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Keywords:

  • Angiotensin AT2 receptor;
  • Cardiac c-kit+ cell;
  • Myocardial infarction

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

The expression pattern of angiotensin AT2 receptors with predominance during fetal life and upregulation under pathological conditions during tissue injury/repair process suggests that AT2 receptors may exert an important action in injury/repair adaptive mechanisms. Less is known about AT2 receptors in acute ischemia-induced cardiac injury. We aimed here to elucidate the role of AT2 receptors after acute myocardial infarction. Double immunofluorescence staining showed that cardiac AT2 receptors were mainly detected in clusters of small c-kit+ cells accumulating in peri-infarct zone and c-kit+AT2+ cells increased in response to acute cardiac injury. Further, we isolated cardiac c-kit+AT2+ cell population by modified magnetic activated cell sorting and fluorescence activated cell sorting. These cardiac c-kit+AT2+ cells, represented ∼0.19% of total cardiac cells in infarcted heart, were characterized by upregulated transcription factors implicated in cardiogenic differentiation (Gata-4, Notch-2, Nkx-2.5) and genes required for self-renewal (Tbx-3, c-Myc, Akt). When adult cardiomyocytes and cardiac c-kit+AT2+ cells isolated from infarcted rat hearts were cocultured, AT2 receptor stimulation in vitro inhibited apoptosis of these cocultured cardiomyocytes. Moreover, in vivo AT2 receptor stimulation led to an increased c-kit+AT2+ cell population in the infarcted myocardium and reduced apoptosis of cardiomyocytes in rats with acute myocardial infarction. These data suggest that cardiac c-kit+AT2+ cell population exists and increases after acute ischemic injury. AT2 receptor activation supports performance of cardiomyocytes, thus contributing to cardioprotection via cardiac c-kit+AT2+ cell population. STEM CELLS 2009;27:2488–2497


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Acute myocardial infarction (MI) leads to loss of cardiomyocytes and impaired pump function of the heart. When the remaining cardiomyocytes are unable to reconstitute with time, heart failure results. A wealth of information indicates that the renin-angiotensin system (RAS) can interfere with the cardiac remodeling process after MI [1]. Angiotensin II (Ang II), the main effector peptide of the RAS, acts through its receptors. At least two angiotensin receptors, AT1 and AT2 receptors, have been detected in the heart [2]. Pharmacological modulation of the RAS through blocking AT1 receptors has clearly been shown to improve the outcome of heart failure. Experimental evidence from AT2 −/− mice or from rats under AT1/AT2 receptor blockers suggests that AT2 receptors, which may be exposed to enhanced Ang II levels after AT1 receptor blockade, afford protection against MI-induced cardiac injury [3, 4]. We recently demonstrated that antiapoptotic and anti-inflammatory mechanisms are associated with AT2 receptor-mediated cardioprotection [5].

There are conflicting observations on AT2 receptor expression in cardiac fibroblasts of patients with heart failure arguing whether AT2 receptors are involved in the regulation of the cardiac fibrotic process [6, 7]. On the other hand, following acute MI, there is clear evidence that myocardial AT2 receptors are dramatically upregulated within the first week [2, 8]. It seems unlikely that, under these circumstances, increased AT2 receptors are involved in fibrotic process, given the fact that fibrosis is an adaptive cardiac event that needs several weeks to develop after the induction of MI [6]. Thus, it is presently not well defined how AT2 receptors are regulated at the cardiac cellular level.

Both AT1 and AT2 receptors belong to the seven transmembrane-domain receptor families but exhibit only 34% amino-acid sequence homology [9], which indicates that the two angiotensin receptors may have substantially different functions. During fetal development, the RAS is active and AT2 receptors are transiently highly expressed in rodent and primate fetuses [1]. The AT2 receptors especially are most abundant in less differentiated mesenchymal cells of the fetus [1], suggesting their potential role in mesenchymal cell differentiation. Dramatic increase of AT2 receptors also occurs during tissue injury, including MI [8], brain ischemia [10], and nerve crush or transaction [11]. This expression pattern of AT2 receptors with predominance in fetal life and upregulation under pathological conditions during tissue injury/repair process indicates that AT2 receptors may exert an important action in adaptive tissue regeneration, which may be different from the known effects of Ang II mediated via AT1 receptors.

Accumulating experimental and clinical evidence indicates that intramyocardial implantation of hematopoietic undifferentiated cells, which express antigens commonly found in bone marrow (BM) precursor cells, such as c-kit, leads to restoration of cardiac function [12, 13], albeit the underlying mechanisms involving cell fusion and transdifferentiation are still debated [12, 14, 15]. Recently, a growing body of evidence highlighted that BM c-kit+ cells lead to an improved cardiac function independent of transdifferentiation into either cardiac muscle or endothelial cells, but rather associated with cardiac release of angiogenic cytokines and neovascularization [16]. The discovery of adult c-kit+ precursor cells in rodent and human heart has sparked intense hope for cardiac regeneration with cells that are from the heart itself and are thereby inherently programmed to reconstitute damaged myocardium [12]. However, the growth factor- receptor systems that regulate the proliferation and differentiation of adult cardiac c-kit+ precursor cells are less well understood. It is also undefined how cardiac c-kit+ precursor cells may influence the cardiomyocyte fate in triggering cardiac regeneration.

