Ca2+ exchanger 1 inhibition abolishes ischemic tolerance induced by ischemic preconditioning in different cardiac models

European Journal of Pharmacology 794 (2017) 246–256

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Na+/Ca2+ exchanger 1 inhibition abolishes ischemic tolerance induced by ischemic preconditioning in different cardiac models Pasqualina Castaldoa,1, Maria Loredana Macrìa,1, Vincenzo Laricciaa, Alessandra Matteuccia, ⁎ Marta Maiolinoa, Santo Gratterib, Salvatore Amorosoa, , Simona Magia a b

Department of Biomedical Sciences and Public Health, School of Medicine, University “Politecnica delle Marche”, Via Tronto 10/A, 60126 Ancona, Italy Department of Health Sciences, University “Magna Graecia”, Viale Europa–Localitá Germaneto, 88100 Catanzaro, Italy

A R T I C L E I N F O

A BS T RAC T

Keywords: Calcium Cardioprotection Heart NCX1 Ischemia/reperfusion Preconditioning

Ca2+-handling disturbances play an important role in the genesis of myocardial ischemia/reperfusion (I/R) injury. Ischemic preconditioning (IPC) is a powerful strategy to induce tolerance against subsequent ischemic episodes. IPC signaling pathways may be triggered by Ca2+ ion. Since Na+/Ca2+ exchanger 1 (NCX1) participates in modulating intracellular Ca2+ homeostasis, here we further defined its role in I/R and investigated its potential involvement in IPC-induced cardioprotection. In isolated ventricular cardiomyocytes, perfused rat heart and H9c2 cardiomyoblasts, I/R produced a significant cell injury, assessed by measuring extracellular lactate dehydrogenase (LDH) and, for the whole heart, also by estimating myocardial infarct size area. Characterization of cell death revealed the involvement of apoptotic processes. Interestingly, I/R challenge induced NCX1 protein upregulation. In NCX1-transfected H9c2 cells, exchanger protein upregulation was accompanied by an increase in its reverse mode activity. The effects of I/R on extracellular LDH and infarct size area were drastically reduced by 1 μM SN-6, a selective NCX1 inhibitor. Moreover, SN-6 also prevented I/Rinduced increase of NCX1 reverse-mode activity and protein upregulation. These results suggested a deleterious role of NCX1 in I/R-induced cell damage. In both isolated cardiomyocytes and perfused heart, IPC followed by I/R afforded cardioprotection, reducing extracellular LDH release and limiting ischemic area extent. Interestingly, NCX1 blockade (1 μM SN-6) completely abolished IPC protection against I/R, leading to exacerbation of cell injury, massive infarct size area and restoration of NCX1 protein expression. These findings suggest that NCX1 is deleterious in I/R, whereas it may be beneficial in promoting IPCinduced cardioprotection.

1. Introduction Myocardial ischemia stems from a reduced blood flow to the heart, causing oxygen and substrates supply deprivation and leading to sudden biochemical and metabolic impairments. Cell metabolism is switched to anaerobic respiration, with accumulation of toxic glycolytic products, ATP depletion, ionic derangements and inhibition of myocardial contractile function (Frank et al., 2012). Time is muscle: the quicker the perfusion is restored, the more heart tissue is saved (Kalogeris et al., 2012). At reperfusion, however, blood flow restoration paradoxically initiates a cascade of events that imposes additional

stress to cardiomyocytes, giving rise to ischemia/reperfusion (I/R) injury (Sanada et al., 2011; Yellon and Hausenloy, 2007). Over the last years different approaches have been explored to limit infarct size and to improve outcomes (Ibanez et al., 2015). One of the most promising interventions is based on the ischemic conditioning, whereby short periods of subcritical ischemic stimuli confer protection from lethal injury when performed before (preconditioning) (Murry et al., 1986), during (perconditioning) (Vinten-Johansen and Shi, 2011) or after (postconditioning) (Penna et al., 2008) the index ischemia. Specifically, ischemic preconditioning (IPC) can be applied minutes or even days before the lethal ischemia, thereby producing two windows of rever-

Abbreviations: [Ca2+]i, Intracellular Ca2+ concentration; HPC, Hypoxic Preconditioning; H/R, Hypoxia/Reoxygenation; IPC, Ischemic Preconditioning; I/R, Ischemia/Reperfusion; LDH, Lactate Dehydrogenase; NCX, Na+/Ca2+exchanger; SN-6, 2-[[4-[(4Nitrophenyl) methoxy] phenyl] methyl]−4-thiazolidinecarboxylic acid ethyl ester; TTC, Triphenyltetrazolium chloride; WT, Wild Type ⁎ Correspondence to: Department of Biomedical Sciences and Public Health, School of Medicine, University Politecnica of Marche, Via Tronto 10/A, 60126 Ancona, Italy. E-mail addresses: [email protected] (P. Castaldo), [email protected] (M.L. Macrì), [email protected] (V. Lariccia), [email protected] (A. Matteucci), [email protected] (M. Maiolino), [email protected] (S. Gratteri), [email protected] (S. Amoroso), [email protected] (S. Magi). 1 These authors equally contributed to this paper. http://dx.doi.org/10.1016/j.ejphar.2016.11.045 Received 2 July 2016; Received in revised form 22 November 2016; Accepted 24 November 2016 Available online 25 November 2016 0014-2999/ © 2016 Published by Elsevier B.V.

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perfusion with HEPES solution, i.e. stabilization period. Pharmacological inhibition of NCX1 was achieved by using 2-[[4[(4Nitrophenyl) methoxy] phenyl] methyl]−4-thiazolidinecarboxylic acid ethyl ester (SN-6) (Iwamoto et al., 2004). Seven different experimental conditions were tested. A more detailed description of the groups is provided below.

sible protection, referred as early and delayed phase (Marber et al., 1993). Several studies have been performed in order to characterize the biology of IPC according to different endogenous mediators that can promote innate protective responses (Cohen and Downey, 2008; Laude et al., 2003; Ohnuma et al., 2002). In this regard, modifications of intracellular Ca2+ concentration ([Ca2+]i) have been recognized as important triggers. Specifically, transient and reversible fluctuations in [Ca2+]i can mimic the protective effect of IPC (Meldrum et al., 1996; Miyawaki and Ashraf, 1997; Przyklenk et al., 1997). Dysregulation of intracellular Ca2+ homeostasis seems to play a critical role in the I/R-induced cell injury. Persistent elevation of [Ca2+]i ultimately causes much of the damage accompanying I/R (Garcia-Dorado et al., 2012), supporting the hypothesis that timing and magnitude of Ca2+ response during I/R and IPC stimuli are key determinant of the cell fate. It is well established that Na+/Ca2+ exchanger 1 (NCX1) is a critical protein contributing to the Ca2+ homeostasis in the heart (Lytton, 2007). NCX1 catalyzes the electrogenic and reversible exchange of 3 Na+ for 1 Ca2+ across the plasma membrane, participating in excitation-contraction coupling (Aronsen et al., 2013), nodal pace-maker activity (Groenke et al., 2013) and cell metabolism (Magi et al., 2013, 2012). Alteration of the exchanger activity seems to contribute to the I/ R-induced cell injury, since pharmacological or genetic inhibition of NCX1 significantly limits cardiac injury induced by I/R (Imahashi et al., 2005; Li et al., 2014; Ohtsuka et al., 2004). In contrast, up to date NCX1 role in IPC is poorly investigated, although Zhang and coworkers reported that a functional NCX1 may be effectively involved in this phenomenon (Zhang et al., 2015). Thus, in the present study we aimed to further define the role of NCX1 in I/R and its specific contribution to IPC-induced protective adaptation in different cardiac models.

