Yand,a,

d Chinese Ministry of Public Health,

ifeng, Henan, China

X. Chen et al. / The International Journal of Biochemistry & Cell Biology 69 (2015) 5261 53

products in mammals, referred to as MEF2A-D (Black and Olson,1998; Molkentin and Olson, 1996). MEF2A and MEF2D are the pri-mary MEF2s expressed in adult heart. Multiple lines of evidencesuggest that MEF2s play prominent roles in the regulation of cardiachypertroph2009; Xu etin responsediomyocyteor MEF2C ieration andthese studieocytes; the cardiac rem

Numero2003, 2013pathways, induced carsignaling paet al., 2001;cardiac rem

We hypohyperglycemliferation anprotective athese hypoexperiment

2. Materia

2.1. Lentivi

A lentivireporter geused to expexpression MEF2A) wacontrol sequ

2.2. Animal

Eight-weused for exzotocin (STZet al., 2014were dividelentiviral vement, 1 1same volumple sites. Afconformed published bethical com

2.3. Primar

CFs werneonatal mcell morphoperformed cells with n

Supplemonline versi

The CFs and shRNA24 h. Then,

glucose, NG), 33 mM d-glucose (high glucose, HG), or 5 mM d-glucose + 27.5 mM mannose (osmotic control; OC) for 48 h. The cellswere observed under a uorescence microscope to conrm thatmore 90% of the cells were GFP-positive.

mun

wereizedboviere i, Cam

-SMAn LaidinagesLSM

ester

teinsrylamyvinyd w

andainst

meinhibAPK,llulaor

adish temscen

anti

priTGGA CATCreened fa reafold cd.

easu

liferaotimnufarything s, Fons.

ll mi

l migion a, scraing 2 h, ell. Tcialy (Kim et al., 2008b; Konno et al., 2010; Pereira et al., al., 2006). MEF2 DNA-binding activities are enhanced

to biomechanical and neurohormonal stimuli in car-s (Nadruz et al., 2003,2005). Overexpression of MEF2An cultured cardiomyocytes induces sarcomere degen-

cardiomyocyte elongation (Xu et al., 2006). However,s mainly focused on the effects of MEF2A on cardiomy-function of MEF2A in CFs in diabetes mellitus-inducedodeling remains unknown.us signaling pathways (Kim et al., 2008a; Li et al.,; She et al., 2012), such as the MAPK and TGF-/Smadare implicated in the mediation of hyperglycemia-diac remodeling. MEF2A is intimately linked with thesethways (Liu et al., 2004; Ornatsky et al., 1999; Quinn

Wales et al., 2014). Therefore, MEF2A may participate inodeling through these signaling pathways in diabetes.thesized that an increased level of MEF2A may facilitateia-induced cardiac remodeling via regulating CF pro-d activation. Knockdown of cardiac MEF2A may have affection in diabetic cardiomyopathy. In order to provetheses, we performed a series of in vitro and in vivos.

ls and methods

ral vectors for RNA interference (RNAi)

ral vector containing a green uorescent protein (GFP)ne and a U6 promoter upstream of the cloning site wasress short-hairpin RNAs (shRNAs) to suppress MEF2Avia RNAi. The target sequence for MEF2A (ShRNA-s 5-GCTTGCCACCTCAGAACTTCT-3 and the negativeence (shRNA-NC) was 5-TTCTCCGAACGTGTCACGT-3.

model and RNAi

ek-old C57BL/6J wild-type (WT) mice (2530 g) wereperiments. Diabetes was induced by injecting strepto-) (Sigma, St. Louis, MO) as described previously (Wang). After induction of diabetes (12 weeks), the miced into 3 groups for treatments: shRNA-MEF2A (n = 10),hicle (n = 10) and shRNA-NC control (n = 10). For treat-07 UT/30 l of lentivector withMEF2A-shRNA or thee of vehicle was injected into the left ventricle at multi-ter 16 weeks, the mice were sacriced. All experimentsto the Guide for the Care and Use of Laboratory Animalsy the US National Institutes of Health and approved bymittee of Shandong University.

y mouse CF culture and treatments

e isolated from ventricular tissues of 1- to 3-day-oldice. CFs were identied by immunouorescence andlogical analysis (Supplementary Fig. S1). CF culture wasas described previously (Dubey et al., 1997). We usedo more than 4 passages.entary Fig. S1 related to this article can be found, in theon, at http://dx.doi.org/10.1016/j.biocel.2015.10.012.were infected with lentiviruses carrying ShRNA-MEF2A-NC at a multiplicity of infection (MOI) of 20 for

the cells were exposed to 5 mM d-glucose (normal

2.4. Im

CFsmeabilin 5% cells w(Abcamanti-(Jackso6-diamand imscope (

2.5. W

Propolyacto polblockefor 2 hies agmatrixtissue p38 Mextracep-Akt, horserat roomlumine

