�-Catenin downregulation attenuates ischemiccardiac remodeling through enhanced residentprecursor cell differentiationLaura C. Zelarayana, Claudia Noacka,1, Belaid Sekkalib,1, Jana Kmecovab,1, Christina Gehrkec, Anke Rengera,Maria-Patapia Zafirioua, Roel van der Nageld, Rainer Dietzc, Leon J. de Windtd, Jean-Luc Balligandb,2,and Martin W. Bergmanna,c,2

cDepartment of Cardiology, Campus-Buch and Campus Virchow-Klinikum, Charite–Universitatsmedizin Berlin, Franz Volhard Klinik, Germany; aMax DelbruckCenter for Molecular Medicine, 13125 Berlin, Germany; bUnit of Pharmacology and Therapeutics, University of Louvain Medical School, 1200 Brussels, Belgium;and dDepartment of Medical Physiology, University Medical Center Utrecht, Royal Netherlands Academy of Sciences, 3524, Utrecht, The Netherlands

Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved September 30, 2008 (received for review August 28, 2008)

We analyzed the effect of conditional, �MHC-dependent genetic�-catenin depletion and stabilization on cardiac remodeling followingexperimental infarct. �-Catenin depletion significantly improved4-week survival and left ventricular (LV) function (fractional shorten-ing: CT�ex3–6: 24 � 1.9%; �-cat�ex3–6: 30.2 � 1.6%, P < 0.001).�-Catenin stabilization had opposite effects. No significant changes inadult cardiomyocyte survival or hypertrophy were observed in eithertransgenic line. Associated with the functional improvement, LV scarcellularity was altered: �-catenin-depleted mice showed a markedsubendocardial and subepicardial layer of small cTnTpos cardiomyo-cytes associated with increased expression of cardiac lineage markersTbx5 and GATA4. Using a Cre-dependent lacZ reporter gene, weidentified a noncardiomyocyte cell population affected by �MHC-driven gene recombination localized to these tissue compartments atbaseline. These cells were found to be cardiac progenitor cells sincethey coexpressed markers of proliferation (Ki67) and the cardiomy-ocyte lineage (�MHC, GATA4, Tbx5) but not cardiac Troponin T (cTnT).The cell population overlaps in part with both the previously de-scribed c-kitpos and stem cell antigen-1 (Sca-1)pos precursor cell pop-ulation but not with the Islet-1pos precursor cell pool. An in vitrococulture assay of highly enriched (>95%) Sca-1pos cardiac precursorcells from �-catenin-depleted mice compared to cells isolated fromcontrol littermate demonstrated increased differentiation toward�-actinpos and cTnTpos cardiomyocytes after 10 days (CT�ex3–6: 38.0 �1.0% �-actinpos; �-cat�ex3–6: 49.9 � 2.4% �-actinpos, P < 0.001). Weconclude that �-catenin depletion attenuates postinfarct LV remod-eling in part through increased differentiation of GATA4pos/Sca-1pos

resident cardiac progenitor cells.

Despite adaptive mechanisms including activation of cardiomy-ocyte survival pathways and hypertrophy, left ventricular (LV)

remodeling often progresses to cardiac dilation and heart failure(1). Recently, the quantitative contribution of endogenous cardiacregeneration was found to account for at least 25% of cardiomy-ocytes in the infarct border zone (2). However, essential charac-teristics of this cardiac precursor cell pool, like signaling pathwaysdirecting differentiation and/or proliferation, are largely unknown.

Transcription factors essential for embryonic cardiac develop-ment also affect adult cardiac remodeling in mice (3). Regulationof the Wnt/�-catenin pathway differentially regulates embryoniccardiac progenitor cells prespecification, renewal, and differentia-tion in the cardiac mesoderm (4–7). Activation of the Wnt/�-catenin pathway specifically stimulates Islet-1 cardiac progenitorcells proliferation while delaying differentiation. Conversely, in-creased expression of Wnt signaling inhibitors in �MHCpos cardiacprecursor cells isolated from embryoid bodies lead to increasedcardiomyocyte differentiation (8).

