Stimulation of the beta2 adrenergic receptor at reperfusion limits myocardial reperfusion injury via IL-10 dependent anti-inflammatory pathway in the spleen

Abstract

Background:

In addition to the airway-relaxing effects, β₂ adrenergic receptor (β₂AR) agonists are also found to have broad anti-inflammatory effects. The current study is to define the role of β₂AR agonists in limiting myocardial ischemia/reperfusion injury (IRI).

Methods and results:

Adult male wild-type (WT) and IL-10 KO mice underwent 40-min left coronary artery ligation and 60-min reperfusion. A selective β₂AR agonist, Clenbuterol at doses of 0.1 μg or 1 μg/per gram weight i.v. 5 min before reperfusion, significantly reduced myocardial infarct size (IS) by 28% and 39% (vs. control, p<0.05) in WT mice, but had no protective effect in IL-10 KO mice. Inhalational therapy with nebulized Clenbuterol, Albuterol, Salmeterol or Arformoterol immediately before ischemia significantly reduced IS (p<0.05) in WT mice respectively. Splenectomy similarly reduced IS as Clenbuterol-treated mice, but intravenous Clenbuterol did not further reduce IS in splenectomized mice. In splenectomized WT mice, acute transfer of isolated splenocytes, not the Clenbuterol-pretreated splenocytes, restored the myocardial IS to the level of intact mice. Intravenous Clenbuterol significantly increased splenic protein levels of β₂AR, phosphorylated Akt and IL-10 and plasma IL-10, and inhibited the expression of proinflammatory mRNAs.

Conclusions:

Both intravenous and inhalational β₂AR agonists exert a cardioprotective effect against IRI by activating the anti-inflammatory β₂AR-IL-10 pathway.

INTRODUCTION

Recently, β₂ adrenergic receptor (β₂AR) agonists were found to exert protective effects against ischemia/reperfusion injury (IRI) in the kidney¹, brain² and spinal cord³. Several studies have also demonstrated that activation of β₂AR can exert cardioprotective effects against ischemia/reperfusion injury^4–6. In these studies, the β₂AR agonist was administered either before ischemia or before reperfusion and the results suggested that the β₂AR agonist acted primarily on cardiomyocytes by activating the cAMP-PKA-pAkt pathway to inhibit cardiomyocyte apoptosis. However, the anti-inflammatory effect of β₂AR in mediating cardioprotection remains unknown. Inflammatory responses during post-ischemic reperfusion play a key role in mediating myocardial reperfusion injury. Innate and adaptive immune systems are both involved in this inflammatory response^{7, 8}. Recently, β₂AR agonists have been shown to exert broad anti-inflammatory effects^9–11. The present study sought to evaluate the role of β₂AR activation in promoting anti-inflammatory responses and in attenuating myocardial IRI. Using an established in vivo mouse model of myocardial IRI, our results demonstrate that β₂AR agonist attenuates myocardial infarct size by activating a pathway involving β₂AR, Akt phosphorylation and IL-10 in splenic leukocytes.

MATERIALS and METHODS

This study conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Eighth Edition, revised 2011) and was conducted under protocols approved by the University of Virginia’s Institutional Animal Care and Use Committee.

Animals and experimental protocols.

C57BL/6 wild type (WT) mice (9–13 weeks of age, purchased from The Jackson Laboratory) and congenic IL-10 knockout (KO) mice (9–13 weeks of age, breeding pairs purchased from The Jackson Laboratory) were randomly assigned to either IR injury groups or sham surgery groups. Compared to the WT mice, the phenotype of IL-10 KO mice showed significantly higher spleen weight (% of body weight, 0.446±0.033% vs. 0.392±0.013%, p<0.05). There was no difference in body weight, heart weight, blood pressure, heart rate. There was no evidence of dermatitis or rectal prolapse as reported in the literature.

In treated mice, Clenbuterol (purchased from Sigma-Aldrich, St. Louis, MO) was administered either as an i.v. bolus 5 min before reperfusion at two different doses, 0.1 μg/g and 1.0 μg/g weight with an injection volume of 2 μl per gram body weight, or nebulized in a solution with 1 mg Clenbuterol in 5 ml saline. Clenbuterol was nebulized using an Ecosonic nebulizer (Medisonic USA Inc., Clarence, NY). The effect of Clenbuterol on heart rate was determined using PowerLab instrumentation (ADInstruments, Colorado Springs, CO). Additional 3 short acting to long acting β₂AR agonists, albuterol, salmeterol and Arformoterol (purchased from Sigma-Aldrich, St. Louis, MO), were also tested individually in 3 groups of mice respectively and administered by inhalation before ischemia.