2.6. Qu

TheGGAGCTAGGCGTGCCSYBR Gwas ususing mean metho

2.7. M

Pro8; Beythe maPhycoecontainsciencedirecti

2.8. Ce

CelmigratBrieycontainAfter 7cells/wan artiouorescence

xed in 4% paraformaldehyde for 15 min and then per- in 0.03% Triton X-100 for 20 min. After being blockedne serum albumin at room temperature for 1 h, thencubated with primary antibodies mouse anti-MEF2Abridge, MA), rabbit anti-p-MEF2A (Abcam), and mouse

(SigmaAldrich). Subsequently, secondary antibodiesboratories) were added. Nuclei were visualized with 4-o-2-phenylindole (5 mg/ml; Beyotime, Haimen, China)

were acquired using a laser scanning confocal micro-710; Carl Zeiss, Germany).

n blot analysis

were separated by 10% sodium dodecyl sulfate-ide gel electrophoresis (SDS-PAGE) and transferredlidene diuoride membranes. The membranes wereith 5% non-fat milk in TBST at room temperature

incubated overnight at 4 C with primary antibod- MEF2A, p-MEF2A, -SMA, collagen I, collagen III,talloproteinase 2 (MMP2), MMP9 (all from Abcam),itor of metalloproteinase 1 (TIMP1), TIMP2, TGF-1,

p-p38 MAPK, Jun NH2-terminal kinase (JNK), p-JNK,r-regulated protein kinase 1/2 (ERK1/2), p-ERK1/2,Akt,-actin (all from Cell Signaling Technology). Then,

peroxidase-conjugated secondary antibody was addedperature for 2 h. Membranes were visualized by chemi-ce reagent.

tative RT-PCR

mers for MEF2A were as follows: forward, 5-AAATAGTCCTGTGG-3, reverse, 5-GGGAGCTTTG-TGACT-3; and for -actin: forward, 5-CACT-TACGA-3, reverse, 5-GTAGTCTGTCAGGTCCCG-3.

RT-PCR master mix (Life Technologies, Carlsbad, CA)or reactions and quantitative assays were performedl-time PCR detection system (Bio-Rad). The relativehange in expression was calculated using the 2CT

rement of CF proliferation and apoptosis

tion was analyzed by using Cell Counting Kit-8 (CCK-e) and Cell-LightTMEdU assay (RiboBio) according tocturers directions. Apoptosis was detected by using arin (PE)-Annexin V Apoptosis Detection Kit (BD559763,7-amino-actinomycin D (7-AAD) marker; BD Bio-ranklin Lanes, NJ) according to the manufacturers

gration assays

ration was examined by scratch assay and Transwellssay as described previously (Ibrahim et al., 2015).tch assay was performed as follows: lentiviral vectorsshRNA-NC or shRNA-MEF2A were transfected into CFs.the cells were seeded into a 6-well plate at 1 105he next day, the cells were treated with HG or OC. Then,

gap was generated by scratching with apipette tip. The

54 X. Chen et al. / The International Journal of Biochemistry & Cell Biology 69 (2015) 5261

Fig. 1. High glucose (HG) induces MEF2A expression increase and nuclear translo-cation in cardiac broblasts (CFs). (A) CFs were treated with HG for various periods,and MEF2A expression was analyzed by western blotting. (B) Immunocytouores-cence of MEF2A translocation. Scale bar: 25 m. MEF2A stained red; nuclei werecounterstained with DAPI (blue). (C) Immunocytouorescence of p-MEF2A. Scalebar: 25 m. P-MEF2A stained red; nuclei were counterstained with DAPI (blue).(D) Western blot analysis of MEF2A and p-MEF2A protein expression in cyto-plasm (D1) and nucleus (D2). NC: 5 mM glucose, HG: 30 mM glucose, OC: 5 mMd-glucose + 27.5 mM mannose. Data are the mean SD of 3 independent experi-ments; *P < 0.05 vs. NC. (For interpretation of the references to color in this gurelegend, the reader is referred to the web version of this article.)