– Group 1. Control (CTR): isolated hearts were perfused with HEPES buffered solution for 150 min – Group 2. Control +1 μM SN-6 (CTR SN-6): isolated hearts were perfused with HEPES solution enriched with 1 μM SN-6 for 150 min – Group 3. Ischemia/reperfusion (I/R): global ischemia was induced by interrupting both oxygen and HEPES solution flows (no-flow ischemia) for 30 min, during which the hearts were submerged into HEPES solution without glucose and maintained at 37 °C to avoid reduction in myocardial temperature. Reperfusion was accomplished by restoring oxygen and HEPES solution flows for 120 min – Group 4. Ischemia/reperfusion +1 μM SN-6 (I/R SN-6): isolated hearts were perfused with 1 μM SN-6 for 10 min. Thereafter, they were subjected to I/R as described for group 3. 1 μM SN-6 was maintained throughout the entire I/R protocol. – Group 5. Ischemic preconditioning control (IPC): isolated hearts were subjected to a sub-lethal stimulus made up of 3 bouts of 2 min no-flow ischemia separated by 3 min of reperfusion (Bulvik et al., 2012). Then, hearts were perfused with HEPES buffered solution for 150 min – Group 6. Ischemic preconditioning followed by ischemia/reperfusion (IPC I/R): isolated hearts were subjected to IPC protocol, as described for group 5, followed by I/R as described for group 3. – Group 7. Ischemic preconditioning +1 μM SN-6 followed by ischemia/reperfusion (IPC SN-6 I/R): isolated hearts were subjected to IPC I/R as described in group 6, but IPC protocol was preceded by 10 min perfusion with 1 μM SN-6, which was maintained throughout the entire IPC protocol. Thereafter, I/R was induced in the absence of the NCX inhibitor.

2. Materials and methods 2.1. Animals

At the end of each protocol, hearts were removed and kept at −20 °C for at least 1 h.

Male Wistar rats (two-month old) were used for both ex-vivo experiments and cardiomyocytes isolation. All the rats were bred inhouse at the University Politecnica of Marche Animal Facility. Animals were housed in a 12 h light–dark cycle at room temperature (22 ± 1 °C) with free access to food and water. The animal protocol was approved by the Ethic Committee for Animal Experiments of the University Politecnica of Marche (Ref no. 721/2015-PR). All the experiments were conducted in strict accordance with the guidelines of the Italian Ministry of Health (D.L.116/92 and D.L.111/94-B) and the Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by the National Institutes of Health (USA). All efforts were made to minimize the number of animals used as well as their suffering.

2.4. Evaluation of myocardial ischemic area Measurement of the ischemic area was performed by using triphenyltetrazolium chloride (TTC) staining (Bohl et al., 2009; Csonka et al., 2010; Palfi et al., 2005). TTC stains all living tissue brick red, leaving the ischemic area unstained (white). Frozen hearts were sliced perpendicularly along the long axis from apex to base in 2mm thick sections and then incubated for 40 min at 37 °C with 1% TTC in 0.1 M phosphate buffer, pH 7.4. Sections were then fixed in 4% formalin for 24 h, rinsed with PBS and then photographed by using a digital camera. Ischemic area was estimated by Photoshop CS6 extended software (Adobe Systems Software, Ireland) and expressed as a percentage of the total heart area.

2.2. Preparation and isolated heart perfusion Rats were anesthetized with 4% isoflurane in 100% O2 and then intraperitoneally injected with 1 ml of heparin (5000 IU/ml). After 10 min, the chest was opened and 1 ml of heparin (160 UI/ml) was injected into the right atrium. Then the heart was quickly excised, attached to a modified Langendorff perfusion system and retrogradely perfused with an O2-saturated HEPES buffered solution containing (in mM): NaCl 140, KCl 4, HEPES 10, Na2HPO4 0.5, MgCl2 1, CaCl2 1.5, glucose 15, pH 7.4 adjusted with NaOH. Perfusion solution was constantly gassed with O2 and the temperature was maintained at 37 °C.

2.5. Isolation of rat adult ventricular cardiomyocytes Cardiomyocytes were isolated by Collagenase type II-CLS2 (Worthington Biochemical Corporation, Lakewood, NJ) digestion using a modified Langendorff perfusion system as previously described (Lariccia et al., 2011; Magi et al., 2015). Briefly, the heart was retrogradely perfused with HEPES buffered solution containing 1.5 mM CaCl2. After 2 min, the solution was switched to nominally Ca2+-free HEPES buffered solution for 5 min, followed by perfusion with the same solution containing 100 μM EGTA for 2 min. After that, the heart was perfused with enzyme solution (100–150 U/ml) containing 30 µM blebbistatin (Sigma, Milan, Italy) until the heart became swollen and turned lightly pale (usually 10 min). The digestion was

2.3. Ex vivo protocols of ischemia/reperfusion (I/R) and ischemic preconditioning (IPC) All the perfused hearts were subjected to 10 min of normoxic 247

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– Group 7. Hypoxic preconditioning +1 μM SN-6 followed by hypoxia/reoxygenation (HPC SN-6 H/R): cardiomyocytes were subjected to HPC in the presence of 1 μM SN-6. Afterwards, H/R was induced in the absence of the NCX inhibitor.

stopped by perfusing HEPES buffered solution containing 10% FBS. Then the heart, deprived of atria and aorta, was put on a sterile petri dish containing the perfusion solution without Ca2+, with 100 μM EGTA and 30 µM blebbistatin. The ventricular tissue was chopped with small scissors, and single cells were isolated by mechanical dispersion. Single cardiomyocytes were harvested after filtration through a nylon mesh. At this point, Ca2+ was gradually reintroduced by incubating the cardiomyocytes in the perfusion solution with increasing concentrations of Ca2+(O'Connell et al., 2007), finally achieving a concentration of 1.8 mM as in our culture medium. Once the cardiomyocytes were equilibrated (Ca2+-tolerant), they were plated at a density of 10 000 cells/cm2 on laminin coated dishes, and cultured in M-199 medium containing L-glutamine, NaHCO3 and Earle's salts, supplemented with 0.2% bovine serum albumin, 1X insulin-transferrin-selenium (Gibco, Grand island, NY, USA), 2 mM L-carnitine, 5 mM creatine, 3 mM taurine, 1% penicillin-streptomycin (Invitrogen, Monza, Italy), at 37 °C in 5% CO2 atmosphere (Guaiquil et al., 2004).

