Comparative Proteomic Analysis Reveals Sex and Estrogen Receptorβ Effects in the Pressure Overloaded HeartGeorgios Kararigas,*,† Daniela Fliegner,†,⊥ Stefanie Forler,‡ Oliver Klein,‡,∥ Carola Schubert,†

Jan-Åke Gustafsson,§ Joachim Klose,‡,∥ and Vera Regitz-Zagrosek†

†Institute of Gender in Medicine, Center for Cardiovascular Research, and DZHK (German Center for Cardiovascular Research),Berlin partner site, Charite University Hospital, 10117 Berlin, Germany‡Institute for Human Genetics, Charite University Hospital, 10117 Berlin, Germany§Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, Texas 77004, United States§Department of Biosciences and Nutrition, Karolinska Institutet, Solnavagen 1, 171 77 Solna, Sweden∥Core Unit Proteomics, Berlin-Brandenburg Center for Regenerative Therapies, Charite University Hospital, 10117 Berlin, Germany

*S Supporting Information

ABSTRACT: In pressure overload (PO), sex differences inhumans and rodents have been well documented and estrogenreceptor (ER) β is considered cardioprotective. However, theunderlying mechanisms are poorly understood. Our aim was toinvestigate sex- and ERβ-specific effects in protein abundancein PO employing a 2-dimensional gel electrophoresis/massspectrometry-based proteomics approach. We hypothesizedmajor sex differences and ERβ-specific alterations consistentwith cardioprotection in females. Two-month old male andfemale wild-type (WT) and ERβ knockout (BERKO) micewere subjected to transverse aortic constriction (TAC) for 9weeks (n = 4/group). In WT mice, hypertrophy wassignificantly more pronounced in males than females, while this sex difference was abolished in BERKO mice. We found 82protein spots modulated between TAC and sham in WT males, 31 in WT females, 114 in BERKO males, and 87 in BERKOfemales (P ≤ 0.05). Our analysis revealed in WT and BERKO females an altered pattern of various proteins involved in structureand suggests a link between female sex and cytoskeletal integrity. In males, a set of proteins was identified that associate withmitochondrial bioenergetics and energy supply. We confirmed protein regulation by immunoblotting analysis. In conclusion, theproteomic response of the heart to PO is significantly modulated by ERβ and sex. We put forward that the observed differencesmay identify sex-specific targets for the treatment of heart failure, contributing toward more personalized medical care.

KEYWORDS: estrogen receptor, hypertrophy, pressure overload, proteomic, sex differences

■ INTRODUCTIONPressure overload (PO) triggers complex biological responsesthat result in left ventricular (LV) hypertrophy characterized byLV tissue remodeling. LV remodeling is underlain bymodifications in both the cellular and extracellular compart-ments. Initially, there is an adaptive process through theincrease in LV wall thickness that is mainly aimed atmaintaining cardiac performance.1 However, in the progressionof this process, maladaptive features emerge culminating in thetransition to heart failure.Sex differences in patients with aortic stenosis have been well

documented. In particular, women develop more concentrichypertrophy with smaller LV diameter, greater relative wallthickness, and a better systolic function, while men demonstratemore pronounced chamber dilation.2,3 In rodents, by employ-ing the transverse aortic constriction (TAC) model to simulatePO and assess the cardiac response, sex differences in cardiacmorphology and function have also been reported.4−6

Estrogen signaling is fundamental in several tissues, and weand others have previously shown that it exerts direct effects inthe heart.7−11 The main actions of estrogen are mediated by theclassical estrogen receptors (ERs) α and β. In particular, ERβhas been shown to mediate cardioprotective effects of estrogenin PO.12−14 Moreover, we have previously investigated theeffects of sex and the role of ERβ in a mouse model of POassessing transcriptomic changes and found that female sex andERβ attenuate LV remodeling.15 However, proteomic analysisof LV tissue to determine global changes in protein abundancedue to sex and ERβ in PO has not been performed.In the present study, we employed a large high-resolution 2-

dimensional polyacrylamide gel electrophoresis/mass spec-trometry-based proteomics approach to investigate sex differ-ences in protein abundance in PO. To gain further mechanistic

Received: July 18, 2014

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insight, we also studied the proteomic response of the heart toPO in mice lacking ERβ. We hypothesized major sexdifferences and ERβ-specific alterations in the LV proteomeconsistent with cardioprotection in females.