We previously reported that downregulation of �-catenin in adultheart is required for adaptive hypertrophy upon chronic angiotensinII challenge (9). Here, we describe the effect of �-catenin depletionon ischemic LV-remodeling. We used two transgenic mouse models

to study the effect of conditional depletion (�-cat�ex3–6) or stabili-zation (�-cat�ex3) of �-catenin upon �MHC-driven gene recombi-nation in the adult heart (9). To monitor recombined cells we usedthe ROSA26 (lacZ) reporter mice (10). Our analysis revealed aspecific subpopulation of cardiac progenitor cells including Sca-1pos

and c-kitpos cells to be affected by �MHC-dependent gene recom-bination. In association with the functional improvement afterinfarct in �-catenin-depleted mice, isolated Sca-1pos cardiac pre-cursor cells exhibited enhanced cell differentiation toward maturecardiomyocytes. In Vivo, early cardiac transcription factors GATA4and Tbx5 were upregulated upon �-catenin depletion in infarctedmice. These data suggest �-catenin depletion to be beneficial inpostinfarct LV remodeling in part through enhanced differentiationof �MHCpos cardiac resident progenitor cells.

Results�MHC-Restricted �-Catenin Depletion or Stabilization Plus lacZ Re-porter Gene Expression in Mice. Mice with conditional �-catenindepletion (�-cat�ex3–6) or stabilization (�-cat�ex3) depending on�MHC-CrePR1 recombination in adult myocardium were gener-ated as described before (9). Creneg littermate mice were used ascontrols and termed CT�ex3–6 or CT�ex3, respectively.

To monitor cells affected by �MHC-directed recombination,ROSA26 reporter mice were bred to �-cat�ex3–6 resulting in�-cat�ex3–6/lacZ mice expressing the lacZ gene in recombined cells.Cre-mediated conditional recombination was induced by mifepris-tone (RU-486) injection [Fig. S1A and see supporting information(SI) Text)]. Using the indicated primers (blue arrows in S1A),successful genomic recombination was confirmed (Fig. S1B). De-pletion of the full-length �-catenin protein and expression of the�-galactosidase (�-gal) reporter were detected by immunofluores-cence and Western blot in adult cardiomyocytes (Fig. S1C andS2A). Decreased expression of �-catenin (*, P � 0.05) and its targetgenes LEF1 (*, P � 0.05) and TCF4 (**, P � 0.01) was confirmedby quantitative real time-PCR 3 weeks after Cre induction(CT�ex3–6 n � 6; �-cat�ex3–6 n � 15; Fig. S1D).

The same strategy was used to obtain mice with �MHC-restricted�-catenin stabilization (9). Excision of exon 3 containing the

Author contributions: L.C.Z., J.-L.B., and M.W.B. designed research; L.C.Z., C.N., B.S., J.K.,C.G., A.R., M.-P.Z., R.v.d.N., and M.W.B. performed research; J.-L.B. and M.W.B. contributednew reagents/analytic tools; L.C.Z., C.N., R.D., L.J.d.W., J.-L.B., and M.W.B. analyzed data;and L.C.Z. and M.W.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1C.N., B.S., and J.K. contributed equally to this work.

2To whom correspondence may be addressed. E-mail: martin.bergmann@charite.de orballigand@mint.ucl.ac.be.

This article contains supporting information online at www.pnas.org/cgi/content/full/0808393105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

19762–19767 � PNAS � December 16, 2008 � vol. 105 � no. 50 www.pnas.org�cgi�doi�10.1073�pnas.0808393105

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GSK3� phosphorylation site blocks proteasome-mediated degra-dation and results in cytoplasmic accumulation of �-catenin (11).The truncated �-catenin product was detected by PCR and Westernblot (Fig. S1E and Fig. S2B), indicating successful recombination.The �-catenin target genes LEF1, TCF4, and Axin2 were upregu-lated as demonstrated by real-time PCR (data not shown) (9).

�-Catenin Depletion Attenuates Left Ventricular Remodeling AfterMyocardial Infarction. To determine whether �-catenin modulatesischemic cardiac remodeling, �-catenin depleted, stabilized, andrespective control littermates were subjected to chronic ligation ofthe left anterior descendant coronary artery (LAD) (Fig. S3A).Mice who died within 2 days of the surgery or had no clear infarctscar in echocardiography and histology were excluded from furtheranalysis (Fig. S3B and S4). At 4 weeks, �-catenin-depleted animalsexhibited improved fractional shortening (Fig. 1A, CT�ex3–6 n � 7:24.0 � 1.9% vs. �-cat�ex3–6 n � 13: 30.2 � 1.6% ***, P � 0.001) andreduced heart weight/body weight ratio (HW/BW) (CT�ex3–6: 6.1 �0.42 vs. �-cat�ex3–6: 5.3 � 0.21 *, P � 0.05) (Table S1). In addition,infarct size was significantly reduced in �-catenin-depleted mice vs.controls (CT�ex3–6 n � 6: 47.2 � 3.7% vs. �-cat�ex3–6 n � 6: 37.2 �2.1% *, P � 0.05) (Fig. 1A and Fig. S3C). �-Catenin depletion alsoled to reduced mRNA expression of heart failure markers ANP andBNP at two weeks (CT�ex3–6 n � 5; �-cat�ex3–6 n � 15, Fig. S6A).