In this study, we explored the potential cellular mechanisms that may account for the AT2 receptor-mediated cardioprotection. Here we demonstrated that cardiac c-kit+AT2+ cell population exists and increases after acute ischemic injury. AT2 receptor activation supports cardiomyocyte performance, hence contributing to cardioprotection via cardiac c-kit+AT2+ cell population.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Induction of Myocardial Infarction and Treatment

Myocardial infarction (MI) was induced in male Wistar rats at 7 weeks of age (200-220 g, Harlan Winkelmann GmbH, Borchen, Germany) by coronary artery ligation as described [5]. Briefly, rats were anesthetized with ketamin/xylazine (Sigma-Aldrich, Taufkirchen, Germany, http://www.sigmaaldrich.com) 80 mg/10 mg/kg intraperitoneal, intubated and ventilated with a Starling Ideal Ventilator (Harvard Apparatus, March-Hugstetten, Germany, http://www.hugo-sachs.de). 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, Toronto, Canada, http://www.visualsonics.com) was performed 24 hours after operation to assess post-MI cardiac function. As described previously, only animals with ejection fraction >35% were used for this study [5].

To determine cardiac effects of AT2 receptor activation in vivo, animals were subjected to AT2 receptor agonist (compound 21, [C21]; 0.03 mg/kg per day, intraperitoneal; kindly provided by A. Hallberg, University of Uppsala, Sweden) [17] and vehicle treatment (0.9% saline) 1 day after MI induction as described before [5]. On day 7 after MI or sham operation, the rats were decapitated and the hearts were rapidly excised.

Animal protocols followed the German laws on animal protection.

Ex Vivo Isolation and Flow Cytometry Analysis of Cardiac Cells

Cardiac cells, including adult cardiomyocytes, were isolated from rat hearts on day 7 after MI or sham operation, according to the protocol produced by Worthington Biochemical Company (Katharinen, Germany, http://www.worthington-biochem.com) with some modifications. Briefly, the heart was transferred to a Petri dish where the blood was removed from the coronary vessels by gently squeezing the heart a few times in ice-cold phosphate buffered saline (PBS) solution. The myocardium was minced into pieces smaller than 1 mm3 and incubated in calcium and magnesium-free Hanks Balanced Salt Solution (CMF-HBSS) containing 0.025% Trypsin solution (PAN) at 4°C for 16 hours. After adding trypsin inhibitor (Sigma-Aldrich), the digested tissue was oxygenated and warmed to 30-37°C. The digested tissue was then incubated in CMF-HBSS medium containing 0.5 mg/mL collagenase II (Sigma-Aldrich) at 37°C for 30 minutes. Tissue suspension was triturated to release a single-cell suspension. Density gradient sedimentation was followed. The top band (myocytes) was collected, and middle layers were respun to pellet down the small cells. Cell pellets were then incubated with rabbit anti-c-kit (Santa Cruz Biotechnology, Inc. Heidelberg, Germany, http://www.scbt.com). After washing, cell pellets were incubated with phycoerythrin (PE) antirabbit Abs (Jackson ImmunoResearch, Hamburg, Germany, http://www.jireurope.com), and then with anti-PE microBeads (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). The c-kit+ cells were positively selected using magnetic activated cell sorting (MACS) system (Miltenyi Biotec). Then, the negative mononuclear cell selection (the c-kit- cell fraction) was depleted from the AT2+ cells by MACS, and collected as cardiac c-kit-AT2- cell population for further experiments. The c-kit+ single cell suspension was also incubated with goat anti-AT2, mouse anti-CD34 (Santa Cruz), mouse anti-CD45 (AbD Serotec, Düsseldorf, Germany, http://www.abserotec.com), and IgG isotype control (Santa Cruz), indirectly labeled to allophycocyanin (APC) antigoat or fluorescein isothiocyanate (FITC) antimouse secondary antibodies (Jackson ImmunoResearch), and subjected to flow cytometry. Analysis and cell acquisition was performed on a fluorescence-activated cell sorting calibur (FACSCalibur) system (Becton, Dickinson and Company, Heidelberg, Germany, http://www.bdeurope.com) or cell sorting (c-kit+AT2+) on FACSAria (Becton, Dickinson and Company, Heidelberg, Germany, http://www.bdeurope.com). The data were analyzed using FlowJo software (TreeStar Inc., Ashland, USA, http://www.treestar.com). At least 1 × 103 events in the c-kit cells region were acquired for each sample.