Fig. 2. MEF2A inhibition reduces the proliferation of cardiac broblasts (CFs). (A)Cell Counting Kit-8 analysis of the cell viability of CFs treated with glucose forvarious periods. (B) Laser confocal microscopy of Edu staining. Nuclei were coun-terstained with DAPI (blue), red staining indicates cells undergoing proliferation.Scale bar: 25 m. (C) The Edu-positive index was expressed as a percentage (posi-tive/total cell number). (D) Quantitative analysis of the phycoerythrin (PE)-positiverate. (E) Flow cytometry using PE and 7-amino-actinomycin D (7-AAD) staining todetermine cell apoptosis. NC: 5 mM glucose, HG: 30 mM glucose, shRNA-NC+HG:cells transfected with MEF2A negative shRNA control before glucose incubation, sh-MEF2A+HG: cells transfected with MEF2A-shRNA before glucose incubation. OC:5 mM d-glucose + 27.5 mM mannose. Data are mean SD of 3 independent exper-iments; *P < 0.05 vs. NC; #P < 0.05 vs. HG. (For interpretation of the references tocolor in this gure legend, the reader is referred to the web version of this article.)


Fig. 3. MEF2Ashowing that Scale bar: 100at different tithat MEF2A kncultured with nal surface ofunder a microthe Transwell sample from 3shRNA-NC+HGcose incubatioincubation. OCindependent ethe referencesof this article.)

number of 12-h interv

Transweswell units from Corninchamber, w inhibition reduces cardiac broblast (CF) migration. (A) Scratch assayMEF2A knockdown inhibited CF migration at different time points.

m. (B) Quantication of the number of cells migrating in the gapme points after scratching. (C) Transwell migration assay showingockdown inhibited CF migration. MEF2A-shRNA-transfected CFs wereHG in Transwell plates (0.8-m pore size) for 8 h. CFs on the exter-

the Transwell were stained with crystal violet and photographedscope. (D) Quantication of the number of migrated CFs per eld ofplate. Data represent the mean SD from 5 separate elds in each

independent experiments. NC: 5 mM glucose, HG: 30 mM glucose,: cells transfected with MEF2A-negative shRNA control before glu-n, sh-MEF2A+HG: cells transfected with MEF2A-shRNA before glucose: 5 mM d-glucose + 27.5 mM mannose. Data are the mean SD of 3xperiments; *P < 0.05 vs. NC; #P < 0.05 vs. HG. (For interpretation of

to color in this gure legend, the reader is referred to the web version

cells in the gap was monitored for the next 2 days atals.ll migration assay was performed using 24-well Tran-with 8-m porous polycarbonate membranes obtainedg. Serum-starved CFs (105) were added to the upperhereas the bottom chamber was lled with DMEM

Fig. 4. Inhibitinto myobrotive analysis (of 3 independimages of immstained with DshRNA-NC+HGcose incubatioincubation. OCerences to colthis article.)

containing with 4% foon the upprandomly scells were c

2.9. Gelatin

Cell cultacrylamidewashing buton X-100)(50 mM Tri37 C. Then250 solutioacid) and sLytic bandsMMP9 (83-

2.10. Non-ifunction

Blood pBP-98Acomues were dion of MEF2A attenuates differentiation of cardiac broblasts (CFs)blasts. (A) Representative western blot (upper panel) and quantita-lower panel) of -SMA expression in CFs. Data are the mean SDent experiments, *P < 0.05 vs. NC; #P < 0.05 vs. HG. (B) Representativeunouorescence staining for -SMA (red) in CFs. Nuclei were counter-API (blue). Scale bar = 25 m. NC: 5 mM glucose, HG: 30 mM glucose,: cells transfected with MEF2A-negative shRNA control before glu-n, sh-MEF2A+HG: cells transfected with MEF2A-shRNA before glucose: 5mMd-glucose + 27.5 mM mannose. (For interpretation of the ref-or in this gure legend, the reader is referred to the web version of

HG or OC. After 12 h of incubation, the cells were xedrmaldehyde and stained with crystal violet. The cellser surface were removed with a cotton swab. Fiveelected elds were photographed and the migratedounted.

zymography

ure supernatants were electrophoresed in a 10% poly- gel containing1 mg/ml gelatin. The gel was washed inffer (50 mM TrisHCl [pH 7.5], 100 mM NaCl, 2.5% Tri-

for 1 h and incubated overnight with enzyme buffers [pH 7.5], 150 mM NaCl, 5 mM CaCl2, 0.02% Brij-35) at, the gel was stained with Coomassie brilliant blue R-n (0.5% Coomassie Blue G-250, 30% methanol, 10% aceticubsequently destained in methanol/acetic acid/H2O.

of gelatin digestion represented MMP2 (68-kDa) andkDa) activity.

nvasive analysis of blood pressure and cardiac

ressure was measured in conscious mice using theputerized tail-cuff system (Softron, Tokyo). Mean val-etermined from at least 3 measurements per mouse.


