Urocortin-1 Mediated Cardioprotection Involves XIAP and CD40-Ligand Recovery: Role of EPAC2 and ERK1/2

Aims Urocortin-1 (Ucn-1) is an endogenous peptide that protects heart from ischemia and reperfusion (I/R) injuries. Ucn-1 is known to prevent cardiac cell death, but its role in the transcription of specific genes related to survival signaling pathway has not been fully defined. The aim of this study was to investigate the molecular signaling implicated in the improvement of cardiac myocytes survival induced by Ucn-1. Methods and Results Ucn-1 administration before ischemia and at the onset of reperfusion, in rat hearts perfused in Langendorff system, fully recovered heart contractility and other hemodynamic parameters. Ucn-1 enhanced cell viability and decreased lactate dehydrogenase (LDH) release in adult cardiac myocytes subjected to simulated I/R. Annexin V-FITC/PI staining indicated that Ucn-1 promoted cell survival and decreased cell necrosis through Epac2 (exchange protein directly activated by cAMP) and ERK1/2 (extracellular signal–regulated kinases 1/2) activation. We determined that Ucn-1 shifted cell death from necrosis to apoptosis and activated caspases 9 and 3/7. Furthermore, mini-array, RT-qPCR and protein analyses of apoptotic genes showed that Ucn-1 upregulated the expression of CD40lg, Xiap and BAD in cells undergoing I/R, involving Epac2 and ERK1/2 activation. Conclusions Our data indicate that Ucn-1 efficiently protected hearts from I/R damage by increasing the cell survival and stimulated apoptotic genes, CD40lg, Xiap and BAD, overexpression through the activation of Epac2 and ERK1/2.


Introduction
Despite the considerable advances that have been made in the field of myocardial protection, ischemic heart disease represents a major public health problem and the main cause of mortality in the industrialized world [1]. Percutaneous transluminal angioplasty, fibrinolysis and cardioplegic solutions are some of the strategies developed to preserve the myocardial viability from ischemia. All these procedures involve myocardial reperfusion/reoxygenation after an ischemic episode. However, the subsequent reperfusion also activates various injury responses leading to necrosis, apoptosis and general heart dysfunction [1,2].
Special interest has been made toward the endogenous protection elicited by the heart as a potent approach to limit heart lesions from I/R insult. In the last two-decade, urocortin peptides (Ucn-1, Ucn-2, Ucn-3), which belongs to the corticotropin-releasing factor (CRF) family [3], have emerged as a potential therapeutic agonist that improves heart performances and protects heart from I/R injuries [4]. In the cardiovascular system, urocortin binding to its G protein-coupled receptor (CRF-R 2 ) is known to enhance cAMP production [5], which is classically related to PKA activation. However, a guanine nucleotide exchange factor (GEF) also activated directly by cAMP, named exchange protein activated by cyclic AMP (Epac) emerged as a new player of several cAMP-regulated processes in heart such as heart inotropism [6], cardiac myocytes hypertrophy [7], and Ca 2+ handling in cardiac myocytes [8]. Previously, we have described that Epac and ERK1/2 are involved in urocortin-induced positive inotropism in rat hearts [9]. However, Epac role in cardioprotection has been barely studied.
Different mechanisms are implicated in the cardioprotection afforded either by Ucn-1 or Ucn-2, involving the rapid activation of protective signaling pathways [10], calcium-independent phospholipase A 2 (iPLA 2 ) and protein kinase C epsilon (PKCε) [11], or ERK1/2 [12,13], among others. Urocortin also regulated cell survival and apoptosis during I/R injury, through caspase 3 inhibition [10], STAT3 [14] or p38MAPK activation [15]. We have shown recently that Ucn-1 administration only at the beginning of the reperfusion preserved heart contractility by the improvement of intracellular Ca 2+ handling, which included the recovery of cells excitability, the inhibition of diastolic Ca 2+ increase and the regulation of Na + /Ca 2+ exchanger [16].
Herein, we explored the molecular pathway involved in Ucn-1 evoked heart protection with special emphasis on Epac and ERK1/2 on their role in cardiac myocytes survival. We also examined the effect of Ucn-1 on cell death pathways and its regulation of apoptotic genes, CD40lg, Xiap and BAD.

