Spironolactone and eplerenone are cardioprotective during early phase of ischemia in rats submitted to acute coronary occlusion

Introduction: Mineralocorticoid receptor antagonists (MRAs) are effective in reducing left ventricle remodeling and sudden death after acute myocardial infarction (AMI). Objectives: MRAs in vitro display cardioprotective effects, independent of MR; however, it is unknown whether the rapid effects of MRAs are cardioprotective in vivo. This study evaluated the acute effects of spironolactone and eplerenone in the first minutes of AMI. Methods: Wistar Rats, submitted or not to bilateral adrenalectomy, were treated orally with spironolactone (20 mg/kg) or eplerenone (10 mg/kg), and submitted to the left coronary ligation, under anesthesia. Electrocardiogram (ECG) recordings were obtained to evaluate ST-T segment, QT, and QTc intervals. Arterial pressure was also measured before (baseline) and after coronary ligation. Results: Spironolactone or eplerenone given, one hour before coronary ligation, prevented STT segment elevation in adrenalectomized and non-adrenalectomized. QT interval analysis showed that MRAs Research, Society and Development, v. 11, n. 3, e24011326498, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i3.26498 2 prevented its prolongation after coronary ligation. QT and QTc intervals remained similar to baseline and were smaller than the values displayed by the non-treated group. Animals treated with spironolactone, regardless of adrenalectomy, showed a 3-fold reduced mortality rates compared to the control group. Conclusion: MRAs display acute cardioprotective effects in early phase of AMI, which are independent of aldosterone.


Introduction
Acute myocardial infarction (AMI) is among the leading causes of death and morbidity worldwide (Moran et al., 2014).
The mineralocorticoid receptor antagonists (MRA), spironolactone and eplerenone, are effective to treat patients with mild or chronic heart failure (HF) and left ventricular (LV) dysfunction after AMI, mainly by reducing morbidity and mortality (Pitt et al., 2003;Zannad et al., 2010). Higher levels of aldosterone after AMI are associated with poor clinical outcomes, including mortality, sudden cardiac death, and heart failure (Beygui et al., 2009;Beygui et al., 2013). Aldosterone is associated with endothelial dysfunction, inflammation, ventricular hypertrophy, fibrosis, and cardiac remodeling (Weber 2001). Still, beneficial effects of early MRAs administration during AMI are supported by both experimental (Fraccarollo et al., 2008;Fraccarollo et al., 2015) and clinical studies (Montalescot et al., 2014).
The classical MRAs mechanism of action is the blockade of aldosterone action in the heart via mineralocorticoid receptors (MR) (Struthers 2004). The most comprehend mechanism of action of aldosterone in the heart is the genomic (slow) pathway, which is mediated by cytosolic intracellular MR receptors. This process involves translocation of aldosterone-MR complex to nucleus that acts as a transcriptional regulator that ultimately leads to protein synthesis (Rogerson et al., 2004).
Moreover, some protective effects attributed to MRAs occur even in the absence of abnormal levels of aldosterone. In the Randomized Aldactone Evaluation Study (RALES) and Eplerenone in post-myocardial infarct heart failure study (EPHESUS) aldosterone levels were physiological and MRA treatment displayed unquestioned benefits in HF patients (Pitt et al., 2003;Zannad et al., 2010). Additionally, low-dose spironolactone and eplerenone reduced the infarcted area and apoptosis in isolated heart of adrenalectomized or non-adrenalectomized rats (Mihailidou et al., 2009;Loan et al., 2012). However, it is not yet known whether the acute use of spironolactone and eplerenone are cardioprotective.
In this study, we evaluated whether MRAs, spironolactone or eplerenone, are cardioprotective during early phase of ischemia in a model of acute cardiac ischemia induced by coronary ligation in rats. Thus, animals were submitted to bilateral adrenalectomy to evaluate the effect of MRAs in absence of circulating endogenous aldosterone. It was then analyzed in vivo electrocardiogram (ECG) parameters, such as ST-T segment elevation, QT, and QTc intervals, as well as blood pressure and cardiac histological alterations of adrenalectomized and non-adrenalectomized rats subjected to cardiac ischemia.