Mice with �-catenin stabilization displayed no major differencescompared to controls in infarct size or LV function (Fig. S5). �-Catenindepletion significantly decreased mortality over the first 4 weeks afterexperimental myocardial infarction (CT�ex3–6 n � 11; �-cat�ex3–6 n � 15*P � 0.05), while �-catenin-stabilized mice showed a nonsignificantincrease of mortality (Fig. 1B). A prominent subendo- and subepicar-dial layer of cTnTpos cardiomyocytes was found in the scar upon�-catenin depletion, while controls showed a fibrotic scar with only fewcTnTpos cells (right panel, Fig. 1A). A similar fibrotic scar was observedin �-cat�ex3 mice and their respective controls (right panel, Fig. S5). Insummary, depletion of �-catenin in the adult heart attenuates ischemicLV remodeling and postinfarct mortality.

Improved Cardiac Function Upon �-Catenin Depletion Is Not Associ-ated With Changes in Adult Cardiomyocyte Hypertrophy or Apoptosis.As a molecular and cellular mechanism of attenuated LV remod-eling upon �-catenin depletion, we hypothesized adult cardiomy-ocyte hypertrophy and/or apoptosis to be altered. We investigated

hypertrophy and apoptosis 2 and 4 weeks after LAD ligation. Wefound no significant difference concerning the hypertrophy mark-ers �-skeletal-actin and �-MHC between �-cat�ex3–6 mice and theircontrols 2 weeks after LAD (Fig. S6A). At 4 weeks, neither genemarkers of cardiomyocyte hypertrophy nor echocardiographic sep-tal or free wall thickness as a measure of global LV hypertrophyshowed any significant differences (Table S1 and Fig. S4). Becausehypertrophy of the noninfarcted myocardium contributes to LVremodeling (1), myofiber area was measured in the remote zone.No significant change was detected in �-cat�ex3–6 mice (Fig. S6B) or�-cat�ex3 mice (data not shown) compared to their control litter-mates both at 2 and 4 weeks after infarct. In addition, we found noevidence for an altered rate of cardiomyocyte apoptosis as detectedby TUNEL assay (Fig. S6C). Therefore, we conclude that theattenuation of cardiac remodeling after ischemia upon �-catenindepletion is not mediated by inhibition of cardiac hypertrophyand/or decreased apoptosis of adult cardiomyocytes.

Resident Cardiac Progenitor Cells Are Targeted by �MHC-DependentGene Recombination. Since neither adult cardiomyocyte apoptosisnor hypertrophy explained the observed phenotype, we askedwhether other cardiac cell types, apart from mature cardiomyo-cytes, were affected by �MHC-dependent gene recombination. TheROSA26 reporter mice allowed for the identification of cellstargeted for Cre recombination through detection of �-gal expres-sion. We aimed to identify �-galpos cells using flow cytometry in acardiomyocyte-depleted cell fraction from adult heart and found�10% �-galpos cells. Next, we tested whether the �-galpos cells havestem cell characteristics and analyzed the coexpresssion of thecardiac progenitor cell marker Sca-1. Of the noncardiomyocytecells, 8.8 � 0.4% were detected to be Sca-1pos (Fig. S7 A–C). Morethan 80% of these Sca-1pos cells (6.6 � 0.8% cells of the totalnoncardiomyocyte cell population) were �-galpos in bothCT�ex3–6/lacZ and �-cat�ex3–6/lacZ. lacZ�MHC-Cre-neg littermate micewere used as negative controls for �-gal detection (Fig. 2A and datanot shown). Similarly, flow cytometry analysis of another heart-specific inducible Cre line, the �MHC-MerCreMer mice (12) matedto the ROSA26 reporter mice showed �60% of the Sca-1pos cellsto coexpress �-gal following Cre-induction (Fig. S8A). Immuno-fluorescence analysis of the noncardiomyocyte cell fraction provedcoexpression of the Sca-1 and c-kit epitope in a subpopulation of�-galpos cells. Moreover, �MHC protein expression was observed in

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Fig. 1. �MHC-dependent �-catenin depletion attenu-ates postinfarct LV remodeling. (A) �-Catenin depletionresults in improved fractional shortening and reducedinfarct size 4 weeks after infarct associated with a prom-inent subendocardial and subepicardial layer of cardio-myocytes as identified by cTnT staining. (B) Kaplan-Meiersurvival curve demonstrated significantly enhanced sur-vival in �-cat�ex3–6 mice compared to CT�ex3–6 mice 4weeks following experimental infarct. In contrast, in-creased mortality was observed in �-cat�ex3 mice com-pared to CT�ex3 mice. CT�ex3–6 n � 11; �-cat�ex3–6 n � 15;CT�ex3 n � 8; �-cat�ex3 n � 9; MI, myocardial infarct; LV,left ventricle; cTnT, cardiac Troponin T; *, P � 0.05.

