The adenosine A2b receptor (Adora2b) has been implicated in cardioprotection from myocardial ischemia. As such, Adora2b was found to be critical in ischemic preconditioning (IP) or ischemia/reperfusion (IR) injury of the heart. Whereas Adora2b is present on various cells types, the tissue-specific role of Adora2b in cardioprotection is still unknown. To study the tissue-specific role of Adora2b signaling on inflammatory cells, endothelia, or myocytes during myocardial ischemia in vivo, we intercrossed floxed Adora2b mice with Lyz2-Cre+, VE-cadherin-Cre+, or myosin-Cre+ transgenic mice, respectively. Mice were exposed to 60 min of myocardial ischemia with or without IP (four times for 5 min) followed by 120 min of reperfusion. Cardioprotection by IP was abolished in Adora2bf/f-VE-cadherin-Cre+ or Adora2bf/f-myosin-Cre+, indicating that Adora2b signaling on endothelia or myocytes mediates IP. In contrast, primarily Adora2b signaling on inflammatory cells was necessary to provide cardioprotection in IR injury, indicated by significantly larger infarcts and higher troponin levels in Adora2bf/f-Lyz2-Cre+ mice only. Cytokine profiling of IR injury in Adora2bf/f-Lyz2-Cre+ mice pointed toward polymorphonuclear neutrophils (PMNs). Analysis of PMNs from Adora2bf/f-Lyz2-Cre+ confirmed PMNs as one source of identified tissue cytokines. Finally, adoptive transfer of Adora2b−/− PMNs revealed a critical role of Adora2b on PMNs in cardioprotection from IR injury. Adora2b signaling mediates different types of cardioprotection in a tissue-specific manner. These findings have implications for the use of Adora2b agonists in the treatment or prevention of myocardial injury by ischemia.

Myocardial infarction (MI) is the leading cause of death worldwide, and according to the World Health Organization, it is responsible for 7.25 million deaths each year. In the United States, about every 44 seconds somebody will have a new heart attack. About 34% of the people who experience a coronary attack in a given year will die of it (1). Despite standard therapies such as early coronary artery reperfusion for the treatment of acute ST elevation MI, morbidity and mortality from MI remain significant. Based on this, the incidence of congestive heart failure continues to increase, and there is a need to provide better therapy that reduces the amount of necrosis that may be coupled with better clinical outcome in the setting of MI (2, 3).

Substantial research efforts have been dedicated to identify agents modulating the inflammatory response after MI, which represent one mechanism in myocardial injury by reperfusion (ischemia/reperfusion [IR] injury). Multiple studies have suggested that adenosine is critical for protection against IR injury (4). The mechanism of adenosine-dependent cardioprotection involves most likely a shift in parenchymal cell metabolism, vasodilatation of coronary arteries, or inhibition of leukocyte-mediated inflammatory responses (4, 5).

Adenosine elicits protective effects through four adenosine receptors (ARs; Adora1, Adora2a, adenosine A2b receptor [Adora2b], and Adora3) (6-10). All ARs have been associated with cardiac tissue protection in different settings. In particular, Adora2b has been implicated in ischemic preconditioning (IP) (11, 12) and postconditioning (13) effects of the heart. Both represent powerful cardioprotective mechanisms where the heart tissue at risk is exposed to short repeated ischemic periods either prior to the onset of ischemia or at the onset of reperfusion (1416).

Although in vivo experiments have shown the cardioprotective effect of Adora2b (11, 13, 1720), these experiments have not dissected the major cellular targets (myocytes, endothelium, or bone marrow–derived cells) responsible for the salutary effect of Adora2b activation in different settings such as cardiac IP or IR injury of the heart.

In the present study, we used state-of-the-art Cre-lox mouse models to generate tissue-specific Adora2b deletion on bone marrow–derived inflammatory cells (Adora2bf/f-Lyz2-Cre+), endothelia (Adora2bf/f-VE-cadherin-Cre+), or myocytes (Adora2bf/f-myosin-Cre+). Exposing those mice to a murine in situ model for IP or IR injury indicated that Adora2b has a differential tissue-specific function in different settings. These findings implicate that tissue-specific targeting of Adora2b seems to be desirable when using Adora2b agonists to prevent or treat myocardial ischemia.

All animal procedures were performed in an American Association for the Accreditation of Laboratory Animal Care–accredited facility in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the University of Colorado Denver Institutional Animal Care and Use Committee. For all studies, we used male mice 8–16 wk old. Studies were in accordance with the National Institutes of Health guidelines for use of live animals. C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Adora2b-floxed (Adora2bf/f) mice were generated by Ozgene (Perth, Australia). Lyz2-Cre+ (B6.129P2-Lyz2tm1(cre)Ifo/J) (21), VE-cadherin-Cre+ (B6.Cg-Tg(Cdh5-cre)7Mlia/J) (22), and tamoxifen-inducible myosin-Cre+ (Tg(Myh6-cre/Esr1*)1Jmk/J) (23) mice were purchased from The Jackson Laboratory. To obtain tissue-specific Adora2b−/− mice, we crossbred Adora2bf/f (Ozgene) mice with the appropriate tissue-specific Cre recombinase mouse. For studies using myosin-Cre+ mice, all mice were induced by treatment with tamoxifen (1 mg/d i.p.) dissolved in peanut oil for 5 d. Experiments were performed after an additional 14 d following tamoxifen administration. Genotyping PCRs for tissue-specific animals were performed by GeneTyper (New York, NY).

Eight- to twelve-week-old myosin-Cre+ or Adora2bf/f -myosin-Cre+ mice were anesthetized and the heart was quickly removed from the chest cavity and immediately placed in ice-cold Krebs–Henseleit buffer (KHB). After weighing, the aorta was cannulated and the heart was perfused with Ca2+-free KHB for 3 min followed by 8–12 min perfusion with Ca2+-free KHB containing collagenase and elastase. After perfusion, ventricles were removed, minced, and incubated with the collagenase/elastase solution for an additional 3–7 min. The cells were filtered through a nylon mesh (300 μm) and collected in a 15-ml sterile tube. Myocytes were washed and calcium was slowly reintroduced in a stepwise fashion. Finally, cells were resuspended in MEM and plated on laminin. Cells were harvested the next day and immediately resuspended in TRIzol for mRNA analysis (19).

Heart tissue from Lyz2-Cre+ or Adora2bf/f -Lyz2-Cre+ mice was minced and digested for 45 min using collagenase solution. Myeloid cells were isolated using EasySep CD11b-PE positive selection (StemCell Technologies), according to the manufacturer’s instructions. After trypan blue staining to confirm cell viability, cells were immediately resuspended in TRIzol for mRNA analysis.

Heart tissue from VE-cadherin-Cre+ or Adora2bf/f -VE-cadherin-Cre+ mice was minced and digested for 45 min using collagenase solution. Endothelial cells were isolated using EasySep CD31-biotin positive selection (StemCell Technologies), according to the manufacturer’s instructions. After trypan blue staining to confirm cell viability, cells were immediately resuspended in TRIzol for mRNA analysis.