Myocardial IR injury and measurement of infarct size.

In previous work, we established that myocardial infarct size (IS) as measured by late-gadolinium enhanced MRI at 60 minutes of reperfusion attains 95% of the size measured by the same method at 24 hours post-reperfusion in mice^{12, 13}. We therefore used 60 minutes of reperfusion in the present study. The left coronary artery (LCA) of WT or IL-10 KO mice was ligated for a duration of 40 minutes followed by 60 minutes of reperfusion as detailed previously^{7, 8, 13, 14}. Briefly, mice were anesthetized with sodium pentobarbital (80mg/kg, i.p.) and orally intubated. An additional dose of pentobarbital (40mg/kg, i.p.) was applied shortly after reperfusion. The adequacy of the anesthesia was confirmed by hind limb pinch reflex every 15 minutes. The heart was exposed through a left thoracotomy. The LCA was identified under a dissecting microscope. An 8–0 Prolene suture was placed around the LCA at a level 1 mm inferior to the left auricle. Ischemia was induced by securing a suture over a piece of PE-60 tubing placed parallel to the LCA, and reperfusion was achieved by removing the tube. Successful ligation of the LCA was confirmed by blanching in the ischemic zone. The mice were euthanized 60 minutes after reperfusion, and the explanted hearts were cannulated through the ascending aorta for perfusion with 3 ml of 1.0% TTC (Sigma-Aldrich, St. Louis, MO). The LCA was then re-occluded with the same suture used for coronary occlusion prior to perfusion with 10% Phthalo blue to determine risk region (RR), which was statistically similar among all groups. The left ventricle was then cut into 5 to 7 transverse slices that were weighed and digitally photographed to determine IS as a percent of RR^{7, 8}.

Splenic leukocyte adoptive transfer (SPAT): Splenocytes are purified from WT mice and then transferred into recipient mice 5 minutes before reperfusion via external jugular vein injection at dose of 5×10⁶ splenocytes cells/mouse in 50 μl. In Clenbuterol-treated splenocytes, the splenocytes were treated with Clenbuterol for 10 min at a dose of 0.01 μg in 20×10⁶ splenocytes cells (200 μl). After treatment, these splenocytes were re-suspended in Clenbuterol-free PBS. Splenocytes were counted using Cellometer (Nexcelom, Lawrence, MA).

Several additional groups of mice without IRI were treated either with PBS or Clenbuterol. The plasma, heart and spleen were harvested at 5, 15, 30 min following treatment. The samples were analyzed for ELISA, RT-PCR and Western Blot analysis. Clenbuterol-induced tachyarrhythmia was evaluated in mice treated with both bolus iv injection and nebulization using a PowerLab physiological monitoring system, Colorado Springs, CO).

Western analysis.

Protein levels of β₂AR, Akt phosphorylation, CD38, FPR1, IL-1β and IL-10 in the spleen and/or plasma were assessed by Western blot analysis as previous described¹⁴. Briefly, left ventricular and splenic tissue were homogenized in PBS. Plasma samples were obtained by centrifuging blood samples at 1600g for 20 minutes. Total protein concentrations were determined by BCA protein assay (ThermoFisher Scientific, Rockford, IL). Twenty micrograms of protein were separated by 10% SDS–PAGE. After transfer, nitrocellulose membranes (Bio-Rad, Hercules, CA) were probed with primary antibodies against β₂AR, total Akt, phosphorylated Akt (anti-Ser473), CD38, FPR1, IL-1β and IL-10 (ThermoFisher Scientific) at a 1:2,000 dilution and with secondary antibodies (Promega, Madison, WI) at a 1:5,000 dilution in blocking solution (0.5% BSA in TBS-T). Proteins were visualized with enhanced chemiluminescent substrate (ThermoFisher Scientific) followed by densitometry using a FluorChem 8900 imaging system (Alpha Innotech, Santa Clara, CA). β-actin was used as a loading control.

Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis.

