Indole-3-carbinol protects against pressure overload induced cardiac remodeling via activating AMPK-α

1680 Mol. Nutr. Food Res. 2013, 57, 1680–1687DOI 10.1002/mnfr.201300012

RESEARCH ARTICLE

Indole-3-carbinol protects against pressure overload

induced cardiac remodeling via activating AMPK-�Wei Deng1,2∗, Jing Zong1,2∗, Zhouyan Bian1,2, Heng Zhou1,2, Yuan Yuan1,2, Rui Zhang1,2,Haipeng Guo3, Yan Zhang1,2, Difei Shen1,2, Hongliang Li1,2 and Qizhu Tang1,2

1 Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, P. R. China2 Cardiovascular Research Institute of Wuhan University, Wuhan, P. R. China3 The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education andChinese Ministry of Health, Qilu Hospital of Shandong University, Jinan, P. R. China

Scope: Indole-3-carbinol (I3C), a monomer component extracted from leaves and stems ofcruciferous vegetables, has inhibitory effects on tumors, obesity, and liver fibrosis, but its effectson the development of cardiac remodeling remain completely unknown. We determined theeffects of I3C on cardiac remodeling and heart function using an aortic banding (AB) mousemodel.Methods and results: Male 8- to10-wk-old wild-type and 5′ adenosine monophosphate-activatedprotein kinase (AMPK)-�2 knockout mice fed with or without I3C were subjected to AB or asham operation and were phenotyped, accordingly. I3C both prevented and reversed cardiacremodeling induced by AB, as assessed by heart weight/body weight, lung weight/body weight,and heart weight/tibia length ratios, echocardiographic and hemodynamic parameters, histo-logical analysis, and gene expression of hypertrophic and fibrotic markers. The inhibitory effectof I3C on cardiac remodeling was mediated by AMPK-� and extracellular signal-regulatedkinases 1/2 (ERK1/2) signaling. Moreover, AMPK-�2 gene deficiency completely blockedthe inhibitory effects of I3C on cardiac remodeling, preventing the improvements in heartweight/body weight, lung weight/body weight, heart weight/tibia length, cardiac function,gene expression of hypertrophic and fibrotic markers, and phosphorylation of mammaliantarget of rapamycin and ERK1/2 signaling components.Conclusion: I3C both prevents and reverses cardiac remodeling by activating AMPK-� signal-ing. I3C is a potential therapeutic drug for heart failure.

Keywords:

AMPK-� / Cardiac remodeling / Indole-3-carbinol

Received: January 6, 2013Revised: February 18, 2013

Accepted: February 19, 2013

� Additional supporting information may be found in the online version of this article atthe publisher’s web-site

Correspondence: Professor Qizhu Tang, Department of Cardiol-ogy, Renmin Hospital of Wuhan University, JieFang Road 238,Wuhan 430060, P. R. ChinaE-mail: [email protected]: +86-27-88083385

Abbreviations: AB, aortic banding; AICAR, 5-aminoimidazole-4-carboxamide 1-�-D-ribofuranoside; ANP, atrial natriuretic pep-tide; ATP, adenosine triphosphate; BRCA, breast cancer; CTGF,connective tissue growth factor; ECM, extracellular matrix; ERK,extracellular signal-regulated kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H&E, hematoxylin-eosin; HW/BW,heart weight/body weight; HW/TL, heart weight/ tibia length;I3C, indole-3-carbinol; JNK, c-Jun N-terminal kinase; KO, knock-out; LV, left ventricular; LVDd, left ventricular end-diastolic di-ameter; LVDs, left ventricular end-systolic diameter; LVPWs, left

1 Introduction

Heart failure is one of the leading causes of mortality not onlyin the Western world but also in China. The key pathophysio-logical process that ultimately leads to heart failure is cardiac

ventricular posterior wall thickness at end-systole; LW/BW, lungweight/body weight; MAPK, mitogen-activated protein kinase;MEK, MAPK or ERK kinases; mTOR, mammalian target of ra-pamycin; NADPH, nicotinamide adenine dinucleotide phosphate;PSR, picrosirius red; SERCA2a, sarcoplasmic reticulum Ca2+-ATPase2a; TGF-�1, transforming growth factor �1; TSC2, tuber-ous sclerosis protein 2; WGA, wheat germ agglutinin; WT, wild-type; �-MHC, � myosin heavy chain∗These authors contributed equally to this work.