Immunofluorescence Staining

Acetone-fixed heart cryosections (5 μm) or free-floating heart cryosections (10 μm) were incubated in 10% donkey serum for 30 minutes. For multiple immunofluorescence staining, the sections were incubated with rabbit anti-c-kit polyclonal Ab (Santa Cruz Biotechnology, Inc.) and also with goat anti-AT2 polyclonal Ab (Santa Cruz Biotechnology, Inc.) overnight at 4°C. Sections were then incubated with donkey Cy3 antigoat or Alexa488 anti-abbit IgG (Jackson ImmunoResearch, Hamburg, Germany) for 1 hour. For p-Stat-3 and p-Akt, paraformaldehyde-fixed heart sections were blocked and incubated with rabbit anti-p-Stat-3 (Tyr705) or rabbit anti-p-Akt (Ser473) (1:100 or 1:75; Cell Signaling) and mouse anti-α-sarcomeric actin antibody (1:400; Sigma-Aldrich), respectively. Sections were then incubated with donkey Cy3 antimouse or Alexa488 antirabbit IgG (Jackson ImmunoResearch). Sections were counterstained for 10 minutes with DAPI (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com). To exclude any cross-reactivity, matched negative controls were used on serial sections from each specimen. Stained sections (5 μm) were examined under a Leica DMIRE2 microscope (Wetzlar, Germany, http://www.leica.de) or stained free-floating sections (10 μm) were analyzed with a Leica LSM confocal microscope (LEICA SP2 with AOBS) and Leica TCS software. For quantitative analysis, stained sections were imaged at ×40 or ×63 magnification on a Leica DMIRE2 fluorescence microscope equipped with appropriate filter cubes and a digital color camera. Fluorescence-labeled cells were counted from digital images using the Density Slicing and Advanced Measurement Modules of OpenLab Imaging Software (Improvision, Tübingen, Germany, http://www.improvision.com). Quantification of c-kit+ cells coexpressing AT2 receptor in myocardial tissue sections was performed by taking a minimum of 8 fields from each tissue section and from 6 tissue sections per sample or animal.

Terminal Transferase-Mediated dUTP-Biotin Nick-End Labeling

Cardiomyocyte apoptosis was detected by fluorescence microscopy on cardiac cryosections (5 μm), which were fixed by 4% paraformaldehyde. Terminal transferase-mediated dUTP-biotin nick-end labeling (TUNEL) was performed with fluorescein-dUTP (In Situ Cell Death Detection Kit; Roche Diagnostics, Mannheim, Germany, http://www.roche-applied-science.com). After TUNEL, the sections were blocked with 10% of donkey serum, in 2% BSA/PBS and incubated with mouse anti-α-sarcomeric actin antibody (1:400; Sigma) to label cardiomyocytes. Cell nuclei were stained with DAPI (Molecular Probe). Slides were visualized on a LEICA-DMIRE2 microscope.

Determination of Apoptosis and Viability of Cardiomyocytes In Vitro

Adult cardiomyocytes and cardiac c-kit+AT2+ cells were isolated from the same rat hearts on day 7 after MI, as described previously. Cardiomyocytes were cocultured with cardiac c-kit+AT2+ cells (5:1) or cultured alone in a 96-well cell culture plates coated with 3 μg/ml laminin (Sigma). The cells were cultured in complete medium (Dulbecco's Modified Eagle's Medium /F12, 0.1 ng/mL LIF, 100 U/mL of penicillin, 100 μg/mL streptomycin) at 37°C with 5% CO2 for 48 hours. Then, the cultures were treated with 10 nM exogenous angiotensin II (Ang II) alone or in combination with 100 nM AT2 receptor antagonist (PD123319), or with 20 nM AT2 receptor agonist (C21) for 24 hours. In addition, the cells that were treated with 0.9% saline alone served as a vehicle group. At day 7 after treatment, apoptosis was examined by ethidium bromide/acridine orange (EB/AO) method (Sigma) as described previously [18]. Cells were centrifuged at 1,000 rpm (129xg) for 5 minutes. A dye mix for the EB/AO staining was prepared by mixing 100 μl of AO and 100 μl of EB in 1 mL PBS. EB/AO dye mix was added and incubated at 37°C for 1 hour. The experiment was performed in triplicate, counting a minimum of 200 total cardiomyocytes in each culture well. Total apoptotic and necrotic cells were quantified. The percentage of apoptotic cardiomyocytes was calculated as: % apoptotic cells = (necrotic + apoptotic cardiomyocytes) × 100/total cardiomyocytes. Additionally, the cardiomyocyte viability was measured in triplicate using the nonfluorescent cell-permeant Calcein AM (Invitrogen GmbH, Karlsruhe, Germany). At day 7 after treatment, the culture medium was aspirated out and the cells were incubated with 2 μM Calcein AM in PBS at 37°C for 3 hours. Following incubation, the cells were washed twice in PBS. Then the labeled cells were observed and counted under a Leica DMIRE2 fluorescence microscope. The apoptotic and viable rates of single cultured cardiomyocytes in treated groups were set at 100%.

RNA Isolation, Reverse Transcription, and Quantitative Real-Time Polymerase Chain Reaction

Cell pellet was lysed in Trizol Reagent (Invitrogen GmbH, Karlsruhe, Germany, http://www.invitrogen.com) and mixed vigorously, and then RNA was extracted following the manufacture's protocol. The RNA was reversed to DNA using a reverse transcription kit (Promega Corporation, Madison WI, http://www.promega.com). The Real-Time Polymerase Chain Reaction (PCR) procedures were performed using TaqMan Universal PCR Master Mix or SYBR Green PCR Master Mix reagents (Applied BioSystems, Deutschland GmbH, Darmstadt, Germany, http://www.appliedbiosystems.com). The primers and probes with fluorescent dye and quencher as listed in supporting information Table 1 were selected to be intron spanning and synthesized by TIB MOLBIOL (Berlin, Germany, http://www.tib-molbiol.de). Results were analyzed using MxPro ET QPCR software (Stratagene, La Jolla, CA, http://www.stratagene.com). Relative level for each gene was calculated using the standard curve method. Expression levels were normalized according to expression of β-actin housekeeping gene.

