Effect of esaxerenone on ischaemia and reperfusion injury in rat hearts

Abstract

Open in new tabDownload slide

OBJECTIVES

In myocardial infarction, the addition of mineralocorticoid receptor blockers to standard therapies, such as angiotensin-converting enzyme inhibitors or beta-blockers, reportedly reduces mortality and cardiac events. We investigated whether the non-steroidal mineralocorticoid receptor blocker esaxerenone has cardioprotective effects and its protective mechanisms.

METHODS

Isolated rat hearts were Langendorff-perfused (constant pressure, 80 mmHg) with oxygenated Krebs–Henseleit bicarbonate buffer and reperfused for 60 min; afterwards, recovery of function (left ventricular pressure, measured with an intraventricular balloon) and myocardial injury were measured. In a preliminary study, we determined the optimal concentration of esaxerenone required for myocardial protection. Next, esaxerenone was administered in the pre- and post-ischaemic phases to determine the optimal timing of administration. In addition, we assessed coronary flow response to acetylcholine with and without esaxerenone. We examined whether esaxerenone-induced cardioprotection was prevented by targeting putative components in the preconditioning manner (the mitochondrial ATP-sensitive potassium [K_ATP] channel).

RESULTS

Myocardial protection by esaxerenone was observed when esaxerenone was administered before ischaemia but not after ischaemia. The coronary flow response to acetylcholine was significantly better in the esaxerenone group than in the control group. The cardioprotective effect of esaxerenone was eliminated by the mitochondrial K_ATP channel blocker, 5-hydroxy decanoate.

CONCLUSIONS

This study confirmed the myocardial protective effect of the pre-ischaemic administration of esaxerenone. Esaxerenone may contribute to coronary endothelial protection and exert pharmacological preconditioning via the mitochondrial K_ATP channel.

INTRODUCTION

Ischaemia-related deprivation of oxygen and nutrient supply results lactate production, a drop in intracellular pH, ATP depletion and inhibition of myocardial contractile function within the myocardium. Furthermore, myocardial reperfusion can induce coronary endothelial dysfunction, microvascular obstruction or cardiomyocyte death, which is known as reperfusion injury. To reduce myocardial injury and improve post-ischaemic haemodynamic function, myocardial protection has aimed to apply potential therapeutic strategies for preventing lethal ischaemia–reperfusion injury discovered in basic research to the clinical setting [1]. The aldosterone/mineralocorticoid receptor pathway plays a crucial role in the pathogenesis of cardiovascular injury by directly exacerbating inflammation [2], oxidative stress [3] and fibrosis [4]. Thus, aldosterone/mineralocorticoid receptor pathway activation is a risk factor for cardiovascular diseases. In patients with heart failure, it has been proven that the prognosis can be improved by blocking the renin–angiotensin–aldosterone system [5]. In large clinical trials, the addition of a nonselective mineralocorticoid receptor blocker (MRB), spironolactone, to angiotensin-converting enzyme inhibitors and a loop diuretic reduces the risk of mortality and morbidity among patients with severe heart failure [6]. Medical therapy for post-myocardial infarction patients with standard treatments, including angiotensin-converting enzyme inhibitors and beta-blockers, and the addition of the selective MRB eplerenone reduced mortality and cardiac accident events [7]. In these trials, the MRBs were administered long after the onset of reperfusion. However, the administration of eplerenone before ischaemia or prior to reperfusion has been demonstrated to reduce myocardial damage in animal studies [8–10]. Spironolactone or eplerenone prevents the mitochondrial production of superoxide anions induced by aldosterone in the myocardium [11]. Additionally, eplerenone pretreatment attenuates cardiac steatosis and apoptosis [12]. This protection against ischaemia–reperfusion injury depends on the phosphatidylinositol 3-kinase (PI3K)/Akt pathway [13]. Furthermore, the PI3K/Akt pathway plays a major role in ischaemic preconditioning [14].

Esaxerenone is a novel MRB with no steroid skeleton and higher selectivity for mineralocorticoid receptors than conventional MRBs [15]. Therefore, esaxerenone is expected to exert a lower effect on other steroid receptors and possess fewer side effects.