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a subpopulation of Sca-1pos cells confirming the activation of theendogenous �MHC promoter and protein expression in the iden-tified cell population (Fig. 2B).

Using magnetic cell sorting (MACS) technology we enriched cardiacSca-1pos cells from �-catenin depleted, stabilized and their respectivecontrol littermates to �95% for further characterization. Independentfrom the genotypes, mRNA quantification showed coexpression ofc-kit. Other known precursor cell markers such as Islet-1 or Oct3/4 werenot coexpressed, suggesting different subsets of cardiac precursor cellsto be present in the adult heart. Sca-1pos cells from �-cat�ex3–6 and�-cat�ex3 showed both Cre recombinase and �MHC gene expressionconsistent with activation of the �MHC promoter in these cells (Fig.2C). Gene expression of the cardiac transcription factors GATA4,Tbx2, Tbx5, and Tbx20 was detected, confirming that these Sca-1pos/�-galpos cells in the noncardiomyocyte fraction are progenitor cells ofthe cardiac lineage (Fig. 2C, Fig. S8 D and E).

Aiming to visualize the identified cardiac progenitor cells in vivo,we analyzed heart sections from �-cat�ex3–6/lacZ and CT�ex3–6/lacZ

mice at baseline. Small intramyocardial �-gal-expressing cells weredetected by enzymatic lacZ reaction. In addition, immunoperoxi-dase detection identified endo- and epicardial �-galpos cells (arrows,Fig. 2D). Heart sections costained with �-gal and cTnT showed asubendo- and subepicardial layer of �-galpos/cTnTneg cells with alarge nucleus to cytoplasmic ratio as expected for cardiac precursorcells (Fig. 2E, see Fig. S9 for controls). Double stainings ofconsecutives slides confirmed �-galpos/Sca-1pos cells to be cTnTneg,GATA4pos, and Tbx5pos (arrows and numbers in Fig. S8 D and E,see S7 for Sca-1 control staining). In conclusion, we identified apopulation of �MHCpos/Sca-1pos/c-kitpos/GATA4pos/cTnTneg car-diac progenitor cells in an endocardial and epicardial compartment.

Cardiac Progenitor Cell Proliferation and Distribution Following Ex-perimental Infarct. We demonstrated �MHCpos/Sca-1pos/cTnTneg

cardiac precursor cells to be targeted for �-catenin depletion. As ahallmark of cardiac precursor cells, a BrdU/Sca-1 and Sca-1pos/Ki67pos double staining identified proliferating Sca-1pos cells.�-Catenin depletion did not affect the number of proliferatingSca-1pos cells (Fig. 3A and Fig. S10B). While these data provideevidence for self-renewal of cardiac resident Sca-1pos precursor cellsno evidence was found that differences in proliferation rate explainsthe observed functional phenotype upon �-catenin depletion.

Next, we asked whether altered migration might contribute to LVremodeling upon �-catenin depletion. We investigated the distri-bution of Sca-1pos/GATA4pos/cTnTneg cardiac progenitor cells usingconfocal microscopy in heart sections 2 and 4 weeks after ischemia.At 2 weeks the distribution of �-galpos/Sca-1pos/GATA4pos/cTnTneg

progenitor cells and a semiquantification of the Sca-1 cells in thescar vs. remote zone showed no major difference between�-cat�ex3–6 and controls (representative pictures in Fig. S10A anddata not shown). A prominent layer of GATA4pos/cTnTneg cells wasdetected along the scar at 4 weeks (white arrows in Fig. 3 B and C).Additionally, a few small GATA4pos/cTnTpos cells were localized inthe ischemic region of �-cat�ex3–6 and control littermates in the prox-imity (subendo- and subepicardium) of the compartment where theSca-1pos/�-galpos cardiac precursor cells were observed initially (endo-and epicardium) (red arrows in Fig. 3 B and C). In summary, we foundno evidence that altered migration of cardiac progenitor cells wouldexplain the observed functional phenotype or the more prominent layerof cTnTpos cells in the scar of �-cat�ex3–6 mice.