2.8. Lactate dehydrogenase (LDH) assay Cell damage was quantified by measurement of lactate dehydrogenase (LDH) activity, released from the cytosol of damaged cells in the experimental media (Jiao et al., 2008; Li et al., 2012), using the LDH Cytotoxicity Detection Kit (Roche Diagnostic, Monza, Italy). After the end of a cell culture experiment, 100 μl of cell culture medium were removed and added to a 96 well plate. Then, 100 μl of the reaction mixture (Diaphorase/NAD+ mixture premixed with iodotetrazolium chloride/sodium lactate) were added to each well and the plate was incubated for 30 min at room temperature, protected from light. LDH activity was assessed by reading the absorbance of the sample medium at 490 nm in a Victor Multilabel Counter plate reader (Perkin Elmer, Waltham, MA, USA). Myocardial tissue damage was assessed by determining LDH activity in the coronary effluent collected after the stabilization period and after 15 min of reperfusion (Granville et al., 2004; Kutala et al., 2006), for all the groups that underwent I/R. For control groups, coronary effluent was collected at comparable time.

2.6. Cell culture H9c2 Wilde Type (WT) cells, a clonal line derived from embryonic rat heart, were obtained from American Type Culture Collection (Manassas, VA). Generation of H9c2 cells stably expressing NCX1 was carried out as previously described (Magi et al., 2013, 2015). H9c2-WT and H9c2-NCX1 (passage 10–18) were cultured as monolayer in polystyrene dishes (100 mm diameter) and grown in DMEM medium (Invitrogen) containing 10% heat inactivated fetal bovine serum (Invitrogen), 1% L-glutamine (200 mM) (Invitrogen), 1% sodium pyruvate (100 mM) (Invitrogen), 100 IU/ml penicillin (Invitrogen), and 100 μg/ml streptomycin (Invitrogen), in a humidified incubator at 37 °C in a 5% CO2 atmosphere (Magi et al., 2013, 2015).

2.9. Protein extraction and western blotting Heart tissue, adult cardiomyocytes and H9c2 cells were lysed using protein lysis buffer containing (in mM): NaCl, 150; Tris-HCl (pH 7.4), 10; EDTA (pH 8.0), 1; SDS 1%, and a protease inhibitor cocktail mixture (Roche Diagnostics). Protein content was determined by the Bradford method (Bio-Rad, Milan, Italy), using bovine serum albumin as standard. Samples containing equal amounts of protein (30 μg) were boiled in 4X Laemmli Sample Buffer with 2–mercaptoethanol for 10 min. Proteins were subjected to 8% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then electro-transferred to polyvinylidine difluoride (PVDF) membranes (Immobilon Transfer Membranes, Millipore Co., Bedford, MA, USA). The membranes were blocked in PBS buffer containing 5% non-fat dry milk for 1 h at room temperature. Thereafter, the membranes were incubated overnight at 4 °C with diluted primary antibodies directed against NCX1 (1:500; R3F1, Swant, Bellinzona, Switzerland) (Magi et al., 2012, 2015), Bax (1:500; Santa Cruz Biotechnology) (Zlatkovic and Filipovic, 2012), Bcl-2 and Caspase-3 (1:1000; Cell Signaling Technology, Danvers, MA, USA) (Song et al., 2016). β-actin was used as loading control (1:10000; A5316, Sigma). Following incubation with primary antibody, membranes were washed 3 times for 10 min each in PBS-T and then incubated on rocker for 1 h at room temperature with horseradish peroxidase conjugated secondary antibody (Santa Cruz Biotechnology, CA, USA) in PBS/milk. An enhanced chemiluminescence detection system (Super Signal West Femto kit, Thermo Scientific, Milano, Italy) was used to detect bound antibodies. Images were captured and stored on a ChemiDoc station (Bio-Rad). Band densities were analyzed with the Quantity One (Bio-Rad) analysis software (Castaldo et al., 2010, 2007; Magi et al., 2015) and normalized to corresponding β-actin band densities.

2.7. In vitro protocols of hypoxia/reoxygenation (H/R) and hypoxic preconditioning (HPC) Hypoxia was induced by incubating the cells in an airtight chamber in which O2 was replaced by N2 with glucose-free Tyrode's solution containing (in mM): NaCl 137, KCl 2.7, MgCl2 1, CaCl2 1.8, Na2HPO4 0.2, NaHCO3 12, pH 7.4, at 37 °C. Reoxygenation was accomplished by changing the medium into Tyrode's solution containing 5.5 mM glucose followed by incubation under normoxic conditions. The general experimental protocols employed are described below: – Group 1. Control (CTR): cells were incubated in Tyrode's solution with 5.5 mM glucose and maintained under normoxia throughout the experimental period. – Group 2. Control +1 μM SN-6 (CTR SN-6): both cardiomyocytes and H9c2 cells were treated with 1 μM SN-6 in Tyrode's solution containing 5.5 mM glucose and maintained under normoxic conditions for the entire experimental period. – Group 3. Hypoxia/reoxygenation (H/R): cells were subjected to 90 min of hypoxia and 120 min of reoxygenation for rat adult cardiomyocytes, and 3 h of hypoxia followed by 5 h of reoxygenation for H9c2 cells. – Group 4. Hypoxia/reoxygenation +1 μM SN-6 (H/R SN-6): both cardiomyocytes and H9c2 cells were subjected to H/R, as described for group 3, in the presence of 1 μM SN-6. – Group 5. Hypoxic preconditioning control (HPC): cardiomyocytes were exposed to a sublethal stimulus made up of 10 min of hypoxia and 30 min of reoxygenation (Jiao et al., 2008), and then maintained in Tyrode's solution containing 5.5 mM glucose under normoxic conditions for the entire experimental period. – Group 6. Hypoxic preconditioning followed by hypoxia/reoxygenation (HPC H/R): cardiomyocytes were exposed to HPC prior to H/R insult as described for group 3.

2.10. Analysis of NCX1 activity [Ca2+]i was measured by single-cell computer-assisted videoimaging using a LSM 510 confocal system (Carl Zeiss, Milan, Italy) as previously described (Magi et al., 2013, 2015). Briefly, H9c2 cells, cultured on 25 mm coverslip, were loaded with 4 µM Fluo-4/AM for 30 min in the dark at room temperature (Molecular Probe, Eugene, OR), in a solution (we named standard solution) containing the following (in mM): NaCl 140, KCl 5, MgCl2 1, CaCl2 2, glucose 10, 248

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3.3. Role of NCX1 in H/R injury in H9c2 cell models

HEPES 20, pH 7.4 adjusted with NaOH. At the end of the Fluo-4/AM loading period, cells were washed and left in standard solution for further 15 min to allow the complete de-esterification of the dye. Then the coverslips were placed into a perfusion chamber mounted onto the stage of an inverted Zeiss Axiovert 200 microscope. NCX1 activity was evaluated as Ca2+ uptake through the reverse mode by switching the standard solution to a Na+-free solution containing (in mM): LiCl 140, KCl 5, MgCl2 1, CaCl2 2, glucose 10, HEPES 20, pH 7.4 adjusted with LiOH. Excitation light was provided by an argon laser at 488 nm and the emission was time-lapse recorded at 505–530 nm. Images were acquired every 5 s. Analysis of fluorescence intensity was performed off-line after images acquisition, by averaging the fluorescence intensity values within selected areas overlying the cell somata as described before (Magi et al., 2013, 2015).