Total RNA was isolated from cells using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, 15596-018). For this purpose, cells were homogenized in the presence of TRIzol reagent and chloroform was added. After spinning at 12,000 × g for 15 min, the aqueous phase was removed and the RNA was precipitated with isopropanol. RNA was pelleted, washed with ethanol, treated with DNase, and the concentration was quantified. The PCR reactions contained a mix of forward and reverse oligonucleotides with SYBR Green (Bio-Rad Laboratories, 170-8880). Each target sequence was amplified using increasing numbers of cycles of 94°C for 1 min, 58°C for 0.5 min, and 72°C for 1 min. Quantification of transcript levels was determined by real-time RT-PCR (iCycler; Bio-Rad Laboratories). The primers were QuantiTect from Qiagen (Mm_Adora2b, QT01543444).

Anesthesia was induced (70 mg/kg i.p.) and maintained (10 mg/kg/h i.p.) with sodium pentobarbital. Mice were placed on a temperature-controlled heated table (RT, Effenberg, Munich, Germany) with a rectal thermometer probe to maintain body temperature at 37°C. The tracheal tube was connected to a ventilator (Servo 900C, DRE Medical, Louisville, KY). Animals were ventilated with a pressure-controlled ventilation mode (pressure of 10 mbar, frequency of 110 breaths/min, positive end-expiratory pressure of 5 mbar, FiO2 of 0.4). After induction of anesthesia, animals were monitored with a surface electrocardiogram (Hewlett-Packard, DRE Medical). Fluid replacement was performed with normal saline (0.2 ml/h intra-arterially) until the onset of reperfusion, and with 1 ml/h intra-arterially during reperfusion. Operations were performed under an upright dissecting microscope (Olympus SZX12). Following left anterior thoracotomy, exposure of the heart, and dissection of the pericardium, the left coronary artery (LCA) was visually identified and an 8.0 nylon suture (Prolene, Ethicon, Somerville, NJ) was placed around the vessel. Atraumatic LCA occlusion for ischemia and IP studies was performed using a hanging weight system (24, 25). Successful LCA occlusion was confirmed by an immediate color change of the vessel from light red to dark violet, and of the myocardium supplied by the vessel from bright red to white (pale), as well as the immediate occurrence of ST elevations in the electrocardiogram.

During reperfusion, the changes of color immediately disappeared when the hanging weights were lifted and the LCA was perfused again. Infarct sizes were determined by calculating the percentage of myocardium that underwent infarction compared with the area at risk (AAR) using a previously described double staining technique with Evans blue and triphenyl-tetrazolium chloride (TTC). AAR and the infarct size were determined via planimetry using the National Institutes of Health software Image 1.0, and the degree of myocardial damage was calculated as the percentage of infarcted myocardium from the AAR (12, 18).

Blood was collected by central venous puncture for cardiac troponin I (cTnI) measurements using a quantitative rapid cTnI assay (Life Diagnostics, West Chester, PA) (1820, 25, 26).

To localize the Adora2b in heart tissue, we analyzed β-galactosidase (β-gal) expression in Adora2b-knockout (KO)/β-gal knock-in mice (27). Hearts were harvested following perfusion fixation in 4% paraformaldehyde/0.1 M phosphate buffer without saline and postfixed in the same fixative for 3 h, followed by cryoprotection in 20% sucrose in 0.1 M phosphate buffer without saline (pH 7.2) overnight at 4°C. Using a cryostat, 16-μm sections were collected onto Superfrost Plus slides (Fisher Scientific). The slides underwent three 10-min washes in 0.1 M PBS before incubation in blocking solution (2% normal goat serum, 1% BSA, 0.3% Triton X-100 in PBS) for 1 h at room temperature. The samples were then incubated overnight at 4°C in guinea pig anti–β-gal (1:1000, Ab was a gift from Prof. Thomas E. Finger at the University of Colorado Denver) diluted in blocking solution. To stain for macrophages and determine whether they colocalize with β-gal staining, the goat anti-rat F4/80 (1:100) (Serotec, Oxford, U.K.) was coincubated with the anti–β-gal Ab diluted in blocking solution. Following three washes in PBS, samples were incubated for 2 h at room temperature with Alexa Fluor 594 goat anti-guinea pig (1:400) (Invitrogen) and Alexa Fluor 488 goat anti-rat (1:400) (Invitrogen) diluted in blocking buffer. Slides were then washed twice in PBS, followed by a 10-min wash in 0.1 M phosphate buffer without saline and coverslipped using Vectashield containing DAPI (Vector Laboratories). Immunofluorescent images were taken using a Zeiss 780 LSM.

To measure cytokine tissue levels after 60 min of ischemia, Lyz2-Cre+ or Adora2f/f-Lyz2-Cre+ mice were euthanized following 120 min of reperfusion. Remaining blood was removed and the myocardial tissue (AAR) was excised after delineation with Evans blue and immediately frozen with liquid nitrogen and stored at −80°C. Tissues were homogenized on ice using a Tissue Master 125 (OMNI International) in T-PER (Thermo Scientific, 78510) containing Pierce protease inhibitors according to the manufacturers’ recommendations (Thermo Scientific, 88665). After spinning at 10,000 RPM for 5 min, the supernatant was removed and diluted to a final concentration of 10 μg/100 μl per well. The cytokine ELISA array (Signosis, EA-4005) was performed according to the manufacturer’s instructions.

Polymorphonuclear neutrophils (PMNs) were harvested from bone marrow according to the product manual for preparing a single-cell suspension (Stemcell Technologies, 19762A). The PMNs were subsequently isolated from the bone marrow according to the EasySep protocol (Stemcell Technologies, 19762A) using the purple EasySep magnet (Stemcell Technologies, 18000). The isolated PMNs were transferred immediately to ice to avoid activation. Total RNA was isolated from PMNs using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, 15596-018). For this purpose, liquid nitrogen–frozen cells were homogenized in the presence of TRIzol reagent and chloroform was added. After spinning at 12,000 × g for 15 min, the aqueous phase was removed and the RNA was precipitated with isopropanol. RNA was pelleted, washed with ethanol, treated with DNAse, and the concentration was quantified. The PCR reactions contained a mix of forward and reverse oligonucleotides with SYBR Green (Bio-Rad, 170-8880). Each target sequence was amplified using increasing numbers of cycles of 94°C for 1 min, 58°C for 0.5 min, and 72°C for 1 min. Quantification of transcript levels was determined by real-time RT-PCR (iCycler; Bio-Rad Laboratories). The primers were Quantitect from Qiagen (TNF-α, QT00104006; IL-6, QT00098875).

Cardiac protein carbonyl was measured using OxiSelect protein carbonyl ELISA kit (Cell Biolabs, San Diego, CA).