�-Catenin Depletion Enhances Cardiac Progenitor Cell DifferentiationAfter Ischemia. We next asked whether depleting �-catenin altersdifferentiation of cardiac-resident progenitor cells toward cTnTpos

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Fig. 2. Activation of the �MHC-promoter in cardiac progenitor cells. (A) Flow cytometry analysis reveals �80% of the noncardiomyocytes to coexpress �-galand Sca-1 at baseline. Creneg mice were used as negative control. IgG-rabbit-APC and IgG-FITC antibodies were used as a control to discard unspecific staining.(B) Representative immunofluorescence picture of �-galpos cells from the cardiomyocyte depleted cell fraction proving coexpression of the Sca-1 and c-kit epitope.A subpopulation of cells coexpresses �MHC and Sca-1. (C) Analysis of Sca-1 cells gene expression by quantitative real-time PCR documenting expression of �MHC,c-kit, and cardiac transcription markers (GATA4, Tbx5). (D and E) Immunohistochemistry of adult heart from lacZ�MHC-Cre mice. Activation of the �MHC promoterwas analyzed by �-gal reporter gene expression. Small cells, with a high nuclei/cytoplasm ratio, positive for �-gal were detected by immunoperoxidase, enzymaticlacZ detection, and fluorescence staining in endo-, epi-, and myocardium.

19764 � www.pnas.org�cgi�doi�10.1073�pnas.0808393105 Zelarayan et al.

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cardiomyocytes. Flow cytometry analysis of the noncardiomyocytecell fraction revealed the fraction of Sca-1pos cells coexpressingGATA4 to significantly increase in �-cat�ex3–6 mice 4 weeks afterinfarct compared to �-cat�ex3–6 mice at baseline and control miceafter infarct (Fig. 4A and S8 B and C). This increased expression ofcardiac differentiation markers was accompanied by a decrease oftotal Sca-1pos cells in �-cat�ex3–6 mice vs. baseline. Control animalsdid not decrease the Sca-1pos cell population after infarct (Fig. 4B).

If progenitor cell differentiation contributes to stabilize the scar, earlycTnTpos-expressing cells with smaller cell size than mature cardiomy-ocytes should be detectable. Therefore, we analyzed the scar cellularity4 weeks after ischemia. �-Catenin-depleted mice showed a significantincrease of GATA4pos/cTnTpos cells (white circle in Fig. 4C) with �16�m2 surface area (matched to DAPIpos cell number). In contrast, micewith stabilized �-catenin did not show any significant difference incomparison to their respective controls (Fig. 4C).

To confirm the hypothesis of enhanced precursor cell differen-tiation upon �-catenin depletion, we performed an in vitro differ-entiation assay. Isolated Sca-1 cells from �-catenin depleted, sta-bilized, and respective control littermates were enriched by MACSto �95% purity and labeled with the cell tracer CM-Dil. The cellswere cocultured with neonatal mouse (FVB WT strain) cardiomy-ocytes for 10 days and the number of double �-sarcomeric actin(�-sr ac)pos/CM-Dilpos cells was calculated in relation to the totalCM-Dilpos cells. Sca-1pos cells isolated from �-cat�ex3–6 mice showedsignificantly increased differentiation capacity in comparison tocells isolated from controls (CT�ex3–6 n � 10: 38 � 1.0% of �-sracpos � CM-Dilpos/total CM-Dilpos; �-cat�ex3–6 n � 10: 49.9 � 2.4%**, P � 0.001, Fig. 4D). Similar to the in vivo situation, we detectedan increased percentage of differentiated GATA4pos/cTnTpos cellsin �-catenin-depleted cells compared to control cells (CT�ex3–6:21.2 � 1.5% vs. �-cat�ex3–6: 34.0 � 1.6% **, P � 0.001, Fig. 4E). Incontrast, Sca-1pos cells isolated from �-catenin-stabilized miceexhibited decreased differentiation capacity (CT�ex3 n � 4: 49.1% �4.8 of �-sr acpos � CM-Dilpos/total CM-Dilpos; �-cat�ex3 n � 6:38.3 � 2.1% *, P � 0.05, Fig. 4D) and decreased differentiatedGATA4pos/cTnTpos cells (CT�ex3: 26.7 � 0.9% vs. �-cat�ex3: 18.3 �0.7% **, P � 0.001, Fig. 4E). Collectively, these data indicate thatfollowing chronic LAD ligation, �-catenin depletion enhancesresident endogenous Sca-1pos cardiac progenitor cell differentiationtoward GATA4pos/cTnTpos/�-sr acpos-expressing cells (scheme inFig. 4G). Enhancing endogenous repair mechanisms contributes toglobal LV remodeling including infarct size extension (1).

