Preconditioning actions of aldosterone through p38 signaling modulation in isolated rat hearts

in Journal of Endocrinology
View More View Less
  • Division of Cardiology, Department of Internal Medicine, The Jikei University School of Medicine, 3-25-8, Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan

Although persistent excessive actions of aldosterone have unfavorable effects on the cardiovascular system, primarily via mineralocorticoid receptor (MR)-dependent pathways, the pathophysiological significance of aldosterone cascade activation in heart diseases has not yet been fully clarified. We herein examined the effects of short-term aldosterone stimulation at a physiological dose on cardiac function during ischemia–reperfusion injury (IRI). In order to study the effects of aldosterone preconditioning, male Wistar rat Langendorff hearts were perfused with 10−9 mol/l of aldosterone for 10 min before ischemia, and the response to IRI was assessed. Although aldosterone did not affect the baseline hemodynamic parameters, preconditioning actions of aldosterone significantly improved the recovery in left ventricular contractility and left ventricular end-diastolic pressure associated with a reduced activity of creatine phosphokinase released into the perfusate after ischemia–reperfusion. Notably, the MR inhibitor eplerenone did not abrogate these beneficial effects. Biochemical analyses revealed that p38MAPK phosphorylation was significantly increased during aldosterone preconditioning before ischemia, whereas its phosphorylation was substantially attenuated during sustained ischemia–reperfusion, compared with the results for in the non-preconditioned control hearts. This dual regulation of p38MAPK was not affected by eplerenone. The phosphorylation levels of other MAPKs were not altered by aldosterone preconditioning. In conclusion, the temporal induction of the aldosterone cascade, at a physiological dose, has favorable effects on cardiac functional recovery and injury following ischemia–reperfusion in a MR-independent manner. Phasic dynamism of p38MAPK activation may play a key role in the physiological compensatory pathway of aldosterone under severe cardiac pathological conditions.

Abstract

Although persistent excessive actions of aldosterone have unfavorable effects on the cardiovascular system, primarily via mineralocorticoid receptor (MR)-dependent pathways, the pathophysiological significance of aldosterone cascade activation in heart diseases has not yet been fully clarified. We herein examined the effects of short-term aldosterone stimulation at a physiological dose on cardiac function during ischemia–reperfusion injury (IRI). In order to study the effects of aldosterone preconditioning, male Wistar rat Langendorff hearts were perfused with 10−9 mol/l of aldosterone for 10 min before ischemia, and the response to IRI was assessed. Although aldosterone did not affect the baseline hemodynamic parameters, preconditioning actions of aldosterone significantly improved the recovery in left ventricular contractility and left ventricular end-diastolic pressure associated with a reduced activity of creatine phosphokinase released into the perfusate after ischemia–reperfusion. Notably, the MR inhibitor eplerenone did not abrogate these beneficial effects. Biochemical analyses revealed that p38MAPK phosphorylation was significantly increased during aldosterone preconditioning before ischemia, whereas its phosphorylation was substantially attenuated during sustained ischemia–reperfusion, compared with the results for in the non-preconditioned control hearts. This dual regulation of p38MAPK was not affected by eplerenone. The phosphorylation levels of other MAPKs were not altered by aldosterone preconditioning. In conclusion, the temporal induction of the aldosterone cascade, at a physiological dose, has favorable effects on cardiac functional recovery and injury following ischemia–reperfusion in a MR-independent manner. Phasic dynamism of p38MAPK activation may play a key role in the physiological compensatory pathway of aldosterone under severe cardiac pathological conditions.

Introduction

Aldosterone plays a pivotal role in the pathophysiology of cardiovascular diseases. There is increasing evidence of unfavorable effects of aldosterone on the cardiovascular system, in experimental models as well as large clinical trials (Pitt et al. 2003), primarily via mineralocorticoid receptor (MR)-dependent actions (Caprio et al. 2008, Matsui et al. 2008, Rickard et al. 2012, Shen & Young 2012, Sherajee et al. 2012, Diaz et al. 2014, Nolly et al. 2014). However, the pathophysiological significance of the increase (even by a small amount) in aldosterone levels both in circulation and local tissue, as well as its cascade activation in severe heart diseases (Mizuno et al. 2001, Nakamura et al. 2004), such as myocardial infarction (Beygui et al. 2006), has not yet been fully clarified.

Preconditioning induced by either brief periods of ischemia or short-term stimulation of neurohumoral factors, such as catecholamines and erythropoietin before ischemia–reperfusion, results in cardioprotective effects against ischemia–reperfusion injury (IRI; Marais et al. 2001, 2005, Sanada et al. 2001, Cai & Semenza 2004, Turrell et al. 2011). The signaling pathways involved in preconditioning have been investigated intensively, and PI3K–Akt–GSK3 as well as the MAPK family (ERK, c-Jun N-terminal kinases (JNK) and p38) has been shown to play a part in major cascades (Mocanu et al. 2000, Tong et al. 2000, Marais et al. 2001, 2005, Sanada et al. 2001, Cai & Semenza 2004, Hausenloy & Yellon 2006, Schmidt et al. 2010, Turrell et al. 2011).

Aldosterone, one of the major neurohumoral factors acting under critical cardiac conditions (such as acute-phase ischemic heart disease), exerts biphasic effects on somatic cells, including those in the myocardium (Connell & Davies 2005). While the prolonged pathological actions of excessive aldosterone are largely mediated through the activation of an MR-dependent pathway, there is a rapid, MR-independent action representing a physiological compensatory mechanism that may function to improve cardiac output during heart failure. In fact, previous studies have demonstrated that the rapid actions of aldosterone exert positive inotropic effects in the heart (Barbato et al. 2002, 2004, Chai et al. 2005, 2006, 2010). Moreover, we recently reported that aldosterone has favorable effects on cardiomyocytes during the early phase in an in vitro model of neonatal rat cardiomyocytes (NRCMs), partly due to the acute activation of a MR-independent cascade, whereas the persistent activity of aldosterone produces pathological effects in a MR-dependent manner (Yamamuro et al. 2006, Nagoshi et al. 2012). Therefore, we herein propose a hypothesis that the rapid MR-independent actions of aldosterone at a physiological dose initially provide cardioprotective effects under critical pathological conditions as a possible compensatory mechanism. In order to test the functional significance of the temporal induction of the aldosterone cascade, we studied the effects of short-term aldosterone stimulation before IRI (namely, ‘aldosterone preconditioning’) and examined the response in the hearts in an ex vivo model of IRI.