Mice were treated with a Ly6G-specific mAb 1A8 (Bio X Cell) (28). This depletes circulating neutrophils but does not affect circulating GR-1+ monocytes, as described previously (28). In a subset of experiments, mice were treated with an anti–G-CSF Ab (PeproTech) that prevents recruitment of endogenous PMNs (29). To perform an adoptive transfer of PMNs into PMN-depleted animals, we first euthanized donor wild-type (WT) mice (C57BL/6J, 6–8 wk) and harvested the bone marrow by flushing the femoral bones. PMNs were separated by negative selection using an EasySep mouse neutrophil enrichment kit (Stemcell Technologies) and counted by a hemocytometer. Then, 1 × 106 cells were injected into the neutrophil-depleted mice via an arterial catheter during 10 min. After a waiting period of 60 min, the mice underwent myocardial ischemia and reperfusion injury as described above.

Data were compared by a Student t test where appropriate. Values are expressed as means ± SD from three to six animals per condition. The chosen numbers of animals per group were based on findings in previous studies and subsequent sample size analyses. The studies are designed to be able to reject the null hypothesis that the population means of the experimental and control groups are equal with probability (power) of 0.8. The type I error probability associated with this test of this null hypothesis is 0.05. Data are expressed as means ± SD. A p value <0.05 was considered statistically significant. For all statistical analyses, GraphPad Prism 5.0 software for Windows was used.

The experimental protocols are displayed in Fig. 1A. For myocardial ischemia and reperfusion injury, we used 60 min of ischemia followed by 120 min of reperfusion (Fig. 1A, model 1). For IP, we performed four cycles of 5 min of ischemia and 5 min of reperfusion prior to 60 min of ischemia and 120 min of reperfusion (Fig. 1A, model 2). After reperfusion, we visualized the infarcted area using Evans blue and TCC. Additionally, we determined troponin I from serum samples.

FIGURE 1.

Overview of the experimental protocol and tissue-specific Adora2b-deficient mice. (A) Experimental protocols used. Model 1: Myocardial ischemia consisted of 60 min of ischemia followed by 2 h of reperfusion. Model 2: For IP studies, four cycles of 5 min of ischemia and 5 min of reperfusion prior to the onset of myocardial ischemia were performed. (B) Overview of tissue-specific Adora2b deletion using a Cre-lox system. (C) Genotyping data. Tissue was collected from the organs as labeled and genotyped using conventional PCR (GeneTyper). Genotyping was analyzed for Adora2b floxed (lox), Cre recombinase (Cre), Adora2b WT, and for the Adora2b KO signal. Left: Representative PCR results from heart or aortic tissue from Adora2bf/f -myosin-Cre+ mice (cardiomyocyte specific); note the positive KO band in cardiac tissue, in contrast to only WT band present in the aortic tissue. Middle: Representative PCR result from Adora2bf/f-VE-cadherin-Cre+ mice (endothelial specific); note the positive KO signal in heart and aorta. Right: Representative PCR results from Adora2bf/f-Lyz2-Cre+ mice (bone marrow–derived cell specific); note the positive KO and WT band in heart and aorta. The Cre signal for myosin-Cre+ or VE-cadherin-Cre+ mice was different from the signal from Lyz2-Cre+ mice, as a different PCR strategy was used (according to The Jackson Laboratory protocols): Adora2b flox signal, 200 bp; Adora2b KO signal, 366 bp, Adora2b WT signal, 1600 bp; myosin-Cre+ and VE-cadherin-Cre+ signals, double band at 270 and 497 bp; Lyz2-Cre+ signal, 260 bp; ladder used is depicted to the right. (DF) RT-PCR data showing Adora2b mRNA transcript levels from the respective tissues of the tissue-specific Adora2b-deleted mice. (D) Cardiomyocytes were isolated from myosin-Cre+ and Adora2bf/f -myosin-Cre+ mice and cultured overnight. (E) Endothelia and (F) myeloid cells were isolated via positive selection (CD31+ for endothelia from VE-cadherin-Cre+/Adora2bf/f -VE-cadherin-Cre+ hearts and CD11b+ for myeloid cells from Lyz2-Cre+/Adora2bf/f -Lyz2-Cre+ hearts) using magnetic beads (EasySep), and bone marrow cells were isolated from the Lyz2-Cre and Adora2bf/f -Lyz2-Cre femurs.

FIGURE 1.

Overview of the experimental protocol and tissue-specific Adora2b-deficient mice. (A) Experimental protocols used. Model 1: Myocardial ischemia consisted of 60 min of ischemia followed by 2 h of reperfusion. Model 2: For IP studies, four cycles of 5 min of ischemia and 5 min of reperfusion prior to the onset of myocardial ischemia were performed. (B) Overview of tissue-specific Adora2b deletion using a Cre-lox system. (C) Genotyping data. Tissue was collected from the organs as labeled and genotyped using conventional PCR (GeneTyper). Genotyping was analyzed for Adora2b floxed (lox), Cre recombinase (Cre), Adora2b WT, and for the Adora2b KO signal. Left: Representative PCR results from heart or aortic tissue from Adora2bf/f -myosin-Cre+ mice (cardiomyocyte specific); note the positive KO band in cardiac tissue, in contrast to only WT band present in the aortic tissue. Middle: Representative PCR result from Adora2bf/f-VE-cadherin-Cre+ mice (endothelial specific); note the positive KO signal in heart and aorta. Right: Representative PCR results from Adora2bf/f-Lyz2-Cre+ mice (bone marrow–derived cell specific); note the positive KO and WT band in heart and aorta. The Cre signal for myosin-Cre+ or VE-cadherin-Cre+ mice was different from the signal from Lyz2-Cre+ mice, as a different PCR strategy was used (according to The Jackson Laboratory protocols): Adora2b flox signal, 200 bp; Adora2b KO signal, 366 bp, Adora2b WT signal, 1600 bp; myosin-Cre+ and VE-cadherin-Cre+ signals, double band at 270 and 497 bp; Lyz2-Cre+ signal, 260 bp; ladder used is depicted to the right. (DF) RT-PCR data showing Adora2b mRNA transcript levels from the respective tissues of the tissue-specific Adora2b-deleted mice. (D) Cardiomyocytes were isolated from myosin-Cre+ and Adora2bf/f -myosin-Cre+ mice and cultured overnight. (E) Endothelia and (F) myeloid cells were isolated via positive selection (CD31+ for endothelia from VE-cadherin-Cre+/Adora2bf/f -VE-cadherin-Cre+ hearts and CD11b+ for myeloid cells from Lyz2-Cre+/Adora2bf/f -Lyz2-Cre+ hearts) using magnetic beads (EasySep), and bone marrow cells were isolated from the Lyz2-Cre and Adora2bf/f -Lyz2-Cre femurs.