C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com

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remodeling in response to chronic pathological stresses,such as hypertension and myocardial ischemia [1]. Cardiacremodeling, which involves myocyte hypertrophy along withinterstitial cell proliferation and extracellular matrix (ECM)remodeling, induces structural and functional changesmainly in the left ventricle. It is initially a beneficial compen-satory process, as it decreases wall stress and increases cardiacoutput, but it ultimately results in the inability of heart tomeet hemodynamic demands [2]. Despite considerable ther-apeutic advances, the prevalence of heart failure is increasingthroughout the world, and the discovery of new therapeuticstrategies to attenuate cardiac remodeling and restore cardiacfunction are urgent goals for the biomedical community.

Indole-3-carbinol (I3C) is a monomer extracted fromleaves and stems of cruciferous vegetables, such as cabbageand flowering Chinese cabbage. I3C has antitumor activity,interfering with multiple oncogenic signaling pathways thatgovern cell-cycle progression, survival, invasion, and otheraggressive phenotypes of cancer cells [3]. In addition, I3Ctreatment can decrease body weight and fat accumulation inobese mice, reduce the number of activated hepatic stellatecells in the liver of rats, accelerate collagen degradation, andpromote the reversal of liver fibrosis [4, 5]. However, the in-fluence of I3C on cardiac remodeling has not been reported.

5′ adenosine monophosphate-activated protein kinase(AMPK) is a serine/threonine protein kinase that plays an im-portant role in the cardiovascular system [6]. Previous studieshave shown that AMPK activation can protect the heart fromischemic injury and pressure overload induced cardiac hy-pertrophy [7, 8]. In hypertrophic hearts subjected to chronicpressure overload, the activity of both AMPK-�1 and AMPK-�2 is increased [9]. AMPK-�2 is the main functional isoformin stress state of heart and is proved to protect against pres-sure overload induced ventricular hypertrophy and dysfunc-tion [8, 10].

We undertook the present study to investigate whetherI3C improves pressure overload induced cardiac remodelingin mice. Our results indicate that oral I3C prevented andreversed cardiac hypertrophy and fibrosis induced by pressureoverload via activating AMPK-� signaling.

2 Methods

A detailed Methods section is given in the online-only Sup-porting Information. All animal procedures were performedin accordance with the ‘Guide for the Care and Use of Lab-oratory’ Animals published by the US National Institutes ofHealth (NIH Publication no. 85–23, revised 1996) and ap-proved by the Institutional Animal Care and Use Commit-tee at Renmin Hospital, Wuhan University, China (protocol00020392). All surgeries and subsequent analyses were per-formed in a blinded fashion.

Adult male C57/BL6 (wild-type, WT) and AMPK-�2knockout (KO; C57BL/6J background) mice (8- to 10-wk old)were used in this study. WT mice received normal mainte-

nance feed (vehicle) or maintenance feed containing 0.05%I3C (dose: 100 mg/kg/day). AMPK-�2 KO mice also receivedfeed with or without I3C (100 mg/kg/day). Before or after1 wk of I3C or vehicle feeding, WT mice were subjected toaortic banding (AB) or a sham operation as described pre-viously [11]. After 1 wk of I3C or vehicle feeding, KO micewere given the same surgery as WT mice. Echocardiographywas performed on anesthetized (1.5% isoflurane) mice, us-ing a MyLab 30CV ultrasound (Biosound Esaote, Inc.) with a10 MHz linear array ultrasound transducer, as previouslydescribed [12]. The invasive hemodynamic measurementswere performed in anesthetized (1.5% isoflurane) mice usingcardiac catheterization, as described previously [13]. Excisedhearts were arrested in diastole with 10% KCl, weighed, fixedby perfusion with 10% formalin, and embedded in paraffinfor histological studies or were frozen for RNA and proteinextraction. The effects of I3C on cardiac hypertrophy and fi-brosis were studied using hematoxylin-eosin (H&E), wheatgerm agglutinin (WGA), and picrosirius red (PSR) staining,as well as real-time quantitative PCR. Kinases phosphoryla-tion was examined using Western blotting.

