Estrogen receptor α supports cardiomyocytes indirectly through post-infarct cardiac c-kit+ cells

Journal of Molecular and Cellular Cardiology 47 (2009) 66–75

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Journal of Molecular and Cellular Cardiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y j m c c

Original article

Estrogen receptor α supports cardiomyocytes indirectly through post-infarct cardiac c-kit+ cells Marie Brinckmann a,1, Elena Kaschina a,1, Wassim Altarche-Xifró a, Caterina Curato a, Melanie Timm a, Aleksandra Grzesiak a, Jun Dong b, Kai Kappert a, Ulrich Kintscher a, Thomas Unger a, Jun Li a,⁎ a b

Center for Cardiovascular Research (CCR) and Institute of Pharmacology, Charité - Universitätsmedizin Berlin, Hessische Str. 3-4, 10115 Berlin, Germany German Rheumatism Research Centre, Clinical Immunology, 10117 Berlin; Germany

a r t i c l e

i n f o

Article history: Received 8 September 2008 Received in revised form 10 March 2009 Accepted 16 March 2009 Available online 31 March 2009 Keywords: Estrogen receptor Cardiac c-kit+ cell Myocardial infarction Cardiomyocyte Cardioprotection

a b s t r a c t Despite previous studies demonstrating a cardioprotective role of estradiol via its estrogen receptor (ER)α, the underlying mechanisms remain unclear. Here we aimed to define ERα-involved mechanisms against cardiac injury. Seven days after myocardial infarction in male rats, cardiac ERα was upregulated in postinfarct cardiac c-kit+ cells accumulating in periinfarct myocardium as shown by Western blotting and immunofluorescence staining. Further, we isolated post-infarct cardiac c-kit+ cell population by modified magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS), and confirmed predominant ERα expression in this post-infarct cardiac c-kit+ cell population by real-time PCR. These postinfarct cardiac c-kit+ cells, characterized by upregulated transcription factors implicated in cardiogenic differentiation (GATA-4, Notch-2) and genes required for self-renewal (Tbx3, Akt), maintained a stable phenotype in vitro for more than 3 months. ERα stimulation supported proliferation but prevented differentiation of undifferentiated myoblast cells. When adult myocytes isolated from infarcted rat hearts were co-cultured with post-infarct cardiac c-kit+ cells, ERα stimulation inhibited apoptosis and enhanced survival of these myocytes. These findings suggest that cardiac ERα supports survival of cardiomyocytes through post-infarct cardiac c-kit+ cells, which may contribute to cardioprotection against cardiac injury. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Estrogen is produced in both females and males, and appreciated as pleitotropic hormone that alters target gene transcription in both reproductive and nonreproductive tissues including the cardiovascular system upon binding nuclear estrogen receptors (ER), ERα and ERβ [1]. In addition to its classical nuclear actions, estrogen can also induce the survival and proliferation of multiple cells that express only a membrane-localized ERα [2]. Despite similar domain structure of these ER, the low homology in the N-terminal transactivation domain raises the possibility that estrogen may play a differential role through ERα or ERβ specific mechanisms. Indeed, the estrogen effects in blood vessels and myocardium depend on the relative abundance of ERα and ERβ. For example, in a murine model of carotid artery injury, estradiol mediates reendothelization in an ERα dependent manner [3]. By contrast, endothelial expression of ERβ but not ERα is induced in response to aortic balloon injury in rats [4], and coronary expression of ERβ is correlated with calcification and atherosclerosis in female patients [5]. ⁎ Corresponding author. Tel.: +49 30 450 525 294; fax: +49 30 450 525 901. E-mail address: [email protected] (J. Li). 1 Both authors contributed equally to this work. 0022-2828/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2009.03.014