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To understand the tissue-specific contribution of Adora2b in cardioprotection, we used state-of-the-art Cre-lox mouse models. Fig. 1B displays the different Adora2b tissue-specific mouse models that were used in the present studies. For Adora2b deletion on cardiomyocytes we generated Adora2bf/f-myosin-Cre+ mice, for deletion on endothelial cells we used Adora2bf/f-VE-cadherin-Cre+ mice, and for a bone marrow–derived cell deletion we used Adora2bf/f-Lys2-Cre+ mice (Fig. 1B). The myosin-Cre+ mice have an inducible Cre-recombinase (Fig. 1B, upper panel), and therefore the Adora2bf/f-myosin-Cre+ and myosin-Cre+ mice received 1 mg tamoxifen i.p. for 5 d to induce Cre recombinase activity. The other Cre strains (VE-cadherin-Cre+, Lyz2-Cre+) express constitutively Cre recombinase and therefore no induction was necessary.

As shown in Fig. 1C, the tissue-specific deletion of the Adora2b in the different tissue-specific mouse models was confirmed using a genotyping PCR analysis from hearts and aortas to understand how the cardiovascular system was affected. Adora2b floxed (lox), Cre recombinase (Cre), Adora2b KO, or Adora2b WT signal is depicted in each case. PCR analysis confirmed that all tissue-specific strains were positive for Cre recombinase and the floxed Adora2b gene. In Adora2bf/f-myosin-Cre+ mice we found, as expected, a KO signal in the heart but a WT signal in the aorta. In Adora2bf/f-VE-cadherin-Cre+ mice, we found a KO signal in the heart and the aorta, as both tissues are abundant in endothelial cells. Adora2bf/f-Lys2-Cre+ mice had a heterozygous signal in the heart and the aorta (KO/WT signal in heart and aorta), probably as a result of resident inflammatory cells in these tissues. To analyze the efficiency of Adora2b gene deletion in the respective tissues of the tissue-specific mice, we isolated mRNA from the cells in question and determined Adora2b transcript levels using real time RT-PCR. Cardiomyocytes were isolated and cultured overnight from Adora2bf/f-myosin-Cre+ or myosin-Cre+ mice. Endothelial cells were isolated from Adora2bf/f-VE-cadherin-Cre+ or VE-cadherin-Cre+ hearts. Myeloid cells or bone marrow cells were isolated from Adora2bf/f-Lyz2-Cre+ or Lyz2-Cre+ hearts or femurs, respectively. As shown in Fig. 1D–F, Adora2b mRNA was significantly depleted in the different cell compartments from our tissue-specific Adora2b-deleted mice. In summary, we generated three state-of-the-art Cre-lox mouse models to assess the tissue-specific contribution of Adora2b to IP or myocardial IR injury.

Based on earlier studies using germline Adora2b−/− mice, Adora2b is crucial for the cardioprotective effects of IP (12). To gain further insight into a tissue-specific function of Adora2b, we recapitulated what cell types express Adora2b in the heart. To obtain a very specific expression profile of the Adora2b receptor in the mouse heart tissue, we used an Adora2b KO/β-gal knock-in reporter mouse (27). As shown in Fig. 2A–C, β-gal staining (green) of representative heart sections from an Adora2b–β-gal reporter mouse revealed dominantly Adora2b+ cells in the vessel walls at baseline and a strong and significant upregulation of Adora2b on endothelia and cardiomyocytes upon IP or IR injury. Although some resident macrophages were detected in the heart (red staining, Fig. 2D, 2E), none coexpressed Adora2b (Fig. 2F). Indeed, expression could be different in circulating versus tissue-resident macrophages (27). Additionally, only IR treatment slightly increased the number of resident macrophages (Fig. 2F). Based on the fact that IP consists of short nonlethal repeated ischemic periods prior to ischemia, and based on our expression profile of cardiac Adora2b receptors, we hypothesized that bone marrow–derived inflammatory cells do not play an important role during IP of the heart. To test this hypothesis, we used Adora2bf/f-Lyz2-Cre+ mice and their respective controls (Lyz2-Cre+) and exposed them to 60 min of ischemia with IP (+IP; four cycles of 5 min of ischemia followed my 5 min of reperfusion) or without IP (−IP) followed by 120 min of reperfusion. Infarct sizes were measured by double staining using Evans blue and TTC, whereas serum troponin I was determined using ELISA. As shown in Fig. 3A and 3C, IP significantly decreased infarct sizes from 39.3 ± 5.5 (n = 5) to 22.4 ± 7.5% (n = 4) and troponin I serum levels from 179.6 ± 65.1 to 68.7 ± 72.7 ng/ml (n = 6/group) in control (Lyz2-Cre+) mice. However, studies of Adora2bf/f-Lyz2-Cre+ mice (Fig. 3B, 3C) also showed significantly decreased infarct sizes from 73.1 ± 3.0 to 48.8 ± 10.2% (n = 5/group) and troponin I serum levels from 396.0 ± 154.4 to 133.9 ± 86.5 ng/ml (n = 6/group) when hearts had been pretreated with IP prior to ischemia.

FIGURE 2.

Effect of IP or IR injury on Adora2b+ and F4/80+ cell expression. (AC) β-gal staining of representative sections of an Adora2b–β-gal reporter mouse at baseline (A), after IP (B), or after IR injury (C). The green stain shows β-gal positivity, indicating Adora2b promoter activation. Note that Adora2b+ cells are dominantly located in the vessel wall at baseline, whereas IP or IR injury significantly upregulates Adora2b gene promoter activity on endothelia but also on myocardial cells. (DF) F4/80 staining (red) at baseline (D), after IP (E), or after IR injury (E and F). Note that whereas F4/80+ cells (indicated by white arrows) are rarely found at baseline or after IP, a slight increase of F4/80+ cells was found after IR injury (F). Also note that no β-gal staining was observed on residential macrophages (F). The green stain shows Adora2b, the red stain shows macrophages, and the blue stain (DAPI) shows nuclei.

FIGURE 2.

Effect of IP or IR injury on Adora2b+ and F4/80+ cell expression. (AC) β-gal staining of representative sections of an Adora2b–β-gal reporter mouse at baseline (A), after IP (B), or after IR injury (C). The green stain shows β-gal positivity, indicating Adora2b promoter activation. Note that Adora2b+ cells are dominantly located in the vessel wall at baseline, whereas IP or IR injury significantly upregulates Adora2b gene promoter activity on endothelia but also on myocardial cells. (DF) F4/80 staining (red) at baseline (D), after IP (E), or after IR injury (E and F). Note that whereas F4/80+ cells (indicated by white arrows) are rarely found at baseline or after IP, a slight increase of F4/80+ cells was found after IR injury (F). Also note that no β-gal staining was observed on residential macrophages (F). The green stain shows Adora2b, the red stain shows macrophages, and the blue stain (DAPI) shows nuclei.

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FIGURE 3.