Splenic mRNA levels of CD38, Egr2, Fpr1, IL-10, IL-1β and Ncf1 were assessed by qRT-PCR. In brief, total RNA was isolated from cells using the RNeasy Mini Kit (QIAGEN USA, Germantown, MD) according to the manufacturer’s instructions. cDNAs were synthesized using iScript™ cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA). qPCR was performed with the SsoAdvanced™ Universal SYBR Green supermix (Bio-Rad Laboratories, Hercules, CA) and monitored using a CFX Real-Time PCR Detection System (Bio-Rad). GAPDH (Unique Assay ID: qMmuCID0027497) was used as a housekeeping gene. The primers were purchased from Bio-Rad Laboratories. (Unique Assay ID: qMmuCID0006259 for CD38, qMmuCID0009042 for Egr2, qMmuCID0015439 for Fpr1, qMmuCID0015452 for IL10, qMmuCID0005641 for IL-1β and qMmuCID0006405 for Ncf1). mRNA levels were quantified using the 2(-ΔΔCt) relative quantification method as previously described¹⁵.

Statistical analysis.

All data are presented as mean ± SEM (standard error of the mean). Changes in heart rates were analyzed using repeated measures ANOVA followed by Bonferroni pairwise comparisons. All other data were compared using one-way ANOVA followed by t-test for unpaired data with Bonferroni correction.

RESULTS

C57BL/6 WT mice and congenic IL-10 KO mice underwent 40 min of LCA occlusion followed by 60 min of reperfusion. A long-acting selective β₂AR agonist, Clenbuterol at doses of 0.1 μg and 1 μg per gram weight, was administered as an i.v. bolus 5 min before reperfusion. Clenbuterol at 0.1 μg and 1 μg per gram weight significantly attenuated myocardial IS by 28% and 39% in WT mice, respectively (IS as %RR; 39.2±2.8 and 33.4±4.0 vs. control 54.7±3.4, p<0.05). There was no significant difference in the infarct-sparing effect of Clenbuterol between these two doses. The infarct-sparing effect of Clenbuterol disappeared in IL-10 KO mice, even at the high dose of 1 μg per gram weight. There was no difference in risk region among these groups (Figure 1). Inhalational therapy with nebulized Clenbuterol (1mg in 5 ml normal saline for 5 min) immediately before ischemia significantly reduced infarct size (40.8±4.9 vs. 53.6±3.0, p<0.05) in WT mice (Figure 2A, 2B & 2C). Intravenous bolus administration of Clenbuterol significantly increased heart rate by 36% in both high-dose and low-dose mice (Figure 3, upper panel). However, inhalational therapy with Clenbuterol only increased heart rate by 11% from the corresponding baseline, a modest effect that did not achieve statistical significance (Figure 3, lower panel). Three more selective β₂AR agonists were tested to evaluate their roles in attenuating myocardial IRI. Albuterol (short-acting at dose of 300 μg/ml), salmeterol (moderate-acting at a dose of 20 μg/ml) and arformoterol (long-acting at a dose of 10 μg/ml) were nebulized to treat mice for 1 minute before LCA occlusion. All these β₂AR agonists exerted similar cardioprotective effect in attenuating myocardial IS (Figure 2D).

Figure 1. Selective β₂AR activation attenuates myocardial ischemia/reperfusion injury in wild type but not IL-10 KO mice.

C57BL/6 mice and congenic IL-10 KO mice (n=animals in each group) were treated with a selective β₂AR agonist, Clenbuterol, at two different doses as an i.v. bolus 5 min before reperfusion, 0.1 μg/g (low dose) and 1.0 μg/g weight (high dose) using a constant injection volume of 2 μl per g weight. IL-10 KO mice were treated with the high dose of Clenbuterol. Infarct size is presented as percentage of risk region (RR). RR is defined as percentage of left ventricle (LV).

Figure 2. Pretreatment of mice with inhalational β₂AR agonist attenuates myocardial ischemia/reperfusion injury.

After evaluating its intravenous formula in myocardial IR injury, inhalational Clenbuterol as well as other selective β₂AR agonists, was further studied. Clenbuterol (long-acting at a dose of 200 μg/ml), Albuterol (short-acting at dose of 300 μg/ml), salmeterol (moderate-acting at a dose of 20 μg/ml) and arformoterol (long-acting at a dose of 10 μg/ml) was individually nebulized to treat mice for 1 minute before LCA occlusion using an Ecosonic nebulizer (2A). Mice received inhalational therapy with saline only or β₂AR agonists for 1 min before undergoing 40 min of ischemia and 60 min of reperfusion. Hearts were harvested at the end of 60 min reperfusion and the myocardial infarct zone, ischemic zone and none ischemic zone were delineated using TTC-blue staining (2B). All these β₂AR agonists exerted similar cardioprotective effect in attenuating myocardial infarct size (Figure 2C to 2D, n represents animal number in each group). (2A).