Although the biological effects of estrogen are less well defined in males than in females, a growing body of clinical evidence demonstrates that endogenous estrogen (17β-estradiol) may protect cardiovascular health not only in female but also in male patients [6–8]. For instance, higher serum estrogen levels are associated with a lower risk for cardiovascular disease events in older men [8]. Interest in a potential role of ERα in cardiac adaptive mechanisms has been aroused by the observation that both ERα and ERβ have been detected in the heart [9] but only ERα mRNA is elevated in both female and male patients with heart failure [10]. Further clinical evidence revealed that a genetic variation in ERα is associated with increased risk of myocardial infarction in male patients [11]. Given the fact that ERα, but not ERβ, is required for estrogen-mediated cardioprotection in rodent models [12], it seems that ERα may account for cardioprotective actions in males [13], albeit the underlying cellular mechanisms remain largely unknown. Accumulating experimental and clinical evidence indicates that intramyocardial implantation of hematopoietic undifferentiated cells, which express antigens commonly found in bone marrow (BM) precursor cells, like c-kit, leads to restoration of cardiac function [14,15], albeit the underlying mechanisms involving cell fusion and transdifferentiation are still debated [14,16,17]. The discovery of adult c-kit+ precursor cells in rodent and human heart

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has sparked intense hope for myocardial regeneration with cells that are from the heart itself and are thereby inherently programmed to reconstitute the damaged myocardium [14]. Recently, experimental evidence reveals that the improvement in cardiac function is associated with cardiac release of angiogenic cytokines and neovascularization [18] independent of myocardial regeneration, challenging the direct transdifferentiation of c-kit+ precursor cells to cardiomyocytes [18,19]. Given the recent observations that estradiol exerts cardioprotective effects via augmented mobilization of BM-derived endothelial progenitor cells and enhanced angiogenesis [20,21], it is also possible that ER may be involved in regulating cardiac performance via c-kit+ precursor cells. To understand the potential adaptive mechanisms underlying ERα-mediated cardioprotection, we examined the regulation of ER in post-infarct cardiac c-kit+ cells and their functional relevance in response to acute ischemic injury in male rats. 2. Methods 2.1. Induction of myocardial infarction in rats Myocardial infarction (MI) was induced in male Wistar rats (Harlan Winkelmann GmbH) by permanent ligation of left anterior descending coronary artery. Briefly, rats were anaesthetized with ketamin/xylazine (Sigma-Aldrich) 80 mg/10 mg/kg i.p., intubated and ventilated with a Starling Ideal Ventilator (Harvard Apparatus). After thoracotomy, a suture was tightened around the proximal left anterior descending coronary artery. Sham-operated rats underwent the same surgical procedure without coronary ligature. Transthoracic Doppler echocardiography (VisualSonics 770) was performed 24 h after operation to verify the presence of MI (Supplementary Fig. 3). To label proliferating cells, animals were treated with BrdU (Sigma-Aldrich) 30 mg/kg i.p., on days 2, 4 and 6 post MI. Animal protocols followed the German law on animal protection. 2.2. Ex vivo cardiac cell isolation and FACS analysis Seven days after MI or sham operation, animals were perfused with phosphate buffered saline (PBS) under anaesthesia to remove

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blood from the heart. Cardiac cell populations were isolated using a Worthington neonatal cardiomyocyte isolation system. Briefly, after digestion with trypsin and collagenase, the tissue fragments were dispersed into a single-cell suspension. Density gradient sedimentation was followed. The top band (myocytes) was collected and middle layers were re-spun to pellet down the small cells. PBS applied during staining and cell sorting procedures was supplemented with 0.5% bovine serum albumin and 2 mM EDTA. Cell pellets were incubated with a rabbit anti-c-kit Ab (1:80; Cat. sc-5535; Lot. J3106; Santa Cruz). After washing, cell pellets were incubated with phycoerythrin (PE)conjugated anti-rabbit secondary Ab (1:200; Cat. 111-116-152; Lot. 67831; Jackson ImmunoResearch), and then with anti-PE microBeads (1:5; Cat. 120-000-294; Lot. 5070605166; Miltenyi Biotec). Post-infarct cardiac c-kit+ cells were positively selected using the MACS system (Miltenyi Biotec). A small fraction of single-cell suspension was labeled with fluorescein isothiocyanate (FITC)-conjugated anti-CD45 (1:40; Cat. R32135F; Lot. E0846AF; EuroBioSciences), or mouse antiCD34 (1:40; Cat. sc-7324; Lot. B2807; Santa Cruz) in combination with allophycocyanin (APC)-conjugated anti-mouse (1:40; Cat. 550826, Lot. 51888; BD Biosciences) Ab, and subjected to flow cytometry. MACS enriched c-kit+ cells were further sorted by a FACSDiva™ (Becton Dickinson). The purity of post-infarct cardiac c-kit+ cells was determined by a FACScalibur™ using CELLQuest™ (Becton Dickinson) or Flowjo (Tree Star) Software. Highly purified post-infarct cardiac c-kit+ cells were maintained and further characterized in vitro. 2.3. Immunofluorescence staining Heart cryosections (5 μm or 10 μm) were incubated with 10% donkey serum for 30 min, followed by mouse anti-ERα (1:100; Cat. sc787; Lot. G1906; Santa Cruz) and goat anti-mouse Sca-1 (1:100; Cat. AF1226; Lot. ICJ01; R&D Systems) or rabbit anti-c-kit (1:150; Cat. sc5535; Lot. J3106; Santa Cruz) polyclonal Abs for overnight at 4 °C. Sections were then incubated with donkey Cy-3 anti-mouse (1:300; Cat. 205-165-108; Lot. 74775, Jackson ImmunoResearch) and FITC anti-goat (1:150; Cat. 715-096-150; Lot. 71204, Jackson ImmunoResearch) or FITC anti-rabbit (1:150; Cat. 711-095-152; Lot. 64654, Jackson ImmunoResearch) for 40 min. Sections were counterstained with DAPI (1:1000; Cat. D21490; Lot. 42240A; Molecular Probes) for 10 min. The sections were also incubated with mouse anti-BrdU (1:300; Cat. 8434; Lot. 057K4859; Sigma-Aldrich), rabbit anti-ERα