■ EXPERIMENTAL PROCEDURES

Mice

We have previously studied the effects of sex and ERβ oncardiac function and the genomic response to PO.15 In thepresent study, we investigated a subgroup of mice taking aproteomic approach (n = 4 mice/group). Two-month old maleand female mice on the C57Bl/6J background were used.BERKO mice were generated from heterozygous mousecolonies16 and compared with WT littermates. The genotypeof the mice was screened using PCR amplification as previouslydescribed.16 The mice were kept on a 12−12-h light−dark cyclein temperature-controlled rooms with water ad libitum. Allexperiments were approved by the Landesamt fur Gesundheitund Soziales, Berlin, Germany.Experimental Design

TAC was performed as previously described15 under anesthesiawith ketamine hydrochloride (80 mg/kg)/xylazine hydro-chloride (12 mg/kg) by i.p. injection. Sham animals underwentan identical surgical procedure without placement of a suture.Animals recovered from anesthesia under warming conditionsand normal ventilation. After 9 weeks, the mice were sacrificedunder CO2 anesthesia. Following weight measurements, heartswere snap frozen in liquid nitrogen and stored at −80 °C untilfurther analysis.Protein Extraction

Protein extracts were prepared from heart tissue as previouslydescribed.17,18 Briefly, frozen tissues were grounded to finepowder in buffer containing: 4% CHAPS, 50 mM TRIZMAbase (Sigma-Aldrich, Steinheim, Germany), 50 mM KCl, and20% w/v glycerol at pH 7.5, as well as proteinase andphosphatase inhibitor cocktail (Complete, PhosStop; RocheDiagnostics, Mannheim, Germany). They were then sonicatedon ice (12× for 20 s), and benzonase, magnesium chloride (5mM), urea (6.5 M), and thiourea (2 M) were added. Theprotein extracts were supplemented with 70 mM dithiothreitol(Bio-Rad, Munich, Germany) and 2% v/w of ampholytemixture Servalyte pH 2−4 (Serva, Heidelberg, Germany) andstored at −80 °C until further analysis.Two-Dimensional Gel Electrophoresis (2DE) and SpotDetection

Protein extracts were separated by large-gel 2DE as previouslydescribed.17,18 The original gel format for proteome analysiswas 40 cm (isoelectric focusing) × 30 cm (SDS-PAGE) × 0.75

mm (gel width). Protein spot patterns were evaluated by theDelta2D imaging software version 4.0 (DECODON, Greifs-wald, Germany) as previously described.19 Briefly, spot patternswere matched to each other using the Delta2D “exact” mode.Subsequently, using “union” mode, a fusion image wasgenerated of the visible spots for each 2DE gel from eachgroup. Only the fusion image was used for spot detectionfollowed by manual control. Percent volume of spot pixelintensities was used for quantitative protein abundance analysis.After background subtraction, normalized spot intensity wasused for statistical analysis. All significantly altered protein spotswere assigned to their respective positions in silver-stained gels.Identification of Protein Spots by Mass Spectrometry

For protein identification by mass spectrometry (MS), 400 μgof protein extract was separated on 2DE gels and stainedaccording to an MS-compatible silver staining protocol.17

Protein spots of interest were excised from 2DE gels andsubjected to in-gel tryptic digestion. Peptides were analyzed bya nanoLC (Proxeon, Denmark) coupled with a LCQ Deca XPion trap instrument (Thermo Finnigan, Waltham, MA, USA).20

ESI-MS data acquisition was performed throughout the LC run.Three scan events(i) full scan, (ii) zoom scan of mostintense ion in full scan, and (iii) MS/MS scan of the mostintense ion in full scanwere applied sequentially. No MS/MSscan on single charged ions was performed. Raw data wereextracted by the TurboSEQUEST algorithm, and trypsinautolytic fragments and known keratin peptides weresubsequently filtered. All DTA files generated by BioWorksversion 3.2 (Thermo Scientific, Waltham, MA, USA) weremerged and converted to MASCOT generic format files(MGF). Mass spectra were analyzed using our in-houseMASCOT software package license version 2.1. MS/MS ionsearches were performed with the following set of parameters:(i) database: Uniprot version 290709 (531,473 sequences;188,463,640 residues), (ii) taxonomy: Mus musculus (16,441sequences), (iii) proteolytic enzyme: trypsin, (iv) maximum ofaccepted missed cleavages: 1, (v) mass value: monoisotopic,(vi) peptide mass tolerance: ±100 ppm and methionineoxidation and acrylamide derived cysteine alkylation (cys-teinyl-S-propionamide) were considered as possible modifica-tions. Only proteins with scores corresponding to P < 0.05 withat least two independent peptides identified were considered.Furthermore, the molecular weight and pI of each proteinidentified by database search were compared to values obtainedfrom our 2DE patterns.Immunoblotting