Effect of IP on myocardial injury in bone marrow–derived cell- and endothelial-specific Adora2b-deficient mice. (AF) Mice underwent 60 min of ischemia with IP (+IP; four cycles of 5 min of ischemia followed by 5 min of reperfusion) or without IP (−IP) followed by 120 min of reperfusion. Infarct sizes were measured by double staining with Evans blue and TTC. Infarct sizes are expressed as the percentage of the AAR that underwent infarction. Serum troponin I concentrations were measured by ELISA. (A and B) Infarct sizes and serum troponin I levels in Lyz2-Cre+ (controls) or Adora2bf/f-Lyz2-Cre+ mice with and without IP. (C) Representative infarct staining from Lyz2Cre (controls) or Adora2bf/f-Lyz2-Cre+ mice (n = 5–6, ± SD). (D and E) Infarct sizes and serum troponin I levels in VE-cadherin-Cre+ (controls) and Adora2bf/f-VE-cadherin-Cre+ mice with and without IP. (F) Representative infarct staining from VE-cadherin-Cre+ (controls) and Adora2bf/f-VE-cadherin-Cre+ mice (n = 5–6, ± SD).

FIGURE 3.

Effect of IP on myocardial injury in bone marrow–derived cell- and endothelial-specific Adora2b-deficient mice. (AF) Mice underwent 60 min of ischemia with IP (+IP; four cycles of 5 min of ischemia followed by 5 min of reperfusion) or without IP (−IP) followed by 120 min of reperfusion. Infarct sizes were measured by double staining with Evans blue and TTC. Infarct sizes are expressed as the percentage of the AAR that underwent infarction. Serum troponin I concentrations were measured by ELISA. (A and B) Infarct sizes and serum troponin I levels in Lyz2-Cre+ (controls) or Adora2bf/f-Lyz2-Cre+ mice with and without IP. (C) Representative infarct staining from Lyz2Cre (controls) or Adora2bf/f-Lyz2-Cre+ mice (n = 5–6, ± SD). (D and E) Infarct sizes and serum troponin I levels in VE-cadherin-Cre+ (controls) and Adora2bf/f-VE-cadherin-Cre+ mice with and without IP. (F) Representative infarct staining from VE-cadherin-Cre+ (controls) and Adora2bf/f-VE-cadherin-Cre+ mice (n = 5–6, ± SD).

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Taken together, these data are in support of our initial hypothesis that Adora2b receptors on bone marrow–derived inflammatory cells do not play an important role in mediating cardioprotection by IP. Because within the heart, Adora2b is mainly expressed in the vessel wall and to some extent also in cardiomyocytes, those tissues might be important for cardioprotection by IP.

Given that there is no apparent role for Adora2b on bone marrow–derived inflammatory cells during cardiac IP, we next investigated the role of the endothelia or myocytes. First, we used our endothelial-specific Adora2b-deficient mice (Adora2bf/f-VE-cadherin-Cre+) and appropriate controls (VE-cadherin-Cre+). As shown in Fig. 3D and 3F, IP significantly decreased infarct sizes from 45.9 ± 8.4 to 26.5 ± 9.6% (n = 5/group) and troponin I serum levels from 178.6 ± 92.4 to 58.2 ± 63.6 ng/ml (n = 6/group) in VE-cadherin-Cre+ mice. In contrast, IP had no significant effect on infarct sizes (−IP, 54.5 ± 8.9% versus +IP, 46.7 ± 12.7%; n = 6/group) or troponin I serum levels (−IP, 219.9 ± 162.1 ng/ml versus +IP, 139.6 ± 36.3 ng/ml; n = 6/group) in Adora2bf/f-VE-cadherin-Cre+, as shown in Fig. 3E and 3F.

Next we analyzed the contribution of the Adora2b on cardiomyocytes as shown in Fig. 4A–D. In this study, we found that although IP significantly decreased infarct sizes from 45.9 ± 3.1 (n = 5) to 20.4 ± 5.1% (n = 4) and troponin I serum levels from 223.1 ± 103.9 to 34.9 ± 17.7 ng/ml (n = 6/group) in control mice (myosin-Cre+), IP in cardiomyocyte-specific Adora2b-deficient mice (Adora2bf/f-myosin-Cre+) significantly increased infarct sizes from 47.5 ± 6.8 to 62.1 ± 4.7% (n = 5/group) or troponin I serum levels from 188.2 ± 55.3 to 327.5 ± 109.3 ng/ml (n = 6/group). As myosin-Cre+ controls had been pretreated with tamoxifen and this could have anti-inflammatory effects (30), we performed infarct size studies in myosin-Cre+ mice without tamoxifen treatment. As shown in Fig. 4E, no significant differences in infarct sizes were observed between tamoxifen-treated and untreated myosin-Cre+ mice.

FIGURE 4.

Effect of IP on myocardial injury in cardiomyocyte-specific Adora2b-deficient mice. (AE) Mice underwent 60 min of ischemia with IP (+IP; four cycles of 5 min of ischemia followed by 5 min of reperfusion) or without IP (−IP) followed by 120 min of reperfusion. Infarct sizes were measured by double staining with Evans blue and TTC. Infarct sizes are expressed as the percentage of the AAR that underwent infarction. Serum troponin I concentrations were measured by ELISA. (A and B) Infarct sizes and serum troponin I levels in myosin-Cre+ (controls) and Adora2bf/f-myosin-Cre+ mice with and without IP. (C and D) Representative infarct staining from myosin-Cre+ (controls) and Adora2bf/f-myosin-Cre+ mice. (E) Infarct sizes in myosin-Cre+ (controls) with and without tamoxifen pretreatment (n = 3–6, ± SD).

FIGURE 4.

Effect of IP on myocardial injury in cardiomyocyte-specific Adora2b-deficient mice. (AE) Mice underwent 60 min of ischemia with IP (+IP; four cycles of 5 min of ischemia followed by 5 min of reperfusion) or without IP (−IP) followed by 120 min of reperfusion. Infarct sizes were measured by double staining with Evans blue and TTC. Infarct sizes are expressed as the percentage of the AAR that underwent infarction. Serum troponin I concentrations were measured by ELISA. (A and B) Infarct sizes and serum troponin I levels in myosin-Cre+ (controls) and Adora2bf/f-myosin-Cre+ mice with and without IP. (C and D) Representative infarct staining from myosin-Cre+ (controls) and Adora2bf/f-myosin-Cre+ mice. (E) Infarct sizes in myosin-Cre+ (controls) with and without tamoxifen pretreatment (n = 3–6, ± SD).

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Taken together, these data show an important role of the Adora2b on endothelial cells and myocytes in mediating cardioprotection by IP.