Statistical Analysis

Results are expressed as mean ± SEM. Multiple comparisons were analyzed with one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test, and two-way ANOVA with Bonferroni post hoc analyses was used for cardiomyocytes apoptosis and viability assays. Two-group comparisons were analyzed by the two-tailed Student's unpaired t test for independent samples. For all procedures, p values of <.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

AT2 Receptors Are Increased in Cardiac c-kit+ Precursor Cells in Response to Ischemic Injury

We first examined the cellular regulation of cardiac AT2 receptor in response to acute ischemic injury. Seven days after MI, intensive infiltration of small c-kit+ precursor cells into the interstitial space of peri-infarct myocardium was observed by immunohistochemical staining (Fig. 1A). In contrast, c-kit+ cells were rarely detected in the heart without MI (Fig. 1B). Notably, AT2 receptors were expressed mainly in cardiac c-kit+ cells (Fig. 1A). In response to acute myocardial infarction, cardiac c-kit+AT2+ cells were significantly increased in the peri-infarct area (from 18 ± 7 cells/mm2 in sham-operated myocardium to 236 ± 79 cells/mm2 in infarcted myocardium. Sham, n = 5; MI, n = 6 animals) (Fig. 1C).1

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Figure 1. Increased c-kit+AT2+ cells in the infarcted myocardium. (A, B): Representative images of stained cardiac free-floating sections (10 μm) showed that specific signals for increased AT2 receptor were detected mainly in accumulating c-kit+ cells distributing in interstitial space of the peri-infarct myocardial infarction (MI) and sham-operated (Sham) myocardium. Cells were stained using anti-c-kit (Alexa488, green), anti-AT2 (Cy3, red) antibodies, and DAPI (blue) for nuclei counterstaining. Arrows point to the c-kit+AT2+ cell clusters under a Leica LSM confocal microscope (LEICA SP2 with Acousto-Optical Beam Splitter (AOBS)). Scale bar, 10 μm. (C): Quantification of cardiac c-kit+AT2+ cells. Data represented the analysis of a minimum of 6 sections per animal from the peri-infarct area of MI tissue (n = 6) and respective area from Sham tissue (n = 5); *p < .05 versus Sham; values are means ± SEM, expressed as cell number per mm2 of 8 randomly chosen fields per section. All images were acquired with ×63 magnification. Abbreviations: Cy3, Cyanine3; DAPI, 4′,6-diamidino-2-phenylindole; MI, myocardial infarction; Sham, sham operated.

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Table 1. Primers sequence for Real-Time PCR
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Isolation and Flow Cytometric Analysis of Cardiac c-kit+AT2+ Cell Population

The presence of cardiac c-kit+AT2+ cells suggested that AT2 receptors are potentially involved in adaptive cardiac regeneration/protection, probably via cardiac c-kit+ precursor cells in response to ischemic injury. We next isolated and characterized this cardiac c-kit+AT2+ cell population from rat hearts 7 days after MI or sham operation. With modified isolation method using MACS plus FACS sorting (Fig. 2A), 2.1 ± 0.7 × 105 c-kit+AT2+ cells per infarcted rat heart were obtained (n = 6), and the purity of these isolated cells was 95 to 99%, as determined by a FACScalibur. In contrast, only around 0.2 ± 0.02 × 105 c-kit+AT2+ cells were isolated from each sham-operated heart (n = 9) (Fig. 2B). The frequency of c-kit+AT2+ cells in infarcted and sham-operated heart represented ∼0.19% and ∼0.02% of the total cardiac cells, respectively (Fig. 2B). From infarcted hearts, more than 95% of enriched cardiac c-kit+ cells expressed AT2 receptor, but ∼10% expressed the leukocyte marker CD45 and ∼4% the endothelial/hematopoietic progenitor marker CD34. From sham-operated hearts, only 4% of enriched cardiac c-kit+ cells expressed AT2 receptor, ∼5% CD45 and ∼2% CD34 (n = 6 for each marker) (Fig. 2C, 2D). Thus, MI leads to an increase in c-kit+AT2+ cell population, specifically within the infarcted myocardium, and mainly with low expression of hematopoietic CD45 and CD34 (versus BM c-kit+AT2+ cell population, supporting information Fig. 1C, 1D). This phenomenon may represent preferential proliferation or recruitment of c-kit+AT2+ cell population of BM origin [16] in the infarcted myocardium in response to acute ischemic injury.