To further explore the role of NCX1 in cell injury induced by H/R, we used an experimental model based on two H9c2 clones, H9c2-WT (devoid of any detectable endogenous NCX1) and H9c2-NCX1 (stably expressing canine NCX1); only H9c2-NCX1 cells display functional exchanger activity (Magi et al., 2012, 2015). When we exposed H9c2WT to H/R we observed a significant increase in extracellular LDH (up to about 89% compared to the respective control) (Fig. 3B). In H9c2NCX1 cells, H/R challenge induced a further increase in extracellular LDH (about 30%) compared to WT cells under similar conditions (Fig. 3B). Notably, when H/R was induced in H9c2-NCX1 in the presence of SN-6, extracellular LDH was significantly reduced to values comparable to H9c2-WT. SN-6 had no effects under normoxic conditions in both cell lines, as well as in H9c2-WT cells subjected to H/R, confirming that the observed protection was not due to SN-6 effects on targets other than NCX1. As shown in Fig. 3C, protein expression analysis revealed that, in H9c2-NCX1 cells, NCX1 levels were increased after H/R. As already observed in cardiomyocytes and in the whole rat heart, NCX1 protein increase was prevented by SN-6 treatment. SN-6 per se was unable to modify NCX1 expression in H9c2-NCX1 cells.

2.11. Statistical analysis All the data are expressed as mean ± S.E.M. Statistical comparisons were performed with ANOVA followed by Dunnet's post hoc test, as appropriate. The threshold for statistical significance was set at P < 0.05. Statistical comparisons were carried out using the GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA).

3.4. Analysis of NCX1 activity following H/R challenge in H9c2NCX1 cells

2.12. Drugs and chemicals We next explored whether any enhancement in NCX1 activity would mirror the increase of exchanger expression induced by H/R. Exchanger activity in H9c2-NCX1 cells was monitored in Fluo-4 loaded cells subjected to isotonic extracellular Na+ removal at the end of the experimental protocol reported in Fig. 3A (see material and methods for more details). As shown in Fig. 4, when NCX1 reverse mode was activated by superfusing a Na+-free extracellular solution, in control cells a rise in [Ca2+]i of about 46% occurred, as revealed by the increase in fluorescence signal. When H/R group was analyzed, we observed that the increase in [Ca2+]i was enhanced (about 70%) compared to what observed in control cells. Of note, such response was completely abolished in H9c2-NCX1 cells that were subjected to H/R in the presence of the NCX1 inhibitor. Notably, SN-6 per se did not modify basal NCX1 activity, thus excluding that an incomplete inhibitor wash out during recordings could have affected our results. Furthermore, no change in fluorescence baseline was observed in H9c2-WT cells when subjected to the Na+ removal protocol, confirming that the Ca2+ responses observed in H9c2-NCX1 were mediated by the reverse mode of NCX1 activity (data not shown).

SN-6 was obtained from Tocris. All other chemicals were of analytical grade and they were purchased from Sigma. 3. Results 3.1. Role of NCX1 in H/R injury in cultured rat adult cardiomyocytes We initially used primary culture of adult rat heart cardiomyocytes and assessed the role of NCX1 in an in vitro model of cardiac ischemia. Cell damage was evaluated by measuring LDH activity retained in the culture medium after H/R protocol (Fig. 1A). As shown in Fig. 1B, in cardiomyocytes exposed to 90 min hypoxia and 120 min reoxygenation, extracellular LDH significantly increased (67% compared to normoxic conditions). When cells were treated with 1 μM SN-6, a concentration which is near closely to its IC50 (Iwamoto et al., 2004), the H/R-induced cell death was completely counteracted. Notably, as shown in Fig. 1C, H/R challenge induced a significant increase in NCX1 protein levels that was completely prevented by SN-6 treatment. SN-6 had no effect on cardiomyocytes survival or NCX1 expression in normoxic conditions (Fig. 1B and C).

3.5. Role of NCX1 in HPC in rat adult cardiomyocytes HPC-induced cardioprotection is a well known phenomenon that has been explained by virtue of several mechanisms (Halestrap et al., 2007; Yang et al., 2010). Our aim was to focus on the role of Ca2+, with main interest on the role of NCX1. HPC protocol (Fig. 5A) was first tested in rat adult cardiomyocytes. As expected, in this model H/R cell injury was completely counteracted by HPC (Fig. 5B), which per se did not affect cell viability (data not shown). Notably, SN-6 treatment completely prevented the beneficial effect exerted by HPC (Fig. 5B). In terms of NCX1 protein expression (Fig. 5C), when HPC was followed by H/R, NCX1 protein expression was upregulated. SN-6 treatment during HPC restored NCX1 expression to the levels observed under normoxia.

3.2. Role of NCX1 in I/R injury in rat adult isolated heart We next verified whether NCX1 blockade in ex-vivo perfused hearts subjected to I/R confirmed the protected phenotype observed in primary cardiomyocytes culture. When isolated hearts were exposed to 30 min ischemia followed by 120 min reperfusion (Fig. 2A), extracellular LDH was markedly increased, up to about 15-folds, compared to normoxic control (Fig. 2B). I/R-induced cell death was significantly reduced by SN-6 (Fig. 2B). When we analyzed the ischemic damage in TTC-stained heart sections, we observed a significant increase in the ischemic area (about 30% compared to the control) that was completely abolished by SN-6 treatment (Fig. 2C). Changes in NCX1 expression during I/R challenge were also analyzed and, again, we observed that I/ R injury was accompanied by an increase in NCX1 protein expression that was fully reversed by SN-6 (Fig. 2D). Again, SN-6 did not affect cell viability (Fig. 2B) or NCX1 protein levels (data not shown) under condition of normoxia, in line with our previous results (Magi et al., 2015).