It was hypothesized that the administration of esaxerenone might attenuate ischaemia–reperfusion injury in the heart and have a protective effect on the myocardium. Therefore, the first experiment described herein was conducted to investigate the efficacy of this agent. Consequently, further experiments were conducted to examine the optimal timing for esaxerenone administration and investigate endothelial preservation by response to acetylcholine response or the involvement of preconditioning phenomena using mitochondrial ATP-sensitive potassium (K_ATP) channel blocker, 5-hydroxydecanoate (5HD).

MATERIALS AND METHODS

Ethics statements

All animals received humane care in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animals’, published by the National Institute of Health (NIH) [NIH publication number (no.): 85-23, revised 1996]. This study was approved by the Animal Ethics Committee of the Nippon Medical School (no.: 2020-079).

Animals

In total, 78 male Wistar rats (weight, 240–300 g; Oriental Yeast Co., Ltd, Tokyo, Japan) were used in this study. The rats were anesthetized with pentobarbital (50 mg/kg, intraperitoneally) and anticoagulated with heparin (1000 IU/kg, intravenously). The exclusion criteria by cardiac function are listed in the next section.

Heart isolation and perfusion

The rat hearts were quickly excised and immersed in the cold (4°C) Krebs–Henseleit bicarbonate buffer (KHB). Afterward, the aorta was cannulated, and the heart was perfused at 37°C in the Langendorff mode with KHB at constant pressure (80 mmHg), as previously described [16]. Next, the heart was prepared by inserting a saline-filled vinyl balloon into the left ventricle via the left atrium. The balloon was connected to a pressure transducer to measure left ventricular pressure. The balloon volume was then adjusted to obtain a left ventricular end-diastolic pressure (LVEDP) of 3–8 mmHg. All hearts were equilibrated with 20 min of aerobic perfusion, and the following baseline readings were recorded: left ventricular systolic pressure (mmHg), LVEDP (mmHg), heart rate (beats/min) and coronary flow (ml/min). Left ventricular developed pressure (LVDP) was calculated (LVDP = left ventricular systolic pressure − LVEDP). At the time of baseline readings, a heart was excluded if the acceptable ranges of LVDP (>100 mmHg), heart rate (>200 beats/min) and coronary flow (10–20 ml/min) were not met. All hearts were reperfused for 60 min after 30 or 40 min of global ischaemia.

Perfusion medium and drugs

The KHB composition was as follows: 118.5 mmol/l of NaCl; 25.0 mmol/l of NaHCO₃; 4.8 mmol/l of KCl; 1.2 mmol/l of MgSO₄; 1.18 mmol/l of KH₂PO₄; 1.4 mmol/l of CaCl₂; and 11.0 mmol/l of glucose. KHB was prepared daily and filtered through a 5-µm cellulose nitrate filter before use.

Prior to this study, a material transfer agreement for esaxerenone was signed between Nippon Medical School and Daiichi Sankyo Co., Ltd Esaxerenone was dissolved in dimethyl sulfoxide (DMSO; final concentration, 0.1% for all drug groups) at concentrations of 0.01, 0.1 and 1.0 μmol/l, among which the concentration that produced the most effective functional recovery was determined in a preliminary study (‘Preliminary study: does esaxerenone affect global ischaemia and reperfusion injury, and what concentration works best?’ section).

Acetylcholine chloride (ACh; Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) was dissolved in distilled water and diluted using oxygenated KHB to obtain 1 µmol/l of ACh-dissolved KHB, as previously described [17].

5HD (Abcam, Tokyo, Japan), a mitochondrial K_ATP channel blocker, was dissolved in deionized water to produce a 50-mmol/l stock solution, which was diluted in the perfusion solution to obtain a final drug concentration of 50 mmol/l immediately before use, as previously described [18].

Perfusion protocol

After equilibration, the hearts were randomly subjected to one of the experimental groups in each perfusion protocol, as shown in Fig. 1.

Preliminary study: does esaxerenone affect global ischaemia and reperfusion injury, and what concentration works best?

The hearts were assigned to 1 of 5 groups (n = 6/group): (i) control (additional 10 min of KHB perfusion), (ii) DMSO (10 min of KHB with DMSO) or (iii–v) Esax (10 min of esaxerenone perfusion at concentrations of 0.01, 0.1 and 1.0 μmol/l) (n = 6/group), after which the hearts were subjected to 40 min of global ischaemia (Fig. 1A).

Study 1: does the timing of esaxerenone administration influence cardioprotection?