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Figure 2. Flow cytometric analysis of cardiac c-kit+ cells. Analysis was performed on c-kit+ cells isolated from rat hearts after myocardial infarction (MI) or sham operation (Sham). c-kit+ cells were enriched by the magnetic activated cell sorting system with phycoerythrin-indirectly labeled to anti-c-kit antibody and anti-phycoerythrin micro beads, and after sorting, ∼95-99% of the cells expressed c-kit. c-kit+ cells were also stained with allophycocyanin to detect anti-AT2 antibody and fluorescein isothiocyanate to detect anti-CD45 antibody, or anti-CD34 antibody. (A): Representative fluorescence-activated cell sorting (FACS) plots of cardiac c-kit+AT2+ cells isolated from MI and sham hearts. (B): FACS plots were quantified as absolute c-kit+AT2+ cell number sorted, or as a percentage of total cardiac cells. **p < .01 versus Sham; values are means ± SEM; (Sham, n = 9; MI, n = 6). (C, D): In enriched cardiac c-kit+ cells, CD34 and CD45 expression were calculated and expressed as percentage. Values are means ± SEM; n = 6. Abbreviations: MI, myocardial infarction; Sham, sham operation.

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Molecular Characterization of the Cardiac c-kit+AT2+ Cells

To evaluate whether cardiac c-kit+AT2+ cells were potential cardiogenic precursors, we next dissected the regulation of transcription factors implicated in cardiogenic fate decision of mesodermal cells (Gata-4, Notch-2, Nkx-2.5) and genes required for cell cycle progress and survival (c-Myc, Akt, Tbx-3, Stat-3) in cardiac c-kit+AT2+ cell population. In comparison to cardiac mononuclear c-kit-AT2- cells, obtained from the same infarcted rat hearts on day 7 after MI, Real-time PCR showed that the mRNA expression of Gata-4, Notch-2, Nkx-2.5, c-Myc, Akt and Tbx-3 was significantly induced 4.8-fold, 1.8-fold, 2.6-fold, 3.5-fold, 1.9-fold and 1.3-fold, respectively (p < .05, n = 5 for each gene), in cardiac c-kit+AT2+ cells, with the exception of Stat-3, which remained unchanged (Fig. 3). These data provided evidence that cardiac c-kit+AT2+ cell population holds self renewal as well as cardiac differentiation potentials during acute ischemic injury.

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Figure 3. Gene expression pattern in cardiac c-kit+AT2+ cells isolated from rat hearts 7 days after myocardial infarction (MI). Gene expressions of Gata-4, Notch-2, Nkx-2.5, c-Myc, Akt, Tbx-3 and Stat-3 in cardiac c-kit+AT2+ cells were compared with that in c-kit-AT2- cells. The relative gene expression levels were measured by TaqMan polymerase chain reaction. Data were normalized to the β-actin levels and represented as relative x-fold of c-kit-AT2- mRNA levels. Values are means ± SEM; *p < .05, **p < .01 versus c-kit-AT2-, n = 5 for each gene. Abbreviation: MI, myocardial infarction.

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In Vitro Roles of AT2 Receptors on Adult Cardiomyocytes via c-kit+AT2+ Cells

To understand how AT2 receptor exerts potential cardioprotective actions, we examined whether AT2 receptor may indirectly influence cardiomyocyte performance via c-kit+AT2+ cells in a coculture system of adult cardiomyocytes and cardiac c-kit+AT2+ cells. First, we observed that angiotensin II (Ang II) stimulation induced apoptosis of adult cardiomyocytes in single culture, whereas the AT2 receptor agonist (C21) or AT2 receptor antagonist (PD123319) failed to exert any apoptotic or antiapoptotic effect on these adult cardiomyocytes (supporting information Fig. 3), supporting the notion that AT1 receptor, but not AT2 receptor, mediates Ang II-induced apoptosis of adult cardiomyocytes [19]. The presence of cocultured cardiac c-kit+AT2+ cells led to a tendency toward some antiapoptotic effects on adult cardiomyocytes (p > .05, n = 6 for each group, Fig. 4B). Notably, when the cells in coculture or single culture were stimulated with 10 nM exogenous Ang II alone or in combination with 100 nM PD123319, or with 20 nM C21, Ang II alone or C21 significantly enhanced this antiapoptotic effect of c-kit+AT2+ cells on cocultured cardiomyocyte, 1.6 (p < .05) and 2.2-folds (p < .01), respectively, whereas PD123319 abolished this effect (1.2-fold) (Fig. 4B).

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Figure 4. In vitro effects of cardiac c-kit+AT2+ cells on the apoptosis of adult cardiomyocytes (CM). (A): Adult cardiomyocytes and cardiac c-kit+AT2+ cells were isolated from the same rat hearts after myocardial infarcition (MI). Apoptotic CM were detected using the fluorescence probe ethidium bromide/acridine orange (EB/AO). An image of the nuclei stained with ethidium bromide and acridine orange was taken and merged into one picture. Scale bar, 100 μm. (B): Quantification of apoptotic cardiomyocyte (CM) showed exogenous angiotensin II (Ang II) or AT2 receptor agonist (C21) significantly reduced apoptosis of CM cocultured with cardiac c-kit+AT2+ cells. **p < .01 versus CM in single culture. The determination of CM apoptosis was performed on a 96-well plate in triplicate. All images were acquired with ×20 magnification. Values are means ± SEM; n = 6 for each group. Abbreviations: A, apoptotic cardiomyocyte; Ang II, angiotensin II; C21, compound 21; CM, cardiomyocyte; EB/AO, ethidium bromide/acridine orange; MI, myocardial infarction; N, necrotic cardiomyocyte; PD, PD123319.