Fig. 1. Increased ERα but unaltered ERβ expression after myocardial infarction (MI). The infarct zone was distinguished from non-infarct myocardium surrounding it by its distinctive pale coloration. The periinfarct myocardium consisting of normal appearing myocardium surrounding the infarct zone was dissected from the left ventricle for protein preparation. Immunoblot analysis of cardiac ERα and ERβ proteins was performed 7 days after MI. Densitometric analysis was performed. n = 7, ⁎P b 0.05 vs. sham operation.

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(1:100; Cat. sc-542; Lot. C0507; Santa Cruz), indirectly labeled to aminomethylcoumarin (AMCA) anti-mouse IgG (1:150; Cat. 715-155150; Lot. 71129; Jackson ImmunoResearch) and Cy-3 anti-rabbit (1:300; Cat. 711-165-152; Lot. 65517, Jackson ImmunoResearch) respectively. For multiple immunofluorescence staining performed in a cell culture plate, mouse anti-α-sarcomeric actin (1:400; Cat. A2172; Lot. 076K4840; Sigma-Aldrich) and rabbit anti-ERα (1:100) were used. To exclude any cross-reactivity, matched negative controls including donkey FITC anti-goat or Cy-3 anti-rabbit or AMCA/Cy-3 anti-mouse IgG were used for background determination on serial sections from each specimen. Stained sections were examined under Leica DMIRE2 (Wetzlar, Germany). For primary cultured cells, they were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.3% Triton X-100 for 5 min on glass coverslips, followed by the same staining procedure. 2.4. Real-time PCR Cardiac tissues or cell pellets were homogenized. After preparing total RNA and first strand cDNA synthesis, PCR amplification was performed using ABI PRISM® 7000 Sequence Detection System. The primers and probes with fluorescent dye and quencher as listed in Supplementary Table 1 were selected to be intron spanning and synthesized by TIB MOLBIOL (Berlin, Germany). Relative level for each gene was calculated using the standard curve method. Expression levels were normalized according to expression of β-actin housekeeping gene. 2.5. Immunoblotting The infarct zone was distinguished from the non-infarct myocardium surrounding it by its distinctive pale coloration. The periinfarct myocardium consisting of normal appearing myocardium surrounding the infarct zone was dissected from the left ventricle for protein preparation. Myocardial samples were homogenized in lysis buffer (50 mM Tris–HCl, 500 mM EDTA, 150 mM NaCl, 0.1% Triton X100) supplemented with 100 μg/ml PMSF and protease-inhibitorcocktail (Roche) followed by centrifugation. Protein concentrations were determined by the method of Bradford using BSA as a standard. 10 mg of total protein per lane was separated on SDS-PAGE and blotted. Equal amount of protein loading was verified by staining the gel and the membrane with Coomassie brilliant blue and Ponceau S, respectively. The membrane was incubated with mouse anti-ERα/ rabbit anti-ERβ polyclonal Abs (1:100, Santa Cruz), followed by horseradish peroxidase-conjugated secondary Abs (1:200; DAKO). Target protein was detected by enhanced chemiluminescence detection system (Amersham Pharmacia). Protein levels were normalized according to the level of GAPDH housekeeping protein. Densitometric analysis of the relevant band was performed and the relative protein level was expressed as arbitrary unit. 2.6. Differentiation of H9c2 myoblast cells H9c2 cells, derived from embryonic rat ventricle, were obtained from the American Type Culture Collection (Manassas, USA) and used for studying the functional roles of ERα activation in self-renewal and differentiation. Permanent H9c2 myoblast cells are characterized by their self-renewal and cardiac differentiation potentials. In addition,

