Cardioprotective effects of mineralocorticoid receptor antagonists at reperfusion

Abstract

Aims: Pre-treatment with mineralocorticoid receptor (MR) antagonists is reported to reduce myocardial infarct size from ischaemia/reperfusion. Here, we tested whether the MR antagonists potassium canrenoate and eplerenone could protect in the more clinically relevant schedule of administration at the end of ischaemia.

Methods and results: In all models, hearts were subjected to 30 min regional ischaemia followed by 120 min (rabbits 4 h) reperfusion. A bolus of canrenoate 5 min prior to reperfusion in open-chest mice decreased infarct size in a dose-dependent manner. Maximum protection was seen at 1 mg/kg where infarction was 18% of that in the control (P < 0.001). Ecto-5'-nucleotidase (CD73) as well as adenosine A(2b) receptor knock-out mice could no longer be protected, suggesting a role for adenosine and the A(2b) receptor in the mechanism. A 1 mg/kg bolus of canrenoate prior reperfusion also reduced infarct size in open-chest rabbits. To explore the underlying mechanisms, we studied isolated rat hearts. Eplerenone (10 microM) at the end of ischaemia was similarly protective in the rat heart and the protection was abolished by co-treatment with inhibitors of the adenosine receptor, protein kinase C, PI3-kinase, and ERK. In addition, eplerenone or canrenoate treatment increased phosphorylation of the pro-survival kinases Akt and ERK1/2 at reperfusion in the rat hearts.

Conclusion: Taken together, MR antagonists when given at the end of ischaemia are highly effective and potent cardioprotective drugs with a signalling similar to that of ischaemic pre-conditioning and, hence, could be a very promising candidate for the treatment of acute myocardial infarction in man.

Figures

Fig. 1

Experimental protocols of the isolated rat heart experiments. All hearts were stabilized for 30 min prior to experiments. Control hearts received 30 min of regional ischemia followed by 2 h of reperfusion. Mineralocorticoid receptor (MR) antagonists were given throughout reperfusion starting 5 min before reperfusion. The blockers were given either alone accordingly or 5 min before the MR antagonist treatment, while aldosterone (ALDO) was given together with the MR antagonist. The arrows indicate the times of the biopsies of the left ventricle. ■ = regional ischemia.

Fig. 2

Results of the in situ open chest mouse experiments. A Potassium canrenoate (CAN) as a bolus given 5 min prior to reperfusion was protective with a concentration as low as 30 μg/kg, whereas 1 mg/kg showed very profound protection. The right panels depict experiments with the use of CAN either given to ecto-5′-nucleotidase (CD73) knock-out (ko) or A2b adenosine receptor (AR) ko mice. In both knock-out animals, CAN’s protection was blunted, suggesting a role for extracellular adenosine and the A2bAR in the protective signaling. Open symbols represent individual experiments while closed symbols are the means ± SD. *p<0.001 vs. control. B Plasma levels of cardiac troponin I (cTnI) at the end of the in situ open-chest mouse experiments. While a bolus of potassium canrenoate 1 mg/kg 5 min before reperfusion reduced cTnI release at the end of the reperfusion period, there was no significant decrease with lower canrenoate concentrations. *p=0.022 vs. control.

Fig. 3

Results of the in situ rabbit heart experiments with infarct size assessment after 4 h of reperfusion (left panel) or 72 h (right panel). Canrenoate was applied as a 1 mg/kg bolus 5 min prior to reperfusion identical to the mouse experiments and clearly reduced infarct size presented at a percentage of the risk zone. *p<0.05 vs. control

Fig. 4

Results of the isolated rat hearts experiments. Drugs were given as depicted in Fig. 1. Eplerenone (EPL) resulted in a significant reduction in infarct size, which could be abolished with the co-treatment of pharmacological inhibitors of known protective signaling elements. [8p-sulfophenyladenosine (SPT) = adenosine receptor blocker, wortmannin (WORT) = PI3 kinase inhibitor, chelerythrine (CHEL) = PKC inhibitor, and U0126 = ERK blocker]. While a low aldosterone (ALDO) concentration did not overcome the eplerenone’s protection, a higher ALDO concentration did. All blockers and ALDO alone had no effect on infarct size. Canrenoate also showed protection in this model. *p

Fig. 5

Fig. 5A, 5B, and 5C Akt and ERK1/2 phosphorylation in isolated rat heart. Myocardial samples were obtained from transmural biopsies of isolated rat hearts of the left ventricle right before ischemia (baseline) and at 10 min of reperfusion following a 30 min period of global ischemia as indicated by the arrows in Fig. 1. Phosphorylation of all tested kinases was clearly increased at reperfusion compared to untreated control when either canrenoate (CAN) or eplerenone (EPL) were present. Protection was abolished with co-infusion of aldosterone (ALDO) or wortmanin (WORT), while ALDO had no effect on its own. Results represent the mean±SD of 6 independent experiments, *p

Fig. 5

Fig. 5A, 5B, and 5C Akt and ERK1/2 phosphorylation in isolated rat heart. Myocardial samples were obtained from transmural biopsies of isolated rat hearts of the left ventricle right before ischemia (baseline) and at 10 min of reperfusion following a 30 min period of global ischemia as indicated by the arrows in Fig. 1. Phosphorylation of all tested kinases was clearly increased at reperfusion compared to untreated control when either canrenoate (CAN) or eplerenone (EPL) were present. Protection was abolished with co-infusion of aldosterone (ALDO) or wortmanin (WORT), while ALDO had no effect on its own. Results represent the mean±SD of 6 independent experiments, *p