Fig. 5. MEF2A plays an important role in HG-induced matrix metalloproteinase(MMP)tissue inhibitor of metalloproteinase (TIMP) interactions and collagenproduction in cardiac broblasts (CFs). (AC) Western blot and quantitative analysisof MMP2 and MMP9 protein expression. (DF) Western blot and quantitativeanalysis of TIMP1 and TIMP2 protein expression. (G) Activity of MMP2 and MMP9was studied by zymography by separating culture supernatants from FCs. (H and I)

Left ventricular dimension and cardiac function were assessed byechocardiography (Vevo770 imaging system) before the mice werekilled. The mice were anesthetized with isourane. M-mode imagesat the level of the papillary muscles were obtained to measurethe interventricular septum (IVS) thickness, left ventricular end-diastolic dimension (LVEDD), LV end-systolic dimension (LVESD),LV posterior wall (LVPW), ejection fraction (EF), and fractionalshortening (FS). Pulsed-wave Doppler echocardiography was usedto measure the ratio of early to late mitral inow velocity (E/A).

2.11. Histology and immunohistochemistry

Massonas describechemistry, with primasecondary lowing the Data were a

2.12. Statis

The resudata are expANOVA waby Tukeys used for an

3. Results

3.1. HG inc

CFs w(5.525 mMhigh as 2of MEF2A(P < 0.05), apressure (Fafter 6 h of(2.83-fold irescence anof CFs cultuMEF2A shotion of the expression These resul

3.2. MEF2Aand is not a

CF prolmyocardialon CF prolCCK-8 assaover 24 h dependent (Fig. 2A).

Quantitative aquantitative aHG: 30 mM glcontrol beforeshRNA beforeare mean SDs trichrome and picrosirius red staining were performedd previously (Bauman et al., 2014). For immunohisto-heart tissue sections were incubated overnight at 4 Cry antibodies (collagen I and III, Abcam). Addition of theantibody and color development were conducted fol-manufacturers instructions (Jingmei, Shenzhen, China).nalyzed using Image-ProPlus6.0 (Media Cybernetics).

tical analysis

lts were from at least 3 independent experiments. Theressed as the mean standard deviation (SD). One-ways used to compare differences among groups, followedtest (2-tailed). SPSS v16.0 (SPSS Inc., Chicago, IL) wasalysis. P < 0.05 was considered statistically signicant.

reases MEF2A expression and translocation in CFs

ere exposed to various concentrations) of glucose for 72 h. A concentration as5 mM glucose signicantly increased the level

as demonstrated by western blot analysisnd these effects were not due to changes in osmoticig. 1A). MEF2A protein expression in the CFs increased

HG treatment (1.87-fold increase) and peaked at 48 hncrease) as compared with NC and OC. Immunouo-alysis revealed that MEF2A is expressed in the nucleired under NG (5.5 mM glucose) (Fig. 1B). Under HG,

wed diffuse cytoplasmic staining, indicating transloca-protein. Further, HG increased only nuclear p-MEF2Aand did not affect p-MEF2A translocation (Fig. 1C).ts were conrmed by western blot analysis (Fig. 1D).

inhibition decreases HG-induced proliferation of CFsssociated with apoptosis

iferation and migration play an important role in brosis. To examine the effect of MEF2A silencingiferation, we investigated the proliferation of CFs byy. Under HG, the CFs showed a fast growth rateand proliferation signicantly increased in a time-manner when compared with NC and OC (P < 0.05)Inhibition of MEF2A by RNAi signicantly reduced

nalysis of MMP2 and MMP9 activity. (J and K) Western blot andnalysis of collagen I and III protein expression. NC: 5 mM glucose,ucose, shRNA-NC+HG: cells transfected with MEF2A-negative shRNA

glucose incubation, sh-MEF2A+HG: cells transfected with MEF2A- glucose incubation. OC: 5 mM d-glucose + 27.5 mM mannose. Data

of 3 independent experiments; *P < 0.05 vs. NC; #P < 0.05 vs. HG.


HG-induced CF proliferation at all tested time points (efciency ofRNAi-mediated knockdown of MEF2A in the CFs is shown in Sup-plementary Fig. S2A2C). The proliferative effect of MEF2A wasconrmed by EdU staining. HG induced an increase in the pro-portion of the HG-ind(Fig. 2B andMEF2A inhE).