Reexpression of Cardiac Developmental Transcription Factors in Isch-emic Adult Heart. During embryogenesis, the cardiac mesodermactivates several transcriptional regulators of the cardiac programincluding GATA4 and members of the T-box family necessary for

ventricular cardiac differentiation in response to inductive signals(13). Tbx5 is specifically expressed in the first heart field, which givesrise to left ventricular cardiomyocytes (14). Therefore, we studiedTbx5 and GATA4 expression following infarct via quantitativereal-time PCR using heart samples obtained from the apex con-taining scar tissue, border zone, and remote area. Four weeks afterinfarct, expression of Tbx2, Tbx5, and GATA4 was significantlyupregulated in �-cat�ex3–6 mice in comparison to controls andTbx20 was downregulated. Gene regulation in heart samples frommice with �-catenin stabilization showed the opposite results (Fig.4F). These data suggest �-catenin depletion in the adult myocar-dium to induce cardiomyocyte differentiation similar to the em-bryonic formation of the left ventricle.

DiscussionOur study describes a resident cardiac progenitor cell populationexhibiting �MHC-promoter activity and expression of cardiactranscription factors GATA4 and Tbx5 as specific markers for thefirst heart field (FHF) giving rise to the left ventricle duringembryonic cardiac development. Enhancing signaling cascadesorchestrating LV embryonic cardiomyocyte differentiation, namelydepletion of �-catenin, positively affects global LV function andsurvival at 4 weeks. We suggest enhanced cardiomyocyte differen-tiation of endogenous cardiac resident stem cells to mediate thiseffect by limiting secondary infarct expansion.

�-Catenin Depletion Attenuates Postischemic Mortality. Infarct mor-tality was ameliorated by �-catenin depletion already during thefirst 2 weeks post infarct. Similarly, ubiquitous overexpression of theWnt signaling antagonist frizzledA, which prevents �-catenin ac-cumulation after infarct, resulted in reduced mortality between 2and 5 days after permanent LAD ligation because of reducedcardiac rupture (15). Fatal arrhythmias resulting from ‘‘vulnerable’’myocardium might be reduced through the effects of �-catenin oncellular scar composition. In association with the improved LVfunction following infarct, we found an increased number ofcTnTpos-expressing cells with small cell size upon �-catenin deple-tion. These data suggest that despite the dramatic size differencebetween the small-identified cTnTpos cells in the scar and the adultcardiomyocytes, these cells might positively affect secondary scar ex-pansion (16) and therefore affect ventricular wall stability early on.

Cardiomyocyte Hypertrophy and Apoptosis Upon �-Catenin Deple-tion. Four weeks after chronic LAD ligation mice with �-catenindepletion exhibited a complete lining of cTnTpos cells along thescar subendocardium and subepicardium, while control animalsshowed only scattered cTnTpos cells. One explanation is thatthese cells might have survived the initial hypoxia through

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Fig. 3. Proliferation and tissue distribution of �-galpos/GATA4pos/cTnTneg cardiac progenitor cells after myocardial infarct. (A) Quantification of Sca-1pos/Ki67pos

cell proliferation in a cardiomyocyte-depleted cell fraction by flow cytometry shows no significant differences between CT�ex3–6 and �-cat�ex3–6 mice neither atbaseline nor after ischemia. Immunofluorescence shows colocalization of Sca-1pos/BrdUpos and Sca-1pos/Ki67pos cells 2 weeks after infarct proving the proliferativecapacity of these cells. (B and C) �-Catenin depleted �-cat�ex3–6 mice exhibit a layer of cTnTpos cardiomyocytes along the scar, which appears less prominent incontrol mice (B and C, Upper Right). The subendocardial and subepicardial layer of the infarct scar is populated with small GATA4pos/cTnTneg cells (white arrows).In addition, small GATA4pos/cTnTpos cells are detected within the scar area of both �-cat�ex3–6 and control mice (red arrows in B and C, Lower Left). No significantdifference in tissue distribution is detected. LV, left ventricle; MI, myocardial infarct.