Materials and methods

Experiments in Langendorff hearts

All animal procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Research Committee at the Jikei University School of Medicine. Male Wistar rats (weight: 250–300 g) were heparinized (1000 IU/kg, i.p.) and anesthetized (pentobarbital, 60 mg/kg, i.p.). The hearts were rapidly excised and the aortae were cannulated onto a Langendorff apparatus, followed by retrograde-perfusion at a constant pressure (80 mmHg) with modified Krebs–Henseleit buffer, as previously described (Nagoshi et al. 2005). A water-filled balloon catheter was introduced into left ventricles to record the hemodynamic parameters.

Ischemia–reperfusion model

The experimental protocols are shown in Fig. 1. After a stabilization period of 15 min, the hearts were perfused with the control samples for another 10 min before ischemia–reperfusion in order to measure the baseline pre-ischemia cardiac function. In the aldosterone preconditioning groups, 10−9 mol/l of aldosterone (Wako Pure Chemical Industries, Osaka, Japan) with or without 10−6 mol/l of the MR antagonist eplerenone (Sigma–Aldrich, the dose used in the previous study using a rat Langendorff perfusion model in order to substantially block the MR-dependent action (Chai et al. 2006), as well as on the basis of the pharmacokinetics of eplerenone after oral administration to humans (Cook et al. 2003)), 10−7 mol/l of the glucocorticoid receptor (GR)/progesterone receptor antagonist RU486 (Sigma–Aldrich), 10−7 mol/l of the G protein-coupled estrogen receptor (GPER, previously known as G protein-coupled receptor 30 (GPR30)) antagonist G15 (Cayman Chemical, Ann Arbor, MI, USA), or 10−5 mol/l of p38MAPK inhibitor SB203580 (Tocris, Bristol, UK) was added to the buffer during the 10-min pre-ischemia perfusion period. Where indicated, 10−6 mol/l of eplerenone alone was also perfused during the 10-min pre-ischemia period only. Water-insoluble reagents, such as eplerenone, RU486, and G15, were dissolved in dimethylsulfoxide (DMSO), and the solvent concentrations were identically maintained in the control groups. The preliminary experiments showed that the presence of equivalent amounts of DMSO in the perfusate did not modify cardiac performance. Subsequently, global ischemia was applied by eliminating flow for 30 min followed by 40 min of reperfusion. In the signaling analysis, the individual perfused hearts were snap-frozen in liquid nitrogen at the indicated time points and stored at −80 °C before protein extraction (Fig. 1).

Figure 1
Figure 1

Experimental protocol showing the duration and time course of ischemia/reperfusion. The hearts were frozen at the points in the protocol indicated by solid arrows. Creatine phosphokinase (CPK) activity released into the perfusate was measured at the points in the protocol indicated by dashed arrows. Aldo, aldosterone; Epl, eplerenone.

Citation: Journal of Endocrinology 222, 2; 10.1530/JOE-14-0067

Cardiac enzyme measurement

Creatine phosphokinase (CPK) levels were measured in the effluent at the time points indicated in the experimental protocols (Fig. 1) using an enzymatic activity assay.

Immunoblotting

Immunoblotting was carried out as described previously (Nagoshi et al. 2005, 2012) using primary antibodies to phospho-Akt (Ser473), phospho-GSK3β (Ser9), Akt (Cell Signaling Technology, Danvers, MA, USA), GSK3β, phospho-p38 (Thr180/Tyr182), p38, phospho-ERK (Thr202/Tyr204), ERK, phospho-JNK (Thr183/Tyr185) or JNK (BD Transduction Laboratories, Franklin Lakes, NJ, USA). The signals were detected using chemiluminescence, and the band intensity was quantified using the Multi Gauge software program (Version 3.1; Fujifilm, Tokyo, Japan).

Statistical analysis

The data are presented as the mean±s.e.m. of at least four independent experiments. The hemodynamic parameters and CPK levels were compared between the groups using a one-way ANOVA followed by post-hoc Bonferroni and Tukey's tests for multiple comparison correction. The phosphorylation levels of intracellular kinase signaling were compared using the Wilcoxon rank-sum test. A value of P<0.05 was considered to be significant.

Results

Effects of aldosterone preconditioning on functional recovery and cardiac injury after IRI

The baseline cardiac function measured at the end of 10 min of pre-ischemia perfusion was not significantly affected by aldosterone preconditioning with or without eplerenone (Table 1). After 30-min global ischemia followed by 40-min reperfusion, aldosterone preconditioning significantly improved the left ventricular developed pressure (LVDP) recovery compared with that observed in the nontreated control hearts (70.5±3.1 vs 55.3±3.0% recovery of baseline, P<0.05, Fig. 2A and B). Left ventricular end-diastolic pressure (LVEDP) during reperfusion was also significantly lower in the aldosterone-preconditioned hearts compared with that observed in the control hearts (29.2±3.7 vs 44.9±2.6 mmHg at the end of IRI, P<0.03, Fig. 2C). Notably the administration of eplerenone, a selective MR antagonist, during aldosterone preconditioning did not abrogate these beneficial effects (Fig. 2A, B and C). The administration of eplerenone alone during the pre-ischemic period only did not significantly affect the recovery of the cardiac function after IRI (Supplementary Figure 1A, see section on supplementary data given at the end of this article). In the context of an MR-independent cascade, the effects of aldosterone in improving the cardiac functional recovery may be mediated via GR-dependent (Rossier et al. 2008) or GPER-dependent (Krug et al. 2011) actions. However, neither RU486 (GR inhibitor) nor G15 (GPER inhibitor) affected the cardiac function at baseline or in terms of LVDP recovery after IRI (Fig. 2D), indicating that the functional effects of aldosterone preconditioning observed in the current study may be mediated via MR-, GR- or GPER-independent actions.