Supplemonline versi

3.3. MEF2A

To evaluscratch andtime-depen(Fig. 3A andtion to thetranswell massay (Fig. 3

3.4. MEF2Amyobrobla

To clarifmyobroblExpression icantly incr(Fig. 4A). M-SMA expimmunou

3.5. MEF2Acollagen syn

To gain brosis, weCFs. We obsprotein expwas signicExpression between thshowed thaMMP2 and OC, and shRMMP2 andaltered MMsignicantlyment inhibi

3.6. MEF2ATGF-1/Sma

Cellular were enhanNC or OC tincreased preduced HG(Fig. 6BD)ERK1/2 andS3).

Supplemonline versi

ignal transduction mechanisms involved in MEF2A and high-glucose (HG)nt in cardiac broblasts (CFs). (AE) Western blot analysis of p-p38, p-Akt,

p-Smad2, and p-Smad3 in CFs. HG activated the p38, Akt, and TGF-1/Smads in CFs. Treatment with MEF2A-shRNA further enhanced the activation ofand reversed the activation of the Akt (B) and TGF-1/Smad (CE) path-uced by HG. NC: 5 mM glucose, HG: 30 mM glucose, shRNA-NC+HG: cells

ted with MEF2A-negative shRNA-control before glucose incubation, sh-HG: cells transfected with MEF2A-shRNA before glucose incubation. OC:-glucose + 27.5 mM mannose. Data are the mean SD of 3 independentents; *P < 0.05 vs. NC; #P < 0.05 vs. HG.EdU-positive CFs as compared with NC or OC, whileuced increase was inhibited after silencing of MEF2A

C). Further, ow cytometric analysis indicated thatibition had no effect on CF apoptosis (Fig. 2D and

entary Fig. S2 related to this article can be found, in theon, at http://dx.doi.org/10.1016/j.biocel.2015.10.012.

inhibition limits HG-induced CF migration

ate the inuence of MEF2A on CF migration, we used Transwell migration assays. HG induced prominentdent chemotaxis of CFs as compared with NC or OC

B). MEF2A inhibition signicantly suppressed CF migra- level of the controls at all time points tested. Theigration assay results conrmed those of the scratchC and D).

inhibition attenuates differentiation of CFs intosts

y the role of MEF2A in the differentiation of CFs intoasts in vitro, we stimulated CFs with HG for 48 h.of -SMA (a myobroblast marker) in CFs was signif-eased by HG stimulation as compared with NC or OCEF2A inhibition signicantly suppressed the increase inression induced by HG. This effect was also observed byorescence analysis (Fig. 4B).

mediates HG-induced imbalance of MMP-TIMP andthesis in CFs

further insights into the potential roles of MEF2A in measured MMP expression and collagen synthesis inerved that HG induced an increase in MMP2 and MMP9ression in CFs as compared with NC or OC, whichantly attenuated by inhibition of MEF2A (Fig. 5AC).of TIMP1 and TIMP2 showed no signicant differencese groups (Fig. 5DF). Similarly, gelatin zymographyt HG treatment signicantly increased the activity ofMMP9 in culture supernatants as compared with NC orNA-MEF2A treatment markedly abrogated HG-induced

MMP9 activity (P < 0.05) (Fig. 5GI). In line with theP activity, collagen I and III protein expression was

increased in the HG group, while shRNA-MEF2A treat-ted the effect (P < 0.05) (Fig. 5J and K).

inhibition alleviates HG-induced Akt andd signaling pathway activation in CFs

levels of activated MAPKs, Akt, TGF-1, and Smad2/3ced at 24 h after HG stimulation as compared withreatment (P < 0.05) (Fig. 6). MEF2A inhibition furtherhosphorylation of p38 in CFs (P < 0.05) (Fig. 6A), while it-induced activation of Akt and TGF-1/Smad (P < 0.05). MEF2A silencing had no effect on the activation of

JNK under HG treatment (P > 0.05) (Supplementary Fig.

entary Fig. S3 related to this article can be found, in theon, at http://dx.doi.org/10.1016/j.biocel.2015.10.012.