Materials and Methods
All the experiments with animals were performed in accordance with the recommendations of the Royal Decree 53/2013 in agreement to the Directive 2010/63/EU of the European Parliament and approved by the local Ethics Committee on human Research of the "Virgen del Rocio" University Hospital of Seville and the Animal Research Committee of the University of Seville.
Chart Powerlab software (ADInstruments) was used for continuous recording throughout the experiments of heart rate, left ventricular developed pressure (LVDP), and maximum positive and negative derivative of left ventricular pressure (±dP/dt). The heart contractility under different treatments was evaluated by the analysis of +dP/dt, which corresponds to % increase of +dP/dt normalized to basal value after the period of stabilization.
The standard protocol of ischemia/reperfusion in perfused hearts was followed as described previously [16]. Group 1 of I/R: After stabilization, rat hearts were exposed to global ischemia (without aorta perfusion) during 40 minutes and 1 hour of reperfusion with freshly oxygenated solution. Group 2 corresponds to the pharmacological preconditioning with Ucn-1 (10 nM). After stabilization, Ucn-1 was applied 20 minutes before ischemia and 30 minutes at the beginning of reperfusion.

Apoptosis and necrosis assays
The level of lactate dehydrogenase (LDH) was detected in supernatant of cells subjected to simulated I/R protocol according to the manufacturer's instructions (Promega). Trypan Blue staining method was also used for detecting cell death in cardiac myocytes undergoing treatment with I/R ± Ucn-1 (10 nM). Briefly, after each treatment cells were washed three times with PBS, then cells were bathed in 0.5% of trypan blue for 10 minutes. Based on the classical cell counting, we acquired 4 random snapshots per each condition using 40x Objective of an Olympus microscope. Maximum projection was recorded and analyzed with "Image J" software to count the different proportion of trypan positive and negative cells by an investigator blinded to the treatment conditions. This process was repeated in triplicate for each condition and the experiment was replicated on 3 separate occasions. 100% indicates total number of trypan stained and unstained cells in each condition.
Double staining with annexin V-FITC and propidium iodide (PI) was also performed to examine apoptosis and necrosis in cultured cardiac myocytes. During apoptosis, an early and ubiquitous event is the exposure of phosphatidylserine at the cell surface, which is detected with annexin V-FITC. PI is used to determine the population of cells that have lost membrane integrity, an indication of late apoptosis or necrosis. Briefly, cardiac myocytes were seeded in a 6-channel μ-Slide (μ-Slide VI 0,4) from IBID. After treatments cardiac myocytes were incubated with 100 μl of the binding buffer supplied in the kit (Cat. 4830-01-K. Trevigen) and 1 μl of annexin-FITC reagent, during 60 minutes at 25°C. Then PI was added and incubated for 15 minutes and later washed before mounting the slides. Images were taken with confocal microscope Leica TCS SP2 microscopy (Leica). 5 snapshots per condition were acquired using a HCX PI Apo CS 40x objective in z-stacks intervals, and maximum projection was recorded and analyzed with "Image J" software to count the different proportion of cells that were labeled only with annexin (early apoptosis), or with PI and/or annexin and PI (necrosis or late apoptosis).

Caspase 9, 8 and 3/7 activity
To determine the caspase activity we used the Caspase-Glo assay kit system from Promega; TB323 for Caspase 3/7, TB332 for Caspase 8, and TB333 for Caspase 9. Protein extracts from cultured cardiac myocytes were quantified by Bradford (Sigma Aldrich) method and were diluted at 1.5 μg/μl of protein. 10 μl of protein extract and 10 μl of Caspase-Glo reagent were added in a 384 flat and white multi-well dish in quadruplicate replicates and incubated for 40 minutes at room temperature. Luminescent signal was detected in a Luminoskan ascent microplate luminometer (Thermo Scientific).

Western Blotting
Protein samples were extracted from cultured and treated cardiac myocytes and 40 μg of protein were subjected to SDS-PAGE (10% acrylamide) and electrotransferred onto PVDF membranes. Membranes were probed overnight at 4°C with specific primary antibodies in TTBS with 1% of BSA. CD40lg antibody was from sigma. BAD, phosphoBAD, Xiap, phosphoERK1/2 and ERK1/2 primary antibodies were from Cell Signaling. Detection was performed with the enhanced chemiluminiscence reagent ECL-plus (Amersham Bioscience) in the ImageQuant LAS 4000 mini (GE Healthcare). For quantification, the images were analyzed with "Image J" software using alpha-tubulin (Sigma) or GAPDH (Genetex) as housekeeping loading control.