After we found an important role of the Adora2b on myocytes and endothelia in mediating cardioprotection from ischemia by IP, we next analyzed their role in IR injury. Surprisingly, as shown in Fig. 5, infarct sizes and troponin I serum values were unchanged between control mice and mice with a tissue-specific Adora2b deletion on endothelia or myocytes (VE-cadherin-Cre+: infarct sizes, 54.4 ± 8.8%, cTnI, 219.9 ± 162.1 ng/ml versus Adora2bf/f-VE-cadherin-Cre+: infarct sizes, 45.9 ± 8.4%, cTnI, 178.60 ± 92.4 ng/ml; n = 6/group or myosin-Cre+: infarct sizes, 45.9 ± 3.1%, cTnI, 223.1 ± 103.9 ng/ml versus Adora2bf/f-myosin-Cre+: infarct sizes, 47.5 ± 6.8%, cTnI, 188.2 ± 55.3 ng/ml; n = 6/group). Taken together, these data indicate that Adora2b-expressing cardiac cells such as endothelia or myocytes do not play a major role in cardiac IR injury.

FIGURE 5.

Effect of myocardial IR injury on myocardial damage in endothelial- or cardiomyocyte-specific Adora2b-deficient mice. (A and B) Mice underwent 60 min of ischemia and 120 min of reperfusion. Infarct sizes were measured by double staining with Evans blue and TTC. Infarct sizes are expressed as the percentage of the AAR that underwent infarction. Serum troponin I concentrations were measured by ELISA. (A) Infarct sizes and serum troponin I levels in VE-cadherin-Cre+ (controls) and Adora2bf/f-VE-cadherin-Cre+ mice with representative infarct staining. Note that data in (A) are from Fig. 3D–F to display and analyze IR injury. (B) Infarct sizes and serum troponin I levels in myosin-Cre+ (controls) and Adora2bf/f-myosin-Cre+ mice with representative infarct staining (middle) (n = 6, ± SD). Note that data in (B) are from Fig. 4A–D to display and analyze IR injury.

FIGURE 5.

Effect of myocardial IR injury on myocardial damage in endothelial- or cardiomyocyte-specific Adora2b-deficient mice. (A and B) Mice underwent 60 min of ischemia and 120 min of reperfusion. Infarct sizes were measured by double staining with Evans blue and TTC. Infarct sizes are expressed as the percentage of the AAR that underwent infarction. Serum troponin I concentrations were measured by ELISA. (A) Infarct sizes and serum troponin I levels in VE-cadherin-Cre+ (controls) and Adora2bf/f-VE-cadherin-Cre+ mice with representative infarct staining. Note that data in (A) are from Fig. 3D–F to display and analyze IR injury. (B) Infarct sizes and serum troponin I levels in myosin-Cre+ (controls) and Adora2bf/f-myosin-Cre+ mice with representative infarct staining (middle) (n = 6, ± SD). Note that data in (B) are from Fig. 4A–D to display and analyze IR injury.

Close modal

After we discovered that mice with a tissue-specific deletion of the Adora2b on endothelia or myocytes do not have increased IR injury when compared with their respective controls, we next analyzed mice with an Adora2b deletion on bone marrow–derived inflammatory cells, which include macrophages, monocytes, and PMNs. As shown in Fig. 6A, Adora2bf/f-Lyz2-Cre+ mice have increased infarct sizes when compared with Lyz2-Cre+ mice (Lyz2-Cre+, 39.2 ± 5.5% versus Adora2bf/f-Lyz2-Cre+, 73.1 ± 3.0%) and troponin I serum levels (Lyz2-Cre+, 179.6 ± 65.1 ng/ml versus Adora2bf/f-Lyz2-Cre+, 396.0 ± 154.4 ng/ml; n = 6/group). To identify potential cell-specific cytokines responsible for the increased damage within the heart tissue during IR injury, we performed a cytokine screen by using a multiplex ELISA. We found that Adora2bf/f-Lyz2-Cre+ mice had higher levels of TNF-α or IL-6 in the AAR after 60 min of ischemia and 120 min of reperfusion. Additionally, lower levels of G-CSF, stem cell factor, IL-10, and resistin were found in Adora2bf/f-Lyz2-Cre+ mice when compared to controls (n = 2/group; Fig. 6B). Based on the cytokine profile, we hypothesized that PMNs could be the major source of these cytokine changes (17). Whereas Lyz2-Cre+ mice affect other bone marrow–derived cells such as macrophages, they have been found to be particularly effective in deleting gene targets on PMNs in a tissue-specific manner (31). We therefore isolated PMNs from Lyz2-Cre+ or Adora2bf/f-Lyz2-Cre+ mice and determined transcript levels of IL-6 or TNF-α at baseline (Fig. 6C). RT-PCR studies revealed that IL-6 baseline transcript values were increased by 3.8 ± 0.3, and TNF-α baseline transcript values were increased by 4.2 ± 0.2-fold in Adora2bf/f-Lyz2-Cre+ mice when compared with controls (Lyz2-Cre+). Values were normalized to the housekeeping gene β-actin (n = 6). Based on the proinflammatory phenotype in Adora2bf/f-Lyz2-Cre+ mice, we next investigated whether Adora2bf/f-Lyz2-Cre+ mice could also have more superoxide production (reactive oxygen species [ROS]) during IR. As shown in Fig. 6D, IR significantly increased ROS levels in the ischemic area of control mice. Interestingly, Adora2bf/f-Lyz2-Cre+ mice had already increased ROS levels at baseline when compared with control animals. This is consistent with earlier studies showing an augmented proinflammatory phenotype in Adora2b−/− mice in conjunction with enhanced leukocyte rolling at baseline (27). Taken together, these data indicate an important anti-inflammatory role of the Adora2b in IR injury.

FIGURE 6.

Myocardial IR injury in bone marrow–specific Adora2b-deficient mice. (A) Mice underwent 60 min of ischemia and 120 min of reperfusion. Infarct sizes were measured by double staining with Evans blue and TTC. Infarct sizes are expressed as the percentage of the AAR that underwent infarction. Infarct sizes in Lyz2-Cre+ (controls) or Adora2bf/f-Lyz2-Cre+ with representative infarct staining are shown. Serum troponin I concentrations were measured by ELISA. Note that data in (A) are from Fig. 3A–C rearranged to display IR injury. (B) Multiplex ELISA from the AAR after 60 min of ischemia and 120 min of reperfusion comparing Lyz2-Cre+ (controls) and Adora2bf/f-Lyz2-Cre+ mice. (C) Isolated PMNs from Lyz2-Cre+ (controls) or Adora2bf/f-Lyz2-Cre+ mice were analyzed for IL-6 or TNF-α transcript levels (n = 5–6, ± SD). (D) Cardiac protein carbonyl, as indicator of ROS production, was measured using an OxiSelect protein carbonyl ELISA kit in control or ischemic heart tissue from Lyz2-Cre+ (controls) or Adora2bf/f-Lyz2-Cre+ mice.

FIGURE 6.