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throughout the myogenic differentiation process, ERα is abundantly expressed in H9c2 myoblast cells. To prevent loss of differentiation potential, H9c2 cells were not allowed to reach confluence at any time. Cells were maintained in undifferentiated myoblast state in DMEM supplemented with 0.2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FBS. After starvation in serum-free medium for 24 h, cells were exposed to 1 nM estradiol (E2; Sigma-Aldrich) or 10 nM ERα selective agonist propyl pyrazole triol (PPT; Tocris) or 10 nM ERβ selective agonist diarylpropionitrile (DPN; Tocris) for 6 days, and cells were then harvested for respective RNA analysis. 2.7. Cell proliferation assay To confirm the undifferentiated state of myoblast cells, alkaline phosphatase staining (Roche) was conducted. Undifferentiated myoblast cells (10,000 cells/well), which maintained alkaline phosphatase activity, were seeded in 96-well plates. Upon exposure to 1 nM E2 or 10 nM PPT or 10 nM DPN for 72 h, the proliferation of undifferentiated myoblasts was assessed. Calcein AM assay (Invitrogen) was combined to identify viable cells, which were distinguished by their intracellular esterase activity, determined by the enzymatic conversion of the nonfluorescent calcein-AM to fluorescent calcein (green). The numbers of viable undifferentiated myoblast cells in each well were directly counted under Leica DMIRE2 with a hemocytometer. Additionally, post-infarct cardiac c-kit+ or c-kit− cells in complete DMEM/F12 medium (PAN Biotech) containing 0.1 ng/ml LIF (Chemicon), 100 U/ml penicillin and 100 μg/ml streptomycin (GIBCO) were treated with 10 nM PPT for 24 h. The cells were then washed and incubated in fresh complete medium for 24 h. At the end of incubation, conditioned media were collected from post-infarct cardiac c-kit+ or c-kit− cells. Cell proliferation was evaluated after exposure of myoblast cells to conditioned media for 24 h. 2.8. Co-culture system of post-infarct cardiac c-kit+ cells and adult myocytes Post-infarct cardiac c-kit+ cells were cultured in serum-free complete Iscove's modified Dulbecco's medium. The cells adherent to the culture flask were maintained for propagation and nonattached cells were discarded by 4 changes of medium. Myocytes (1000 cells/well) were co-cultured with post-infarct cardiac c-kit+ (vs. c-kit−) cells (5000 cells/well) derived from the same rat heart in 96-well plates. Co-cultured cells were stimulated with 1 nM E2 or 10 nM PPT or 10 nM DPN for 6 days. Apoptotic myocytes were evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) kit (Roche) and counterstained with DAPI. Apoptotic myocytes were scored for TUNEL-positive nuclei per total number of myocytes detected by DAPI. The percentage of TUNELpositive cells was evaluated by viewing 500–700 cells in 10 randomly chosen fields from each well at × 10 magnification. The apoptosis rate of co-cultured myocytes in vehicle treated group was set at 100%. Calcein AM assay (Invitrogen) was used to evaluate viable myocytes rate (green) under Leica DMIRE2. 2.9. Statistical analysis Results are expressed as mean ± SEM. Multiple comparisons were analyzed with one-way ANOVA followed by Bonferroni post-hoc test.