3 Results

3.1 I3C prevents cardiac hypertrophy, fibrosis, and

dysfunction induced by pressure overload when

administered before AB

To investigate the effects of I3C on cardiac remodeling, all WTmice were subjected to AB surgery or a sham operation withor without 1 wk of I3C feeding before surgery. In the preven-tion experiment, I3C inhibited the development of cardiac hy-pertrophy after 8 wk of AB, which was indicated by increasedleft ventricular (LV) ejection fraction and LV fractional short-ening and decreased LV end-diastolic diameter (LVDd), LVend-systolic diameter (LVDs), interventricular septal thick-ness at end-diastole, interventricular septal thickness at end-systole, LV posterior wall thickness at end-diastole, LV poste-rior wall thickness at end-systole (LVPWs), heart weight/bodyweight (HW/BW), lung weight/body weight (LW/BW), andHW/tibia length (HW/TL) compared with the vehicle group(Supporting Information Fig. S1A and B). Gross heart, H&E,and WGA staining indicated that the mice fed I3C before ABexhibited significantly decreased cardiac hypertrophy and my-ocyte cross-sectional areas (Fig. 1A and B). The AB-mediatedinduction of hypertrophic markers, including atrial natri-uretic peptide (ANP), � myosin heavy chain (�-MHC), andsarcoplasmic reticulum Ca2+−ATPase2a (SERCA2a), wasseverely blunted in these mice (Fig. 1B).

In addition, to determine the extent of fibrosis in the heart,paraffin-embedded sections were stained with PSR. Markedperivascular and interstitial fibrosis was detected using PSRstaining in vehicle-fed and I3C-fed mice that were subjectedto AB (Fig. 1C and D). However, the extent of cardiac fi-brosis and the LV collagen volume fraction was remarkably


1682 W. Deng et al. Mol. Nutr. Food Res. 2013, 57, 1680–1687

reduced in I3C-fed mice (Fig. 1C and D). Subsequent analy-sis of the mRNA levels of fibrotic mediators, such as trans-forming growth factor �1 (TGF-�1), TGF-�2, connective tis-sue growth factor (CTGF), and collagen I and collagen III,demonstrated a blunted response to fibrosis in I3C-fed mice(Fig. 1D). These results suggest that I3C negatively regulatesthe extent of cardiac hypertrophy, fibrosis, and dysfunctioninduced by pressure overload.

3.2 I3C increases myocardial AMPK-� activation and

attenuates mTOR and ERK1/2 activation in

response to pressure overload when

administered before AB

To explore the molecular mechanisms of I3C-mediated repairin the hypertrophic response, we investigated the activationof AMPK-� and mitogen-activated protein kinases (MAPK)signaling in response to pressure overload in I3C-fed andvehicle-fed hearts. The phosphorylation of AMPK-� was sig-nificantly increased in I3C-fed hearts compared with vehicle-fed hearts after AB (Fig. 2A). The AB-mediated increasesin p-mammalian target of rapamycin (mTOR), p-p70S6K,p-S6K, p-4E-BP1, p-eIF4E, p-MEK (MAPK or ERK kinases),and p-extracellular signal-regulated kinases 1/2 (ERK1/2)were inhibited by I3C (Fig. 2A–D). However, the phospho-rylation of c-Jun N-terminal kinase (JNK) and p38 were notsignificantly different between I3C-fed hearts and vehicle-fedones (Fig. 2C and D). These data suggest that I3C signifi-cantly inhibits cardiac hypertrophy by stimulating AMPK-�activity and inhibiting mTOR and MEK-ERK1/2 signaling.