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Viability assay was also performed with calcein AM to measure the percentage of living cardiomyocytes cocultured with c-kit+AT2+ cells. After 7 days in coculture, c-kit+AT2+ cells appeared to increase the viability of cocultured cardiomyocytes when compared to individually cultured cardiomyocytes (p > .05). When both cultures were stimulated with exogenous Ang II alone, or in combination with PD123319, or C21, we observed a significant improvement of cardiomyocyte viability in the coculture, over the single culture, after Ang II or C21 stimulation (p < .001, n = 6 for each group) (data not shown). Thus, these findings suggested that AT2 receptor activation supports adult cardiomyocyte performance indirectly via cardiac c-kit+AT2+ cells.

In Vivo Effects of AT2 Receptor Activation on Cardiac c-kit+AT2+ Cell Population

To extend the role of cardiac AT2 receptors from in vitro to in vivo, we investigated the effects of AT2 receptor activation on cardiac c-kit+AT2+ cells. Seven days after MI, in vivo AT2 receptor stimulation with C21 led to an increased accumulation of c-kit+AT2+ cell population into the infarcted myocardium. Flow cytometry analysis of isolated cardiac cell populations showed that both the number and frequency of cardiac c-kit+AT2+ cells were significantly increased after C21 treatment (Fig. 5A, 5B). Immunohistochemical staining also revealed a 2.5-fold increment of c-kit+AT2+ cells (Fig. 5C). These findings suggested that AT2 receptor activation attracts c-kit+AT2+ cells into the infarcted area of the heart by cardiac proliferation of these cells and/or mobilization from bone marrow derived c-kit+AT2+ cells [16], which were also increased after MI (from ∼6% to ∼26% of bone marrow c-kit+ cells, supporting information Fig. 1A, 1B).

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Figure 5. In vivo effect on cardiac c-kit+AT2+ cells by compound 21 treatment (MI+C21; 0.03 mg/kg per day intraperitoneal) or vehicle (MI+vehicle [V]; 0.9% saline). (A): Representative fluorescence-activated cell sorting (FACS) plots of cardiac c-kit+AT2+ cells (B): Quantification of cardiac c-kit+AT2+ cells was presented as absolute cell number sorted, or as percentage of total cardiac cells. **p < .01 versus MI+V; values are means ± SEM; (MI+C21, n = 7; MI+V, n = 6). (C): Quantification of c-kit+AT2+ cells using immunofluorescence microscopy images taken from (MI+V) and (MI+C21) heart sections. Data represented the analysis of at least 6 sections per animal (n = 6 per group) from the infarct border area of heart tissue. *p < .05 versus MI; values are means ± SEM expressed as cell number per mm2 of 8 randomly chosen fields in each tissue section. (D): Effect of AT2 receptor activation on mRNA expression of c-Myc, Akt, Ki67, Gata-4, and Notch-2 in cardiac c-kit+AT2+ cells by C21 treatment. Data were presented as relative level to the β-actin; *p < .05 versus MI+V; values are means ± SEM; (MI+C21, n = 6; MI+V, n = 8). Abbreviations: C21, compound 21; MI, myocardial infarction; n.s., not significant; V, vehicle.

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To elucidate whether a selective AT2 receptor stimulation directly influences the proliferation and/or differentiation potentials of cardiac c-kit+AT2+ precursor cells, we determined the expression of relevant genes related to proliferation and differentiation. Real-time PCR showed that the oncogene c-Myc and proliferation specific marker Ki67 [20] in cardiac c-kit+AT2+ cell population were significantly downregulated 2.8-fold and 2.6-fold, respectively, in MI rats after C21 treatment in comparison with vehicle treatment (Fig. 5D). In contrast, transcription factors Gata-4 and Notch-2, which are required for cardiac differentiation [21, 22], were significantly upregulated 5.0-fold and 1.9-fold, respectively (Fig. 5D). No significant change was observed in the expression of prosurvival Akt gene (Fig. 5D) (MI+C21, n = 6; MI+ vehicle [V], n = 8). Together, these data suggested that AT2 receptor mediates differentiation but not proliferation potential of cardiac c-kit+AT2+ cell population. Thus, rather than cardiac proliferation, enhanced cardiac mobilization may help explain the increased c-kit+AT2+ cell population in the infarcted heart after AT2 receptor activation.