Analysis of mRNA expression
Total RNA was extracted from cultured cardiac myocytes using the RNeasy kit (Qiagen) according to the manufacturer's instructions. RNA purity and concentration were assessed by measuring absorption at 260 nm and 280 nm. 1 μg of RNA were retro-transcribed to cDNA with the RT quantitec kit (Qiagen). qRT-PCRs were performed with the use of a Prism 7900HT Sequence Detection System, Taqman primers and probes technologies (Applied Biosystems). Fold change in gene expression was calculated using the comparative cycle threshold CT (ΔΔCT) method.
For apoptosis PCR array, we used plates from SA Biosciences (Cat. PAHS-012). Total RNA from Ucn-1 treated and no-treated cardiac myocytes (as described in cell treatment protocols) submitted to I/R was extracted and reverse transcribed into cDNA with the use of an RT2 First Strand Kit (SA Biosciences). The templates were combined with an RT2 SYBR Green qPCR Master Mix (SA Biosciences), and then equal aliquots of this mixture (25 μl) were added to each well of the same PCR Array plate that contained the pre-dispensed gene-specific primer sets. The qRT-PCR quantification was performed as described above by ΔΔCT method.

Statistical analysis
Data analysis was carried out using SigmaPlot software, version 11.0. A sample size calculation was performed prior the start of this study. Group data are presented as mean ± S.E.M. Single or paired Student's t test was used to determine the statistical significance of the obtained data. The significance between multiple groups was evaluated using ANOVA followed by Tukey multiple comparison post hoc tests for comparing different groups. Data marked by Ã , ÃÃ , ÃÃÃ were considered significantly different at p<0.05, p<0.01 and p<0.001 respectively.