3.3 I3C reverses cardiac hypertrophy, fibrosis, and

dysfunction induced by pressure overload when

administered during AB

Next, we observed whether I3C could reverse cardiacremodeling and dysfunction after 8 wk of AB. We found thatI3C, which was added to the chow after 1 wk of AB, alsoattenuated the development of cardiac hypertrophy. Mainte-nance I3C-fed mice exhibited weakened cardiac hypertrophy

� Figure 1. I3C prevents cardiac hypertrophy, fibrosis, and dys-function induced by pressure overload.(A) Histology results fromgross hearts, H&E staining, and WGA staining after 8 wk of ABor sham with or without I3C feeding (n = 6). (B) The myocytecross-sectional areas and the mRNA expression of hypertrophicmarkers in the LV myocardium after 8 wk of AB or sham withor without I3C feeding (n = 6). (C) PSR staining of histologicalsections of the LV was performed for each group after 8 wk of AB(n = 6). (D) The fibrotic areas in the histological sections and themRNA expression of fibrosis markers in the LV myocardium after8 wk of AB or sham with or without I3C feeding (n = 6). Valuesrepresent means ± SEM. †p < 0. 05 and ‡p < 0.01 compared withthe vehicle-sham group; *p < 0.05 and **p < 0.01 compared withthe vehicle-AB group.


Mol. Nutr. Food Res. 2013, 57, 1680–1687 1683

and dysfunction compared with vehicle-treated mice, asmeasured by increased LV ejection fraction and LV fractionalshortening and decreased LVDd, LVDs, LVPWs, HW/BW,LW/BW, and HW/TL (Supporting Information Fig. S2A andB). Gross hearts, H&E staining, and WGA staining showedconsistent results and decreased myocyte cross-sectionalareas (Fig. 3A and B). Meanwhile, I3C significantly inhibitedthe expression of cardiac hypertrophy markers ANP, �-MHC,and SERCA2a in heart from the I3C-fed AB group comparedwith the vehicle-fed AB group (Fig. 3B).

In the reversal experiment, we also observed that I3Cmarkedly reduced the extent of cardiac fibrosis and LVcollagen volume fraction in mice that were subjected to AB(Fig. 3C and D). The subsequent analysis of the mRNA levelsof fibrotic mediators was consistent with the pathologicalresults: TGF-�1, TGF-�2, CTGF, collagen I and collagenIII demonstrated blunted responses to pressure overload inI3C-fed mice (Fig. 3D). These results further suggest thatI3C negatively regulates the extent of cardiac hypertrophy,fibrosis, and dysfunction induced by pressure overload.

3.4 I3C increases myocardial AMPK-� activation and

attenuates MEK-ERK1/2 activation in response

to pressure overload when administered

during AB

In the reversal experiment, I3C had similar effects on signalpathway proteins as detected in the prevention experiment.I3C increased the phosphorylation levels of AMPK-� com-pared with vehicle-fed hearts after AB (Fig. 4A and B). I3Cinhibited the AB-mediated increases in p-mTOR, p-p70S6K,p-S6K, p-eIF4E, p-4E-BP1, p-MEK, and p-ERK1/2 (Fig. 4A–D).The phosphorylation of JNK and p38 were not significantlydifferent between I3C-fed hearts and vehicle-fed ones (Fig. 4Cand D).

3.5 I3C exhibits no protective effects on cardiac

hypertrophy, fibrosis, or dysfunction induced by

pressure overload in AMPK-�2 knockout mice

To further investigate whether I3C antagonizes the hy-pertrophic response to pressure overload through AMPK,

� Figure 2. The preventive effect of I3C on AMPK-�-mTOR andMAPK signaling. (A) Phosphorylation levels of proteins relatedto AMPK-�-mTOR signaling in the LV myocardium of AB or shammice with or without I3C feeding (n = 6). (B) Quantitative mea-surements of p-AMPK-�, p-mTOR, p-4E-BP1, p-eIF4E, p-p70S6K,and p-S6K protein relative to glyceraldehyde 3-phosphate dehy-drogenase (GAPDH). (C) Phosphorylation of proteins related toMAPK signaling in the LV myocardium of AB or sham mice withor without I3C feeding (n = 6). (D) Quantitative measurements ofp-MEK, p-ERK1/2, p-JNK, and p-P38 protein relative to GAPDH.Values represent means ± SEM. ‡p < 0.01 compared with thevehicle-sham group; **p < 0.01 compared with the vehicle-ABgroup.