Methodology
All the experimental procedures were approved by the Ethics Committee for Animal Use at the Federal University of Ouro Preto. (Protocol N o . 2011/88). All procedures performed in studies involving animals were in accordance with the ethical standards of Brazilian animal welfare laws and guidelines.

Animals
Male Wistar rats (200 to 250 g) were obtained from the Vivarium of the Federal University of Ouro Preto (UFOP, Brazil). The animals were kept into polypropylene cages with free access to food and water with light and dark periods of 12/12 hours.

Bilateral adrenalectomy and dosage of serum aldosterone
The rats were anesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg) intraperitoneally (i.p). After shaving the lumbar region, a longitudinal skin and muscle incision was performed, the adrenal glands were removed, and the incision sutured. After that, the animals received flunixin (Banamine® -Intervet/Matercorp, Rio de Janeiro, RJ), an anti-inflammatory and analgesic drug, at 1 mg/kg/day. The animals were kept in cages with free access to food and water containing 0.9% NaCl [Drazen et al., 2004]. Only after a recovery period of 48 h, the animals underwent to left coronary ligation procedure. At the end of the experiment, blood samples were collected, and serum aldosterone was measured by radioimmunoassay (RIA aldosterone Assay, Cisbio Assays, Codolet, France).

Experimental Protocol
The rats were treated orally (per gavage) with spironolactone (20 mg/kg) or eplerenone (10 mg/kg). All the drugs were purchased from Sigma-Aldrich, USA. The doses used for all drugs were based on previous studies (Kobayashi et al., 2006;Silvestre et al., 1999). The treatment was given to awaken animals 30 minutes before anesthesia for coronary ligation surgery.
The animals were tracheostomized and connected to a small rodent respirator (SAR-840, USA) to allow artificial ventilation with room air (volume 1.5-2.5 ml/cycle and frequency of 45-50 cycles/min) (Bobryshev et al., 2009). The femoral artery and vein were catheterized to obtain the signal from the arterial pressure (AP) and to administer pancuronium bromide (1 mg/kg), a muscle relaxant, to assist with mechanical ventilation.
After these procedures, hypodermic needles were positioned subcutaneously to obtain ECG signal lead II and the femoral artery catheter was connected to a pressure transducer (TruWave; Edwards Life Sciences, USA). The method used to Research, Society andDevelopment, v. 11, n. 3, e24011326498, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i3.26498 4 induce myocardial ischemia was the left descending coronary ligation (Selye et al., 1960). The Sham group was submitted to the same surgical procedure without coronary ligation. The control group was submitted to coronary ligation and treated with vehicle. The entire procedure from the drug treatment until coronary ligation took approximately 60 to 70 minutes. Then, the ECG signal was continuously obtained in anesthetized rats before (baseline) and during the 60 minutes after left coronary ligation. After one hour of ligation procedure, the animals were euthanized, and the heart was removed for histological analysis.

Cardiovascular parameters determination
To obtain the arterial pressure (AP) and electrocardiography (ECG) signal parameters, a real-time system of signal conditioner was used at a frequency of 1200 Hz, using an analog-digital converter board of 12-bit resolution (Daqboard 2000, USA). To analyze the systolic blood pressure (SBP), diastolic blood pressure (DBP), and ECG parameters used 2 seconds long segments, that correspond to 8 to 12 heart beats, before (baseline), and 60 minutes after coronary ligation was extracted. ECG signal, baseline, 10, 30, and 50 minutes after coronary ligation, was used to measure RR interval to obtain the heart rate (HR), QT interval and QTc interval, that is the QT corrected by HR according to Fridericia's formulae: QTc = QT/(RR) 0.33 . The QT interval is widely used in experimental and clinical studies to identify cardiac risk, as susceptibility to arrhythmias and cardiac ischemia (Baillard et al., 2000). The ST segment elevation, a parameter that indicates the myocardium ischemia, was also evaluated. This parameter was calculated by the area under the curve of ST-T segment. From lead II ECG signal a horizontal line was drawn between the beginning of the inclination of S wave and the end of T wave when the latter reaches the isoelectric point (Resende et al., 2012). To determine the area, the software Analyzer 7.0/Hemolab Data Acquisition software 14.2 was used.