3.6. Role of NCX1 in IPC in rat adult isolated heart Similarly to what observed in cardiomyocytes, IPC protocol (Fig. 6A) significantly reduced I/R cell damage (Fig. 6B). IPC per se did not affect LDH release (data not shown). Surprisingly, inhibition of NCX1 during IPC, not only abolished its protective effect, but also 249

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Fig. 1. Role of NCX1 in H/R injury in isolated rat adult cardiomyocytes. (A) Schematic diagram depicting the experimental protocol of H/R in isolated rat adult cardiomyocytes, in the presence or in the absence of 1 µM SN-6. (B) Extracellular LDH measured after H/R insult, as indicated by the arrow. Cell death was expressed as percentage of extracellular LDH from damaged cells versus control. Each column represents the mean ± S.E.M. of 7 independent experiments performed in triplicate. *P < 0.001 versus CTR and CTR SN-6; P < 0.05 versus H/ R SN-6; #P < 0.05 versus H/R. (C) Quantitative densitometry showing NCX1 protein expression. β-actin was used as loading control. Normalized optical density values are expressed as percentage of the respective control. Each column represents the mean ± S.E.M. of 4 independent experiments. *P < 0.01 versus CTR, P < 0.05 versus H/R SN-6, P < 0.001 versus CTR SN-6; #P < 0.05 versus H/R. A representative western blot image is shown below. CTR = control; H/R = hypoxia/reoxygenation.

Interestingly, both SN-6 treatment during the whole I/R protocol and IPC were associated with a significant prevention of the apoptosis progression. Of note, NCX1 inhibition during IPC, which resulted in the loss of IPC-conferred cardioprotection against I/R, was also accompanied by Caspase-3 processing. The ratio of Bcl-2 to Bax proteins was also tested. The cellular commitment to apoptosis is regulated by the Bcl-2 family of proteins, which includes both anti-apoptotic and pro-apoptotic members (Murphy et al., 2000). Bcl-2 is a protein that blocks programmed cell death without affecting cellular proliferation (Hockenbery et al., 1990), whereas Bax protein is a member of the Bcl-2 family that promotes apoptosis (Oltvai et al., 1993). The ratio of Bcl-2 to Bax determines the susceptibility of a cell to undergo apoptosis (Oltvai et al., 1993; Yang and Korsmeyer, 1996). As shown in Fig. 7B, consistently with Caspase3 activation, we found that Bcl-2/Bax ratio was significantly lowered during I/R. Interestingly, SN-6 treatment was associated with a significant increase of Bcl-2/Bax ratio, suggesting an anti-apoptotic effect of this inhibitor. IPC treatment restored the Bcl-2/Bax ratio observed under normoxia. In line with Caspase-3 activation, the Bcl-2/ Bax ratio was dramatically reduced when IPC was carried out in the presence of SN-6.

caused an exacerbation of the I/R-induced cell injury. As a matter of fact, as shown in Fig. 6B, extracellular LDH was about 2.4-folds higher than that observed in I/R group. Moreover, these findings were confirmed by TTC staining (Fig. 6C), which revealed that ischemic area did not develop when I/R was preceded by IPC, whereas SN-6 presence during IPC led to a dramatic extent of the ischemic area, with a trend that was similar to what observed in LDH assay. In terms of protein expression (Fig. 6D), western blot analysis was in line with data obtained in cardiomyocytes. When IPC was followed by I/R, NCX1 protein levels were still upregulated. SN-6 treatment during IPC restored NCX1 expression to the levels observed under normoxia. 3.7. Characterization of cell death events induced by I/R injury Apoptosis and necrosis are two major forms of cell death observed in normal and disease pathologies. Even though LDH leakage is an indicator of necrosis, it may also be an indicator of late-stage apoptosis (Kaja et al., 2015; Parhamifar et al., 2013). Therefore, to further characterize the cell death events induced by I/R in our experimental conditions, we studied the activation and expression of apoptosisrelated factors, such as Caspase-3, Bcl-2 and Bax (Elmore, 2007) in the whole rat heart, which can be considered the most physiological model. We first examined processing of Caspase-3 to the p17-20 fragment, which forms an essential part of the activated enzyme, the final executioner of apoptosis. As shown in Fig. 7A, we found that Caspase-3 was processed when rat hearts were challenged with I/R insult, suggesting a strong involvement of the apoptotic process.

4. Discussion Findings from our study strongly suggest that, despite NCX1 has a deleterious role in the I/R induced cell damage, its presence during IPC seems to be crucial in the mechanisms leading to ischemic tolerance. 250

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Fig. 2. Role of NCX1 in I/R injury in perfused rat adult hearts. (A) Schematic diagram depicting the experimental protocol of myocardial I/R injury in perfused rat hearts, in the presence or in the absence of 1 µM SN-6. (B) Extracellular LDH measured after 15 min of reperfusion, as indicated by the arrow. Each column represents the mean ± S.E.M. of LDH values obtained from 5 different hearts. Data are presented as percentage relative to control group. *P < 0.001 versus all groups; #P < 0.001 versus CTR and IR, P < 0.01 versus CTR SN6. (C) Myocardial ischemic area determined by TTC staining, evaluated at the end of I/R protocol (arrow), as a percentage of the whole heart (left). Each column represents the mean ± S.E.M. of 5 independent experiments. *P < 0.001 versus CTR, P < 0.01 versus CTR SN6 and IR SN-6; #P < 0.01 versus IR. On the right, representative heart sections of each experimental group are shown. Red-stained areas indicate viable tissue and unstained pale areas indicate infarct tissue. (D) Quantitative densitometry showing NCX1 protein expression. β-actin was used as loading control. Each column represents the mean ± S.E.M. of 4 different hearts. Data are presented as percentage relative to control group. *P < 0.001 versus CTR; P < 0.01 versus I/R SN-6; #P < 0.01 versus IR. A representative western blot image is shown below. CTR = control; I/R = ischemia/reperfusion.

Namekata et al., 2006; Seki et al., 2002), more recently a lack of selectivity of these compounds towards NCX1 has been reported (Barrientos et al., 2009; Birinyi et al., 2008; Niu et al., 2007; Reuter et al., 2002). Therefore, we used the benzyloxyphenyl derivative SN-6 (Niu et al., 2007) at the concentration of 1 µM, which has been demonstrated to be selective for NCX1, in condition of ATP depletion, as in the case of ischemia (Iwamoto et al., 2004). As expected, when injury was assessed by LDH assay, SN-6 treatment was effective in limiting cell damage, which was fully counteracted in isolated cardiomyocytes and almost abrogated in the whole heart. When I/R injury was assessed in TTC stained ventricular slices, we found that the development of the infarcted area was completely prevented by SN-6. The discrepancy in the extent of the protection induced by SN-6, evaluated by LDH assay or by TTC staining, may depend on differences between methods and sampling procedures. The analysis of apoptotic events revealed that SN-6 abolished Caspase-3 activation and, interestingly, the balance of anti-apoptotic and pro-apoptotic effectors, expressed by the Bcl-2/Bax ratio, was significantly upset in favor of the former. To further confirm NCX1 involvement in the I/R-induced cell injury, we used an additional experimental model based on two cell