AMPK-�2 KO mice were fed I3C or vehicle before 1 wk ofAB surgery or sham operation. After 4 wk of AB, the devel-opment of cardiac remodeling was detected in the two ABgroups compared with the sham groups, as evidenced byincreased LVDd, LVDs, interventricular septal thickness atend-diastole, interventricular septal thickness at end-systole,LVPWs, LV posterior wall thickness at end-diastole, HW/BW,LW/BW, and HW/TL, and no significant differences were de-tected between the I3C-fed and vehicle-fed groups (Support-ing Information Fig. S3A and B). Gross hearts, H&E, andWGA staining and the myocyte cross-sectional area yieldedconsistent results (Supporting Information Fig. S3C and D).The induction of the hypertrophic markers ANP, �-MHC,and SERCA2a was dramatically increased in both I3C-fedand vehicle-fed KO mice in response to AB, but the mRNAexpression of these markers was not distinguishable betweenthe two AB groups (Supporting Information Fig. S3D).

Marked perivascular and interstitial fibrosis were detectedby PSR staining in both I3C-fed and vehicle-fed KO micesubjected to AB, and there was no obvious difference be-tween the two groups (Supporting Information Fig. S3E andF). Subsequent analysis of the mRNA levels of fibrotic me-diators TGF-�1, TGF-�2, CTGF, collagen I, and collagen IIIalso demonstrated similar results (Supporting InformationFig. S3F). These results suggest that I3C does not protectagainst cardiac hypertrophy, fibrosis, and dysfunction in re-sponse to pressure overload in the absence of AMPK-�2.

3.6 I3C loses its inhibitory effect on pressure

overload induced mTOR and MEK-ERK1/2

activation in AMPK-�2 knockout mice

Subsequently, we investigated the activation of mTOR,p70S6K, 4E-BP1, MEK, and ERK1/2 in heart in response topressure overload in I3C-fed and vehicle-fed KO mice. Thephosphorylation levels of mTOR, p70S6K, 4E-BP1, MEK, andERK1/2 were not different between I3C-fed and vehicle-fedKO mice (Supporting Information Fig. S4; data of quantita-tive measurements not shown). These findings indicate thatI3C loses its inhibitory effect on mTOR and MEK-ERK1/2activation induced by pressure overload in the absence ofAMPK-�2.

� Figure 3. I3C reverses cardiac hypertrophy, fibrosis, and dysfunc-tion induced by pressure overload. (A) Histology results fromgross hearts, H&E staining, and WGA staining after 8 wk of ABor sham with or without I3C feeding (n = 6). (B) The myocytecross-sectional areas and the mRNA expression of hypertrophicmarkers in the myocardium after 8 wk of AB with or without I3Cfeeding (n = 6). (C) PSR staining of histological sections of the LVwas performed for each group after 8 wk of AB (n = 6). (D) Thefibrotic areas in the histological sections and the mRNA expres-sion of fibrosis markers in the LV myocardium after 8 wk of AB orsham with or without I3C feeding (n = 6). Values represent means± SEM. †p < 0. 05 and ‡p < 0.01 compared with the vehicle-shamgroup; *p < 0.05 and **p < 0.01 compared with the vehicle-ABgroup.