Table 1

Data on baseline cardiac function of the ex vivo perfused hearts

Control (n=23)Aldo (n=20)Aldo+Epl (n=9)
LVSP (mmHg)150.0±3.3146.5±3.7156.9±4.3
LVEDP (mmHg)7.4±0.46.4±0.48.4±0.5
LVDP (mmHg)142.6±3.4140.1±3.9148.5±4.2
LV +dp/dt (mmHg/s)4985.2±137.44757.3±155.94941.0±135.0
LV −dp/dt (mmHg/s)−2839.9±75.3−2751.0±75.0−2955.2±78.8
Heart rate (bpm)229.8±4.5233.9±4.4226.2±4.1
Coronary flow (ml/min)11.7±0.311.7±0.411.5±0.3

LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; LVDP, left ventricular developed pressure.

Figure 2
Figure 2

Cardiac functional recovery after ischemia–reperfusion injury. LVDP profiles during ischemia–reperfusion (A), LVDP recovery (percentage of baseline value) measured at the indicated time points (B), and LVEDP profiles during ischemia–reperfusion (C) in the control hearts (white squares and bars; n=23), aldosterone-preconditioned hearts (Aldo, black squares and bars; n=20) and aldosterone-preconditioned hearts treated with eplerenone infusion (Aldo+Epl, gray squares and bars; n=9). (D) LVDP recovery (percentage of baseline value) measured at the end of the 40-min reperfusion period in the aldosterone-preconditioned hearts treated with or without either RU486 or G15 (Aldo+RU486 or G15; n=7 each). *P<0.05, control vs Aldo; P<0.05 control vs Aldo+Epl; P<0.05 control vs Aldo+RU486; #P<0.05 control vs Aldo+G15.

Citation: Journal of Endocrinology 222, 2; 10.1530/JOE-14-0067

The activity of CPK released into the perfusate during reperfusion was measured as an index of myocardial injury. During baseline pre-ischemia perfusion, no CPK activity was detectable. After IRI, the CPK activity was significantly reduced in the aldosterone-preconditioned hearts compared with that observed in the control hearts at 10 min of reperfusion (16.9±3.1 vs 48.6±13.7 U/l, P<0.02, Fig. 3A). Moreover, the total amount of CPK released during the entire reperfusion period, as indicated by the area under the curve (AUC) for the CPK level, was significantly decreased by aldosterone preconditioning (84.5±16.6 vs 146.0±24.8, P<0.05, Fig. 3B). Again, the administration of eplerenone did not influence the reduction in CPK achieved by aldosterone preconditioning (Fig. 3A and B).

Figure 3
Figure 3

(A) CPK profiles in the effluent collected during the reperfusion period. (B) Area under the curve (AUC) was calculated from the CPK profile shown in (A) (n≥7 for each group). *P<0.05 control vs Aldo; P<0.05 control vs Aldo+Epl.

Citation: Journal of Endocrinology 222, 2; 10.1530/JOE-14-0067

Together, these data indicate that aldosterone preconditioning improves cardiac functional recovery and reduces myocardial injury after IRI in a MR-independent manner.

Effects of aldosterone preconditioning on intracellular kinase signaling activation

In order to explore possible mechanisms underlying these results, we next examined signaling regulated by aldosterone preconditioning. It has been reported that the acute activation of Akt signaling during IRI induced by ischemic preconditioning or other factors is cardioprotective (Tong et al. 2000, Cai & Semenza 2004, Nagoshi et al. 2005, Hausenloy & Yellon 2006). Moreover, we recently demonstrated that aldosterone has favorable effects on cardiomyocytes due to the acute activation of a MR-independent cascade through PI3K–Akt in vitro (Nagoshi et al. 2012). Therefore, we first examined Akt signaling and found that aldosterone perfusion for 10 min significantly increased phosphorylation of Akt and that this was inhibited by eplerenone, while no significant change was observed in phosphorylation of GSK3β, one of the downstream effectors of Akt (Fig. 4). Aldosterone preconditioning with or without eplerenone did not exert any significant effects on Akt–GSK3β phosphorylation during either the early or late phase of reperfusion.

Figure 4
Figure 4

Phosphorylation of Akt and GSK3β was evaluated in hearts treated with or without ischemia–reperfusion for the indicated periods shown in Fig. 1. Representative immunoblots obtained using the indicated antibodies are shown. Averaged densitometry data normalized to the control at the same time points are shown in the bar graphs (n=3–4 for each group). *P<0.05, control vs Aldo; P<0.05 Aldo vs Aldo+Epl.

Citation: Journal of Endocrinology 222, 2; 10.1530/JOE-14-0067

MAPKs also play critical roles in cardioprotective effects during IRI (Mocanu et al. 2000, Marais et al. 2001, 2005, Sanada et al. 2001, Hausenloy & Yellon 2006, Schmidt et al. 2010, Turrell et al. 2011). Among the three major MAPK signaling pathways, we found that p38MAPK activation is dramatically altered by aldosterone preconditioning during IRI (Fig. 5). Phosphorylation of p38 was significantly increased by aldosterone at the end of the 10-min baseline period of pre-ischemia perfusion. In contrast, phosphorylated p38 levels were markedly reduced in the aldosterone-preconditioned hearts compared with those observed in the nontreated controls during both the early and late phases of reperfusion. This rapid dual p38 regulation was not affected by eplerenone, which was consistent with the functional data. The administration of eplerenone alone during the pre-ischemic period only did not change phosphorylated p38 level significantly (Supplementary Figure 1B). The phosphorylation of ERK and JNK was not significantly altered by aldosterone preconditioning.

Figure 5
Figure 5

Phosphorylation of MAPKs signaling pathways was evaluated in hearts treated with or without ischemia–reperfusion for the indicated periods shown in Fig. 1. Representative immunoblots obtained using the indicated antibodies are shown. Averaged densitometry results normalized to the control at the same time points are shown in the bar graphs (n=4 for each group). *P<0.05, control vs Aldo; P<0.05 control vs Aldo+Epl.

Citation: Journal of Endocrinology 222, 2; 10.1530/JOE-14-0067

In order to better understand the significance of the rapid pre-ischemic induction of p38MAPK signaling induced by aldosterone preconditioning, we examined the effects of SB203580 (a potent p38 inhibitor) perfusion during aldosterone preconditioning on cardiac functional recovery after IRI. Perfusion with 10−6 mol/l of SB203580 alone or SB203580+aldosterone during 10-min pre-ischemic period caused a rapid positive inotropic effect, although there were no synergistic effects (138.8% increase in LVDP). After IRI, the recovery achieved with aldosterone preconditioning was blunted by SB203580 to a level similar to that of the control (Fig. 6), indicating that transient p38MAPK activation during aldosterone preconditioning plays a key role in cardioprotection.