First of all, in agreement with previously published data (Jiao et al., 2008), we found that, when rat adult isolated cardiomyocytes were subjected to H/R, a significant cytotoxic damage occurred, as revealed by LDH assay. Similar results were obtained when the whole heart was exposed to I/R. Notably, in these conditions, I/R protocol induced a more severe extracellular LDH release compared to what observed in isolated cardiomyocytes. We can assume that the higher vulnerability of the whole heart could be related to its complexity compared to the isolated cardiomyocytes (Bell et al., 2011; Diaz and Wilson, 2006). In the whole perfused heart we also evaluated the extent of the ischemic area by TTC staining and, in line with extracellular LDH measurements, we found a significant injured area (30% compared to the control). Characterization of cell death events demonstrated the involvement of apoptotic processes, as revealed by Caspase-3 cleavage and the decrease of the Bcl-2/Bax ratio. In order to thoroughly investigate the contribution of NCX1 to I/R damage, we used a selective pharmacological tool. Several compounds afford to inhibit the reverse mode of NCX1 such as the benzyloxyphenyl derivatives KBR7943 and SEA0400. Although many studies have been performed by using these inhibitors (Hagihara et al., 2005; Matsumoto et al., 2003; 251

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Fig. 3. Role of NCX1 in H/R injury in H9c2 cell lines. (A) Schematic diagram depicting the experimental protocol of H/R in H9c2 cell lines, in the presence or in the absence of 1 µM SN-6. (B) Extracellular LDH measured after H/R insult, as indicated by the arrow. Cell death was expressed as percentage of extracellular LDH from damaged cells versus control. In H9c2-WT cells group, each column represents the mean ± S.E.M. of 6 independent experiments performed in triplicate. *P < 0.001 versus CTR WT and CTR SN-6 WT. In H9c2-NCX1 cells group, each column represents the mean ± S.E.M. of 7 independent experiments performed in triplicate. #P < 0.001 vs all groups both of H9c2-NCX1 and H9c2-WT; §P < 0.001 versus H/R NCX1 and versus CTR NCX1, P < 0.01 versus CTR SN-6 NCX1. (C) Quantitative densitometry showing NCX1 protein expression. β-actin was used as loading control. Normalized optical density values are expressed as percentage of the respective control. Each column represents the mean ± S.E.M. of 5 independent experiments. *P < 0.001 versus CTR and CTR SN6, P < 0.01 versus H/R SN-6. A representative western blot image is shown below. CTR = control; H/R = hypoxia/reoxygenation.

susceptibility toward H/R insult suggested that the genesis of ischemic damage may also involve NCX1-independent mechanisms (Kalogeris et al., 2012). Notably, failure of SN-6 to induce any effect on cell survival in H9c2-WT (where NCX1 is virtually absent) confirmed the specificity of this inhibitor. In line with these findings, we assessed whether the critical contribution of NCX1 to the ischemic damage could be paralleled by substantial changes in its expression. As a matter

lines: H9c2-WT and H9c2-NCX1, which differ in the expression of the dog cardiac NCX1 cDNA (Magi et al., 2013, 2015). When cells were exposed to H/R protocol we observed that cell viability was affected in both phenotype. However, in H9c2-NCX1 cell injury was of a much greater extent than in H9c2-WT cells. Of note, SN-6 was ineffective in H9c2-WT, whereas in H9c2-NCX1 cells the inhibitor reduced H/R damage up to the levels observed in H9c2-WT cells. H9c2-WT

Fig. 4. Real time [Ca2+]i analysis in H9c2-NCX1 cell lines subjected to H/R injury. (A) NCX1 reverse mode activity was evaluated by monitoring the Ca2+ response to a Na+-free protocol (see materials and methods for further details) in controls and in H/R-challenged cells in the presence or in the absence of 1 µM SN-6. Representative traces of each experimental conditions are reported. Fluo-4 fluorescence intensity was expressed as F/F0-ratio, where F is the background subtracted fluorescence intensity and F0 is the background subtracted mean fluorescence value measured from each cell at rest. (B) NCX1 reverse mode activity expressed as percentage of resting condition (Δ%) after H/R protocol. For Δ% calculation, we used the maximal value of fluorescence attained after stimulation and, as baseline, the mean of fluorescence recorded during the 60 s preceding the Na+-free challenge. Each column represents the mean ± S.E.M. of more than 50 cells recorded in 3 different experimental sessions. *P < 0.001 versus all groups. CTR = control; H/R = hypoxia/reoxygenation.

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Fig. 5. Role of NCX1 in HPC followed by H/R in isolated rat adult cardiomyocytes. (A) Schematic diagram depicting the experimental protocol of HPCH/R in isolated rat adult cardiomyocytes, in the presence or in the absence of 1 µM SN-6. (B) Extracellular LDH measured after HPCH/R protocol, as indicated by the arrow. Cell death was expressed as percentage of extracellular LDH from cells versus control. Each column represents the mean ± S.E.M. of 7 independent experiments performed in triplicate.*P < 0.001 versus CTR and HPC H/R; #P < 0.001 versus HPC H/R, P < 0.01 versus CTR. (C) Quantitative densitometry showing NCX1 protein expression. β-actin was used as loading control. Normalized optical density values are expressed as percentage of the respective control. Each column represents the mean ± S.E.M. of 4 independent experiments. *P < 0.001 versus CTR and HPC H/R; #P < 0.001 versus all groups. A representative western blot image is shown below. CTR = control; H/R = hypoxia/reoxygenation; HPC = hypoxic preconditioning.

cardiac I/R injury (Imahashi et al., 2005; Namekata et al., 2006; Seki et al., 2002), more likely through its reverse mode activity which promotes an [Ca2+]i increase that is deleterious to the cell survival, as it can cause cytoxicity and trigger cell death pathways (Orrenius et al., 2003). In contrast to this negative role of NCX1 in I/R, our data revealed a beneficial contribution exerted by the exchanger during IPC. As expected, preconditioning was effective in limiting cell damage. In particular, cell injury was abolished in isolated cardiomyocytes and significantly reduced in the whole heart. We should underline that when IPC was evaluated in TTC stained ventricular slices, the development of the infarcted area was completely prevented. Such results were consistent with data obtained from the evaluation of apoptotic processes. Caspase-3 activation was abolished by IPC and the Bcl-2/ Bax ratio was comparable to what observed under normoxia. Intriguingly, in cardiomyocytes the protective effect of HPC was fully abolished by SN-6. It is interesting to note that in perfused hearts, the same experimental conditions, not only abolished IPC cardioprotective effect, but also caused an exacerbation of the I/R-induced cell injury, as assessed in terms of infarct size extent and extracellular LDH. Again, the analysis of apoptosis-related factors was in line with LDH results. Indeed, in this experimental conditions, Caspase-3 cleavage was activated and the Bcl-2/Bax ratio significantly dropped. These results point out that IPC cardioprotection may rely upon a beneficial NCX1 activity. In this regard, it has been shown that prior stimulation of NCX1 reverse mode activity can reduce the deleterious effects of a subsequent I/R, thereby mimicking IPC (El-Ani et al., 2011; Khan et al., 2006; Li et al., 2007). Furthermore, pharmacological inhibition