Mol. Nutr. Food Res. 2013, 57, 1680–1687 1685

4 Discussion

Much progress has been made in understanding the molecu-lar and cellular processes of heart failure, which has led to thedevelopment of effective therapies, but this disease remainsa major cause of illness and death in our aging society. Newtreatments that target disease mechanisms at the cellular andwhole-organ level are needed to halt and reverse the devastat-ing consequences of this disease [14]. The present study useda pressure overload induced cardiac remodeling paradigmwith WT and AMPK-�2 KO mice. The major findings are asfollows: (i) I3C both prevented and reversed cardiac hypertro-phy, fibrosis, and dysfunction induced by pressure overload;(ii) I3C increased myocardial AMPK-� activation and atten-uated mTOR and ERK1/2 activation in response to pressureoverload in both prevention and reversal experiments; (iii)I3C lost its inhibitory effects on pressure overload inducedcardiac hypertrophy, fibrosis, and heart function in AMPK-�2KO mice. These findings demonstrate that I3C can effectivelyinhibit cardiac remodeling and heart failure.

I3C can exert its pharmacological effects through mul-tiple diseased states. Solo or combination treatment withI3C can target multiple aspects of cancer: cell-cycle regula-tion, survival and apoptosis (including Akt-NF�B signaling),caspase activation, cyclin-dependent kinase activities, estro-gen receptor signaling, ER stress, and breast cancer geneexpression [15, 16]. I3C supplementation significantly ame-liorates high fat diet induced obesity and metabolic disor-ders by decreasing adipogenesis and inflammation, alongwith activating thermogenesis [4, 17]. In addition, I3C in-hibits proliferation and stimulates apoptosis in hepatic stel-late cells by blocking the nicotinamide adenine dinucleotidephosphate oxidase/reactive oxygen species/p38 MAPK path-way and the inhibitor of �B kinase �/inhibitor of �B-�/NF-�Bsignal pathway, ultimately preventing and ameliorating liverfibrosis [5,18]. Our findings demonstrate that I3C also affectsthe myocardium and attenuates pressure overload inducedcardiac remodeling and dysfunction via activating AMPK-�signaling.

AMPK has been considered only a sensor of cellularenergy status that is activated under conditions of low in-tracellular adenosine triphosphate following stresses suchas nutrient deprivation and hypoxia. AMPK activation alsoaffects protein metabolism. By phosphorylating tuberous

� Figure 4. The reversal effect of I3C on AMPK-�-mTOR and MAPKsignaling. (A) Phosphorylation of proteins related to AMPK-�-mTOR signaling in the LV myocardium of AB or sham mice (n= 6). (B) Quantitative measurements of p-AMPK-�, p-mTOR, p-4E-BP1, p-eIF4E, p-P70S6K, and p-S6K protein relative to GAPDH.(C) Phosphorylation of proteins related to MAPK signaling in theLV myocardium of AB or sham mice (n = 6). (D) Quantitativemeasurements of p-MEK, p-ERK1/2, p-JNK, and p-P38 proteinrelative to GAPDH. Values represent means ± SEM. ‡p < 0.01compared with the vehicle-sham group; **p < 0.01 comparedwith the vehicle-AB group.



sclerosis protein 2 and raptor, AMPK blocks the mTOR path-way, a major controller of protein synthesis and biomassgeneration [19]. This control of nonmetabolic targets is re-lated to cardiac hypertrophy and heart failure [10]. Tian et al.found that chronic changes in hypertrophied hearts inducedby pressure overload were accompanied by significantly el-evated AMPK-� activity and isoform-specific alterations inAMPK expression [9]. Pharmacological activation of AMPK-� by metformin and 5-aminoimidazole-4-carboxamide 1-�-D-ribofuranoside (AICAR) in hypertrophied hearts resultsin decreased p70S6 kinase phosphorylation, increased EF2phosphorylation, and decreased protein synthesis and hyper-trophic growth in the cardiac myocyte [20]. AMPK-�2 genedeficiency in mice significantly augments the transverse aor-tic constriction-induced increase of p-p70S6 kinase, p-S6, p-eIF4E, and p-4E-BP1 in myocardium and also significantlyexacerbates the increase of ventricular hypertrophy and my-ocardial fibrosis while decreasing heart function [8]. Consis-tent with previous studies, our results show that I3C inhib-ited the phosphorylation of mTOR, p70S6K, S6, 4E-BP1, andeIF4E by activating upstream protein kinase AMPK-�.