In Vivo Effects of AT2 Receptor Activation on Cardiomyocyte Survival

To extrapolate our observations in cocultured cardiomyocytes to in vivo settings, we studied whether AT2 receptor activation had any impact on cardiomyocyte performance in rats with acute MI. Cardiomyocyte apoptosis was analyzed by TUNEL staining on cardiac sections after C21 and vehicle treatment (Fig. 6A). At high-power magnification (×40) of the peri-infarct myocardium, the percentage of TUNEL-positive nuclei reached 1.6 ± 0.9% in C21 treated group versus 3.5 ± 1.1% in the vehicle-treated group (p = .01; MI, n = 6; MI+C21, n = 7) (Fig. 6B). Cardiomyocyte identity of TUNEL-positive nuclei was determined by costaining with α-sarcomeric actin. To analyze the potential signaling mechanisms in supporting adult cardiomyocytes, we examined the expression levels of a number of genes, including prosurvival genes such as Bcl-2, Stat-3, and Akt, and proapoptotic gene Bax, using isolated cardiomyocytes derived from C21 or vehicle-treated infarcted hearts. Real-time PCR showed that Akt and Stat-3 genes were significantly upregulated threefold and 1.6-fold, respectively, in the isolated cardiomyocytes of the C21-treated group when compared with the vehicle-treated group (MI+C21, n = 7; MI+V, n = 6; Sham, n = 6) (Fig. 7A). Importantly, the activation of Akt and Stat-3 signaling pathways was confirmed by quantitative immunohistochemical staining showing increased expression of p-Stat-3 (Fig. 7B, 7C) and p-Akt (Fig. 7D, 7E) proteins in rat cardiomyocytes after MI under C21 treatment (MI+C21, n = 6; MI+V, n = 6; Sham, n = 5). Thus, these data suggested that AT2 receptor activation in vivo supports cardiomyocyte survival, probably through Stat-3 and Akt involved signal pathways.

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Figure 6. Cardiomyocyte apoptosis in myocardial infarction (MI) rats after treatment with compound 21 (MI+C21) or vehicle (MI+V) for 7 days in comparison to rats with sham operation (Sham). (A): Apoptotic cardiomyocytes were detected by TUNEL staining on cardiac sections from MI rats. Cardiomyocytes (Cy3, red) underwent TUNEL staining with Fluorescein-labeled dUTP (green) and nuclei counterstaining with DAPI (Blue). The arrows indicate terminal transferase-mediated dUTP-biotin nick-end labeling-positive nuclei. Scale bar, 50 μm. (B): Quantitative analysis revealed reduced percentage of apoptotic cardiomyocytes (% of total cardiomyocytes counted) following compound 21 (C21) treatment. *p < .05; **p < .01; ***p < .001; values are means ± SEM (Sham, n = 5; MI+V, n = 6; MI+C21, n = 7). Abbreviations: Cy3, Cyanine3; C21, compound 21; DAPI, 40,6-diamidino-2-phenylindole; MI, myocardial infarction; Sham, sham operation; TUNEL, terminal transferase-mediated dUTP-biotin nick-end labeling; V, vehicle.

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Figure 7. Modulation of survival genes in adult cardiomyocytes (CM) by treatment with compound 21 (MI+C21). (A): Real-time polymerase chain reaction showed that C21 treatment led to an upregulation of Akt and Stat-3 genes in adult cardiomyocytes (CM) isolated from myocardial infarction (MI) rats treated with compound 21 (MI+C21) or vehicle (MI+V) for 7 days in comparison to CM from rats with sham operation (Sham). Data were presented as relative level to the β-actin; *p < .05; **p < .01; ***p < .001 (Sham, n = 6; MI+V, n = 6; MI+C21, n = 7). (B, D): Immunohistochemical analysis of the phosphorylated Stat-3 or Akt proteins in cardiac sections. The arrows indicate p-Stat-3-positive or p-Akt-positive nuclei (Alexa488, green), CM (Cy3, red), and nuclei counterstaining with DAPI (blue). Scale bar, 50 μm. (C, E): Quantitative analysis revealed that C21 treatment increased activated p-Stat-3 and p-Akt proteins in cardiomyocytes from MI rats when compared to vehicle treatment. Data represented the analysis of 6 to 9 sections per animal. n.s: not significant; *p < .05; **p < .01 (Sham, n = 5; MI+V, n = 6; MI+C21, n = 6). Abbreviations: C21, compound 21; CM, cardiomyocyte; Cy3, Cyanine3; DAPI, 40,6-diamidino-2-phenylindole; MI, myocardial infarction; n.s., not significant; Sham, sham operation; V, vehicle.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Despite previous studies demonstrating a cardioprotective role of angiotensin AT2 receptor, the underlying mechanisms remain unclear. In this study, we provide the evidence that cardiac c-kit+AT2+ precursor cells exist and increase in response to acute myocardial infarction (MI) in rats. Further evidence showed that AT2 receptor stimulation increased c-kit+AT2+ cell population in the infarcted myocardium and supported cardiomyocyte performance, which were associated with improved cardiac function and reduced infarct size as shown recently by our group [5]. These findings afford a direct explanation for the protective actions of AT2 receptor against cardiac injury.

The renin-angiotensin system (RAS) is an important player in the control of the cardiovascular homeostasis. Most of the actions of angiotensin II (Ang II), such as blood pressure- and osmotic control, are mediated by AT1 receptors [1]. By contrast, the upregulation of AT2 receptors, which has also been observed in the ischemic heart after acute myocardial infarction [8] or in the ischemic brain [10], speaks in favor of a general role of these receptors in the repair of injured tissue. Looking into the cardiac cellular localization, however, the conflicting observations on AT2 receptor expression in cardiac fibroblasts of patients with heart failure raise the argument whether AT2 receptors are involved in the regulation of the fibrotic process [6, 7]. By applying a single-cell RT-PCR technique, we have previously shown that a small fraction of cardiomyocytes (<10%) express AT2 receptor mRNA under physiologic conditions, but that the number of AT2 receptor expressing cardiomyocytes was not increased 1 day after MI, indicating that noncardiomyocytes may be responsible for the enhanced AT2 receptors [2]. In the present study, we could provide the first evidence that the post-MI expression of AT2 receptors was mainly induced in cardiac c-kit+ precursor cells, raising a potential role of AT2 receptors in adaptive cardiac differentiation and regeneration.