Figure 3. Positive chronotropic effect of Clenbuterol in mice.

Panel A: Heart rate was monitored in mice (3–4 animals in each group) following treatment with intravenous Clenbuterol at 2 different doses. Heart rate rapidly increased by 36% within one min following injection and then slowly drifted down over the course of 20 min. The tachycardiac response was slightly less, but without statistical difference, in mice treated with low dose Clenbuterol. Panel B: Tachycardia was reduced in mice treated with inhalational Clenbuterol (3 animals). Note that the increase in heart rate over baseline (11%) was modest and did not reach statistical significance.

Splenectomy before ischemia attenuated infarct size to a similar extent as in Clenbuterol-treated mice (32.7±4.9 vs. control of 53.6±3.0, p<0.05). However, Clenbuterol at a dose of 1 μg per gram weight did not provide for any additional reduction in infarct size in splenectomized mice (Figure 4A). In splenectomized WT mice, splenic leukocytes adoptive transfer (SPAT) from WT mice 5 min at a dose of 5×10⁶ before reperfusion restored the myocardial infarct size to the level of that of intact mice (52.1±37 vs. intact WT control 53.6±3.0, p<0.05). Clenbuterol-pretreated splenocytes failed to increase the infarct size in splenectomized mice (Figure 4B). Without re-suspension of the isolated WT splenocytes, PBS-treated splenocytes had a 30% loss at 10 minutes and a 34% loss at 30 min, whereas, the Clenbuterol-treated splenocytes had 15% higher live cells than the untreated at both 10 min (p<0.05) and 30 min (p=0.08) (Figure 4C).

Figure 4. Splenectomy (SPLX) before ischemia attenuates myocardial infarct size. Clenbuterol is ineffective in reducing infarct size in splenectomized mice.

Splenectomy was performed 5 min before occlusion of LCA. Clenbuterol was administered at a dose of 1.0 μg/g weight (i.v.) 5 min before reperfusion or incubated with splenic cell at a dose of 0.01 μg in 20×10⁶ splenocytes cells (200 μl). A: Myocardial infarct size was significantly reduced and to a similar extent in Clenbuterol-treated (Figure 1) and splenectomized mice. However, Clenbuterol did not further reduce infarct size in splenectomized mice (n=animal number). B: splenic leukocytes adoptive transfer (SPAT) from WT mice 5 min before reperfusion restored the myocardial infarct size to the level of that of intact mice. However, Clenbuterol-pretreated splenocytes failed to increase the infarct size in splenectomized mice (n=animal number). C: Treated and control splenocytes without re-suspension were dispensed into 3 vials (n) in each group and evaluated at 10 minutes and 30 minutes using Cellometer (Nexcelom, Lawrence, MA). The live splenocytes were significantly reduced in both control and Clenbuterol-treated groups. But the reduction of cell counts in treated group was significantly less than that in control group.

In WT mice without IRI, intravenous Clenbuterol at a dose of 1 μg per gram weight significantly increased splenic protein levels of phosphorylated Akt, β₂AR and CD38 as early as 5 min post-administration, and these levels stayed elevated between 5 and 30 min following Clenbuterol treatment (Figure 5 upper 3 panels). FPR1 level was significantly reduced following Clenbuterol treatment (Figure 5 lowest panel). IL-10 levels in the spleen and plasma were only transiently elevated at 5 and 15 min following Clenbuterol treatment and then returned to normal levels at 30 min following treatment (Figure 6). IL-1β level was measured but could not detected in the spleen or the plasma (data not shown).

Figure 5. Protein levels of phosphorylated Akt, β₂AR, CD38 and FPR1 in the spleen by Western Blot.

Levels of phosphorylated Akt, β₂AR, CD38 and FPR1 in the spleen were serially evaluated in mice following i.v. injection of Clenbuterol at a dose of 1.0 μg/g weight. Phosphorylated Akt and β₂AR levels were significantly and steadily increased within the 30-min period following treatment. At the same time, levels of CD38 also significantly elevated in the spleen but started to decline at the 30-min after treatment. Levels of FPR1 was significantly reduced within the 30-min period following treatment. In the Western blots shown at the top of each graph, n = 3 animals per group except for the 30 min group where n = 4 animals. The calculation was based on equal β-lactin levels.

Figure 6. IL-10 levels in the spleen and plasma by Western Blot.

