Cardioprotective effects of mineralocorticoid receptor antagonists at reperfusion

mineralocorticoid is to reduce infarct Here, we tested whether the and relevant administration at the end of ischaemia. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Keywords concentration less than 0.01%. All other inhibitors were dissolved in the buffer directly. concentration


Introduction
Aldosterone causes sodium and water retention by a genomic action of the hormone on the kidney. These effects involve the translocation of the steroid-mineralocorticoid receptor (MR) complex to the nucleus, where it acts as a transcriptional regulator. Several hours later, the newly expressed proteins induce their effect. More recently, rapid, non-genomic effects of aldosterone occurring within minutes have also been described. 1,2 Interestingly, MR blockade also exerts rapid effects on calcium metabolism and the sodium hydrogen exchanger. 3 Chai et al. 4 reported that MR blockade prior to ischaemia in the isolated rat heart reduced ischaemic injury, suggesting that the MR antagonists might somehow cause pharmacological pre-conditioning. 5 Because preconditioning is of little clinical value, there has been an extensive search for a safe drug that reduces infarct size when given at reperfusion. 6 Here, we test two selective MR blockers, potassium canrenoate, the i.v. compatible metabolite of spironolactone, and eplerenone, in several different animal models to see whether either might also be protective at the end of ischaemia. Finally, we test whether the protection from MR blockade might use any of the signalling pathways that are used by ischaemic preand post-conditioning. 7 We tested the MR antagonists in three different species and a powerful anti-infarct effect was seen in all.

Methods
The experiments in rats and mice were conducted in Greifswald, Germany, while rabbits were used in Maisons-Alfort, France. All experiments were conducted in accordance with The Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC, 1996). The experimental protocols used in this study were either approved by the local authorities of the state of Mecklenburg-Vorpommern, Germany (rat and mouse), or according to French official regulations (rabbit).

Open-chest in situ mouse heart
We used the open-chest in situ mouse heart model described by Eckle et al. 8 Briefly, mice were anaesthetized with pentobarbital sodium (70 mg/kg i.p.) and additional anaesthesia was administered as needed throughout the experiment. Animals were ventilated with room air supplemented with oxygen (peak inspiratory pressure of 10 mbar, positive end-expiratory pressure of 3 mbar). The ventilation frequency was set at 110 b.p.m. and a tidal volume of 200 -250 mL. To administer drugs, a butterfly needle was placed in the tail vein. After a left thoracotomy, a prominent branch of the left coronary artery was surrounded with a 7-0 nylon suture to form a snare. The mice were allowed to stabilize for 15 min after surgery before the protocols were begun. In all cases, the coronary branch was occluded for 30 min and reperfused for 2 h.

Experimental protocol
Six groups were studied in control wild-type CD1 mice (Charles River, Kisslegg, Germany). Control mice had only the index occlusion followed by reperfusion. In drug-treated mice with potassium canrenoate was started i.v. 5 min before the onset of reperfusion. Canrenoate was given in different concentrations as a bolus. Control animals received the corresponding amount of saline. Two additional treatments (vehicle and 1 mg/kg BW canrenoate) were performed in CD73 knock-out and adenosine A 2b receptor knock-out mice. 9

Measurement of risk zone and infarct size
After completion of the protocol, the coronary artery was re-occluded, and Evans blue was injected retrogradely through the aortic root to demarcate the ischaemic zone (region at risk zone). Hearts were excised, perfused with 0.9% saline, weighed, frozen, and then cut into 1 mm thick transverse slices. The slices were incubated in 1% triphenyltetrazolium chloride (TTC) in sodium phosphate buffer (pH 7.4) at 388C for 20 min. Triphenyltetrazolium chloride stains the non-infarcted myocardium brick-red indicating the presence of dehydrogenase enzymes. The slices were then immersed in 10% formalin to enhance the contrast between stained (viable) and unstained (necrotic) tissue. The areas of infarct and risk zone were determined by planimetry of each slice and volumes were calculated by multiplying each area by the slice thickness and summing the areas for each heart. Infarct size was expressed as a percentage of the risk zone.