Myocardial IR injury in bone marrow–specific Adora2b-deficient mice. (A) Mice underwent 60 min of ischemia and 120 min of reperfusion. Infarct sizes were measured by double staining with Evans blue and TTC. Infarct sizes are expressed as the percentage of the AAR that underwent infarction. Infarct sizes in Lyz2-Cre+ (controls) or Adora2bf/f-Lyz2-Cre+ with representative infarct staining are shown. Serum troponin I concentrations were measured by ELISA. Note that data in (A) are from Fig. 3A–C rearranged to display IR injury. (B) Multiplex ELISA from the AAR after 60 min of ischemia and 120 min of reperfusion comparing Lyz2-Cre+ (controls) and Adora2bf/f-Lyz2-Cre+ mice. (C) Isolated PMNs from Lyz2-Cre+ (controls) or Adora2bf/f-Lyz2-Cre+ mice were analyzed for IL-6 or TNF-α transcript levels (n = 5–6, ± SD). (D) Cardiac protein carbonyl, as indicator of ROS production, was measured using an OxiSelect protein carbonyl ELISA kit in control or ischemic heart tissue from Lyz2-Cre+ (controls) or Adora2bf/f-Lyz2-Cre+ mice.

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To further understand the role for Adora2b on PMN cells in cardioprotection from IR injury, we next tested whether adoptive transfer of Adora2b−/− PMNs into PMN-depleted animals would increase infarct sizes when compared with a transfer of WT PMNs (see details of experimentally setup in Fig. 7A). First, we treated WT animals with 1A8 Ly6G-specific Abs 24 h prior to the experiment. This treatment leads to a significant reduction of peripheral PMNs but not circulation monocytes in mice (28). On the day of experiment, we isolated PMNs from WT or Adora2b−/− bone marrow and reinfused these cells via a carotid catheter prior to the onset of myocardial ischemia. As seen in Fig. 7B, Adora2b−/− PMN infusion significantly increased infarct sizes when compared with the WT PMN infusion (41.2 ± 6.1 [WT] versus 57.5 ± 7.2% [Adora2b−/−]; n = 4/group). To control for possible effects of the 1A8 Ly6G-specific Ab on transferred PMNs, we repeated the experiment with an anti–G-CSF Ab (Fig. 7C). This Ab prevents the recruitment of endogenous PMNs but does not interfere with transferred PMNs (29). As shown in Fig. 7D, a similar infarct size was observed with transferred Adora2b−/− PMNs. Therefore, in contrast to IP, the Adora2b on bone marrow–derived inflammatory cells, seemingly PMNs, represents an important therapeutic target during IR injury.

FIGURE 7.

Effects of adoptive transfer of PMNs from Adora2b−/− into WT mice in IR injury. (A and C) Model. WT PMNs or Adora2b−/− PMNs were isolated and transferred into PMN-depleted (1A8 Ly6G-specific Ab treatment 24 h prior to ischemia) or anti–G-CSF-treated (24 h prior to ischemia) animals. (B and D) Mice underwent 60 min of ischemia and 120 min of reperfusion. Infarct sizes were measured by double staining with Evans blue and TTC. Infarct sizes are expressed as the percentage of the AAR that underwent infarction. Infarct sizes in PMN-depleted WT mice that received either WT PMNs (controls) or Adora2b−/− PMNs with representative infarct staining shown (n = 3–4, ± SD).

FIGURE 7.

Effects of adoptive transfer of PMNs from Adora2b−/− into WT mice in IR injury. (A and C) Model. WT PMNs or Adora2b−/− PMNs were isolated and transferred into PMN-depleted (1A8 Ly6G-specific Ab treatment 24 h prior to ischemia) or anti–G-CSF-treated (24 h prior to ischemia) animals. (B and D) Mice underwent 60 min of ischemia and 120 min of reperfusion. Infarct sizes were measured by double staining with Evans blue and TTC. Infarct sizes are expressed as the percentage of the AAR that underwent infarction. Infarct sizes in PMN-depleted WT mice that received either WT PMNs (controls) or Adora2b−/− PMNs with representative infarct staining shown (n = 3–4, ± SD).

Close modal

Adora2b signaling has been shown to effectively protect the myocardium from ischemia in various settings such as IP or IR injury (1113). In the present study, we investigated the cellular source of Adora2b-dependent cardioprotection. Studies using state-of-the-art Cre-lox mouse models for the Adora2b revealed an important role of myocytes or endothelial-expressed Adora2b in IP of the heart, as it was abolished in Adora2bf/f-VE-cadherin-Cre+ or Adora2bf/f-myosin-Cre+ mice. In contrast, protection from IR injury was primarily mediated by Adora2b signaling on bone marrow–derived inflammatory cells. Characterization of the postischemic inflammatory response in Adora2bf/f-Lyz2-Cre+ mice revealed a PMN-driven cytokine profile. In proof of principle studies, an adoptive cell transfer of Adora2b−/− PMNs confirmed the hypothesis that Adora2b signaling on PMNs had dominantly been responsible for the observed phenotype in Adora2bf/f-Lyz2-Cre+ mice. Taken together, these studies suggest that activation of Adora2b on different tissues represents different therapeutic strategies.

The extent of myocardial cell death determines patient outcome after myocardial ischemia (32). Thus, it is not surprising that protective strategies to make the heart more resistant to ischemia or limit the damage during IR are an area of intense investigation (6, 16, 17, 19, 3338). It is accepted that a number of G protein–coupled receptors can activate cardioprotective mechanisms (32). These receptors include the adenosine, opioid, and bradykinin families (39). During the past 20 y, substantial evidence indicates that adenosine, administered either prior to ischemia or during reperfusion, reduces myocardial injury (32). These effects are mediated via the activation of one or more of the four known AR subtypes (Adora1, Adora2a, Adora2b, and Adora3). All four ARs have been associated with cardiac tissue protection in different settings (12, 40, 41). Experimental studies in different species and models implicated that activation of Adora1 or Adora3 prior to ischemia is cardioprotective (42, 43). Other studies revealed that the administration of Adora2a or Adora2b agonists during reperfusion can reduce MI (41, 44). However, although all ARs have been found to mediate cardioprotection from ischemia, Adora2b might be the only one that was found to play a role in almost all known cardioprotective settings. As such, Adora2b has been implicated in postconditioning, which protects the reperfused heart from infarction (44). Other studies have shown the importance of Adora2b signaling for cardioprotection mediated by IP (12), which seems to be associated with the initiation of a metabolic program to make the heart more oxygenic efficient (19). Further mechanistic studies on Adora2b-mediated cardioprotection revealed the involvement of hypoxia inducible factor 1 (HIF1), an important transcription factor implicated in tissue adaptation to hypoxia (18, 19). Other studies indicated reduction of superoxide generation from mitochondria through ERK, PI3K, and NOS as Adora2b mediated, all of which have been implicated in protection from ischemia (45). A very elegant study recently discovered that the Adora2b is present in or near mitochondria, suggesting that Adora2b signaling results in inhibition of mitochondrial transition pores (46). Because it is thought that cardioprotective signaling pathways converge on the mitochondria, inhibition of mitochondrial transition pores is thought to be the “holy grail of cardioprotection” (47). However, note that Adora2b is the only one of the four ARs whose cardiac expression was found to be induced by ischemia in both mice and humans and whose function is implicated in IP and postconditioning of the heart (19).