Histological Analysis
The hearts were fixed into a 10 % formalin solution. The ventricles were separated and sectioned into three transverse sections; dehydrated with alcohol in increasing concentrations; diaphonized with xylene and embedded in paraffin. Paraffin sections of approximately four micrometers thick were obtained in a microtome, fixed on glass slides, and then stained with hematoxylin and eosin (Acikel et al., 2005).

Statistical Analysis
For the analysis of ECG parameters and AP the comparison between the groups at different times was performed using one-way ANOVA followed by Bonferroni post-test. When the analysis was conducted within different groups at the same time point, two-way ANOVA followed by Tukey posttest was used. Fisher test was used to identify mortality differences. Statistical analysis was performed with GraphPad Prism Project (version 6.0) software. Significant differences at p value < 0.05 were considered.

Serum aldosterone after adrenalectomy
Serum aldosterone levels were dramatically reduced after the adrenal glands removed compared to the Sham group (93 ± 25.7 ng/dl). No difference was observed between the groups submitted solely to coronary ligation (2 ± 0.6 ng/dl) compared to groups submitted to the same procedure and treated with either spironolactone (1 ± 0.7 ng/dl) or eplerenone (1 ± 0.5 ng/dl). Table 1 presents SBP and DBP data of all experimental groups, both baseline and after 60 minutes of coronary ligation.

Blood pressure and ECG parameters
In non-adrenalectomized rats, we observed a significant reduction of SBP and DBP in the vehicle-treated group after coronary ligation. No significant difference between SBP and DBP was observed in spironolactone-or eplerenone-treated groups submitted to coronary ligation compared to baseline of the same group or vehicle treatment; thus, showing the ability of both drugs to prevent arterial pressure reduction observed after ischemia. In adrenalectomized rats, SBP and DBP were significantly lower in all groups submitted to coronary ligation despite different treatments compared to baseline and compared to nonadrenalectomized with the same treatment (Table 1). ECG signal lead II was used for the quantitative analysis of ST segment elevation. Figure 1 shows the percentage variation of area under the curve ST-T during one hour after coronary ligation procedure. Coronary ligation of nonadrenalectomized rats without treatment promoted ST-T segment elevation compared to the Sham group ( Figure 1A). The MRAs, spironolactone or eplerenone orally administered one hour before ligation, prevented ST segment elevation ( Figure 1A) compared with the vehicle treated the coronary ligation group and the response was also similar to the Sham group. The response profile observed in adrenalectomized animals subjected to coronary ligation without treatment and treated with spironolactone or eplerenone was also similar ( Figure 1B), showing clearly that MRAs drugs could prevent ischemia alterations of ECG signal even in the absence of aldosterone. Values are expressed as mean ± SEM two-way ANOVA followed by Bonferroni Posttest. *p < 0.05 compared to coronary ligation group vehicle treated; λ p < 0.05compared to the Sham group. N = 3 -6. Source: Authors.
Regarding HR, significant bradycardia was observed one hour after coronary occlusion in all groups compared to baseline values. However, it was not observed difference in HR between any groups. Considering that prolonged QT interval of ECG as an arrhythmia and sudden cardiac death predictor (Schwartz & Wolf 1978), this parameter was analyzed, being corrected by HR. QT and QTc intervals of ECG analysis were performed at baseline 10, 30, and 50 minutes after coronary ligation ( Figure 2). Baseline values of QT and QTc intervals were reduced when rats were treated with spironolactone (60 ± 3.1 ms and 101 ± 5.1 ms, respectively, for QT and QTc) or eplerenone (62 ± 2.5 ms and 99 ± 3.5 ms, respectively, for QT and QTc) compared to vehicle treatment (73 ± 3.3 ms and 125 ± 6.1 ms, respectively, for QT and QTc). Coronary ligation of vehicle-treated rats induced QT interval prolongation observed 50 minutes after and this effect was not observed for any treatment or for QTc interval, thus showing the ability of MRA treatment to prevent this alteration. Although we observed a decreasing trend, the lack of QTc alteration was probably due to intense bradycardia induced by the surgical procedure. Values are expressed as mean ± SEM. Two-way ANOVA followed by Bonferroni post-test. *p < 0.05 compared to baseline (zero). # p < 0.05 differences compared to vehicle treatment. N = 4 -6. Source: Authors.
The mortality after coronary ligation was 30.6 % and 37.5 % of vehicle treated non-adrenalectomized and adrenalectomized animals, respectively, in less than one hour after (Table 2). Animals treated with eplerenone regardless of adrenalectomy showed similar mortality rate compared to the vehicle treated group (Table 2). Non-adrenalectomized and adrenalectomized rats treated with spironolactone showed approximately 3-fold reduction of mortality rate within one hour after ligation. Groups were submitted to coronary ligature and mortality evaluated until one hour after. *Fisher test indicates the statistical differences. Source: Authors.