Results
Urocortin-1 improves the hemodynamic performances in isolated rat hearts submitted to ischemia and reperfusion protocol The protective effects of Ucn-1 on heart contractility were analyzed in ex vivo Langendorff-perfused rat hearts submitted to I/R. Fig 1A-1C shows that hearts exposed to 40 minutes of ischemia and 60 minutes of reperfusion (I/R) suffered a significant decrease in the left ventricular developed pressure (LVDP) as well as in the positive and negative maximum derivative of left ventricular pressure (±dP/dt), indicating significant loss in heart function. The addition of Ucn-1 (10 nM) 20 minutes before ischemia and 30 minutes at the beginning of reperfusion recovered significantly LVDP and ±dP/dt as shown in Fig 1B and 1C. The comparison of superimposed LVDP after I/R obtained either from untreated hearts or from hearts treated with Ucn-1, revealed that the peptide decreased the time-to-peak of LVDP, recovered fully its amplitude and accelerated the relaxation comparing to untreated hearts submitted to I/R ( Fig  1D and 1E).
Furthermore Fig 2 shows that Ucn-1 administration recovered completely +dP/dt which was sustained in the maximum level in reperfusion (Fig 2A), and improved others hemodynamic parameters such as the left ventricular end diastolic pressure (LVEDP) (Fig 2B), and the coronary vascular resistances whose progressive rise observed in reperfusion was prevented significantly by Ucn-1 ( Fig 2C). These cardioprotective effects of Ucn-1 were even maintained 30 minutes after washing out the peptide.
Urocortin-1 promotes cell survival, regulates apoptosis and necrosis in cardiac myocytes exposed to I/R To further evaluate the cardioprotection exerted by Ucn-1, we examined cardiac myocytes viability using several approaches. First, we estimated trypan blue staining in cardiac myocytes subjected to I/R. Fig 3A shows a significant increase in trypan blue positive staining in cells submitted to I/R as compared to control. However, cells pretreatment with Ucn-1 (10 nM) decreased significantly the number of trypan blue positive cells. Next, to verify the trypan blue finding cell death was also assessed by measurement of LDH release. Cells exposition to I/R resulted in significant increase of LDH levels, meanwhile Ucn-1 (10 nM) significantly reduced the release of LDH during I/R (Fig 3B).
In addition, we used annexin V-FITC staining to label apoptotic cells in combination with red-fluorescent PI (propidium iodide) that stains late apoptotic/necrotic cells. Representative images and data analysis in Fig 4A and 4B show that cells subjected to I/R resulted in a significant loss of cell viability and a marked increase of the PI positive population, reflecting higher level of cell death by necrosis. Whereas, Ucn-1 (10 nM) treatment of cardiac myocytes exposed to I/R increased the amount of viable cells. Interestingly, Ucn-1 promoted a significant decrease in PI positive cells (necrosis) but it increased early apoptosis as indicated by annexin V positive    -1 (10 nM). B) Bar graph shows the level of LDH release in cardiac myocytes from 5 independent cultures, in control and after I/ R (30 minutes/18 hours each) ± Ucn-1 (10 nM). Ucn-1 was also applied in the presence of ERK inhibitor, 5 μM PD 098059 (Ucn-1 + PD), or with Epac2 inhibitor, 5 μM ESI-05 (Ucn-1+ESI). C) Western blot and D) summary data of ERK 1/2 phosphorylation in control cardiac myocytes and in cells exposed to I/R (30 minutes/18 hours each). Cells were treated with Ucn-1 (10 nM) or with 8CPT (10 μM) alone or in the presence of 5 μM PD 098059 indicated as Ucn-1 + PD, 8CPT + PD respectively. Data are given as means ± s.e.m. from three independent cardiac myocytes cultures. "*" and "**" indicate significance at p<0.05, and p<0.01 respectively. staining, suggesting that Ucn-1 enhanced cell viability and shifted cell death from necrosis to apoptosis in cells exposed to I/R. Next, we examined caspases activity using an enzymaticbased assay. Fig 4C shows that Ucn-1 enhanced significantly caspase 9 and 3/7 activity in cells exposed to I/R, consistent with annexin-FITC V/PI observations. However, the activation of caspase 8 was not affected by Ucn-1 as shown in S1 Fig.   Fig 4. Urocortin-1 effects on cell death and apoptosis. A) Representative images of isolated adult cardiac myocytes submitted to I/R (left) and treated with Ucn-1 (right), stained with annexin V-FITC (green) and PI (red). B) Shows the summary data from untreated cells (control) and cells exposed to I/R (30 minutes/18 hours each) ± Ucn-1 (10 nM) alone or in presence of 5 μM PD 098059 (Ucn-1 + PD) or 10 μM ESI-05 (Ucn-1 + ESI). 8CPT bars are for cells exposed to I/R (30 minutes/18 hours each) treated with 8CPT (10 μM). White bars indicate viable cells; black bars are for necrosis; and grey bars indicate apoptosis. n = 5 cultures. "*" indicates significance at p<0.05 of I/R vs control. "#" indicates significance at p<0.05 of Ucn-1 and 8CPT comparing to I/R. "&" indicates significance at p<0.05 of Ucn-1 comparing to Ucn-1+PD or to Ucn-1+ESI. C) Graphs show caspase 9 and 3/7 activation in similar experiments as in "B. n = 5 cultures. Data are given as means ± s.e.m. "*", "**" and "***" indicate significance at p<0.05, p<0.01 and p<0.001 respectively.
Because Epac activates the Ras1-ERK1/2 pathway [18], we also investigated whether Epac signals ERK1/2 activation in Ucn-1 treated cardiac myocytes. Fig 3C and 3D shows that cells incubation with 8CPT (10 μM), the specific agonist of Epac [19], also stimulated ERK1/2 phosphorylation in I/R, which was inhibited significantly by PD 098059. Fig 4B also shows that 8CPT (10 μM) mimicked the effect of Ucn-1 on cell viability, apoptosis and necrosis. To further confirm Epac role in Ucn-1 action we tested ESI-05 (10 μM), considered as the most specific inhibitor of Epac2 [20]. As shown in Figs 3B and 4B, cells pretreatment with ESI-05 abolished Ucn-1 cytoprotective effects on LDH release cell viability, apoptosis and necrosis. The addition of PD 098059 or ESI-05 in cardiac myocytes subjected to I/R, but in the absence of Ucn-1, didn't change cell viability, apoptosis and necrosis as illustrated in S2 Fig.