Splenic and plasma IL-10 levels were serially evaluated in mice following i.v. injection of Clenbuterol at a dose of 1.0 μg/g body weight. Both splenic tissue and plasma IL-10 levels were significantly elevated within 5 min following Clenbuterol treatment, and then trended back down to baseline levels at 30 min following treatment. In the Western blots shown at the top of each graph, n = 3 animals per group except for the 30 min group where n = 4 animals.

The qRT-PCR assays demonstrated that Clenbuterol reduced the expression of CD38, Egr2 and Fpr1 mRNAs in splenic tissue at 15 min following treatment, but that CD38 and Fpr1 mRNAs returned to baseline levels by 30 min after treatment. In contrast, Clenbuterol enhanced the expressions of mRNAs for Ncf1, IL-1β and IL-10 at 30 min following treatment (Figure 7).

Figure 7. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis of splenic tissue following β₂AR activation.

Splenic mRNA expression was serially evaluated in mice following i.v. injection of Clenbuterol at a dose of 1.0 μg/g weight. Clenbuterol decreased the levels of CD38 and Fpr1 mRNAs within 15 min following treatment, but these levels recovered to baseline by 30 min after treatment. Clenbuterol enhanced the expression of mRNAs encoding Ncf1, IL-1β and IL-10 by 30 min following treatment. Three mice per group were used.

DISCUSSION

Our work and others have demonstrated that the inflammatory response contributes importantly to post-ischemic reperfusion injury in mice^{8, 16}. The present study investigated the effects of β₂AR activation in inhibiting inflammatory responses and thereby reducing myocardial infarct size. We found that treatment with Clenbuterol, a long-acting β₂AR agonist just prior to reperfusion attenuates myocardial infarct size by inhibiting inflammatory responses in the spleen through the activation of a pathway involving β₂AR, Akt and IL-10. Furthermore, our results show that individual inhalational therapy with 4 β₂AR agonists with different acting durations exerts similar cardioprotection against IRI as intravenous treatment. Since the inhalational therapy has significantly less chronotropic effect, this might enhance its translational potential for patients with acute coronary syndromes.

Recently, several studies have demonstrated that activation of β₂AR attenuates myocardial ischemia/reperfusion injury^4–6. These studies explore the direct effect of β₂AR agonist on cardiomyocytes. However, the anti-inflammatory effects of β₂AR in mediating cardioprotection remain to be explored. β₂ARs are extensively expressed in the pulmonary system, cardiac muscle and immune cells. Among the adrenergic receptors in immune cells, T and B cells exclusively express β₂ARs instead of β₁ARs¹⁷. In immune cells, β₂AR stimulation of CD4⁺ T cells is known to inhibit T cell proliferation via the cAMP-dependent inhibition of nuclear factor κB activation^{9, 18, 19} and thus favors a Th2 polarization. β₂ARs are also the predominant β-adrenergic receptors in macrophages and neutrophils. β₂AR stimulation on macrophages induces an M2 phenotype among other anti-inflammatory effects^{9, 20, 21}. β₂AR agonists also inhibit neutrophils by increasing cAMP levels and reducing the production of ROS and pro-inflammatory cytokines^{22, 23}. Thus activation of β₂AR exerts a broad spectrum of inhibition of inflammatory cells and this inhibitory mechanism is entirely distinct from the direct inhibition of bronchoconstriction²³. In the current study, by administering a selective β₂AR agonist (Clenbuterol) upon reperfusion, we found that Clenbuterol attenuated myocardial infarct size by 30% and its infarct-limiting effect was not due to its effect on cardiomyocytes but rather to its inhibitory effect on inflammatory responses. This conclusion is supported by evidence that: 1) Clenbuterol did not afford additional reduction of myocardial infarct size in mice without spleen (Figure 4A), and 2) Clenbuterol did not reduce myocardial infarct size in IL-10 knockout mice (Figure 1). Splenic leukocytes adoptive transfer before reperfusion restored the myocardial infarct size in splenectomized mice to the level of that in intact mice. However, Clenbuterol-treated splenocytes before transfer abolished this infarct exacerbating effect of the splenocytes (Figure 4B). As the Clenbuterol washed away from the splenocytes, the systemic role of the Clenbuterol was avoided. The results indicate that the infarct-limiting effect of β₂AR agonist is likely mediated by way of the spleen. Clenbuterol enhanced protein expressions of CD38, IL-10 and p-Akt and decreased expression of FPR1 (Figure 6) support the notion that activation of β₂ARs modify the spleen into an anti-inflammatory phenotype. In light of previous work from our lab and others showing that CD4+ T-cells play an equally critical role in acute reperfusion injury^{8, 24, 25}, these results suggest a pathway by which β₂AR agonists stimulate β₂ARs on CD4+ T-cells (probably regulatory T-cells), thus enhancing the release of IL-10, which in turn dampens the activation of immune cells (primarily neutrophils) in the spleen. This pathway then suffices to inhibit the mobilization of neutrophils into the circulatory system, followed by their homing to the infarct zone where they exacerbate infarct size during reperfusion.