Even though there is increasing evidence for Adora2b signaling in mediating cardioprotection, no study has evaluated the tissue-specific contribution yet. IP has been consistently demonstrated to be a potent protective mechanism in freshly isolated and cultured cardiomyocytes across multiple species, indicating that much of the innate protection of IP resides in cardiomyocytes (48). However, studies using state-of-the-art Cre-lox mouse models have not yet been performed. Based on earlier studies in germline Adora2b−/− mice (12, 17), we now generated tissue-specific Adora2b KO mice. Comparing an Adora2b tissue-specific deletion in myocytes, endothelia, or bone marrow–derived inflammatory cells indeed confirmed studies hypothesizing that cardiomyocytes are crucial for IP of the heart. Additionally, Adora2b on the endothelium was also found to be important for the mechanism of IP. In fact, hypoxic preconditioning to model IP in vitro has repeatedly been found to protect endothelial cells from subsequent long-term hypoxia (19, 49, 50). In contrast, Adora2b signaling on inflammatory cells seems less important during IP. However, this is not surprising, as IP consists of short nonlethal ischemic periods that are most likely not able to attract a significant amount of inflammatory cells (17). Additionally, although we found some resident macrophages in the heart, Adora2bs were dominantly expressed on cardiomyocytes and endothelial cells, supporting our findings in IP of the heart.

Interestingly, the finding that Adora2b-elicited cardioprotection by IP involves vascular endothelial cells is also reflected in a recent study examining the role of HIF1A in cardioprotection. This study demonstrated convincingly, by using tissue-specific HIF1A-deficient mice, that vascular endothelial HIF1A is required for mediating the cardioprotective effects of IP. The authors concluded that HIF1A is functioning as a transcriptional activator, despite the acute nature of the response (51). As discussed above, HIF1A is a critical transcriptional enhancer of Adora2b signaling during IP (18). Therefore, it is conceivable that the transcriptional induction of the Adora2b via HIF1A is a critical component of cardioprotection elicited by IP.

Although IP seems to be mainly linked to endothelial cells and cardiomyocytes, compelling evidence from both animal and clinical studies has indicated that leukocytes are the principal effector cells of IR injury (52). Reperfusion induces a vigorous inflammatory response and a dramatic increase in neutrophil adherence to the reperfused endothelium (17, 53). As Adora2bs are widely distributed in hematopoietic cells (27, 37, 54, 55), studies using in vivo animal models have shown that Adora2b deficiency is associated with enhanced inflammation (20, 27, 37, 56). Other studies indicated that Adora2b-mediated protection from vascular injury is based on anti-inflammatory processes (37, 57).

Based on its anti-inflammatory role, it is convincing that Adora2b signaling could dampen IR injury by interaction with bone marrow–derived inflammatory cells. In fact, in the present study, we have established an important role for the Adora2b on bone marrow–derived cells in mediating cardioprotection against IR injury. Earlier studies using germline Adora2b−/− mice showed that plasma levels of the proinflammatory cytokine TNF-α was elevated at baseline (27) and in mice subjected to femoral artery or myocardial IR injury (17, 57). Thus, our findings on a TNF-α–guided proinflammatory phenotype in tissue-specific deletion of Adora2b on bone marrow–derived cells (Adora2bf/f-Lyz2-Cre+) during cardiac IR are consistent with these previous findings.

In this study, as a proof of concept, we isolated Adora2b−/− PMNs from germline Adora2b−/− mice and transferred them into neutropenic mice. Following ischemia, we found significantly increased infarct sizes when compared with mice that were transferred with WT PMNs. These studies support our findings that only Adora2bf/f-Lyz2-Cre+ mice had larger infarcts when compared with controls. Because bone marrow–derived cells include neutrophils and macrophages, it seems compelling that PMNs play the dominant role, as they are the dominant cell type during the first hours of reperfusion (17). However, it also indicates that our studies cannot completely rule out a role of Adora2b on macrophages in IR injury. Future studies using tissue-specific mice for different inflammatory cell types will be necessary to further elucidate the detailed mechanisms.

Interestingly, apart from the Adora2b dependent regulation of proinflammatory TNFα, we also found significantly lower levels of stem cell factor in Adora2bf/f-Lyz2-Cre+ mice. Stem cell factor is a cytokine that improves myocardial function (58) and enhances cardiac healing after myocardial injury (59). Additionally, it has been found that adenosine can enhance stem cell factor signaling in vitro (60). Thus, apart from a reduction of the postischemic inflammation, Adora2b signaling might act as a switch to start cardiac repair early in reperfusion. Indeed, pharmacological studies have indicated an important role of Adora2b in cardiac healing after IR injury (61).

Taken together, using a novel tissue-specific approach for Adora2b signaling during IP or IR, we found different functions for the Adora2bs in different tissues. Whereas Adora2b signaling in vascular endothelial cells and cardiac myocytes was critical for mediating IP-elicited cardioprotection, the extent of IR injury was determined by Adora2b signaling on inflammatory cells. These findings indicate that when using Adora2b as a therapeutic target it might be best when done in a tissue-specific manner. One possible approach could be the administration of an Adora2b agonist into a coronary artery prior to a high-risk intervention to “precondition” the heart, whereas systematic administration of an Adora2b agonist may be preferable during reperfusion. Future studies using specific Adora2b agonists in clinical trials will be necessary to gain insight into tissue-specific therapies for myocardial ischemia.

We thank the Rocky Mountain Taste and Smell Center for providing the guinea pig anti–β-gal Ab (AB_2316450).

This work was supported by National Institutes of Health/National Heart, Lung, and Blood Institute Grants 1K08HL102267-01 and 1R01HL122472-01 (to T.E.) and R01 DK097075, R01 HL0921, R01 DK083385, R01 HL098294, and POIHL114457-01, as well as by a grant from the Crohn’s and Colitis Foundation of America (to H.K.E.) and a Deutsche Forschungsgemeinschaft research fellowship (to M.K.).

Abbreviations used in this article:

AAR

area at risk

Adora2b

adenosine A2b receptor

AR

adenosine receptor

cTnI

cardiac troponin I

β-gal

β-galactosidase

HIF1

hypoxia inducible factor 1

IP

ischemic preconditioning

IR

ischemia/reperfusion

KHB

Krebs–Henseleit buffer

KO

knockout

LCA

left coronary artery

MI

myocardial infarction

PMN

polymorphonuclear neutrophil

ROS

reactive oxygen species

TTC

triphenyl-tetrazolium chloride

WT

wild-type.

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The authors have no financial conflicts of interest.