To verify the modulation of candidate proteins, immunoblot-ting was performed. Each of the four biological replicates foreach group was run separately on SDS-PAGE gels and

Table 1. Morphometric Data of the Animals Included in the Studya

WT BERKO

Male Female Male Female

Sham TAC Sham TAC Sham TAC Sham TAC

Body weight (g) 28.8 ± 0.3 27.2 ± 0.7 24.0 ± 1.0b 24.0 ± 0.2b 29.9 ± 0.5 29.8 ± 0.5 22.7 ± 0.5b 22.4 ± 0.6b

Heart weight (mg) 118 ± 1 272 ± 10d,e 103 ± 6 159 ± 10d 132 ± 5 199 ± 15d 103 ± 2 162 ± 9d

Liver weight (g) 1.362 ± 0.04c 1.122 ± 0.09 0.937 ± 0.10 1.018 ± 0.07 1.319 ± 0.01c 1.323 ± 0.09c 1.037 ± 0.01 1.029 ± 0.07Tibia length (mm) 17.3 ± 0.04 17.5 ± 0.06 17.1 ± 0.26 16.9 ± 0.14 17.6 ± 0.21 17.2 ± 0.06 17.0 ± 0.11 17.2 ± 0.05Heart weight/tibialength

6.8 ± 0.1 15.5 ± 0.5d,e 6.0 ± 0.3 9.4 ± 0.5d 7.5 ± 0.3 11.5 ± 0.9d 6.1 ± 0.2 9.4 ± 0.5d

aData present mean ± SEM; n = 4/group. bP < 0.001 vs male. cP < 0.05 vs female. dP < 0.001 vs respective sham group. eP < 0.001 vs female.

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transferred to nitrocellulose membranes using standardprocedures. Membranes were probed with primary antibod-iesAldh2 (1:200; sc-100496, Santa Cruz), Hadhb (1:400; sc-55661, Santa Cruz), Vcl (1:15000; V9264, Sigma-Aldrich), Cfl2(1:1000; cs-3318, Cell Signaling), Pkm (1:1000; cs-4053, CellSignaling), and tubulin serving as loading control (1:10000;T9026, Sigma-Aldrich). Immunoreactive proteins were de-tected using ECL Plus (GE Healthcare) and quantified by theImageJ 1.41 version software (http://rsbweb.nih.gov/ij/).

■ STATISTICAL ANALYSIS

Data are shown as mean ± SEM. Comparisons between twogroups were made with Mann−Whitney U tests and amongmultiple groups with 2-way ANOVA and Tukey’s post hoc test,adjusting for multiple comparisons with the R version 2.14.2software, considering P ≤ 0.05 significant.

■ RESULTS

Morphometric Data

The morphometric data of the animals included in the studyare shown in Table 1. As expected, all female mice had lowerbody weight than all male mice (P < 0.001). Absolute heartweight was significantly increased in all TAC groups comparedwith the respective sham groups, and there was a significantlymore pronounced effect in WT male versus female mice (P <0.001). In agreement, the heart-weight-to-tibia-length ratio wassignificantly increased in all TAC groups compared with therespective sham groups. Notably, this difference was signifi-cantly more pronounced in WT male mice compared with WTfemale mice. However, this sex difference was abolished inBERKO mice.

Sex- and ERβ-Dependent Regulation of the CardiacProteomic Response to Pressure Overload

Representative 2-dimensional electrophoresis images from asham and a TAC group are shown in Figure 1. We identifiedand analyzed a total of 795 LV protein spots (on average, up totwo proteins per spot). There was no significant difference inthe number of detected spots or proteins among the groups.Our proteomic analysis revealed a sex-specific cardiac responseto PO. In particular, we found 82 spots modulated between theTAC and sham groups in WT male mice, while in WT femalemice we identified 31 modulated spots due to TAC (P ≤ 0.05,Table 2). The overlap between male and female mice was

limited to 6 spots (Figure 2A). On the other hand, the samesurgical intervention had a larger effect on the proteomicresponse of the heart to PO in BERKO mice than in WT mice,as 114 spots were modulated between the TAC and shamgroups in male mice and 87 spots in female mice (P ≤ 0.05,Table 2). In this case, we identified 18 spots commonlymodulated between male and female mice (Figure 2B). Wefurther inquired into the modulated spots that both WT andBERKO mice would share for each sex separately and found 3

Figure 1. Representative 2-dimensional electrophoresis gels. Images from a male sham (A), a male TAC (B), a female sham (C), and a female TAC(D) extract are shown.