Fig. 2. (a) Cardiac ERα in post-infarct cardiac Sca-1+ cells. The representative immunofluorescence stainings on the same cardiac free-floating section (10 μm) showed that specific signals for increased ERα (red) were detected mainly in accumulating Sca-1+ cells (green) distributing along the periinfarct zone. Arrow heads are pointing at the ERα expressing Sca-1+ cells and arrow is pointing to an unstained cardiomyocyte. The presence of several DAPI stained nuclei reflect the nuclei of overlapping cells, which were not exactly at the cardiomyocyte level. Scale bar, 50 μm. (b) Cardiac ERα in post-infarct cardiac c-kit+ cells. Multiple immunofluorescence staining was carried out on cardiac free-floating section. The representative fluorescence stainings showed that specific signals for increased ERα (red) were detected mainly in accumulating c-kit+ cells (green) distributing in the periinfarct myocardium. Arrow heads are pointing at the colocalized signals (merged). Scale bar, 50 μm. (c) The representative fluorescence stainings showed that ERα or c-kit or Sca-1 positive cells were not detected in negative control sections from each specimen. Scale bar, 50 μm.

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Fig. 3. Cardiac ERα in BrdU+ proliferating cells after myocardial infarction. Periinfarct zone analyzed (infiltrated T lymphocytes in red, DAPI staining in blue) is marked at low magnification on the coronal section of infarct myocardium. The area indicated (in the coronal view) is just indicative of the region stained. The representative immunofluorescence stainings on the same section revealed that ERα signals (red) were colocalized at BrdU+ (blue) proliferating cells in the periinfarct zone. Scale bar, 50 μm.

Two-group comparisons were analyzed by the two-tailed unpaired ttest. Probability values of b0.05 were considered statistically significant. 3. Results 3.1. Upregulated ERα in post-infarct cardiac c-kit+ cells We first examined the regulation of cardiac ERα and ERβ in response to acute ischemia injury. Seven days after MI, Western blot analysis showed that ERα protein in the periinfarct myocardium was increased 1.9-fold when compared to sham operation (Fig. 1). However, there was no significant change of cardiac ERβ protein

after MI (Fig. 1). We performed immunofluorescence staining on heart sections using ERα specific Abs in combination with cardiac cell markers, including common stem cell surface markers Sca-1 and c-kit. In the periinfarct myocardium, ERα was mainly expressed in small infiltrating Sca-1+ or c-kit+ cells (Figs. 2a, b). By contrast, ERα, c-kit and Sca-1 positive cells were not detected in negative control sections from each specimen (Fig. 2c). In addition, ERα was not detected in cardiomyocytes (Fig. 2a). Using the same staining procedures, we could not clearly detect ERβ positive cells in the myocardium. To understand whether ERα is induced in new proliferating cells (with active DNA synthesis) we treated rats with BrdU (an analog of the DNA precursor thymidine) after MI. New proliferating cells, which have incorporated BrdU into DNA synthesis, can be later tracked using

Fig. 4. Post-infarct cardiac c-kit+ cells in vitro culture. Morphology and immunofluorescence staining of post-infarct cardiac c-kit+ cells were observed in long-term culture. One day and three months after cell isolation, cells were stained with stem cell markers, Sca-1 (green) and c-kit (red or blue). During image capture the stained cells were still viable and moving slowly. Scale bars, 50 μm.

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a mAb against BrdU. By double immunofluorescence staining, we found that cardiac ERα was mainly detected in BrdU+ proliferating cells in the periinfarct myocardium (Fig. 3). 3.2. Ex vivo isolation and characterization of post-infarct cardiac c-kit+ cells The enhanced ERα expression in proliferating post-infarct cardiac c-kit+ cells suggests that ERα may participate in cardiac adaptive

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mechanisms involving post-infarct cardiac c-kit+ cells in response to ischemic injury. We next isolated and characterized these post-infarct cardiac c-kit+ cells from rat hearts 7 days after MI. With modified isolation method using MACS plus FACS sorting, around 2 × 105 c-kit+ cells per infarcted rat heart were obtained, and the purity of these cells was 95–99%, as determined by a FACScalibur™ (Supplementary Fig. 1A). About 10% and 5% of post-infarct cardiac c-kit+ cells expressed CD45 (Supplementary Fig. 1B) and CD34, respectively. Immunocytochemical stainings showed that most post-infarct cardiac c-kit+ cells

Fig. 5. Post-infarct cardiac c-kit+ cell population with ERα predominance and self-renewal/differentiation potentials. Gene expression of ERs, self-renewal and differentiation markers in post-infarct cardiac c-kit+ and c-kit− cell populations was analyzed by real-time PCR. (a), ERα and ERβ; (b), GATA-4 and Notch-2 (transcription factors for cardiac differentiation); (c), Tbx3 and c-Myc (transcription factors for self-renewal and proliferation); (d), Akt and STAT3 (mediators of proliferation and survival). Expression levels were normalized to the expression of β-actin housekeeping gene. n = 4; ⁎P b 0.05, ⁎⁎P b 0.01 vs. c-kit− cell population.