Figure 6
Figure 6

Effects of the p38MAPK inhibitor (SB203580) on cardiac functional recovery after ischemia–reperfusion injury. LVDP recovery (percentage of baseline value) measured at the end of the 40-min reperfusion period in aldosterone-preconditioned hearts treated with or without SB203580 is shown (Aldo+SB203580; n=7) *P<0.05 control vs Aldo; P<0.05 Aldo vs Aldo+SB203580.

Citation: Journal of Endocrinology 222, 2; 10.1530/JOE-14-0067

Discussion

In the present study, we found that the temporal induction of the aldosterone cascade, at a physiological dose, results in increased cardiac functional recovery and decreased myocardial injury after ischemia–reperfusion in a MR-independent manner. Our results indicate a critical role of the phasic dynamism of p38MAPK activation in aldosterone preconditioning of the heart during IRI. A transient increase in p38 phosphorylation was observed during the pre-ischemic period, while attenuation of its phosphorylation was observed during ischemia–reperfusion; these effects may be deeply involved in the cardioprotective effects of aldosterone preconditioning.

It has been well documented that the persistent excessive actions of aldosterone, commonly by a combination of salt loading, cause adverse cardiovascular effects primarily via MR-dependent pathways (Matsui et al. 2008, Shen & Young 2012). On the other hand, ourselves and others have recently reported that aldosterone exerts transient but favorable effects on cardiocytes in vitro, mainly via rapid MR-independent actions (Yamamuro et al. 2006, Bunda et al. 2009, Nagoshi et al. 2012). In agreement with these findings, the present ex vivo study demonstrated the rapid, favorable effects of aldosterone on cardiac functional recovery and injury during ischemia–reperfusion, although aldosterone did not affect the baseline cardiac function (Table 1), congruent with the findings of previous reports by other groups (Fujita et al. 2005, Matsui et al. 2007). However, our results do not completely agree with the findings of several other studies showing that rapid, MR-independent actions of aldosterone induce positive inotropic effects at baseline (Barbato et al. 2002, 2004, Chai et al. 2005, 2006, 2010), while the administration of aldosterone results in no significant effects (Chai et al. 2005, Schmidt et al. 2010), but rather results in detrimental effects (Fujita et al. 2005, Mihailidou et al. 2009) on cardiac functional recovery and injury after ischemia–reperfusion. This discrepancy may be explained by the differences in the experimental conditions and the time course of aldosterone stimulation: temporal induction of the aldosterone cascade, at a physiological dose (a relatively lower dose than that commonly used in previous studies), during the pre-ischemic period only (not during the entire IRI protocol) is crucial for the ability of aldosterone to exert favorable effects on the heart during IRI.

Mihailidou and colleagues previously demonstrated that spironolactone protects the heart from IRI, not merely by excluding aldosterone and/or glucocorticoids from MR but also via its inverse agonist activity against MR (Mihailidou et al. 2009). It is also possible that eplerenone acts as a partial agonist under ligand-free conditions, such as ex vivo isolated heart models. In this study, however, neither LVDP recovery nor p38MAPK signaling was affected by the administration of eplerenone alone during the 10-min pre-ischemic period only (Supplementary Figure 1A). The MR antagonists displayed cardioprotective effects when administered under pathological conditions, such as IRI, not at baseline, whereas the purpose of applying eplerenone in this study was to block MR during aldosterone preconditioning only. Therefore, the recovery of cardiac function as well as signaling activation, including that involving p38MAPK to MR antagonists, may differ when eplerenone is administered for 10 min before ischemia in addition to the entire 40-min reperfusion period. Moreover, the cardioprotective effects of MR antagonists may be more salient in vivo, where other MR ligands (e.g. glucocorticoids) and miscellaneous endogenous factors that modulate MR (e.g. salt loading, high glucose) exist. Taken together, the lack of effects of eplerenone in the current ex vivo study does not exclude the well-documented cardioprotective effects of MR antagonists demonstrated in both experimental and clinical studies.

The preconditioning effects of aldosterone may also involve GR or GPER. However, neither GR nor GPER inhibitors affected the cardiac function, at least in the current protocol used to investigate the pre-ischemic rapid actions of aldosterone at its physiological dose (Fig. 2D). It has been reported that GR is activated by aldosterone in cardiomyocytes, but at a relatively high dose (over 10−8 M) for long-term stimulation, compared with that observed in the current study (Rossier et al. 2008). It is more likely that rapid MR-independent actions are mediated via GPER signaling (Deschamps & Murphy 2009, Krug et al. 2011), although the aldosterone–GPER pathway may be activated in a cell-type-specific manner (vascular cells rather than cardiomyocytes) (Gros et al. 2011, 2013). Moreover, this pathway dramatically activates ERK rather than p38MAPK, thus indicating that aldosterone–GPER signaling is not the major cascade, at least in the present model.

We recently reported that short-term aldosterone stimulation has favorable effects on cardiomyocytes due to the acute activation of a MR-independent cascade via PI3K–Akt signaling in an in vitro NRCMs model (Nagoshi et al. 2012). Consistent with this finding, there was a significant increase in phosphorylation of Akt during aldosterone preconditioning in the current study, although the increased level of GSK3β observed at the same time point was variable (Fig. 4). Moreover, this increased Akt phosphorylation was inhibited by eplerenone, consistent with the results recently reported by others (Nolly et al. 2014). Furthermore, there were no significant differences in Akt–GSK3 phosphorylation levels between the groups during the reperfusion period. A variety of important differences between the in vitro NRCMs and ex vivo adult whole-heart tissues (containing cardiomyocytes as well as cardiac fibroblasts, endothelial cells, vascular smooth muscle cells, and tissue-resident macrophages) may have contributed to the differences in observations. In addition, it has been recently reported that the cardioprotective effects of aldosterone achieved via Akt signaling activation are observed exclusively under high-glucose conditions (Fazal et al. 2014), consistent with results from our previous study (Nagoshi et al. 2012). Taken together, even if Akt signaling is partially involved in the cardioprotective effects of aldosterone preconditioning, its contribution was minor in the current study.