In study 1, esaxerenone concentration was fixed at 0.1 μmol/l. The hearts were assigned to 1 of 3 groups (n = 6/group): (i) control, no treatment; (ii) pre-Esax, 10 min of perfusion with 0.1 μmol/l esaxerenone before 30 min of global ischaemia; and (iii) post-Esax, 30 min of global ischaemia followed by 10 min of perfusion at the beginning of the reperfusion with 0.1 μmol/l esaxerenone (Fig. 1B).

Study 2: does administration of esaxerenone affect the coronary flow response to acetylcholine?

In study 2, the hearts (n = 6/group) were subjected to the (i) Sham (perfusion only); (ii) control (additional 10 min of perfusion followed by 30 min of global ischaemia); or (iii) Esax intervention (10 min of perfusion with 0.1 μmol/l esaxerenone before 30 min of global ischaemia). After 60 min of reperfusion, coronary flow was measured. Afterward, 1-μmol/l acetylcholine was infused for 1 min, and coronary flow changes in the next 10 min were recorded. Increases in coronary flow were considered to be relaxation of the coronary vasculature, and reductions in coronary flow were considered to be constriction of the coronary vasculature (Fig. 1C).

Study 3: does 5-hydroxdecanoate influence the effect of esaxerenone?

In study 3, the hearts (n = 6/group) were subjected to the (i) control (no treatment); (ii) 5HD [10 min of KHB perfusion containing 5HD (50 μmol/l)]; (iii) Esax (10 min of perfusion with 0.1 μmol/l esaxerenone; or (iv) Esax + 5HD [10 min of perfusion with 0.1 μmol/l esaxerenone containing 5HD (50 μmol/l)] (Fig. 1D).

Expression of results

Post-ischaemic recoveries of LVDP, heart rate and coronary flow were expressed as percentages of the baseline values, and LVEDP was expressed as an absolute value (mmHg). To evaluate myocardial damage, coronary effluents were collected during reperfusion, and the total troponin T level (expressed as ng/g heart wet weight, ng/g wt) was measured using an electrochemiluminescence immunoassay (Roche Diagnostics K.K., Konan, Minato-ku, Tokyo, Japan). Coronary flow changes after ACh infusion were expressed as percentages of baseline values.

Statistical analysis

All data are expressed as means ± standard deviation. Continuous variables were compared between the groups using one-way analysis of variance or two-way repeated-measures analysis of variance with correction by linear regression analysis, as appropriate. If significance was established, post hoc analysis was performed using the Tukey test, which allows for multiple comparisons.

All statistical tests were two-tailed; a P-value of <0.05 was considered to indicate statistical significance. All statistical analyses were performed using JMP, version 10.0 (SAS Inc., Cary, NC, USA).

RESULTS

Five hearts were excluded because of the unacceptable range at the time of baseline reading.

Preliminary study: does esaxerenone affect global ischaemia, and what concentration works best?

The preliminary dose–response study established the optimal concentration of esaxerenone at 0.1 μmol/l. The esaxerenone dose–response curve for LVDP recovery (Fig. 2) was bell-shaped with increasing concentrations of esaxerenone. The baseline and final recovery of all parameters are shown in Table 1.

Study 1: does the timing of esaxerenone administration influence cardioprotection?

The mean baseline values of LVDP, coronary flow, heart rate, and LVEDP after the equilibration period are shown in Table 2. There were no significant differences in any of these values between the groups. The post-ischaemic recovery of LVDP in the control group was low (reaching a plateau value at ∼20% of the baseline). In contrast, rats that received esaxerenone prior to global ischaemia recovered gradually and reached a value of ∼40% (Fig. 3A). Interestingly, the recovery of LVDP following the administration of esaxerenone after global ischaemia was not different from that in the control. Elevated post-ischaemic LVEDP in the control group tended to be higher during the reperfusion period and was significantly higher at the end of reperfusion than in the pre-Esax group (Table 2 and Fig. 3B). The recovery of heart rate and coronary flow was similar in all groups (Table 2). The total troponin T leakage is shown in Fig. 3C. Troponin T leakage was significantly lower in the pre-Esax group than in the other 2 groups. The administration of esaxerenone after global ischaemia resulted in a higher troponin T leak, which did not differ from that in the control.

Study 2: does the administration of esaxerenone affect the coronary flow response to acetylcholine?