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coexpressed Sca-1 (Fig. 4) but did not express other cardiac cell markers, including CD3, CD8, ED1, von Willebrand factor and αsarcomeric actin. The pluripotency of stem cells is maintained during self-renewal through the prevention of differentiation and promotion of proliferation. To test the self-renewal potential, post-infarct cardiac c-kit+ cells were cultured in conditions that prevent their differentiation and maintained a stable phenotype under in vitro conditions for more than 3 months after their isolation (Fig. 4). To confirm the ERα predominance in post-infarct cardiac c-kit+ cells, we examined ERα and ERβ mRNA expression of these post-infarct cardiac c-kit+ cells in comparison to that of cardiac c-kit− cells. ERα mRNA was upregulated

2.1-fold in post-infarct cardiac c-kit+ cells, compared to c-kit− cells, but ERβ mRNA remained unaltered in post-infarct cardiac c-kit+ cell population isolated from infarcted myocardium as shown by real-time PCR analysis (Fig. 5a). Furthermore, we dissected the regulation of transcription factors implicated in cardiogenic fate decision of mesodermal cells (GATA-4, Notch-2) and genes required for selfrenewal and survival (Tbx3, c-Myc, Akt, STAT3) in ex vivo post-infarct cardiac c-kit+ cells, in comparison to c-kit− cells. The mRNA expression of GATA-4, Notch-2, Tbx3, c-Myc and Akt was significantly induced 1.8-fold, 2.0-fold, 2.0-fold, 2.3-fold and 1.6-fold, respectively, in these post-infarct cardiac c-kit+ cells, with the exception of STAT3, which remained unchanged (Fig. 5b–d). These data provided ex vivo

Fig. 6. (a) ERα promotes proliferation of undifferentiated myoblast cells. Cell proliferation was evaluated on undifferentiated myoblast cells. The representative immunofluorescence stainings and corresponding quantification are showing that undifferentiated myoblast cells (small, round, in red) proliferate after stimulation of E2 (1 nM) or PPT (10 nM) or DPN (10 nM). n = 6, ⁎P b 0.01 vs. vehicle; #P b 0.01 vs. DPN. No statistical significant differences were observed between E2 and PPT treatment or between vehicle and DPN treatment. Scale bar, 50 μm. (b). Conditioned media from post-infarct cardiac c-kit+ cells, but not c-kit− cells, treated with PPT significantly stimulated proliferation of undifferentiated myoblast cells. n = 7, ⁎P b 0.001 vs. conditioned media from c-kit+ cells treated with vehicle or from c-kit− cells treated with PPT or vehicle. No statistical significant differences were observed between conditioned media from PPT treated c-kit− cells and conditioned media from vehicle treated c-kit+ or c-kit− cells. Scale bar, 50 μm.

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Fig. 7. ERα mediates self-renewal but prevents differentiation in myoblast cells. Real-time PCR shows upregulated Tbx3 and Akt but downregualted GATA-4 genes in undifferentiated myoblast cells upon stimulation with E2 (1 nM) or PPT (10 nM). Expression levels were normalized to the expression of β-actin housekeeping gene. n = 7; ⁎P b 0.05, ⁎⁎P b 0.01, vs. vehicle. No statistical significant differences were observed between E2 and PPT treatment.