Table 2. Number of Spots Modulated between TAC andSham

Modulatedspots

TAC-inducedspots (%)

TAC-repressedspots (%)

WT male 82 49 51WT female 31 65 35BERKO male 114 44 56BERKO female 87 56 44

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spots in male mice (Figure 2C) and 11 spots in female mice(Figure 2D).In WT male mice, the intensity of a similar proportion of

spots was either higher or lower in the TAC group comparedwith the sham group (49% and 51%, respectively). Classi-fication of the identified proteins revealed isomerases, trans-ferases, and oxidoreductases, among others (Figure 3A).Protein categorization into pathways demonstrated regulation

of glycolysis, cytoskeleton, and ATP synthesis (Figure 4A). Inparticular, the list of the regulated proteins included several

mitochondrial proteins, such as aldehyde dehydrogenase,mitochondrial (Aldh2) and trifunctional enzyme subunit beta,mitochondrial (Hadhb), which were all repressed in the TACgroup (Supporting Information Table 1).In WT female mice, the intensity of 65% of the modulated

spots was higher in the TAC group compared with the shamgroup, while the remaining spots, 35% of the total, had a lowerintensity. Protein classes included cytoskeletal and structuralproteins and oxidoreductases (Figure 3B). Functional catego-rization revealed regulation of angiogenesis, cytoskeleton, and

Figure 2. Numbers and comparisons of modulated spots betweensham and TAC. Venn diagrams showing the number of spots differentbetween sham and TAC in each genotype and sex and those incommon. (A) WT male vs WT female; (B) BERKO male vs BERKOfemale; (C) WT male vs BERKO male; (D) WT female vs BERKOfemale.

Figure 3. Classes of regulated proteins. Schematic representation of categorization into classes for proteins differentially regulated following TAC inmale and female WT and BERKO mice. (A) WT male; (B) WT female; (C) BERKO male; (D) BERKO female.

Figure 4. Protein categorization into pathways. Major functionalclassification for proteins that are differentially regulated followingTAC in male and female WT and BERKO mice. (A) WT male; (B)WT female; (C) BERKO male; (D) BERKO female.

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the integrin signaling pathway (Figure 4B). Among theregulated proteins, we found myosin regulatory light chain 2(Myl2), vimentin (Vim), and vinculin (Vcl), which were allinduced in the TAC group (Supporting Information Table 2).Of the spots modulated in BERKO male mice, the intensity

of 44% was higher and 56% was lower in the TAC groupcompared with the sham group. Several of the identifiedproteins in this case were cytoskeletal proteins, transferases, andoxidoreductases (Figure 3C). Protein categorization intopathways revealed apoptosis, inflammation, and cytoskeletalregulation (Figure 4C). This was mainly due to theconsiderable number of spots identified as myosin heavychain 6 (Myh6), which was repressed in the TAC group(Supporting Information Table 3).On the other hand, in BERKO female mice, we found the

intensity of 56% of the modulated spots higher in the TACgroup compared with the sham group and that of the remaining44% lower. Protein classification identified oxidoreductases andcytoskeletal and structural proteins, as well as transferases(Figure 3D). Protein categorization into pathways revealedglycolysis, p38 MAPK signaling, and cytoskeletal regulation(Figure 4D). Among the regulated proteins, we found cofilin 2(Cfl2) and pyruvate kinase 2 (Pkm2), which were induced inthe TAC group (Supporting Information Table 4).

Validation of the 2-Dimensional Gel ElectrophoresisAnalysis

Due to the prominent effects in metabolic, mitochondrial, andcytoskeletal pathways, we selected candidates within thesebiological processes for the validation of our proteomic analysisby means of immunoblotting analysis. In addition, we includedin our selection proteins representing different levels of up- ordown-regulation. Immunoblotting analysis confirmed that theprotein levels of aldehyde dehydrogenase, mitochondrial(Aldh2) and trifunctional enzyme subunit beta, mitochondrial(Hadhb) were lower in WT male TAC mice compared withtheir respective sham control group (Figure 5A and B).Moreover, in agreement with our comparative proteomicapproach, immunoblotting analysis demonstrated that theprotein levels of vinculin (Vcl) were higher in female WTTAC mice compared with the sham control group (Figure 5C).In line with the 2-dimensional gel electrophoresis analysis,immunoblotting also verified higher levels of cofilin 2 (Cfl2)and pyruvate kinase 2 (Pkm2) in female BERKO TAC micecompared with the sham control group (Figure 5D and E).