Cardiac enzyme measurement
After removing the heart, blood was collected from the abdominal aorta and centrifuged for measurement of cardiac troponin I (cTnI) in serum using a CTNI reagent kit and a Dimension Vista 1500, Integrated Analytics System (Siemens Healthcare Diagnostics, Deerfield, IL, USA).
Open-chest in situ rabbit heart Male New Zealand White rabbits (2.7-3.3 kg) were anaesthetized with zolazepam and tiletamine (20 -30 mg/kg i.v. each). Animals were ventilated with 100% oxygen. Anaesthesia was thereafter maintained by i.v. pentobarbital as need to maintain a surgical plane. Arterial pressure was measured in a catheter in a marginal ear artery. An electrocardiogram was also recorded. A left thoracotomy was performed at the fourth intercostal space and a 3/0 Prolene suture was passed beneath an anterior branch of the left coronary artery to form a snare. Ischaemia was confirmed by the occurrence of ST segment deviation in the electrocardiogram. Reperfusion was induced by releasing the snare. The chest was closed during reperfusion to prevent cardiac cooling.

Experimental protocol
All rabbits were subjected to a 30 min coronary artery occlusion. Five minutes before reperfusion, they randomly received an i.v. bolus administration of saline (control) or potassium canrenoate (1 mg/kg). In a first set of experiments, rabbits were euthanized after 4 h of reperfusion for infarct size assessment. In a second set of experiments, the open-chest surgery was done in sterile conditions and rabbits underwent 72 h reperfusion. During those 72 h, rabbits returned to animal room and received buprenorphine for analgaesia (0.02 mg/kg/12 h s.c.).

Measurement of risk zone and infarct size
After completion of the 4 or 72 h of reperfusion, risk zone and infarct size were measured according to the above mouse protocol.

Isolated rat heart
Wistar rats were anaesthetized with pentobarbital sodium (60 mg/kg i.p.) after which the heart was excised and perfused on a Langendorff Figure 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 ischaemia 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. Filled square, regional ischaemia. apparatus with Krebs -Henseleit bicarbonate buffer containing (mM) 118.5 NaCl, 24.8 NaHCO 3 , 4.7 KCl, 1.2 MgSO 4 , 1.2 KH 2 PO 4 , 2.5 CaCl 2 , and 10 glucose, and bubbled with 95% O 2 /5% CO 2 to a pH of 7.35-7.45 at 388C. A snare as above was passed around an epicardial coronary arterial branch.

Experimental protocol
Fourteen experimental groups were studied as in Figure 1. The coronary branch was occluded for 30 min and reperfused for 2 h in all groups. In the control group, no other treatment was given. In the eplerenone-treated groups, 10 mM eplerenone was added to the perfusate starting 5 min prior to reperfusion. In the following inhibitor groups, one of four inhibitors was co-infused with eplerenone including the adenosine receptor (AR) blocker 8-p-sulphophenyltheophylline (SPT, 100 mM), the PKC inhibitor chelerythrine (CHEL, 2.8 mM), the ERK inhibitor U0126 (500 nM), or a PI3-kinase inhibitor wortmannin (100 nM). Each inhibitor was tested alone to exclude independent effects of the blockers. In two additional groups, together with eplerenone, aldosterone was given in two different concentrations in order to compete with receptor binding. Aldosterone (500 nM) was finally given alone to exclude any direct effect. Finally, canrenoate was given (50 mM) to confirm its protective properties also in the isolated rat heart model. We delineated the risk zone with 2 -9 mm green fluorescent microspheres and infarction with TTC staining as described above for in situ mouse hearts.

Biochemical studies
Isolated rat hearts were perfused as described earlier. Transmural biopsies of the left ventricle were obtained right before ischaemia (baseline) and at 10 min of reperfusion following a 30 min period of global ischaemia as indicated by the arrows in Figure 1. Seven groups were studied: control hearts without any further treatment, treatment with potassium canrenoate (50 mM) alone and in the presence of aldosterone, treatment with eplerenone (10 mM) alone and in the presence of either aldosterone or the PI3-kinase blocker wortmannin, and aldosterone alone. Finally, the level of phosphorylated Akt and phosphorylated ERK1/2 in the heart at reperfusion was determined by western blotting as described previously. 10 Level of phosphorylation was measured as fold of total Akt and ERK1/2, respectively, and normalized to the baseline sample (n ¼ 6 per group).