The cardioprotective effects of aldosterone preconditioning observed in this study were associated with the phasic dynamism of p38MAPK phosphorylation in a MR-independent manner (Fig. 5). A similar phasic pattern of p38 phosphorylation (activation) has been reported by other groups in miscellaneous cardiac preconditioning models induced by short-term repetitive ischemia as well as catecholamines associated with improvements in cardiac functional recovery and reduced cardiac injury (Marais et al. 2001, 2005, Sanada et al. 2001). p38 exerts both favorable and unfavorable effects on the heart, which probably depend on the intensity and duration of p38 activation (Mocanu et al. 2000). Previous studies using other systems of cardiovascular cells have demonstrated that p38 can be phosphorylated and activated by aldosterone via both MR-dependent and rapid MR-independent pathways, consistent with the findings from this study (Callera et al. 2005a,b, Lopez-Andres et al. 2008). It has been reported that the short-term induction of p38 phosphorylation in cardiomyocytes contributes to the activation of sarcolemmal KATP channels, which is a major common mechanism responsible for preconditioning (Turrell et al. 2011). Thus, the pre-ischemic rapid induction of p38 phosphorylation by aldosterone preconditioning could be beneficial for cardiac functional recovery after IRI. In fact, in this, the inhibition of the p38 activity during aldosterone preconditioning was found to hinder the improvement in LV functional recovery after IRI (Fig. 6). In contrast, p38 phosphorylation has been shown to be reduced by short-term aldosterone stimulation in NRCMs (Karmazyn et al. 2003), indicating the possibility that aldosterone preconditioning attenuates the maladaptive p38 activation induced by IRI. The 27-kDa small heat-shock protein (HSP27) is a major downstream regulator of p38MAPK signaling cascades, whose phosphorylation has been shown to protect against IRI (Sanada et al. 2001, Marais et al. 2005). However, HSP27 phosphorylation levels were not constantly elevated at any point of IRI in this study (data not shown). Although it is difficult to definitively establish causality, our results, together with those of previous studies by others (Mocanu et al. 2000, Marais et al. 2001, 2005, Sanada et al. 2001), revealed that the transient activation of p38 during preconditioning has a trigger action, while the deactivation of p38 after the onset of sustained ischemia–reperfusion acts as a mediator of cardioprotection. Further studies are required to explore the MR-independent aldosterone–p38 signaling pathways and p38-dependent downstream signaling cascades in more detail, including the pathways involved in HSP27 and/or ATF, and elucidate the precise regulatory mechanisms underlying the conversion of the dual activity of p38 induced by aldosterone preconditioning.

Overall, this study highlights the pathophysiological significance of the rapid induction of the cardiac aldosterone cascade in response to critical cardiac conditions, especially during the acute phase. We previously reported increased levels of aldosterone production/secretion from failing human hearts. Moreover, our recent study demonstrated that there is a transient decrease in serum potassium level during ischemic attacks of acute coronary syndrome, which is at least partially mediated through the activation of the aldosterone cascade (Sekiyama et al. 2013). Although these clinical findings provide evidence for local cardiac aldosterone synthesis/actions, this issue remains very controversial, and in fact, some reports have offered negative findings regarding the question of local aldosterone synthesis in the heart (Gomez-Sanchez et al. 2004, Fiebeler et al. 2005, Chai et al. 2010). In any case, the results of the current study confirm the speculation that, while aldosterone generally has little effect on the cardiovascular system under baseline normal conditions (Oliver et al. 1975), an elevated local/systemic aldosterone level (even within the physiological range, from the low-normal range to the high-normal or just above the normal range, which does not generally exceed a pharmacological dose of over 10−8 mol/l) in rapid response to severe cardiac conditions exerts substantial effects on cardiac function, which initially act in a cardioprotective manner. Future studies using aldosterone synthase inhibitors in in vivo models of IRI will contribute significantly to defining the critical role of the induction of the pre-ischemic aldosterone cascade, which actually ‘preconditions’ the heart for subsequent serious of cardiac events. Altogether, this present study provides new insights into the physiological compensatory role of aldosterone under severe cardiac pathological conditions, while still emphasizing the usefulness of MR inhibitors for suppressing unfavorable MR-dependent actions, without blocking favorable MR-independent actions.

Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/JOE-14-0067.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported in part by grants-in-aid for the Ministry of Education, Culture, Sports, Science and Technology (to T N and M Y), the Uehara Memorial Foundation and the Banyu Foundation International Research Grant (to T N).

Author contribution statement

T Y and T N designed, performed researched, and wrote the paper; R A and Y K performed research and data interpretation; K I, D K, M F and Y K performed research and collected the data; T D and K H analyzed and interpreted data M Y designed, and conducted the study, and wrote the paper.

References

  • Barbato JC, Mulrow PJ, Shapiro JI & Franco-Saenz R 2002 Rapid effects of aldosterone and spironolactone in the isolated working rat heart. Hypertension 40 130135. (doi:10.1161/01.HYP.0000025879.29822.24)

    • Search Google Scholar
    • Export Citation
  • Barbato JC, Rashid S, Mulrow PJ, Shapiro JI & Franco-Saenz R 2004 Mechanisms for aldosterone and spironolactone-induced positive inotropic actions in the rat heart. Hypertension 44 751757. (doi:10.1161/01.HYP.0000144466.11568.7e)

    • Search Google Scholar
    • Export Citation
  • Beygui F, Collet JP, Benoliel JJ, Vignolles N, Dumaine R, Barthelemy O & Montalescot G 2006 High plasma aldosterone levels on admission are associated with death in patients presenting with acute ST-elevation myocardial infarction. Circulation 114 26042610. (doi:10.1161/CIRCULATIONAHA.106.634626)

    • Search Google Scholar
    • Export Citation
  • Bunda S, Wang Y, Mitts TF, Liu P, Arab S, Arabkhari M & Hinek A 2009 Aldosterone stimulates elastogenesis in cardiac fibroblasts via mineralocorticoid receptor-independent action involving the consecutive activation of Gα13, c-Src, the insulin-like growth factor-I receptor, and phosphatidylinositol 3-kinase/Akt. Journal of Biological Chemistry 284 1663316647. (doi:10.1074/jbc.M109.008748)

    • Search Google Scholar
    • Export Citation
  • Cai Z & Semenza GL 2004 Phosphatidylinositol-3-kinase signaling is required for erythropoietin-mediated acute protection against myocardial ischemia/reperfusion injury. Circulation 109 20502053. (doi:10.1161/01.CIR.0000127954.98131.23)

