Stimulation of the Beta2 Adrenergic Receptor at Reperfusion Limits Myocardial Reperfusion Injury via an Interleukin-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 was conducted to define the role of β₂AR agonists in limiting myocardial ischemia/reperfusion injury (IRI).

Methods and results: Adult male wild-type (WT) and interleukin (IL)-10 knockout (KO) mice underwent a 40-min left coronary artery ligation and 60-min reperfusion. A selective β₂AR agonist, Clenbuterol, at doses of 0.1 μg or 1 μg/g 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 respectively, 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. 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 pro-inflammatory mRNAs.

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

Keywords: Clenbuterol; Heart; Ischemia/reperfusion; Myocardial infarction; β2AR.

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.

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.

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.

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.