Urocortin-1 alters apoptotic gene expression
To get more insight into the mechanism involved in Ucn-1 cytoprotection and apoptosis, we analyzed the expression of 84 key genes involved in apoptosis using a PCR based micro-array technique. S3 Fig shows that the expression of several genes changed in cells undergoing the same protocol of I/R and incubated with Ucn-1 (10 nM) comparing to untreated cells. Next, we verified by RT-PCR analysis the impact of I/R on CD40lg, Xiap and BAD genes expression and their possible modulation by Ucn-1. We observed that mRNA expression of CD40lg ( Fig  5A), Xiap ( Fig 5B) and BAD (Fig 5C) were significantly decreased in cells exposed to I/R, whereas cells treatment with Ucn-1 recovered completely these genes expression. The effect of Ucn-1 on CD40lg and Xiap were abolished by ERK1/2 inhibition with PD 098059 (5 μM). Meanwhile, Epac2 inhibition with ESI-05 (10 μM) blocked the effect of Ucn-1 on Xiap but not on CD40lg expressions. Furthermore, the evoked upregulation of BAD by Ucn-1 was inhibited in the presence of ESI-05 (10 μM) but not when cells were treated with PD 098059 (5 μM).
Next, the analysis of protein expression showed that CD40lg, Xiap and Bad levels were not significantly affected by I/R, however the administration of Ucn-1 enhanced significantly the expression of CD40lg and Xiap protein in ERK1/2 dependent manner, since treatment with PD 098059 (5 μM) prevented Ucn-1 effects as shown in Fig 5D, 5F and 5G). At the same time, Ucn-1 enhanced potently BAD protein expression comparing to untreated cells exposed to I/R (Fig 5E and 5H), which was inhibited by ESI-05 (10 μM) but not by PD 098059 (5 μM) confirming the implication of Epac2 but not ERK1/2. 8CPT (10 μM) cells treatment also mimicked the effect of Ucn-1 by enhancing BAD expression. Since BAD activity depends on its phosphorylation state, we further examined the amount of serine phosphorylated protein in Ucn-1 treated cardiac myocytes. Fig 5E and summary data in S4 Fig show that Ucn-1 didn't affect BAD phosphorylation, although the amount of free activated protein was significantly increased in cells treated with Ucn-1 and 8CPT compared to cells submitted only to I/R.