■ DISCUSSION

Our study provides novel mechanistic insight into theregulation of the proteomic response of the heart to PO bysex and ERβ. In particular, our comparative proteomic analysisrevealed that there are major sex differences in the response ofthe heart to PO. In WT mice, 2.6 times as many spots weremodulated in males than in females between the sham andTAC groups. Interestingly, there is only a small overlap ofmodulated spots between the sexes. On the other hand, ourcomparative proteomic analysis also identified major differencesdue to ERβ, as several more spots were modulated in both maleand female BERKO mice compared with male and female WTmice. Again, there is only a small overlap between the sexes inBERKO mice. Interestingly, the extent of commonly modulatedspots between WT and BERKO mice was limited to merely 3for the male mice and 11 for the female mice, revealing that thelack of ERβ leads to a highly different proteomic response of

the heart to PO. The latter also indicates that the identifiedproteins from these commonly modulated spots may beassociated with the development of PO-induced LV hyper-trophy independent of ERβ but in a sex-specific manner.The number of modulated spots was greater in BERKO

female mice compared with WT female mice. In agreement, wepreviously showed that the absence of ERβ led to a highernumber of genes and pathways modulated in response to POthan in WT mice.21 On the basis of this finding, we suggest thatthe presence of ERβ is essential for the strict control of theexpression of numerous genes in PO, indicating a repressiverole of gene expression for ERβ. Here, the much larger numberof modulated spots in the absence of ERβ in response to POindicates that ERβ is crucial for the strict regulation of cardiacprotein abundance that may offer protection against theprogression of LV hypertrophy and the transition to heartfailure. Although we assessed the whole transcriptome in ourprior study but here a proportion of the proteome, we considerthis concurring pattern of regulation between transcriptome

Figure 5. Validation of 2-dimensional gel electrophoresis. Immuno-blotting analysis confirmed repression of aldehyde dehydrogenase,mitochondrial (Aldh2) (A) and trifunctional enzyme subunit beta,mitochondrial (Hadhb) (B) in WT male TAC mice and induction ofvinculin (Vcl) (C) in female WT TAC mice and of cofilin 2 (Cfl2)(D) and pyruvate kinase 2 (Pkm2) (E) in female BERKO TAC micecompared with the respective sham groups. The band shown is arepresentative single sample. Representative images of identified spotsin the 2-dimensional electrophoresis gels are also shown. Tubulinserved as loading control. Data present mean ± SEM; n = 4/group.

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and proteome of great importance, confirming ERβ as a masterregulator in the cardiac adaptation to PO.Following protein identification and classification, we

discovered that in WT male mice the levels of several metabolicand mitochondrial proteins, such as aldehyde dehydrogenase,mitochondrial (Aldh2) and trifunctional enzyme subunit beta,mitochondrial (Hadhb), were decreased in the TAC groupcompared with the sham control group. A repression inmitochondrial function and bioenergetics in a high energy-demanding organ, such as the heart, would determine anegative outcome. Along this line, aldehyde dehydrogenase(Aldh2) has been shown to play a major role incardioprotection and maintenance of contractile function inalcohol-induced LV hypertrophy and ischemia/reperfusioninjury.22,23 Therefore, reduced levels of this protein could bea critical factor contributing to impaired cardiac function andgreater dilation observed in male mice under PO conditions.On the other hand, a direct involvement of trifunctionalenzyme subunit beta (Hadhb) in the development of LVhypertrophy and the transition to heart failure has not beenreported. However, a mutation in this protein has been shownto cause cardiac fibrosis and hepatic steatosis.24 Consequently,further investigation of the role of trifunctional enzyme subunitbeta (Hadhb) could uncover novel mechanisms active in PO-induced hypertrophy.In WT female mice, on the other hand, proteins that might