Fig. 6. StreatmeTGF-1,pathwayp38 (A) ways indtransfecMEF2A+5 mM dexperim


Fig. 7. MEF2A inhibition prevents cardiac collagen deposition in diabetic mice. (Aand B) Western blot and mRNA analysis of MEF2A in normal and diabetic mousehearts. (C) Typical Masson trichrome (scale bars = 25 m) and picrosiriusredstaining of the heart in diabetic mice (scale bars = 25 m). (D) Quantitativeanalysis of the collagenous area in hearts of diabetic mice. (E) Typical examplesof collagen I and collagen III immunostaining of hearts of diabetic mice. (F and G)

3.7. MEF2A inhibition attenuates diabetes-induced myocardialbrosis and cardiac dysfunction

To ascertain the role of MEF2A in myocardial brosis and dys-function associated with diabetes, we investigated these disorders adiabetic mouse model after shRNA-mediated MEF2A in vivo knock-down. STZ induced rapid hyperglycemia in mice as compared withcitrate treatment beginning at 1 week after injection (data notshown). Blood glucose, systolic blood pressure, mean blood pres-sure, diastolic blood pressure, and heart rate were comparableamong the 4 groups at the end of the study. We observed an increasein blood gluin the diabmice did nogroup (P > 0ited lower dvehicle conwas found

Hypergl(1.627 0.10.562 0.05compared downregula1.689 0.23as compareB and Supp

Massoncollagen devs. 7.462 1gen depositvs. 30.67 chemical anof brotic mwhereas shdecreased ttreatment (

Cardiac We observeLVEF, FS, anhigher in dIn diabetic dysfunctionnegative co

4. Discussi

This stuMEF2A andattenuated ation into mproductionactivation iheart improbetic mice. clarify the eof HG.

We foundecreased a

Quantitative aof mice. Conmice intramyoMEF2A: DM mmean SD of cose and blood pressure, and a decrease in heart rateetic mice (P < 0.05). Injection of shRNA-NC in diabetict change these parameters as compared with diabetic.05). However, mice injected with shRNA-MEF2A exhib-iastolic blood pressure and a higher heart rate than thetrol group (P < 0.05), while no effect on blood glucose(Table 1).ycemia signicantly increased MEF2A mRNA15-fold increase) and protein (0.835 0.042 vs.4, P < 0.05) expression in diabetic mice hearts aswith healthy controls. ShRNA-MEF2A treatmentted the myocardial MEF2A mRNA (0.301 0.027 vs.4) and protein (0.323 0.025vs. 1.008 0.093) levelsd with vehicle treatment in diabetic mice (Fig. 7A andlementary Fig. 2DF).s trichrome staining showed that diabetes increasedposition as compared with control mice (26.80 5.53%.16%, P < 0.05). ShRNA-MEF2A treatment reduced colla-ion as compared with vehicle treatment (15.12 3.50%4.20%, P < 0.05) (Fig. 7C and D). Similarly, immunohisto-alysis showed that diabetes enhanced the expressionarkers collagen I and III as compared with the control,RNA-MEF2A transfection of diabetic mice signicantlyhe levels of collagen I and III as compared with vehicleFig. 7EG).function was assessed at 24 weeks after STZ injection.d an increase in LVEDD and LVESD, and a decrease ind E/A in diabetic mice. IVS thickness and LVPWd wereiabetic than in control mice, although no signicantly.mice, shRNA-MEF2Atreatment prevented these cardiacs (LVEDD, LVESD, LVEF, FS and E/A) as compared withntrol shRNA (P < 0.05) (Table 2).

on

dy demonstrated that HG increased the expression of induced nuclear translocation in CFs. MEF2A inhibitionHG-induced CF proliferation, migration, and differenti-yobroblasts, and reduced MMP activity and collagen

by blocking Akt and TGF-1/Smad signaling pathwayn CFs. Further, it was shown that MEF2A knockdown inved cardiac dysfunction and collagen deposition in dia-To the best of our knowledge, this is the rst report toffect of MEF2A on the function of CFs in the condition

d that HG induced MEF2A expression peaked at 48 h butt 72 h in CFs. We hypothesize that higher expression of

nalysis of the collagen I- and collagen III-stained areas in heartstrol: normal mice. DM: diabetic mellitus. DM+shRNA-NC: DMcardially injected withMEF2A-negative shRNA-control. DM+shRNA-ice intramyocardially injected with MEF2A-shRNA. Data are the

3 independent experiments; *P < 0.05 vs. control; # P < 0.05 vs. DM.


Table 1Blood glucose, blood pressure, and heart rate measurements.