Ischaemia–reperfusion impaired the acetylcholine-induced increase in coronary flow (percentage pre-acetylcholine-infusion value; maximal changes: Sham, 107.5 ± 5.3%; control, 95.8 ± 3.1%; P < 0.001), and this impairment was attenuated by esaxerenone-infusion before ischaemia (maximal change: Esax, 103.7 ± 2.4%; versus control, P = 0.008) (Fig. 4).

Study 3: does 5-hydroxydecanoate influence the effect of esaxerenone?

The recovery profile of LVDP is shown in Fig. 5. These results are similar to those observed in study 1. The hearts in the control group recovered slowly, reaching a plateau at 60 min. In the Esax group, the recovery of LVDP was gradual, reaching a plateau of ∼35% by 30 min, which was significantly higher than that in the control group. 5HD eliminated the cardioprotective effect of esaxerenone, and the hearts in the Esax + 5HD group recovered to a peak of approximately 20%, similar to the control group (Table 3). Similar to the results of study 1, LVEDP was significantly elevated from baseline at the onset of reperfusion, indicating contracture development. In the control group, the LVEDP increased rapidly, peaked in 10 min and then gradually declined throughout reperfusion. The LVEDP decreased slowly in the hearts of rats in the Esax group, with a continued gradual decline, reaching ∼60 mmHg.

DISCUSSION

Herein, we characterized the concentration-dependent cardioprotective efficacy of esaxerenone, an MRB, against myocardial global ischaemia and reperfusion injury using isolated Langendorff-perfused rat hearts; we found that pre-ischaemic treatment with esaxerenone had a protective effect compared to the administration of esaxerenone at the time of reperfusion, thereby confirming our hypothesis. In addition, this study revealed that esaxerenone prevents ischaemia–reperfusion injury in the coronary endothelium. Furthermore, the protective effect of esaxerenone was eliminated by the mitochondrial K_ATP channel blocker 5HD.

Esaxerenone is a non-steroidal selective and highly potent MRB that inhibits ³H-aldosterone binding to the mineralocorticoid receptor with a half maximal inhibitory concentration (IC₅₀) of 9.4 nM in a radioligand-binding assay. Its potency was superior to that of spironolactone and eplerenone, whose IC₅₀ values were 36 and 713 nM, respectively [15]. Arai et al. [19] demonstrated that the antihypertensive effect of esaxerenone (0.5 mg/kg) was equivalent to that of spironolactone (100 mg/kg) and eplerenone (100 mg/kg) in rats.

Chai et al. [8] reported that spironolactone prior to ischaemia in isolated rat hearts reduced ischaemic injury. Similarly, eplerenone was shown to protect the heart during ischaemia and reperfusion by reducing infarct size and improving left ventricular pressure recovery in the Langendorff-perfused rat model, suggesting that the MRB might induce pharmacological preconditioning [9]. Schmidt et al. demonstrated that a bolus of potassium canrenoate, which acts like other mineralocorticoid receptor antagonists and is classified as a potassium-sparing diuretic, reduced infarct size when administered before reperfusion in mouse and rabbit hearts. Similarly, eplerenone exerted a cardioprotective effect against infarction when administered prior to reperfusion in isolated rat hearts [10]. In the present study, the myocardial protective effect of esaxerenone was confirmed when administered prior to ischaemia but not when infused within the first 10 min of reperfusion. There is a narrow therapeutic window of most postconditioning agents, and Schmidt et al. [10] demonstrated the efficacy of eplerenone in preventing regional ischaemia when administered 5 min before reperfusion and administered throughout reperfusion. Global ischaemia is more invasive than regional ischaemia, and it is necessary to search for the optimal concentration and administration time of esaxerenone to exert its postconditioning effect [20].

Aldosterone is associated with inflammation, vascular smooth muscle cell proliferation, and endothelial dysfunction. Furthermore, aldosterone inhibits the activation of nitric oxide synthase [21], which is considered one of the causes of vascular endothelial damage induced by aldosterone. Nitric oxide has an inhibitory effect on factors that cause myocardial damage, such as cytokinesis, vascular smooth muscle proliferation, endothelial injury and coronary artery relaxation. In addition, spironolactone administration has been reported to increase the bioactivity of nitric oxide and improve vascular endothelial dilatation defects [22]. Acetylcholine-induced vasorelaxation was significantly weaker in a high-cholesterol diet group than in a normal diet group; however, the endothelium-dependent vasorelaxation was strengthened in eplerenone-treated groups [23]. Herein, we evaluated the degree of endothelial damage after 30 min of global ischaemia by measuring coronary flow response to acetylcholine. Pretreatment with esaxerenone resulted in a significantly superior response to that of the control without esaxerenone. This indicates that esaxerenone suppresses endothelial damage, which may preserve the nitric oxide-producing capacity of the vascular endothelium. Matsumoto et al. [24] suggested that esaxerenone ameliorated endothelial function by increasing endothelium-derived hyperpolarizing signalling and suppressing endothelium-derived contracting factor signalling in diabetic rats. Similarly, esaxerenone attenuates diabetes-induced endothelial dysfunction by improving the endothelial nitric oxide synthase phosphorylation in C57B/6 mice [25].