of fact, in the heart, NCX1 expression can undergo to significant modifications during many pathophysiological conditions, such as myocardial infarction (Quinn et al., 2003), heart failure (Studer et al., 1994) and hypertrophy (Magi et al., 2015; Sipido et al., 2000). It is noteworthy that, in all our experimental models, NCX1 protein expression was upregulated after I/R. Intriguingly, SN-6 abolished NCX1 protein upregulation, suggesting that under stressful conditions the exchanger may have the ability to regulate its own turnover, as previously observed during LPS-induced cardiac hypertrophy (Magi et al., 2015). Similar findings were observed for the Ca2+calmodulindependent protein kinase IIδ, whose expression appears to be modulated by its inhibitor KN-93 (Adameova et al., 2012). Although we did not further investigate the molecular mechanisms underlying this increased exchanger expression, we can assume that the observed changes could also rely on promoter-independent mechanisms, considering that in H9c2-NCX1 cell line the exchanger expression is constitutive and not regulated by endogenous factors, since it depends upon citomegalovirus promoter (Magi et al., 2015). Available data on NCX1 protein upregulation under stressful conditions often report an alteration of the exchanger activity (Hudecova et al., 2007; Magi et al., 2015; Quinn et al., 2003). To explore this hypothesis in our system, we studied NCX1 activity in H9c2-NCX1 cells after H/R insult. Interestingly, we observed that NCX1 protein upregulation was accompanied by an increased exchanger reverse mode activity, revealed by monitoring [Ca2+]i after the exposure to a Na+-free solution. Notably, SN-6, that was protective against H/R damage, also suppressed the increased NCX1 activity. Collectively, our data strengthened the critical involvement of NCX1 in the mechanisms underlying the genesis of 253

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Fig. 6. Role of NCX1 in IPC followed by I/R in perfused rat adult hearts. (A) Schematic diagram depicting the experimental protocol of IPC-I/R in perfused rat hearts, in the presence or in the absence of 1 µM SN-6. (B) Extracellular LDH measured after 15 min of reperfusion in the IPCIR protocol, as indicated by the arrow. Each column represents the mean ± S.E.M. of LDH values obtained from 5 different hearts. Data are presented as percentage relative to control group. *P < 0.001 versus CTR and IPC SN6 I/R, P < 0.01 versus IPC I/R; #P < 0.001 versus CTR; §P < 0.001 versus all groups. (C) Myocardial ischemic area determined by TTC staining, evaluated at the end of IPCI/R protocol (arrow), as a percentage of the whole heart. Each column represents the mean ± S.E.M. of 5 independent experiments. *P < 0.001 versus CTR and IPC SN6 I/R, P < 0.05 versus IPC I/R; #P < 0.001 versus all groups. On the right, representative heart sections of each experimental group are shown. Red-stained areas indicate viable tissue and unstained pale areas indicate infarct tissue. (D) Quantitative densitometry showing NCX1 protein expression. β-actin was used as loading control. Each column represents the mean ± S.E.M. of 4 different hearts. Data are presented as percentage relative to control group. *P < 0.05 versus CTR; #P < 0.001 versus CTR, §P < 0.01 versus IPC I/R. A representative western blot image is shown below. CTR = control; I/R = ischemia/ reperfusion; IPC = ischemic preconditioning.

NCX1-mediated Ca2+ influx may directly activate mitoKCa channels, therefore conferring cardioprotection (Zhang et al., 2015). Finally, we observed that despite IPC prevented the I/R-induced cell damage, NCX1 upregulation persisted and SN-6 treatment restored exchanger expression to the level shown under normoxia. These results lead us to tentatively hypothesize that the loss of NCX1 upregulation induced by SN-6 could be responsible for the failure of IPC to afford cardioprotection. Collectively, our data suggest a dual role of NCX1 in ischemic settings: on the one hand its inhibition during I/R is crucial to cell survival, on the other hand its activity during IPC serves as a trigger for the induction of ischemic tolerance.

of NCX1 has been reported to abrogate the cardioprotective effect of different preconditioning stimuli, such as sevoflurane exposure and metabolic inhibition (Bouwman et al., 2006; Li et al., 2007; Zhang et al., 2015), supporting the hypothesis of a beneficial NCX1 role in IPC. We tentatively speculate that the short ischemic episodes accompanying IPC can activate NCX1 reverse mode, giving rise to sublethal spikes in [Ca2+]i that could act in a protective manner. In line with this hypothesis, it has been shown that transient and reversible raises in [Ca2+]i can reproduce the protective effect of IPC (Meldrum et al., 1996; Miyawaki and Ashraf, 1997; Przyklenk et al., 1997). Possible candidates as downstream effectors in mediating Ca2+-induced IPC cardioprotection may be the mitochondrial Ca2+-sensitive K+(mitoKCa) channels, which have been described to mediate the protective effect of different preconditioning stimuli, including IPC (Frassdorf et al., 2010; Shintani et al., 2004; Xu et al., 2002). Additionally, it has been reported that inhibition of the mitoKCa channels with paxilline attenuates the ischemic tolerance induced by NCX1 stimulation, suggesting that