Histological Analysis
Histological sections were stained with hematoxylin and eosin, and we observed that the cardiac muscle fibers from the groups submitted to coronary ligation display abnormalities, presence of necrosis, interstitial edema, and absence of crosscutting striations. These changes were observed in all groups irrespective of treatment and adrenalectomy. No edema or necrosis was observed in the Sham group (Figures 3 and 4).

Discussion
This study demonstrated the effects of MRAs, spironolactone and eplerenone, at first hour after myocardial ischemia in rats. These two drugs could prevent ST-T increase and QT interval prolongation induced by coronary ligation in the presence or absence of circulating endogenous aldosterone.
The elevation of the ST-T segment is an ECG marker of cardiac ischemia, and the reduction of this parameter indicates cardioprotection (Coppola et al., 2013;Birnbaum & Drew 2003). Our data showed that treatment with spironolactone or eplerenone prevents ST-T segment elevation in non-adrenalectomized and adrenalectomized rats submitted to ischemia, suggesting a possible reduction of infarct area. No significant changes were found in hematoxylin and eosin histological analysis, but these data are not conclusive. Our AMI model has permanent left anterior descending artery ligation for 1 h. The time required for completely necrosis of cardiac cells is about 30-90 minutes in rats (Jugdutt 1993). HE staining analysis is not the most precise method to quantify ischemia injuries in AMI. The infarct size and the area at risk should be performed in the future. Previously, studies demonstrated that spironolactone or eplerenone infusion before and immediately after ischemia could reduce the infarcted area in isolated rat heart (Mihailidou et al., 2009;Loan et al., 2012;Chai et al., 2005). This protective action can be explained since MRAs prevent cardiac apoptosis by inhibiting the degradation of apoptosis repressor with caspase-recruiting domain, a multifunctional inhibitor of apoptosis (Loan et al., 2012). These findings provide direct evidence that MRAs have effects independent of MR blockade.
Another electrocardiography alteration associated with AMI is a prolongation of QT interval duration. The prolonged QT interval leads to electrical conduction disorder that alters the ventricular repolarization and increases the likelihood of ventricular tachyarrhythmia and sudden death (Passman & Kadish 2001). MRAs are well-known drugs that reduce the rate of sudden cardiac death in heart failure patients (Pitt et al., 2003). Eplerenone treatment also showed significant reductions in sudden death and total mortality in the early post-MI period (Pitt et al., 2005). Spironolactone improved QT interval dispersion (Yee et al., 2001) and, along with the angiotensin-converting-enzyme inhibitor, reduced the arrhythmic score post-myocardial infarction (Beck et al., 2001). In fact, aldosterone has pro-arrhythmic effects. MR activation leads to arrhythmia by increasing tissue remodeling and fibrosis as well as alteration in the electrophysiology of the cardiomyocytes, and leads to early and delayed after-depolarization, which is a major cause of life-threatening arrhythmia (Gravez et al., 2013). Therefore, these data may explain, sudden death decrease of MRA treated patients, but do not explain why MRAs display cardioprotective effects in patients with normal levels of aldosterone. In fact, our study demonstrated antiarrhythmic effects of MRA in the first phase of AMI that prevents QT interval prolongation fifty minutes after coronary ligation, which suggests that MRAs have acute arrhythmogenic effects.