    • Search Google Scholar
    • Export Citation
  • Callera GE, Montezano AC, Yogi A, Tostes RC, He Y, Schiffrin EL & Touyz RM 2005a c-Src-dependent nongenomic signaling responses to aldosterone are increased in vascular myocytes from spontaneously hypertensive rats. Hypertension 46 10321038. (doi:10.1161/01.HYP.0000176588.51027.35)

    • Search Google Scholar
    • Export Citation
  • Callera GE, Touyz RM, Tostes RC, Yogi A, He Y, Malkinson S & Schiffrin EL 2005b Aldosterone activates vascular p38MAP kinase and NADPH oxidase via c-Src. Hypertension 45 773779. (doi:10.1161/01.HYP.0000154365.30593.d3)

    • Search Google Scholar
    • Export Citation
  • Caprio M, Newfell BG, la Sala A, Baur W, Fabbri A, Rosano G, Mendelsohn ME & Jaffe IZ 2008 Functional mineralocorticoid receptors in human vascular endothelial cells regulate intercellular adhesion molecule-1 expression and promote leukocyte adhesion. Circulation Research 102 13591367. (doi:10.1161/CIRCRESAHA.108.174235)

    • Search Google Scholar
    • Export Citation
  • Chai W, Garrelds IM, Arulmani U, Schoemaker RG, Lamers JM & Danser AH 2005 Genomic and nongenomic effects of aldosterone in the rat heart: why is spironolactone cardioprotective? British Journal of Pharmacology 145 664671. (doi:10.1038/sj.bjp.0706220)

    • Search Google Scholar
    • Export Citation
  • Chai W, Garrelds IM, de Vries R & Danser AH 2006 Cardioprotective effects of eplerenone in the rat heart: interaction with locally synthesized or blood-derived aldosterone? Hypertension 47 665670. (doi:10.1161/01.HYP.0000205831.39339.a5)

    • Search Google Scholar
    • Export Citation
  • Chai W, Hofland J, Jansen PM, Garrelds IM, de Vries R, van den Bogaerdt AJ, Feelders RA, de Jong FH & Danser AH 2010 Steroidogenesis vs. steroid uptake in the heart: do corticosteroids mediate effects via cardiac mineralocorticoid receptors? Journal of Hypertension 28 10441053. (doi:10.1097/HJH.0b013e328335c381)

    • Search Google Scholar
    • Export Citation
  • Connell JM & Davies E 2005 The new biology of aldosterone. Journal of Endocrinology 186 120. (doi:10.1677/joe.1.06017)

  • Cook CS, Berry LM, Bible RH, Hribar JD, Hajdu E & Liu NW 2003 Pharmacokinetics and metabolism of [14C]eplerenone after oral administration to humans. Drug Metabolism and Disposition: The Biological Fate of Chemicals 31 14481455. (doi:10.1124/dmd.31.11.1448)

    • Search Google Scholar
    • Export Citation
  • Deschamps AM & Murphy E 2009 Activation of a novel estrogen receptor, GPER, is cardioprotective in male and female rats. American Journal of Physiology. Heart and Circulatory Physiology 297 H1806H1813. (doi:10.1152/ajpheart.00283.2009)

    • Search Google Scholar
    • Export Citation
  • Diaz RG, Perez NG, Morgan PE, Villa-Abrille MC, Caldiz CI, Nolly MB, Portiansky EL, Ennis IL & Cingolani HE 2014 Myocardial mineralocorticoid receptor activation by stretching and its functional consequences. Hypertension 63 112118. (doi:10.1161/HYPERTENSIONAHA.113.01726)

    • Search Google Scholar
    • Export Citation
  • Fazal L, Azibani F, Bihry N, Coutance G, Polidano E, Merval R, Vodovar N, Launay JM, Delcayre C & Samuel JL 2014 Akt-mediated cardioprotective effects of aldosterone in type 2 diabetic mice. FASEB Journal 28 24302440. (doi:10.1096/fj.13-239822)

    • Search Google Scholar
    • Export Citation
  • Fiebeler A, Nussberger J, Shagdarsuren E, Rong S, Hilfenhaus G, Al-Saadi N, Dechend R, Wellner M, Meiners S & Maser-Gluth C 2005 Aldosterone synthase inhibitor ameliorates angiotensin II-induced organ damage. Circulation 111 30873094. (doi:10.1161/CIRCULATIONAHA.104.521625)

    • Search Google Scholar
    • Export Citation
  • Fujita M, Minamino T, Asanuma H, Sanada S, Hirata A, Wakeno M, Myoishi M, Okuda H, Ogai A & Okada K 2005 Aldosterone nongenomically worsens ischemia via protein kinase C-dependent pathways in hypoperfused canine hearts. Hypertension 46 113117. (doi:10.1161/01.HYP.0000171184.84077.80)

    • Search Google Scholar
    • Export Citation
  • Gomez-Sanchez EP, Ahmad N, Romero DG & Gomez-Sanchez CE 2004 Origin of aldosterone in the rat heart. Endocrinology 145 47964802. (doi:10.1210/en.2004-0295)

    • Search Google Scholar
    • Export Citation
  • Gros R, Ding Q, Sklar LA, Prossnitz EE, Arterburn JB, Chorazyczewski J & Feldman RD 2011 GPR30 expression is required for the mineralocorticoid receptor-independent rapid vascular effects of aldosterone. Hypertension 57 442451. (doi:10.1161/HYPERTENSIONAHA.110.161653)

    • Search Google Scholar
    • Export Citation
  • Gros R, Ding Q, Liu B, Chorazyczewski J & Feldman RD 2013 Aldosterone mediates its rapid effects in vascular endothelial cells through GPER activation. American Journal of Physiology. Cell Physiology 304 C532C540. (doi:10.1152/ajpcell.00203.2012)

    • Search Google Scholar
    • Export Citation
  • Hausenloy DJ & Yellon DM 2006 Survival kinases in ischemic preconditioning and postconditioning. Cardiovascular Research 70 240253. (doi:10.1016/j.cardiores.2006.01.017)