Materials
Eplerenone was provided by Pfizer (Karlsruhe, Germany). Polyclonal antibodies against Akt and its phosphorylated form (Ser473), ERK (p44/42 MAPK) and its phosphorylated form (Thr202/Tyr204), HRP-linked anti-rabbit IgG antibody used on western blot analysis, and cell lysis buffer were purchased from Cell Signaling Technology (Beverly, MA, USA). CHEL and U0126 were from Calbiochem (San Diego, CA, USA), while all other chemicals were from Sigma-Aldrich Chemical Co. Eplerenone, wortmannin, and U0126 were dissolved in dimethyl sulfoxide (DMSO) before being diluted in Krebs-Henseleit buffer resulting in a DMSO concentration less than 0.01%. All other inhibitors were dissolved in the buffer directly.

Statistics
Data are presented as means + SD. Differences in infarct size, troponin I levels, Akt, and ERK1/2 phosphorylation among groups were compared by one-way ANOVA with Fisher LSD post hoc testing using SigmaStat 3.0 software. For the rabbit experiments, heart rate and mean blood pressure were compared between groups using a two-way ANOVA for repeated measures. A value of P , 0.05 was considered significant.

Infarct size measurements in open-chest mice
We measured infarct size after 30 min regional ischaemia and 2 h of reperfusion. Figure 2A reveals that the treatment with a bolus of potassium canrenoate (CAN) caused a dose-dependent reduction in infarct size with a peak effect at 1 mg/kg body weight (7.3 + 4.7% compared with 37.8 + 7.5% in control, P , 0.001).
In order to test our hypothesis that the MR blockers protect through pre-conditioning-like signalling involving extracellular adenosine interacting with the A 2b AR, we used cd73 2/2 or A 2b AR 2/2 mice. It has previously been shown that CD73 an exo-nucleotidase is the source of pre-conditioning's protective adenosine, 11 which then presumably works through the A 2b AR. 9 As shown in Figure 2A, the CD73 and A 2b AR knock-out animals could no longer be protected with CAN.
The infarct size data were mirrored by the levels of cardiac troponin I (cTnI) measured in the serum at the end of reperfusion. Treatment with canrenoate significantly reduced the amount of cTnI release (single measurements expressed as means + SD) again with a peak at 1 mg/kg ( Figure 2B).

Infarct size measurements in open-chest rabbits
We next tested 1 mg/kg canrenoate 5 min prior reperfusion in an in vivo rabbit model. Similarly, canrenoate was able to reduce infarct size in these hearts as shown in Figure 3. Importantly, this beneficial effect was observed when reperfusion was extended up to either 4 or 72 h. Canrenoate had no haemodynamic effect on heart rate or blood pressure ( Table 1).

Infarct size measurements in isolated rat hearts
We next performed mechanistic studies in isolated rat hearts subjected to 30 min regional ischaemia. As seen in Figure 4, eplerenone at 10 mM throughout reperfusion reduced infarct size from 40.8 + 5.3% in the control group to 10.9 + 7.2% (P , 0001), while only 1 mM eplerenone had no effect (data not shown).
Pharmacological inhibition of the AR (SPT), PKC (CHEL), PI3-kinase (WORT), or ERK (U0126) each abolished eplerenone's protection, indicating that protection depended on the same signalling elements as pre-conditioning. The inhibitors alone had no effect.
Eplerenone is a competitive inhibitor of aldosterone. To rule out any non-MR mechanism, aldosterone was given simultaneously with the eplerenone treatment to repopulate MR. Co-infusion of 50 nM aldosterone blunted eplerenone's protection and 500 nM aldosterone abolished it. Aldosterone alone had no effect on infarct size. Canrenoate (50 mM) similarly worked in the isolated rat heart model and reduced infarct size from 40.8 + 5.3% in the control group to 10.2 + 3.8% (P , 0001), while only 10 mM had no effect (data not shown).

Phosphorylation of Akt and ERK1/2
Akt and ERK1/2 activation by phosphorylation at reperfusion are central to protection by pre-conditioning. We found a marked increase in Akt and ERK1/2 phosphorylation following MR blockade at reperfusion (Figure 5A-C ). Both canrenoate and eplerenone increased Akt and ERK1/2 phosphorylation and that was abolished with co-treatment with aldosterone. Aldosterone alone had no effect, and the PI3-kinase inhibitor wortmannin totally blocked eplerenone-induced phosphorylation.