Cardiac c-kit+ precursor cells are characterized by their self-renewal and cardiogenic differentiation potentials [12]. There is a growing body of evidence showing that AT2 receptors are involved in the control of cell proliferation and differentiation in various cell types. For example, previous observations have shown that the AT2 receptor, in contrast to the AT1 receptor, can exert antiproliferative effects and inhibit cell growth in microvascular endothelial cells [23], vascular smooth muscle cells [24], and fibroblasts [25, 26]. Antiproliferation can be a cellular program of its own, for example, in cases where excessive growth induced by growth factors needs to be controlled in the development. On the other hand, growth arrest can also constitute a cellular program subsiding cell differentiation. Indeed, AT2 receptors are most abundant in less differentiated mesenchymal cells of rodent and primate fetuses [1], highlighting their potential role in mesenchymal cell differentiation. In this study, our data further indicate a role of the AT2 receptor in mediating differentiation, but not proliferation of cardiac c-kit+AT2+ cell population. However, it remains to be clarified as to how and whether AT2 receptors direct the differentiation of c-kit+AT2+ cells into cardiomyocytes and/or noncardiomyocytes. Given the fact that the AT2 receptor has a stimulating role in cell migration [27, 28] and MI-induced production of BM c-kit+AT2+ cell pool was observed in the present study, it appeared that AT2 receptor-mediated mobilization/migration of BM c-kit+AT2+ cells into the infarcted myocardium may contribute to an increase in cardiac c-kit+AT2+ cell population following AT2 receptor activation.

Despite previous experimental findings that c-kit+ precursor cells, derived from both BM and adult mammalian heart, can be transdifferentiated into functional cardiomyocytes and help recover injured myocardium [12, 29], there is little evidence supporting cardiac muscle regeneration following BM precursor cell therapy in patients [30, 31]. Recent experimental evidence indicates that an improved cardiac performance is largely independent of transdifferentiation of c-kit+ precursor cells into either cardiac muscle or endothelial cells, but associated with an angiogenic cytokine melieu and neovascularization in the myocardium [16]. Using a coculture system of adult cardiomyocytes and cardiac c-kit+ cells from the infarcted hearts, the present data begin to shed light on an additional role of cardiac c-kit+AT2+ cell population in supporting cardiomyocyte survival. Interesting questions emerging from this study involve the identification of potential survival factors secrected by cardiac c-kit+AT2+ cells or by the interaction of c-kit+ cells and adult cardiomyocytes.

Experimental evidence from AT2 −/− mice or from rats under AT2 receptor blockers suggest that cardiac AT2 receptors exert protective actions in reponse to cardiac ischemic injury [3–5]. It is well established that cardiomyocyte apoptosis remains a major contributor to the loss of myocardium and impaired pump function of the affected heart [32]. Our recent study indicates that antiapoptotic and anti-inflammatory mechanisms are linked to AT2 receptor-mediated cardioprotection [5]. In the present study, we found that AT2 receptor stimulation attenuated apoptosis of cardiomyocytes indirectly by cardiac c-kit+AT2+ cell population, hence unraveling novel aspects of AT2 receptors and offering a plausible explanation for their cardioprotective actions. Support for current findings comes from a recent report showing that AT2 receptor stimulation is required for BM precursor cell-mediated cerebroprotection against ischemia-reperfusion injury in mice [33]. In identifying intracellular signaling pathways that trigger cardiomyocyte survival, we found that in vivo AT2 receptor activation led to phosphorylation of Stat-3 and Akt proteins in cardiomyocytes after MI, which further strengthens the importance of Stat-3 and Akt pathways for the inhibition of apoptosis in affected cardiomyocytes [34, 35]. Given that AT2 receptor mediates anti-inflammatory effects [5] and IL-10 released by BM precursor cells contributes to cardioprotection after experimental MI [36], a novel interpretation of AT2 receptor-mediated cardioprotection seems that AT2 receptor activation may render a favorable local cytokine milieu [37] via cardiac c-kit+AT2+ cell population, triggering Stat-3 and Akt-involved signal pathways, and hence supporting cardiomyocyte performance.

Collectively, this study shows that cardiac c-kit+AT2+ cell population exists and increases after acute ischemic injury. AT2 receptor activation supports cardiomyocyte performance, hence contributing to cardioprotection via cardiac c-kit+AT2+ cell population. The present findings are also clinically relevant because therapeutic strategies involving selective AT2 receptor agonists may help improve adaptive cell differentiation and regeneration effectively during tissue injury/repair events.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

This work was supported in part by a kick-off grant and first grant to J.L. and T.U. from the Berlin-Brandenburg Center for Regenerative Therapies. We thank Melanie Timm for her excellent technical assistance.

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  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

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