confer cardioprotection seem to be activated, such ascytoskeletal and structural proteins, in response to PO. Inparticular, the levels of vinculin (Vcl) were induced in the TACgroup compared with the sham control group. Vinculin (Vcl) isan important protein of the cytoskeleton and is an actin-bindingprotein linking the contractile apparatus to the cardiomyocytesarcolemma. Mutations in this protein are the cause of dilatedcardiomyopathy in humans characterized by ventricular dilationand impaired systolic function,25 while cardiac deletion in miceleads to LV hypertrophy and early death by ventriculartachycardia.26 As a result, the increase in this protein’sabundance in WT female TAC mice strongly suggests a rolein cardioprotection and supports the hypothesis that femaleshave a better structural adaptation in PO, which is expected toslow down the transition to heart failure.Our previous functional characterization of BERKO male

mice in PO indicated that these mice are at higher risk with aworse prognosis than female mice.15 In the present study, wediscovered that there was an extensive decrease in the levels ofseveral myosin heavy chain isoforms in the BERKO male TACgroup compared with the sham control group. The importantrole of the myosin heavy chain in cardiac structure andcontractile function is well-known. Therefore, a decrease in thelevels of this protein strongly suggests an increasedsusceptibility of BERKO male mice to impairments in thefunctional and structural adaptation to PO and to the transitionto heart failure.On the other hand, although BERKO female mice have

worse function and outcome than WT female mice in PO, theydo not seem to have as pronounced effects as BERKO malemice.15 This points to partial cardioprotective mechanismspresent in females independent of ERβ. Along this line, wefound that the protein cofilin 2 (Cfl2) was induced in theBERKO female TAC group compared with the sham controlgroup. Cofilin 2 (Cfl2) is a cytoskeletal protein, which plays animportant role in the regulation of sarcomeric actin filamentsand, when mutated, causes an autosomal recessive form of

congenital myopathy with features of both nemaline andmyofibrillar myopathy.27 In addition, BERKO female miceinduced the levels of pyruvate kinase 2 (Pkm2) in response toPO. This protein is involved in glycolysis, ultimately generatingATP. A direct link between regulation of cofilin 2 (Cfl2) andpyruvate kinase 2 (Pkm2) and LV hypertrophy has not beenestablished. We put forward that the induction of these proteinsmight underlie a better structural and metabolic adaptation ofBERKO female mice to PO.There are several complex relationships between ERα and

ERβ, including actions of each that complement those of theother, antagonistic and synergistic.28,29 Due to the fact that wehave not studied mice lacking ERα, we cannot exclude indirectactions of ERα leading to the observed effects in the heart.Furthermore, several factors may influence the expression levelsof the ERs, including aging, gonadectomy, and aromataseinhibition.30−33 Although there were no major differences inERβ expression levels within the WT or within the BERKOgroups, we cannot exclude that different activation orrepression mechanisms of ER might be active between maleand female mice contributing to the differences observed here.Our sample size was rather small, which might have

compromised the identification of modulated spots with smallvariation in their intensities. In addition, 2-dimensional gelelectrophoresis is a highly efficient approach to detect variousintact proteins, as well as posttranslational modifications andsplice variants. However, proteins of less than 10 kDa andstrongly hydrophobic proteins, such as membrane proteins, arenot sufficiently resolved by 2-dimensional gel electrophoresis.The proteomic response of the heart to PO is highly

modulated by sex. The higher number of regulated proteins inBERKO mice suggests that the presence of ERβ is crucial forthe strict regulation of mechanisms active in the developmentof LV hypertrophy. In addition, the present analysis identifiedpreviously unrelated proteins to the development andprogression of PO-induced LV hypertrophy. We believe thatseveral of these regulated proteins deserve further functionalanalysis to identify their possible role in LV hypertrophy and todetermine whether their exploitation can have therapeuticpotential.

■ ASSOCIATED CONTENT

*S Supporting Information

Supplementary Tables 1, 2, 3, and 4: modulated protein spotsbetween sham and TAC in WT males, WT females, BERKOmales, and BERKO females, respectively. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author

*Tel: +49-30-450525355. Fax: +49-30-450525943. E-mail:georgios.kararigas@charite.de.

Present Address⊥D.F.: Bayer HealthCare Pharmaceuticals, Wuppertal, Ger-many.

Author Contributions

G.K. and D.F. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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■ ACKNOWLEDGMENTSWe thank K. Duft and J. Thomas for technical assistance. Thiswork was supported by the German Research Foundation(DFG) and by the DZHK (German Centre for CardiovascularResearch).

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