Blood glucose (mM) SBP (mmHg) MBP (mmHg) DBP (mmHg) HR (pm)

Control 7.35 0.68 119 7 86 7 69 8 598 16DM 33.89 0.97* 135 5* 102 7* 94 7* 545 22*DM+shRNA-NC 35.65 1.56* 138 5* 106 9* 96 9* 524 26*DM+shRNA-MEF2A 34.95 1.08 129 3 96 5 74 3# 594 15#

The parameters were tested at 24 weeks after diabetes induction. SBP: systolic blood pressure; MBP: mean blood pressure; DBP: diastolic blood pressure; and HR:heart rate. Control: normal mice; DM: diabetic cardiomyopathy; DM+shRNA-NC: DM mice intramyocardially injected with shRNA-control. DM+shRNA-MEF2A: DM miceintramyocardially injected with MEF2A shRNA. All results are presented as the mean SEM. n = 10 per group.

* P < 0.05 vs. control.# P < 0.05 vs. vehicle control.

MEF2A is mainly because of increased phosphorylated p-MEF2Aexpression. P-MEF2A is located in the nucleus, and HG inducedtranslocation of MEF2A from the nucleus to the cytoplasm; thus, thep-MEF2A in the nucleus decreased at 72 h, resulting in the suddendecrease in MEF2A at 72 h.

The results that HG induced MEF2A nuclear translocation wereconsistent with previous reports of increased MEF2A expressionin STZ-induaddition, Mshowed thaSTZ-inducefrom the nIn line witMEF2 to thedensity (Wacytoplasm activity of a(Link et al., 2(Xu et al., 2relocalizatiowhich MEF

HG-indupathophysiremodelingtion reduceto curative function in

The diffeincreased inby expressidisorganizeincreases swith increa2014; Yuendifferentiatour study. uated diffesuggested t

remodeling by activating the differentiation of broblasts intomyobroblasts.

MMPs play important roles in the maintenance and degradationof the ECM, thus contributing to the process of cardiac remodeling(Li et al., 2011). Studies have shown upregulation of MMP9 andMMP2 activity in diabetic heart (Li et al., 2011, 2012). Inhibition ofMMP activity has been suggested to have a cardioprotective role

etes. activwas

a lin ECMich fd othn acn. Wand d

knosreg

the actiontive rF2A ther,es-in

actare antiatalsh, bser

Akt, nd t/Smas a dmanied

d pat Simi

Table 2Echocardiogra

IVS; d (mm)LVEDD (mmLVESD (mm)LVPWd (mmEF (%) FS (%) E/A

IVS; d, intervediastolic postemice. DM: diawith shRNA-M

* P < 0.05 vs# P < 0.05 vsced diabetic mouse heart (Feng et al., 2008, 2010). Inora et al. (Mora and Pessin, 2000; Mora et al., 2001)t MEF2A was decreased in heart nuclear extracts fromd diabetic rats, suggesting that MEF2A translocateducleus to the cytoplasm after HG treatment in CFs.h our observations, TGF--induced relocalization of

cytoplasm was seen in myogenic cells grown to highng et al., 2011). Shuttling between the nucleus and theis a rapid and efcient mechanism for regulating the

transcription factor, as has been observed for FOXO009; Zanella et al., 2008), NFAT (Wolff et al., 2010), p53008), etc. Therefore, we hypothesize that HG-inducedn of the MEF2A in the CFs might be a mechanism by

2A regulates CF function under HG.ced CF proliferation and migration are the mainological mechanism underlying diabetes-induced ECM. In the present study, we showed that MEF2A inhibi-d CF proliferation and migration, which may contributetreatment targeting ECM remodeling and cardiac dys-diabetes.rentiation of broblasts into myobroblasts is strongly

the myocardium of failing hearts and is characterizedon of -smooth muscle actin and increased formation ofd collagen matrix (Cucoranu et al., 2005). HG treatmentpontaneous differentiation of CFs to myobroblastssing passage when compared with LG (Shamhart et al.,

et al., 2010). In line with these observations, enhancedion of myobroblasts was seen in HG-treated CFs inIn addition, we found that MEF2A inhibition atten-rentiation of CFs into myobroblasts. These resultshat MEF2A might play a role in diabetes-induced ECM

in diabMMP9which strates

TheIII, whrial anCollagefunctioin CFs MEF2Aand dyble fordysfunprotecfor ME

Furdiabetthat HGwhich differeand Wthese oMAPK,we fouTGF-MEF2 iand hu2009; W/Sma2001).

phic assessment.