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increased expression of survival genes. However, we have notobserved significant differences in the TUNEL analysis. Asecond explanation is that �-catenin influences proliferation but�-catenin depletion did not alter proliferation as quantified byanalysis of Sca-1pos/Ki67pos cells. Accordingly, �-catenin deple-tion in the early mesoderm decreased proliferation of cardiacprecursor cells in the embryonic FHF (17). Upregulation of�-catenin enhances expansion of Islet-1 cardiac stem cells (7,18). Lastly, hypertrophy of surviving cardiomyocytes was dem-onstrated to be unaffected upon �-catenin depletion. Thus, noneof the cellular mechanisms involving �MHC-dependent gene

recombination in adult cardiomyocytes sufficiently explains thephenotype observed here.

Identification of �MHCpos Cardiac Resident Precursor Cells. Employ-ing the ROSA26 reporter mice in which the expression of the lacZgene depends on Cre activation, we documented �MHC-dependentgene recombination, mRNA, and protein expression in a cTnTneg

cell population. Cardiac stem cells were previously shown to express�MHC during early cardiac developmental stages in embryoidbodies in vitro (8). As the adult cardiac cell population underinvestigation here expresses markers of cell proliferation (Ki67,

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Fig. 4. �-Catenin depletion enhances cardiomyocyte differentiation of Sca-1pos precursor cells following experimental infarct. (A) Significant increase of the Sca-1pos/GATA4pos cell fraction in �-cat�ex3–6 mice 4 weeks after infarct vs. baseline and sham mice, which is not observed in CT�ex3–6 animals. It was accompanied by a nonsignificantdecrease of total ScaIpos cells in �-cat�ex3–6 mice while control mice tend to upregulate the total Sca-1 cells (B). (C) In Vivo quantification of GATA4pos/cTnTpos cells in the scarwithacellarea�16�m2 showsasignificant increaseof thesecells in�-cat�ex3–6 micecomparedtocontrols.Arepresentativeconfocal image is shown(whitecircle). (D) InVitroincreaseddifferentiationofisolatedSca-1poscellstoward�-sracposcardiomyocytesfrom�-cat�ex3–6micecomparedtoisolatedSca-1cellsfromCT�ex3–6mice.Theoppositeeffectwas observed in �-catenin-stabilized mice. A representative picture of a 10-day CM-Dil-labeled coculture stained with �-sr ac is shown. (E) Similarly, in vitro differentiationof isolated Sca-1 cells toward GATA4pos/cTnTpos cells increases in �-cat�ex3–6 compared to cells from CT�ex3–6 mice. The same cell population is decreased in �-cat�ex3 mice. (F)Quantitative real-time PCR of heart samples 4 weeks after infarct shows an increased expression of markers for ventricular cardiomyocyte differentiation Tbx5, Tbx2, andGATA4in�-cat�ex3–6micecomparedtoCT�ex3–6.Reciprocalregulationwasobservedin�-cat�ex3miceincomparisontoCT�ex3mice.(G)Hypotheticalschemeof�MHCposcardiacprecursor cells differentiation in vivo triggered by ischemia and amplified by �-catenin depletion. *, P � 0.05; **, P � 0.001.

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BrdU) and cardiomyocyte lineage markers (GATA4, Tbx5) itfulfills the criteria of a cardiac endogenous precursor cell. Itoverlaps in part with the Sca-1 and c-kit positive cardiac stem cellpopulation; however, the cells are negative for Islet-1 and Oct3/4.

Two distinct mesodermal populations participate in the formation ofthe heart. The earliest progenitor cell population corresponds to theFHF, which expresses Tbx5 during differentiation and gives rise to theLV (14). The second population identified by Islet-1 expression corre-sponds to the second heart field (SHF) from which right ventricle, atria,andoutflowtractwill arise (17,19–21).Our findingsof thecoexpressionof �MHC and specific cardiac transcription factors like GATA4, andTbx5 characteristic for the LV, suggest the existence of an �MHCpos/cTnTneg cardiac committed FHF stem cell population in the adult heart.This population was identified in the endo- and epicardial compartmentresembling the localization of tissue-resident cardiac precursors alreadydescribed in zebrafish (22). Similarly, subepicardial and subendocar-dial cardiac precursor cells have been identified in mammaliancardiac development (23, 24).