Our results clearly demonstrate that I3C also markedlyblocked ERK1/2 activation in AB heart. The MAPK signalingpathways, including ERK1/2, JNK, and p38, directly mod-ify transcription factors that promote alterations in cardiacgene expression and result in cardiac hypertrophy; in par-ticular, ERK1/2 is an essential regulator of the hypertrophicresponse [21–23]. However, I3C lost its inhibitory effect onERK1/2 activation and its protective effect on cardiac hyper-trophy induced by pressure overload in AMPK-�2 KO mice.Our observations are consistent with several previous stud-ies. In cultures of cardiac myocytes, adiponectin activatesAMPK and inhibits norepinephrine- or AngII-stimulated hy-pertrophy, and ERK1/2 phosphorylation, while transductionwith a dominant-negative form of AMPK reverses these ef-fects [24]. AICAR, an AMPK activator, reverses the inhibitionof PPAR-� and the activation of ERK1/2 in hypertrophic my-ocardium of rats induced by transaortic constriction, whereasepidermal growth factor, an ERK1/2 activator, blocks the up-regulation of PPAR-� induced by AICAR [25]. These resultsdemonstrate that AMPK activation inhibits cardiac hypertro-phy partly through ERK1/2.

Fibrosis is another classical feature of pathological car-diac hypertrophy [26]. Increased fibrosis decreases myocardialcompliance, impairs diastolic relaxation, and causes cardiacdysfunction, which is characterized by an increase in colla-gens and other ECM components in the interstitium andperivascular regions of the myocardium [27, 28]. The mostabundant collagen types in the heart are collagen I and col-lagen III, accounting together for over 90% of the total colla-gen [28]. TGF-� is a crucial regulator of ECM metabolism in awide range of organ systems. Also in heart, ECM productionis importantly regulated by TGF-�, as has been consistentlydemonstrated by transgenic and KO mouse models [29, 30].In the pressure-overloaded rats, blockade of TGF-�1 functionusing specific neutralizing antibodies prevented the induc-

tion of collagen mRNA, myocardial fibrosis, and diastolicdysfunction [31]. CTGF is a downstream target of TGF-� andessential for TGF-�-induced collagen synthesis and cardiacfibrosis [32]. Pharmacological activation of AMPK by AICARand adiponectin inhibits TGF-�-induced Smad3-dependenttranscription and the secretion of ECM proteins collagen I,collagen IV, and fibronectin in myofibroblasts, but dominant-negative AMPK-�2 blocks these effects [33]. ERK is also oneof the key protein kinases that regulate the growth and prolif-eration of cardiac fibroblasts. AMPK activation inhibits ERKand its downstream substrate p70S6K in neonatal rat cardiacfibroblasts [34]. Our study indicates that I3C decreases themRNA express of TGF-� and CTGF, and inhibits the exces-sive synthesis of collagen I and collagen III, at last attenuatescardiac fibrosis induced by pressure overload via activatingAMPK-�.

Several novel regulators and putative drug targets of car-diac hypertrophy have been found by using gene-modifiedand acquired models of cardiac hypertrophy [35]. The presentstudy reveals that I3C both prevents and reverses cardiachypertrophy and fibrosis induced by pressure overload viaactivating AMPK-� signaling pathway. We also show thatgenetic deletion of AMPK-�2 leads to the loss of the pro-tective effect of I3C against cardiac hypertrophy and fibro-sis. Our results should prove valuable for the application ofI3C in the prevention and treatment of cardiac remodeling,and I3C may become a potential therapeutic drug for heartfailure.

This work was supported by the National Nature Sci-ence Foundation of China [81270303]; and the Fundamen-tal Research Funds for the Central Universities of China[20103020201000193, 302274021/121097]. The AMPK-�2 KOmice were provided by Lianfeng Zhang from the Institute of Lab-oratory Animal Science at the Chinese Academy of Medical Sci-ences.

The authors have declared no conflict of interest.

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Indole-3-carbinol protects against pressure overload induced cardiac remodeling via activating AMPK-α