evidence that post-infarct cardiac c-kit+ cell population expresses predominant ERα and holds self-renewal as well as cardiac differentiation potentials after acute ischemic injury. 3.3. Functional relevance of ERα in undifferentiated myoblast cells To further determine whether ERα activation influences selfrenewal and/or differentiation potentials, we chose a cardiac cell differentiation model, H9c2 myoblast cells derived from rat embryo heart. Upon serum withdrawal, undifferentiated myoblast cells can differentiate into cardiomyocyte-like cells, which are characterized by the expression of alpha-sarcomeric actin and loss of alkaline phosphatase activity (Supplementary Fig. 2). Throughout the myogenic differentiation process, ERα is abundantly expressed in myoblast cells (Supplementary Fig. 2). A dose-dependent downregulation of E2 on GATA-4 expression was observed in myoblast cells at concentrations ranging from 0.1–10 nM (data not shown). The effect of ERα activation on the proliferation of undifferentiated myoblast cells was apparent. The proliferation of myoblast cells was enhanced 1.8-fold and 1.7-fold after exposure to E2 and PPT, respectively, but remained unaltered after DPN treatment (Fig. 6a). In addition, conditioned media from post-infarct cardiac c-kit+ cells treated with PPT also significantly stimulated the proliferation of these undifferentiated myoblast cells, indicating a potential involvement of autocrine/paracrine mechanisms upon ERα activation in post-infarct cardiac c-kit+ cells (Fig. 6b). By contrast, conditioned media from post-infarct cardiac c-kit− cells treated PPT did not influence

proliferation of undifferentiated myoblast cells, excluding the potential effect of residual PPT in the conditioned media. Furthermore, E2 and PPT significantly reduced transcription factor genes implicated in cardiac differentiation of mesodermal cell, like GATA-4 to 43.7% and 34.2%, respectively (Fig. 7). By contrast, E2 and PPT further upregulated genes required for self-renewal/survival, such as Tbx3 (2.4-fold and 2.2-fold) and Akt (1.8-fold and 2.2-fold) (Fig. 7). These results suggest a role of ERα in promoting proliferation but preventing differentiation in post-infarct cardiac c-kit+ cells. 3.4. ERα activation in co-culture system of post-infarct cardiac c-kit+ cells and adult myocytes To understand how ERα exerts potential cardioprotective actions in the absence of cardiac differentiation, we explored whether ERα may indirectly influence cardiomyocyte fate via post-infarct cardiac ckit+ cells (independent of transdifferentiation to myocytes) in a coculture system of adult myocytes and post-infarct cardiac c-kit+ cells or c-kit− cells. Using TUNEL labeling in combination with αsarcomeric actin staining, we observed that E2 and PPT reduced apoptosis of adult myocytes to 77.2% and 76.5%, respectively (P b 0.05; Fig. 8), while they increased survival of adult myocytes to 147% and 144.6%, respectively (P b 0.01; Fig. 8) when adult myocytes were cocultured with post-infarct cardiac c-kit+ cells derived from the same rat heart. However, neither E2 nor PPT influenced apoptosis and survival of adult myocytes when they were co-cultured with cardiac ckit− cells derived from the same rat heart (Fig. 8). ERβ selective

Fig. 8. ERα stimulation reduces apoptosis and supports survival in adult myocytes via post-infarct cardiac c-kit+ cells. Apoptosis of adult myocytes was evaluated when they were cocultured with post-infarct cardiac c-kit+ cells (CM + c-kit+) or c-kit− cells (CM + c-kit−) derived from the same infarcted rat heart using TUNEL in combination with α-sarcomeric actin staining. The survival of co-cultured adult myocytes was assessed by Calcein AM assay. Either E2 (1 nM) or PPT (ERα agonist, 10 nM) reduced apoptosis of myocytes and increased survival of myocytes. Neither apoptosis nor survival of myocytes was influenced upon stimulation with DPN (ERβ agonist, 10 nM). Both E2 (1 nM) and PPT (10 nM) failed to influence the apoptosis and survival in myocytes co-cultured with c-kit− cells. n = 6; ⁎P b 0.05, ⁎⁎P b 0.01, vs. c-kit− cells.