�-Catenin Downregulation Improves Cardiac Function After Experi-mental Infarct. Previous studies suggest a negative role for Wnt/�-catenin during cardiac differentiation (7, 25, 26). Active �-cateninsignaling supports stemness and delays differentiation of cardiac pre-cursors in vivo and in vitro (27). Gene expression analysis from differ-entiating �MHCpos embryonic stem-derived cells in comparison tonondifferentiating cells showed upregulation of negative regulators ofthe Wnt signaling (8). We identified a subpopulation of cardiac residentprogenitors committed to the cardiac lineage to be targeted for �-cate-nin depletion, which results in enhanced differentiation. Similar obser-vations have been described for Islet-1pos cardiac precursors: differen-tiation decreased upon �-catenin stabilization while proliferation of theundifferentiated cells is enhanced (7). Moreover, upregulation of Tbx5and GATA4 gene expression was observed after ischemia, suggestinga reactivation of the LV cell differentiation program in adult heart inadaptation to injury.

We suggest that endogenous cardiac regeneration contributes toLV remodeling following chronic ischemia through differentiationof resident precursor cells, amplified by �-catenin downregulation.Similarly, increased differentiation of Sca-1pos resident cardiacprecursor cells was observed upon upregulation of FGF2. Deple-tion of FGF2 was found to worsen LV remodeling by enhancingsecondary infarct expansion (7, 25, 26). We propose limited sec-ondary infarct expansion to contribute to the improved cardiacfunction after infarct in our study.

We cannot exclude the participation of other precursor cell popu-lations or cellular mechanisms. However, the data suggest endogenouscardiac regeneration to significantly contribute to adult cardiac remod-elinguponstress inaddition toothermechanismspreviously recognizedas adult cardiomyocyte apoptosis or hypertrophy.

Materials and MethodsMouse Strains. The different mouse lines, �-cat�ex3–6, �-cat�ex3, and the ROSA26reporter mice (Jackson Laboratory) were described before (9, 28). Mice were bredinamixedC57BL/6�FVBbackground(�-cat �ex3–6)orFVBbackground(�-cat�ex3).All of the procedures involving animals were approved by the Berlin animalreview board (Reg. 0135/01 and Reg. 0338/05).

Flow Cytometry. Mice hearts were dissected and a myocyte-depleted populationwas prepared by mincing, homogenizing, and filtering the myocardium subse-quently through 40-�m (BDFalcon) and 30-�m mesh (Miltenyi Biotec). Isolatedcells were labeled with Sca-1-FITC (eBioscience); GATA4 (Santa Cruz); Ki67 (Ab-cam) and �-gal (MP Biomedicals) antibodies. The �MHC-specific antibody waskindly provided by J. Robbins, Cincinnati. Isotype controls were used in eachexperiment. Each mouse had 104 cells subjected to flow cytometry. Cell fluores-cence signals were detected with a FACScalibur flow cytometer (Becton Dickinson).

Differentiation Assay. Sca-1pos cells were enzymatically dissociated as describedpreviously (4) from adult murine heart. Cell suspension was filtered and incu-bated with FITC-conjugated anti-Sca-1 antibody (20 min at �4 °C). Next, cellswere incubated with anti-FITC microbeads (30 min at �4 °C). Sca-1pos cells werepurified by two cycles of MACS column (Miltenyi). The purity was confirmed byflow cytometry (�95%). The cells were labeled with the CM-Dil cell tracer (Mo-lecular Probes) and cocultured with neonatal cardiomyocytes in fibronectin-coated chamber slides at a ratio of 1⁄4 in DMEM/10%FBS/antibiotics (37 °C, 5%CO2). After 10 days, cocultures were fixed with 4% PFA and stained. Cells werequantified under fluorescence microscope.

Statistical Analysis. Statistical analyses were performed using unpaired, two-tailed Student’s t or ANOVA tests followed by posthoc analysis employing theBonferroni tests. Data are reported as mean �SEM. P � 0.05 values were consid-ered significant.

For more detail and primers see SI Materials and Methods.

ACKNOWLEDGMENTS. The authors thank Barbel Pohl and Martin Taube [MaxDelbruck Center (MDC)] for technical assistance. This study was funded in part bythe Deutsche Forschungsgemeinschaft grant BE 8–1 and 8–2 and Juergen Man-chot Foundation (to M.W.B.); by the Ernst und Berta Grimmke Foundation (toL.C.Z.); by the Foundation J. Leducq and the FP6-I.P ‘‘EuGeneHeart’’ (UE LSHM-CT05–018833) (to J.L.B.). We thank M.D. Schneider (Imperial College, London) forproviding the �MHC-CrePR1 mice, W. Birchmeier (MDC, Berlin) for providing the�-catflox/floxex3–6, M. Taketo (Kyoto, Japan) for providing �-catflox/floxex3, and J.Molkentin (Cincinnati) for providing �MHC-MerCreMer mice. We especiallythank J. Robbins (Cincinnati) for providing the specific �MHC antibody.

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