Additionally, the group treated with spironolactoneregardless of adrenalectomydisplayed a 3-fold reduction in the mortality rates compared to the non-treated and eplerenone-treated groups, probably due to an antiarrhythmic effect.
Previous studies have shown that animals subjected to coronary ligation and treated with spironolactone during 7 days show reduced mortality compared to the control group (Song et al., 2011). This cardioprotective effect was attributed to the inhibition of ischemia-induced ventricular premature beats by reducing the expression hyperpolarization-activated cyclic nucleotide-gated (HCN) isoform 4 (Song et al., 2011). It is unclear why eplerenone did not reduce the mortality rate. This result is contrary to the one found in mice submitted to coronary ligation as survival rate was significantly increased in the eplerenone group compared with the spironolactone group (Fraccarollo et al., 2008).
The acute cardioprotective mechanisms of spironolactone and eplerenone are yet unknown. Previously, we have demonstrated in vitro that adding spironolactone or eplerenone for 30 min increases the intracellular levels Ca 2+ , cAMP (only for spironolactone), cGMP, and increase cardiomyocyte proliferation, as well as reduce fibroblast proliferation (Hermidorff et al., 2015). The activation of these mediators can be associated with unknown membrane-receptor (Hermidorff et al., 2015;Hermidorff et al., 2017). Rapid effects of aldosterone have been associated with GPER-1 receptor and it been demonstrated that MRAs inhibited partially GPER-mediated aldosterone effects in the vascular system (Gros et al., 2011;Gros et al., 2013); however, in the heart, this antagonism was not observed (Ashton et al., 2015).
In addition, eplerenone or canrenoate treatment increased phosphorylation of AKT and ERK1/2 at reperfusion in the isolated rat hearts and the protection was inhibited by co-treatment with inhibitors of the adenosine receptor, protein kinase C, PI3-kinase, and ERK (Schmidt et al., 2010). Adenosine appears to have an important role in these pathways. Stimulation of membrane-bound adenosine receptors (A1, A2A, A2B, and A3) reduces isquemia-reperfusion injury (Paiva et al., 2009) and prevents cardiac arrhythmias (Riksen & Rongen 2012). However, in a clinical study, eplerenone does not augment extracellular adenosine formation in healthy human subjects (van den Berg et al., 2014). Another line of thought to explain our findings is the fact that MR antagonists may act as inverse agonists, occupying this receptor, triggering opposite responses of what an agonist would do (Mihailidou et al., 2009). Thus, it is still early for any definitive conclusion, as more studies need to be performed regarding the direct pathways activated by MRAs in the cardiovascular system.

Conclusion
We demonstrated in this work that both eplerenone and spironolactone prevent ECG changes induced by ischemia in adrenalectomized rats. It has also been shown that these effects are independent of the antagonistic action of these drugs on the aldosterone receptor (MR). Our data, added to what exists in the literature, clearly demonstrate the beneficial interference of MRAs already in the initial phase of acute myocardial infarction. Seeking to understand more about the mechanisms of action of these pharmacos, at the molecular level, is a necessary task. This understanding will allow the design of new pharmacos, more specific, more efficient and certainly with fewer unwanted effects. Jugdutt, B.I. (1993). Prevention of Ventricular Remodeling Post Myocardial-Infarction -Timing and Duration of Therapy. Can J Cardiol 9, 103-114.