    • Search Google Scholar
    • Export Citation
  • Karmazyn M, Liu Q, Gan XT, Brix BJ & Fliegel L 2003 Aldosterone increases NHE-1 expression and induces NHE-1-dependent hypertrophy in neonatal rat ventricular myocytes. Hypertension 42 11711176. (doi:10.1161/01.HYP.0000102863.23854.0B)

    • Search Google Scholar
    • Export Citation
  • Krug AW, Pojoga LH, Williams GH & Adler GK 2011 Cell membrane-associated mineralocorticoid receptors?: new evidence. Hypertension 57 10191025. (doi:10.1161/HYPERTENSIONAHA.REF35=10.1161/01.CIR.0000143072.36782.51)

    • Search Google Scholar
    • Export Citation
  • Lopez-Andres N, Inigo C, Gallego I, Diez J & Fortuno MA 2008 Aldosterone induces cardiotrophin-1 expression in HL-1 adult cardiomyocytes. Endocrinology 149 49704978. (doi:10.1210/en.2008-0120)

    • Search Google Scholar
    • Export Citation
  • Marais E, Genade S, Huisamen B, Strijdom JG, Moolman JA & Lochner A 2001 Activation of p38 MAPK induced by a multi-cycle ischaemic preconditioning protocol is associated with attenuated p38 MAPK activity during sustained ischaemia and reperfusion. Journal of Molecular and Cellular Cardiology 33 769778. (doi:10.1006/jmcc.2001.1347)

    • Search Google Scholar
    • Export Citation
  • Marais E, Genade S, Salie R, Huisamen B, Maritz S, Moolman JA & Lochner A 2005 The temporal relationship between p38 MAPK and HSP27 activation in ischaemic and pharmacological preconditioning. Basic Research in Cardiology 100 3547. (doi:10.1007/s00395-004-0495-7)

    • Search Google Scholar
    • Export Citation
  • Matsui S, Satoh H, Kawashima H, Nagasaka S, Niu CF, Urushida T, Katoh H, Watanabe Y & Hayashi H 2007 Non-genomic effects of aldosterone on intracellular ion regulation and cell volume in rat ventricular myocytes. Canadian Journal of Physiology and Pharmacology 85 264273. (doi:10.1139/Y07-017)

    • Search Google Scholar
    • Export Citation
  • Matsui H, Ando K, Kawarazaki H, Nagae A, Fujita M, Shimosawa T, Nagase M & Fujita T 2008 Salt excess causes left ventricular diastolic dysfunction in rats with metabolic disorder. Hypertension 52 287294. (doi:10.1161/HYPERTENSIONAHA.108.111815)

    • Search Google Scholar
    • Export Citation
  • Mihailidou AS, Loan Le TY, Mardini M & Funder JW 2009 Glucocorticoids activate cardiac mineralocorticoid receptors during experimental myocardial infarction. Hypertension 54 13061312. (doi:10.1161/HYPERTENSIONAHA.10REF43=10.1186/1475-2840-12-4)

    • Search Google Scholar
    • Export Citation
  • Mizuno Y, Yoshimura M, Yasue H, Sakamoto T, Ogawa H, Kugiyama K, Harada E, Nakayama M, Nakamura S & Ito T 2001 Aldosterone production is activated in failing ventricle in humans. Circulation 103 7277. (doi:10.1161/01.CIR.103.1.72)

    • Search Google Scholar
    • Export Citation
  • Mocanu MM, Baxter GF, Yue Y, Critz SD & Yellon DM 2000 The p38 MAPK inhibitor, SB203580, abrogates ischaemic preconditioning in rat heart but timing of administration is critical. Basic Research in Cardiology 95 472478. (doi:10.1007/s003950070023)

    • Search Google Scholar
    • Export Citation
  • Nagoshi T, Matsui T, Aoyama T, Leri A, Anversa P, Li L, Ogawa W, Del Monte F, Gwathmey JK & Grazette L 2005 PI3K rescues the detrimental effects of chronic Akt activation in the heart during ischemia/reperfusion injury. Journal of Clinical Investigation 115 21282138. (doi:10.1172/JCI23073)

    • Search Google Scholar
    • Export Citation
  • Nagoshi T, Date T, Fujisaki M, Yoshino T, Sekiyama H, Ogawa K, Kayama Y, Minai K, Komukai K & Ogawa T 2012 Biphasic action of aldosterone on Akt signaling in cardiomyocytes. Hormone and Metabolic Research 44 931937. (doi:10.1055/s-0032-1316343)

    • Search Google Scholar
    • Export Citation
  • Nakamura S, Yoshimura M, Nakayama M, Ito T, Mizuno Y, Harada E, Sakamoto T, Saito Y, Nakao K & Yasue H 2004 Possible association of heart failure status with synthetic balance between aldosterone and dehydroepiandrosterone in human heart. Circulation 110 17871793. (doi:10.1161/01.CIR.0000143072.36782.51)

    • Search Google Scholar
    • Export Citation
  • Nolly MB, Caldiz CI, Yeves AM, Villa-Abrille MC, Morgan PE, Amado Mondaca N, Portiansky EL, Chiappe de Cingolani GE, Cingolani HE & Ennis IL 2014 The signaling pathway for aldosterone-induced mitochondrial production of superoxide anion in the myocardium. Journal of Molecular and Cellular Cardiology 67C 6068. (doi:10.1016/j.yjmcc.2013.12.004)

    • Search Google Scholar
    • Export Citation
  • Oliver WJ, Cohen EL & Neel JV 1975 Blood pressure, sodium intake, and sodium related hormones in the Yanomamo Indians, a "no-salt" culture. Circulation 52 146151. (doi:10.1161/01.CIR.52.1.146)

    • Search Google Scholar
    • Export Citation
  • Pitt B, Remme W, Zannad F, Neaton J, Martinez F, Roniker B, Bittman R, Hurley S, Kleiman J & Gatlin M 2003 Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. New England Journal of Medicine 348 13091321. (doi:10.1056/NEJMoa030207)

    • Search Google Scholar
    • Export Citation
  • Rickard AJ, Morgan J, Bienvenu LA, Fletcher EK, Cranston GA, Shen JZ, Reichelt ME, Delbridge LM & Young MJ 2012 Cardiomyocyte mineralocorticoid receptors are essential for deoxycorticosterone/salt-mediated inflammation and cardiac fibrosis. Hypertension 60 14431450. (doi:10.1161/HYPERTENSIONAHA.112.203158)