Discussion
The MR antagonists potassium canrenoate and eplerenone are both cardioprotective against infarction when administered prior to reperfusion. Their rapid action suggests a non-genomic effect   Figure 4 Results of the isolated rat hearts experiments. Drugs were given as depicted in Figure 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 signalling elements. 8p-sulfophenyladenosine (SPT), adenosine receptor blocker; wortmannin (WORT), PI3 kinase inhibitor; chelerythrine (CHEL), PKC inhibitor; 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 , 0.001 vs. control. Figure 5 (A -C) 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 ischaemia (baseline) and at 10 min of reperfusion following a 30 min period of global ischaemia as indicated by the arrows in Figure 1. Phosphorylation of all tested kinases was clearly increased at reperfusion compared with untreated control when either canrenoate (CAN) or eplerenone (EPL) was present. Protection was abolished with co-infusion of aldosterone (ALDO) or wortmannin (WORT), while ALDO had no effect on its own. Results represent the mean + SD of six independent experiments, *P , 0.01, **P , 0.001 vs. control.
of MR blockade. Further, we could show that the observed infarct-reducing properties of MR blockade involve key elements of the signalling pathway used by pre-and post-conditioning.
Finally, similar protection was demonstrated in three species. Ischaemia-induced infarction can be reduced either with preconditioning, an intervention prior to ischaemia, or by postconditioning, an intervention at the onset of reperfusion. While Chai et al. 4 recently reported that MR blockade was cardioprotective when applied as a pre-conditioning stimulus, we found that MR antagonists are also protective in the more clinically relevant setting of administration at the end of ischaemia. Both phenomena, ischaemic pre-and post-conditioning share a common signalling pathway that includes PKC, ARs, PI3-kinase/Akt, and ERK. 12 This signalling is thought to protect by suppressing the opening of mitochondrial permeability transition pores at reperfusion. In the present study, we showed that protection from MR blockade prior to reperfusion depends on these same signalling elements suggesting a common mechanism.
Fujita et al. 13 demonstrated that aldosterone worsens injury from ischaemia by a rapid, non-genomic effect. Here, we could not see any difference between the untreated control group and aldosterone-treated isolated rat hearts. In contrast to our Langendorff-perfused heart model, Fujita et al. used an in situ dog model which could explain these differences.
Most post-conditioning agents have a narrow therapeutic window, and delaying the intervention for only a few minutes after the onset of reperfusion eliminates the protection. 14,15 Canrenoate-induced protection was also absent when the bolus was given within 1 or 15 min after onset of reperfusion (data not shown), confirming a non-genomic mechanism due to the rapidity of the protection with MR blockers.
In large-scale clinical trials (RALES and EPHESUS, respectively), 16,17 treatment of post-myocardial infarction patients with MR antagonists reduced mortality most likely via the prevention of remodelling. In these trials, MR antagonists were given long after the onset of reperfusion, and, therefore, it is not likely that the observed benefits were due to infarct size reduction. Since mortality after AMI is directly correlated with infarct size, the antiinfarct effect of immediate MR blockade shown in the present study could have additional beneficial effects in patients with AMI.
The protection could either be due to the inhibition of detrimental effects of aldosterone itself, to a unique receptor response to antagonist binding, or an MR-independent non-specific effect. Since we could overcome the cardioprotective effect of the competitive MR inhibitors eplerenone and canrenoate with a high concentration of aldosterone, it is most likely that elimination of aldosterone's effect at the MR was involved. Nevertheless, any additional non-specific drug effects of canrenoate or eplerenone cannot be fully excluded. Since ischaemia itself leads to a release of aldosterone by the heart, 18 we can speculate that occupying MR may inhibit the cell's natural protection against ischaemia. If the inhibition were suppressed with MR blockade or overcome by pre-or post-conditioning, the endogenous protective signalling could then promote cell survival.
The powerful cardioprotection of pre-and post-conditioning must be made available to patients with ST-segment elevation AMI (STEMI). Several forms of post-conditioning, either repetitive short ischaemic episodes at reperfusion 19 or pharmacological preconditioning with either cyclosporine A 20 or atrial natriuretic peptide, 21 have been tested in STEMI patients and the results were encouraging. Unfortunately, none of these interventions is ideally suited for routine clinical use. Our demonstration of good protection prior to reperfusion across species lines encourages us that potassium canrenoate is a good candidate for a clinical trial in patients with STEMI. Canrenoate is routinely used clinically as a diuretic with an initial dose of 200 mg. Canrenoate caused a profound infarct size reduction in animal models of AMI in mice and rabbits at a much lower, single bolus, dose of 1 mg/kg.