Pharmacological inhibition of the adenosine receptor, protein kinase C, PI3K or extracellular signal-regulated kinase eliminated this cardioprotective effect, indicating that protection depends on the same signalling pathway as preconditioning. Additionally, Mahajan et al. [13] reported a myocardial protective effect of eplerenone during myocardial ischaemia–reperfusion via the PI3K/Akt and GSK-3β pathways in diabetic rats. Similarly, the co-administration of 5HD, a mitochondrial K_ATP channel blocker, with esaxerenone markedly abolished the myocardial protective effect of esaxerenone administered before ischaemia in this study. This finding suggests that the mitochondrial potassium channel-opening effect of esaxerenone may be involved in its cardioprotection.

Limitations

The current study has several limitations. An isolated heart preparation that does not possess collateral circulation was used. Thus, the validity of our results relies on the specificity of the mitochondrial K_ATP channel blocker 5HD. Myocardial ischaemic disease is a multifactorial condition involving a spectrum of injuries that affect myocardial protection. However, the hearts used in this experimental study were obtained from healthy rats under normal feeding conditions; hence, it is likely that the protective effect of esaxerenone might be different in jeopardized hearts with ischaemic injury or older hearts. Any such heart is likely to require prolonged periods of ischaemia in a clinical setting, whereas a short duration of ischaemia was adopted in this experimental study. As no primary analysis was defined, P-values may not be interpreted as confirmatory but rather descriptive.

CONCLUSIONS

This is the first study to demonstrate the cardioprotective effects of esaxerenone. This novel pharmacological preconditioning effect of esaxerenone on isolated rat hearts warrants further study to determine the efficacy of this potentially beneficial alternative to conventional preconditioning procedures.

ACKNOWLEDGMENTS

We thank Dr Kazutora Mizukami (Ph.D. in Statistical Science, President of Medical Data Management, Fukuoka, Japan) for statistical assistance and Ms. Akina Kimura for assistance with the experiments. We are grateful to Dr David J. Chambers (Cardiac Surgical Research, the Rayne Institute, St Thomas Hospital, London, UK) for providing academic advice. We would also like to thank Editage (www.editage.com) for English language editing.

FUNDING

This study was supported by the Japan Society for the Promotion of Science KAKENHI (grant number: JP22K08965). Esaxerenone was provided by Daiichi Sankyo Co., Ltd under the material transfer agreement.

Conflict of interest: none declared.

DATA AVAILABILITY

Data are available on request.

Author contributions

Hiromasa Yamashita: Funding acquisition; Investigation; Writing—original draft. Masahiro Fujii: Conceptualization; Formal analysis; Methodology; Writing—review & editing. Ryuzo Bessho: Validation; Writing—review & editing. Yosuke Ishii: Supervision.

Reviewer information

European Journal of Cardio-Thoracic Surgery thanks Claudia Heilmann, Nishant Saran and the other, anonymous reviewer(s) for their contribution to the peer review process of this article.

Presented at the 86th Annual Scientific Meeting of the Japanese Circulation Society, 11–13 March 2022, held at Kobe, Japan.

REFERENCES

ABBREVIATIONS

ABBREVIATIONS
 
• ACh

  Acetylcholine chloride

 
• DMSO

  Dimethyl sulfoxide

 
• 5HD

  5-Hydroxydecanoate

 
• K_ATP

  ATP-sensitive potassium

 
• KHB

  Krebs–Henseleit bicarbonate buffer

 
• LVDP

  Left ventricular developed pressure

 
• LVEDP

  Left ventricular end-diastolic pressure

 
• MRB

  Mineralocorticoid receptor blocker

 
• PI3K

  Phosphatidylinositol 3-kinase