Control DM

0.77 0.04 0.79 0.06 ) 3.28 0.15 3.83 0.13*

2.02 0.14 2.88 0.26*

) 1.05 0.18 1.10 0.20

71.99 4.45 55.33 2.58*40.96 3.29 31.83 1.96*1.45 0.12 0.96 0.08*

ntricular septum thickness (diastole); LVEDD, left ventricular end-diastolic dimension; LVrior wall thickness; EF: ejection fraction, calculated. FS: fractional shortening, calculatebetic cardiomyopathy. DM+shRNA-NC: DM mice intramyocardially injected with shRNEF2A. n = 10, data are presented as the mean SD.. control.. DM. In the present study, we demonstrated that MMP2 andities were increased in response to HG in broblasts,

reversed by MEF2A inhibition. This nding demon-k between MEF2A and MMPs in diabetes.

mainly consists of molecules of collagen types I andorm brils and provide most of the connective mate-er structures in the myocardium (Pelouch et al., 1993).cumulation results in the development of heart dys-e found that HG promoted collagen I and III synthesisiabetic mouse hearts, which was partially inhibited byckdown. CF migration, proliferation, and differentiation,ulation of the MMPs were considered to be responsi-bnormal collagen deposition, which resulted in cardiac

in diabetic mice. However, MEF2A inhibition played aole. Taken together, our ndings suggest a general rolein modulating ECM remodeling in the diabetic heart.

we studied the molecular mechanism of MEF2A induced ECM remodeling. Previous studies have outlinedivates MAPK, Akt, and TGF-/Smad signaling pathways,ssociated with cell growth, proliferation, migration, andion (Al-Khalili et al., 2004; Sharma et al., 2012; Shiojima2002; Verrecchia and Mauviel, 2002). In agreement withvations, we demonstrated that HG activated the p38and TGF-/Smad signaling pathways in CFs. Meanwhile,hat MEF2A inhibition abated activation of the Akt andd signaling pathways. Previous studies have shown thatownstream effector of the PI3K/Akt pathway in neurons

umbilical vein endothelial cells (HUVECs) (Sako et al.,mann et al., 2005), and a critical component of the TGF-hway in C2C12 myoblasts (Liu et al., 2004; Quinn et al.,larly, MEF2A might play a role in CFs via the Akt and

DM+shRNA-NC DM+shRNA-MEF2A

0.76 0.07 0.78 0.033.97 0.16* 3.20 0.122.71 0.18* 1.93 0.15

1.15 0.14 1.08 0.15

51.62 3.15* 64.73 4.6226.85 3.47* 37.46 2.870.98 0.12* 1.32 0.10#

ESD: left ventricular end-systolic dimension; LVPW: left ventriculard; E/A: ratio of early to late mitral inow velocity. Control: normalA-control. DM+shRNA-MEF2A: DM mice intramyocardially injected


TGF-/Smad signaling pathways. However, in our study, MEF2Ainhibition increased the phosphorylation of p38 in CFs, which maybe partially explained by the fact that MEF2A is a nuclear targetof the p38 MAPK signaling pathway (Chang et al., 2002; Ornatskyet al., 1999;mechanism

In conclin CFs. MEFpartially referentiationsignaling pinhibition, betic cardiotypes involvand endoth

Conict of

The auth

Acknowled

The studgram of ChFoundationState PrograInnovative

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Inhibition of MEF2A prevents hyperglycemia-induced extracellular matrix accumulation by blocking Akt and TGF-1/Smad activ...1 Introduction2 Materials and methods2.1 Lentiviral vectors for RNA interference (RNAi)2.2 Animal model and RNAi2.3 Primary mouse CF culture and treatments2.4 Immunofluorescence2.5 Western blot analysis2.6 Quantitative RT-PCR2.7 Measurement of CF proliferation and apoptosis2.8 Cell migration assays2.9 Gelatin zymography2.10 Non-invasive analysis of blood pressure and cardiac function2.11 Histology and immunohistochemistry2.12 Statistical analysis

3 Results3.1 HG increases MEF2A expression and translocation in CFs3.2 MEF2A inhibition decreases HG-induced proliferation of CFs and is not associated with apoptosis3.3 MEF2A inhibition limits HG-induced CF migration3.4 MEF2A inhibition attenuates differentiation of CFs into myofibroblasts3.5 MEF2A mediates HG-induced imbalance of MMP-TIMP and collagen synthesis in CFs3.6 MEF2A inhibition alleviates HG-induced Akt and TGF-1/Smad signaling pathway activation in CFs3.7 MEF2A inhibition attenuates diabetes-induced myocardial fibrosis and cardiac dysfunction

4 DiscussionConflict of interestAcknowledgementsReferences

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