    • Search Google Scholar
    • Export Citation
  • Rossier MF, Lenglet S, Vetterli L, Python M & Maturana A 2008 Corticosteroids and redox potential modulate spontaneous contractions in isolated rat ventricular cardiomyocytes. Hypertension 52 721728. (doi:10.1161/HYPERTENSIONAHA.108.114223)

    • Search Google Scholar
    • Export Citation
  • Sanada S, Kitakaze M, Papst PJ, Hatanaka K, Asanuma H, Aki T, Shinozaki Y, Ogita H, Node K & Takashima S 2001 Role of phasic dynamism of p38 mitogen-activated protein kinase activation in ischemic preconditioning of the canine heart. Circulation Research 88 175180. (doi:10.1161/01.RES.88.2.175)

    • Search Google Scholar
    • Export Citation
  • Schmidt K, Tissier R, Ghaleh B, Drogies T, Felix SB & Krieg T 2010 Cardioprotective effects of mineralocorticoid receptor antagonists at reperfusion. European Heart Journal 31 16551662. (doi:10.1093/eurheartj/ehp555)

    • Search Google Scholar
    • Export Citation
  • Sekiyama H, Nagoshi T, Komukai K, Matsushima M, Katoh D, Ogawa K, Minai K, Ogawa T & Yoshimura M 2013 Transient decrease in serum potassium level during ischemic attack of acute coronary syndrome: paradoxical contribution of plasma glucose level and glycohemoglobin. Cardiovascular Diabetology 12 4. (doi:10.1186/1475-2840-12-4)

    • Search Google Scholar
    • Export Citation
  • Shen JZ & Young MJ 2012 Corticosteroids, heart failure, and hypertension: a role for immune cells? Endocrinology 153 56925700. (doi:10.1210/en.2012-1780)

    • Search Google Scholar
    • Export Citation
  • Sherajee SJ, Fujita Y, Rafiq K, Nakano D, Mori H, Masaki T, Hara T, Kohno M, Nishiyama A & Hitomi H 2012 Aldosterone induces vascular insulin resistance by increasing insulin-like growth factor-1 receptor and hybrid receptor. Arteriosclerosis, Thrombosis, and Vascular Biology 32 257263. (doi:10.1161/ATVBAHA.111.240697)

    • Search Google Scholar
    • Export Citation
  • Tong H, Chen W, Steenbergen C & Murphy E 2000 Ischemic preconditioning activates phosphatidylinositol-3-kinase upstream of protein kinase C. Circulation Research 87 309315. (doi:10.1161/01.RES.87.4.309)

    • Search Google Scholar
    • Export Citation
  • Turrell HE, Rodrigo GC, Norman RI, Dickens M & Standen NB 2011 Phenylephrine preconditioning involves modulation of cardiac sarcolemmal KATP current by PKC delta, AMPK and p38 MAPK. Journal of Molecular and Cellular Cardiology 51 370380. (doi:10.1016/j.yjmcc.2011.06.015)

    • Search Google Scholar
    • Export Citation
  • Yamamuro M, Yoshimura M, Nakayama M, Abe K, Shono M, Suzuki S, Sakamoto T, Saito Y, Nakao K & Yasue H 2006 Direct effects of aldosterone on cardiomyocytes in the presence of normal and elevated extracellular sodium. Endocrinology 147 13141321. (doi:10.1210/en.2005-1244)

    • Search Google Scholar
    • Export Citation

If the inline PDF is not rendering correctly, you can download the PDF file here.

 

      Society for Endocrinology

Sept 2018 onwards Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 574 78 7
PDF Downloads 291 50 4
  • View in gallery

    Experimental protocol showing the duration and time course of ischemia/reperfusion. The hearts were frozen at the points in the protocol indicated by solid arrows. Creatine phosphokinase (CPK) activity released into the perfusate was measured at the points in the protocol indicated by dashed arrows. Aldo, aldosterone; Epl, eplerenone.

  • View in gallery

    Cardiac functional recovery after ischemia–reperfusion injury. LVDP profiles during ischemia–reperfusion (A), LVDP recovery (percentage of baseline value) measured at the indicated time points (B), and LVEDP profiles during ischemia–reperfusion (C) in the control hearts (white squares and bars; n=23), aldosterone-preconditioned hearts (Aldo, black squares and bars; n=20) and aldosterone-preconditioned hearts treated with eplerenone infusion (Aldo+Epl, gray squares and bars; n=9). (D) LVDP recovery (percentage of baseline value) measured at the end of the 40-min reperfusion period in the aldosterone-preconditioned hearts treated with or without either RU486 or G15 (Aldo+RU486 or G15; n=7 each). *P<0.05, control vs Aldo; P<0.05 control vs Aldo+Epl; P<0.05 control vs Aldo+RU486; #P<0.05 control vs Aldo+G15.

  • View in gallery

    (A) CPK profiles in the effluent collected during the reperfusion period. (B) Area under the curve (AUC) was calculated from the CPK profile shown in (A) (n≥7 for each group). *P<0.05 control vs Aldo; P<0.05 control vs Aldo+Epl.

  • View in gallery

    Phosphorylation of Akt and GSK3β was evaluated in hearts treated with or without ischemia–reperfusion for the indicated periods shown in Fig. 1. Representative immunoblots obtained using the indicated antibodies are shown. Averaged densitometry data normalized to the control at the same time points are shown in the bar graphs (n=3–4 for each group). *P<0.05, control vs Aldo; P<0.05 Aldo vs Aldo+Epl.

  • View in gallery

    Phosphorylation of MAPKs signaling pathways was evaluated in hearts treated with or without ischemia–reperfusion for the indicated periods shown in Fig. 1. Representative immunoblots obtained using the indicated antibodies are shown. Averaged densitometry results normalized to the control at the same time points are shown in the bar graphs (n=4 for each group). *P<0.05, control vs Aldo; P<0.05 control vs Aldo+Epl.

  • View in gallery

    Effects of the p38MAPK inhibitor (SB203580) on cardiac functional recovery after ischemia–reperfusion injury. LVDP recovery (percentage of baseline value) measured at the end of the 40-min reperfusion period in aldosterone-preconditioned hearts treated with or without SB203580 is shown (Aldo+SB203580; n=7) *P<0.05 control vs Aldo; P<0.05 Aldo vs Aldo+SB203580.