Interestingly, we found that ex vivo treatment of the splenocytes with Clenbuterol, the reduction in splenocytes was significant less than that in PBS-treated splenocytes (Figure 4C). Thus, activation of β₂ARs increased the tolerance of the splenocytes to anoxic injury.

Considerable evidence demonstrates that acute inflammatory responses contribute importantly to reperfusion injury^{7, 8, 26, 27}. Furthermore, the spleen plays a central role in mediating this inflammatory response^{12, 16}. The current study demonstrates that activation of β₂AR exerts a potent anti-inflammatory effect by inhibiting splenic inflammatory responses. As the Clenbuterol was administered shortly before reperfusion, the infarct-limiting effect of Clenbuterol is most likely due to its effect in inhibiting inflammatory response during post-ischemic reperfusion, rather than to its effect on the heart or on preconditioning.

A number of clinical studies have included studies on the effect of β₂AR agonists in patients with ACS^{28, 29}. However, the positive chronotropic effect of β₂AR agonists might argue against its acute application in the setting of ACS³⁰. In the present study, we found that intravenous administration of Clenbuterol increased heart rate by 36% in mice. This led us to examine the alternative administration route that has previously been used in humans with asthma (inhalational administration). Using this approach, Clenbuterol had only a modest effect on heart rate, yet with a comparable reduction in myocardial infarct size (Figures 1–3). These results raise the question of whether there might be potential for the clinical application of β₂AR agonists in ACS patients. Towards this end, additional studies may be warranted to investigate the potential of inhalational therapy of β₂AR agonists against myocardial IRI in animal models of different species, and perhaps human beings. Of note, β₁AR is the most abundant receptor subtype³¹ in cardiomyocytes. Activation of β₁ARs exerts pro-apoptotic effects, while activation of β₂AR on cardiomyocytes produces preconditioning^4–6 and anti-apoptotic effects^{5, 32, 33}. In the current study, inhalational therapy of individual Clenbuterol, Albuterol, Salmeterol and Arformoterol was administered before index ischemia. Thus it is possible that the cardioprotective effects of inhalational β₂AR agonist in the current study may involve both the preconditioning of cardiomyocytes and inhibition of pro-inflammatory responses.

Although we found that Clenbuterol inhibited the exacerbation of infarct size by splenic leukocytes and reduced the expression of pro-inflammatory cytokine mRNAs, such as Egr2 and Fpr1, it also enhanced the expression of other pro-inflammatory mRNAs like Ncf1 and IL-1β (Figure 7). We found consistent expression of protein and mRNA in FPR1 and IL-10. However, opposite trend of changes was found in CD38 and IL-1β (Figures 6 & 7). IL-1β protein in the splenic tissue and the plasma was not detectable (data not shown). Of note, all these changes in mRNA levels were transient. It is also important to mention that these results are limited by small sample sizes. The overall net effect of Clenbuterol administration was anti-inflammatory in nature given the reduction in infarct size. Since both pro-inflammatory and anti-inflammatory cytokine mRNAs were enhanced, it is possible that Clenbuterol might have differential effects on different inflammatory cell populations residing in the spleen.

In summary, using a mouse model of acute myocardial ischemia/reperfusion injury, the present study shows that a selective β₂AR agonist, Clenbuterol, attenuates myocardial infarct size when it is administered just prior reperfusion. We propose that Clenbuterol inhibits pro-inflammatory responses during post-ischemic reperfusion by promoting the release of IL-10, thereby attenuating the activation of the splenic leukocytes that exacerbate infarct size upon reperfusion. The bio-effects of Clenbuterol were also evidenced by the overexpression of β₂ARs on the splenic leukocytes and activation of pAkt via IL-10. Mice treated with inhalational Clenbuterol have significantly less positive chronotropic effect than with intravenous Clenbuterol, but show a similar attenuation in myocardial infarct size. These results underscore the potential for β₂AR agonists in inhibiting the pro-inflammatory responses that contribute importantly to reperfusion injury. The study also has potential clinical implications because it suggests the possibility of using inhalational β₂AR agonists to reduce infarct size in patients suffering acute myocardial infarction.