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agonist DPN had also no effect on the apoptosis and survival of adult myocytes co-cultured with post-infarct cardiac c-kit+ cells. These findings suggest that ERα activation supports adult cardiomyocyte survival indirectly via post-infarct cardiac c-kit+ cells. 4. Discussion Despite previous experimental and clinical data demonstrating a cardioprotective role of estrogen via its ERα, the underlying mechanisms remain unclear. In this study, we provide the evidence that cardiac ERα is mainly induced in post-infarct cardiac c-kit+ cells in response to acute myocardial infarction (MI) and that ERα stimulation promotes the proliferation of undifferentiated myoblast cells. Further evidence demonstrates that ERα activation supports survival of adult cardiomyocytes via post-infarct cardiac c-kit+ cells which affords a direct explanation for protective actions of ERα against cardiac injury. ERs are transcription factors that alter gene expression in both reproductive and non-reproductive target tissues, including the cardiovascular system [7]. Both ERα and ERβ are expressed in the heart [9,10,22], but only ERα is elevated in the cardiomyocytes of patients with end-stage heart failure [10]. The physiological importance of cardiac ERα is underscored by other previous observations showing that ERα knockout in male mice leads to severe myocardial damage after ischemic injury [23] and that ERα gene variation is closely correlated with increased risk of MI in men [11]. Adaptive cardioprotection could be one of the earliest cell regulatory events in response to cardiac injury, such as MI. We focused here on the early cellular regulation of ERα and ERβ in the acute remodelling phase after MI and showed that cardiac ERα is exclusively induced in postinfarct cardiac c-kit+ cells, suggesting that cardiac ERα is likely to be involved in the adaptive cardioprotective mechanisms by stem-like cells. Post-infarct cardiac c-kit+ cells are characterized by their selfrenewal and cardiac differentiation potentials [14,24]. Notably, sexrelated differences occur indeed in muscle-derived stem cells. For instance, female muscle-derived stem cells tend to maintain the undifferentiated phenotype or resist differentiation, which allows for their in vivo expansion, in comparison to male muscle-derived stem cells [25]. In this regard, our data further indicate a role of ERα in the promotion of proliferation and prevention of differentiation in myoblast cells. Consistent with these ERα actions, it has been previously shown that ERα controls the balance of proliferation and differentiation in epithelial cells [26], neonatal cardiomyocytes [27] and smooth muscle cells [28], in favour of stimulating cell proliferation. PI3K/Akt activation through direct interaction of ligandactivated ERα and p85 regulatory subunit of PI3 kinase may be involved in the underlying signalling pathways [27]. Cardiomyocyte apoptosis remains a major contributor to the loss of myocardium after cardiac ischemic injury. Although estrogen has been shown to prevent cardiomyocyte apoptosis [13] and recover cardiac performance [29], the potential cellular mechanisms by which estrogen exerts anti-apoptotic actions in adult cardiomyocytes are not well understood. Using a co-culture system of post-infarct cardiac c-kit+ cells and adult myocytes from the same infarcted heart, we here provide direct evidence that ERα activation supports survival of adult myocytes via post-infarct cardiac c-kit+ cells. Along with other findings from this study, it appears that ERα affords cardioprotection by paracrine or cell–cell contact mechanisms via post-infarct cardiac c-kit+ cells independent of cardiomyocyte differentiation. Interesting questions emerging from this study involve the identification of potential survival factors secreted by post-infarct cardiac c-kit+ cells or by the interaction of post-infarct cardiac c-kit+ cells and adult myocytes. It should be noted that this study does not exclude other possible mechanisms, for instance that ERα activation may improve cardiac repair process through mobilization and homing of endothe-

lial progenitor cells [20]. On the other hand, given the localization of ERβ to mitochondria [30] and mitochondrial actions of estrogen in cardiomyocyte [13], we cannot exclude that the potential ERβmediated antioxidative mechanisms may also contribute to antiapoptotic effects on cardiomyocytes by the reduction of calcium influx [31], and inhibition of apoptosis signal-regulating kinase 1 activity and its downstream targets including c-Jun N-terminal kinase and p38 mitogen-activated protein kinase [13]. Further subcellular studies of ERα and ERβ at both mitochondria and nucleus may certainly enhance our current understanding of estrogen-related cardioprotection. In conclusion, these findings provide new biological insight into ERα-involved adaptive mechanisms in the heart by supporting cardiomyocytes survival via post-infarct cardiac c-kit+ cells. A potential important implication of this study is, that the manipulation of cardiac stem-like cells using a selective ERα modulator may be helpful in preserving tissue and recovering functional performance after cardiac tissue injury. Acknowledgments This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (DFG; GK754-III) to J.L. and T.U. and the Berlin-Brandenburg Center for Regenerative Therapies (BCRT) to J.L. and T.U. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yjmcc.2009.03.014.

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