Discussion
Clinical search for new heart protection strategies and new endogenous cardioprotector is still of major relevance. There is continuous interest in developing novel therapeutic targets that potentially protect the heart against I/R injuries and complications [21,22]. Several studies have demonstrated that different pre or post-conditioning strategies effectively protect the heart and reduce infarct size after an ischemic insult [23]. Urocortin is recognized as a potential therapeutic agent thanks to its powerful cardioprotective properties, involving different intracellular signaling pathways [24,25].
Our experimental data from Langendorff-perfused rat hearts showed that Ucn-1, given both before and after a global ischemic insult, was able to recover heart contractility, to preserve vascular resistance, and to improve the ventricular diastolic pressure, indicative of its prevention of hypercontracture and rigor. Ucn-1 effectively sustained rat hearts hemodynamics properties and protected it from I/R injury consistent with the effect of Ucn-2 described in previous studies [24,25].
The degree of cardiac dysfunction after I/R injury reflects the level of cardiac myocytes lesion related to subsequent cell death, scare formation and a worse long-term outcome. Here, we demonstrated that Ucn-1 exerted significant cytoprotective effects as it efficiently decreased trypan blue cell staining, inhibited LDH release, and enhanced cell viability by the modulation of apoptosis and necrosis. Ucn-1 administration decreased significantly necrotic cells but it increased apoptotic cells number. In fact, while Ucn-1 increased cell viability it shifted cell death from necrosis to apoptosis, a more programmed and controlled cell death. Ucn-1 promotion of apoptosis in detriment of necrosis is supposed more beneficial to reduce the impact of I/R lesions as discussed earlier [26]. Apoptosis is a known form of cell death mediated through sequential activation of the caspase cascade. Consistently, Ucn-1 stimulated caspase 3/7 and caspase 9, but not caspase 8 that is associated to death receptors-mediated apoptosis [27]. These data is contrary to Ucn-2 reduction of caspase 3 levels due to a significant induction of natriuresis and decreased tissue mediators of inflammation [10,28]. Ucn-1 stimulation of caspase 3/7 and 9 findings was supported with Ucn-1 regulation of apoptotic genes expression. Actually, we observed that cardiac myocytes incubation with Ucn-1 preserved the expression of the well known anti-apoptotic genes CD40lg and Xiap [29,30], as well as the pro-apoptotic BAD [29], which were down-regulated in cells exposed to I/R. Furthermore, at protein levels Ucn-1 also upregulated Xiap, CD40lg, and BAD. The levels of BAD phosphorylation known to allow its confinement to the cytoplasm and the inhibition of BAD-dependent death [31,32], was not affected in Ucn-1 treated cells. However, the increase in total amount of BAD protein under Ucn-1 treatment was accompanied also by an increase in the amount of dephosphorylated (free) BAD protein, which might explain the observed apoptosis annexin/PI and caspase experiments. Similarly, the activation of BAD by Ucn-1 and Ucn-2 was previously described in macrophage [33] but not yet in heart. On the other hand, Ucn-1 upregulation of anti-apoptotic genes CD40lg and Xiap explains its beneficial effects on cell survival and necrosis. To our knowledge, this study is the first to involve CD40lg and Xiap in Ucn-1 cardioprotective effects. In this study, we noticed that the changes in mRNA levels of apoptotic genes during cells exposition to in I/R were more evident than at the protein levels, which could be due to the complex mechanism of post-transcriptional mechanisms involved in turning mRNA into protein [34].
Moreover, we demonstrated that Ucn-1 implicated Epac2 and ERK1/2 in its cardioprotective actions. ERK1/2 role as a survival pathway in cardiac cells is widely accepted [10][11][12], nevertheless only few reports directly investigated its activation during reperfusion [35,36]. In this study, we demonstrated that under I/R, Ucn-1 activated significantly ERK1/2, whose inhibition by PD 098059 dramatically prevented Ucn-1 protective effects in Langendorff perfused hearts (data not shown), and abolished Ucn-1 cytoprotection action supporting the importance of ERK1/2 in mediating urocortin effects, in accordance with others studies [12,13]. Indeed, we demonstrated that ERK1/2 are also implicated in Ucn-1 inhibition of LDH release, cell viability, caspase activation, and in the modulation of CD40lg and Xiap expression. Furthermore, very few studies have focused on the role of Epac in urocortin signaling pathway [9,37,38]. However, the role of Epac in cardioprotection is still under debate. Recently, a cardioprotective role of β-adrenergic signaling via cAMP/Epac/Rap1/Rac/ERK pathway has been described in mice [39]. Epac activation was also associated with cardiac myocytes protection from nitric oxide-induced apoptosis [39,40]. Here, we provided different data indicating Epac2 involvement in Ucn-1 cardioprotective effects by the use of ESI-05 as a selective inhibitor for Epac2 isoform (for review see [20]). We found that ESI-05 inhibited the effects of Ucn-1 on LDH release and prevented Ucn-1 restoration of Xiap and BAD expression. Our data suggest for the first time an important role for Epac2 in Ucn-1 heart improvement from I/R lesions, which is consistent with the increasing numbers of studies that propose an important role of Epac in heart [6][7][8]41]. Finally, Epac as a GTP exchange factor is known to activate Rap1-GTPase by catalyzing the formation of Rap1-GTP and further stimulation of ERK. Herein, we demonstrated that Epac activation with 8CPT mimicked the effect of Ucn-1 on ERK1/2 activation in I/R, which suggested that Epac is upstream of ERK1/2 signaling pathway.
In conclusion, we demonstrated that the administration of Ucn-1 before ischemia and during the first minutes of re-oxygenation produced significant long-lasting protective effects, mediated by a complex signaling pathway that involve Epac2 and ERK1/2. Ucn-1 promotion of cell survival and apoptosis might be beneficial since apoptosis is considered a highly regulated energy-consuming process, required for controlled and programmed cell death, which will limit cardiac cell loss and posterior inflammation processes in I/R. Together with the other cardioprotective actions of urocortin including vasorelaxation of human coronary artery [42], the positive inotropism [9], and the decrease in renin activity, aldosterone, vasopressin, endothelin1 and atrial and B-type natriuretic peptides [43], all hallmark events shown to diminish the destroying effect of I/R injury, certainly confirm urocortin as a valuable target for the treatment of diseases associated with cardiac dysfunction under post-ischemic insults.