Authorship contributions

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proteasomal degradation of iron proteins. PloS One 7, e48947. Castaldo, P., Magi, S., Cataldi, M., Arcangeli, S., Lariccia, V., Nasti, A.A., Ferraro, L., Tomasini, M.C., Antonelli, T., Cassano, T., Cuomo, V., Amoroso, S., 2010. Altered regulation of glutamate release and decreased functional activity and expression of GLT1 and GLAST glutamate transporters in the hippocampus of adolescent rats perinatally exposed to Delta(9)-THC. Pharmacol. Res. 61, 334–341. Castaldo, P., Magi, S., Gaetani, S., Cassano, T., Ferraro, L., Antonelli, T., Amoroso, S., Cuomo, V., 2007. Prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 increases glutamate uptake through overexpression of GLT1 and EAAC1 glutamate transporter subtypes in rat frontal cerebral cortex. Neuropharmacology 53, 369–378. Cohen, M.V., Downey, J.M., 2008. Adenosine: trigger and mediator of cardioprotection. Basic Res Cardiol. 103, 203–215. Csonka, C., Kupai, K., Kocsis, G.F., Novak, G., Fekete, V., Bencsik, P., Csont, T., Ferdinandy, P., 2010. Measurement of myocardial infarct size in preclinical studies. J. Pharmacol. Toxicol. Methods 61, 163–170. Diaz, R.J., Wilson, G.J., 2006. Studying ischemic preconditioning in isolated cardiomyocyte models. Cardiovasc. Res. 70, 286–296. El-Ani, D., Stav, H., Guetta, V., Arad, M., Shainberg, A., 2011. Rapamycin (sirolimus) protects against hypoxic damage in primary heart cultures via Na+/Ca2+ exchanger activation. Life Sci. 89, 7–14. Elmore, S., 2007. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35, 495–516. Frank, A., Bonney, M., Bonney, S., Weitzel, L., Koeppen, M., Eckle, T., 2012. Myocardial ischemia reperfusion injury: from basic science to clinical bedside. Semin. Cardiothorac. Vasc. Anesth. 16, 123–132. Frassdorf, J., Huhn, R., Niersmann, C., Weber, N.C., Schlack, W., Preckel, B., Hollmann, M.W., 2010. Morphine induces preconditioning via activation of mitochondrial K(Ca) channels. Can. J. Anaesth. 57, 767–773. Garcia-Dorado, D., Ruiz-Meana, M., Inserte, J., Rodriguez-Sinovas, A., Piper, H.M., 2012. Calcium-mediated cell death during myocardial reperfusion. Cardiovasc. Res. 94, 168–180. Granville, D.J., Tashakkor, B., Takeuchi, C., Gustafsson, A.B., Huang, C., Sayen, M.R., Wentworth, P., Jr., Yeager, M., Gottlieb, R.A., 2004. Reduction of ischemia and reperfusion-induced myocardial damage by cytochrome P450 inhibitors. Proc. Natl. Acad. Sci. USA 101, 1321–1326. Groenke, S., Larson, E.D., Alber, S., Zhang, R., Lamp, S.T., Ren, X., Nakano, H., Jordan, M.C., Karagueuzian, H.S., Roos, K.P., Nakano, A., Proenza, C., Philipson, K.D., Goldhaber, J.I., 2013. Complete atrial-specific knockout of sodium-calcium exchange eliminates sinoatrial node pacemaker activity. PloS One 8, e81633. Guaiquil, V.H., Golde, D.W., Beckles, D.L., Mascareno, E.J., Siddiqui, M.A., 2004. Vitamin C inhibits hypoxia-induced damage and apoptotic signaling pathways in cardiomyocytes and ischemic hearts. Free Radic. Biol. Med. 37, 1419–1429. Hagihara, H., Yoshikawa, Y., Ohga, Y., Takenaka, C., Murata, K.Y., Taniguchi, S., Takaki, M., 2005. Na+/Ca2+ exchange inhibition protects the rat heart from ischemiareperfusion injury by blocking energy-wasting processes. Am. J. Physiol. Heart Circ. Physiol. 288, H1699–H1707. Halestrap, A.P., Clarke, S.J., Khaliulin, I., 2007. The role of mitochondria in protection of the heart by preconditioning. Biochim Biophys. Acta 1767, 1007–1031. Hockenbery, D., Nunez, G., Milliman, C., Schreiber, R.D., Korsmeyer, S.J., 1990. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348, 334–336. Hudecova, S., Kubovcakova, L., Kvetnansky, R., Kopacek, J., Pastorekova, S., Novakova, M., Knezl, V., Tarabova, B., Lacinova, L., Sulova, Z., Breier, A., Jurkovicova, D., Krizanova, O., 2007. Modulation of expression of Na+/Ca2+ exchanger in heart of rat and mouse under stress. Acta Physiol. (Oxf.) 190, 127–136. Ibanez, B., Heusch, G., Ovize, M., Van de Werf, F., 2015. Evolving therapies for myocardial ischemia/reperfusion injury. J. Am. Coll. Cardiol. 65, 1454–1471. Imahashi, K., Pott, C., Goldhaber, J.I., Steenbergen, C., Philipson, K.D., Murphy, E., 2005. Cardiac-specific ablation of the Na+-Ca2+ exchanger confers protection against ischemia/reperfusion injury. Circ. Res. 97, 916–921. Iwamoto, T., Inoue, Y., Ito, K., Sakaue, T., Kita, S., Katsuragi, T., 2004. The exchanger inhibitory peptide region-dependent inhibition of Na+/Ca2+ exchange by SN-6 [2[4-(4-nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic acid ethyl ester], a novel benzyloxyphenyl derivative. Mol. Pharmacol. 66, 45–55. Jiao, J.D., Garg, V., Yang, B., Hu, K., 2008. Novel functional role of heat shock protein 90 in ATP-sensitive K+ channel-mediated hypoxic preconditioning. Cardiovasc. Res. 77, 126–133. Kaja, S., Payne, A.J., Singh, T., Ghuman, J.K., Sieck, E.G., Koulen, P., 2015. An optimized lactate dehydrogenase release assay for screening of drug candidates in neuroscience. J. Pharmacol. Toxicol. Methods 73, 1–6. Kalogeris, T., Baines, C.P., Krenz, M., Korthuis, R.J., 2012. Cell biology of ischemia/ reperfusion injury. Int. Rev. Cell Mol. Biol. 298, 229–317. Khan, S., Salloum, F., Das, A., Xi, L., Vetrovec, G.W., Kukreja, R.C., 2006. Rapamycin confers preconditioning-like protection against ischemia-reperfusion injury in isolated mouse heart and cardiomyocytes. J. Mol. Cell Cardiol. 41, 256–264. Kutala, V.K., Khan, M., Mandal, R., Ganesan, L.P., Tridandapani, S., Kalai, T., Hideg, K., Kuppusamy, P., 2006. Attenuation of myocardial ischemia-reperfusion injury by trimetazidine derivatives functionalized with antioxidant properties. J. Pharm. Exp. Ther. 317, 921–928. Lariccia, V., Fine, M., Magi, S., Lin, M.J., Yaradanakul, A., Llaguno, M.C., Hilgemann, D.W., 2011. Massive calcium-activated endocytosis without involvement of classical endocytic proteins. J. Gen. Physiol. 137, 111–132. Laude, K., Favre, J., Thuillez, C., Richard, V., 2003. NO produced by endothelial NO synthase is a mediator of delayed preconditioning-induced endothelial protection. Am. J. Physiol. Heart Circ. Physiol. 284, H2053–H2060.

Fig. 7. Characterization of cell death. (A) Immunoblot analysis showing cleaved Caspase-3 in the different experimental groups. β-actin was used as loading control. The image is representative of at least 4 independent experiments. (B) Quantification of Bcl-2/Bax protein expression ratio. Each column represents the mean ± S.E.M. of 4 different hearts. Data are presented as percentage relative to control group. *P < 0.05 versus CTR, P < 0.001 versus I/R SN-6, P < 0.01 versus IPC I/R; §P < 0.01 versus CTR, P < 0.05 versus IPC IR, P < 0.001 versus IPC SN-6 I/R; #P < 0.001 versus CTR and IPC I/ R. CTR = control; I/R = ischemia/reperfusion; IPC = ischemic preconditioning.

Participated in research design: Amoroso, Castaldo, Magi. Conducted experiments: Castaldo, Lariccia, Macrì, Maiolino, Matteucci. Performed data analysis: Gratteri, Magi. Contributed to the writing of the manuscript: Amoroso, Castaldo, Lariccia, Magi. All the authors read and approved the final manuscript. Acknowledgements This work was supported by ‘‘Ricerca Scientifica di Ateneo’’ (RSA) Grant (2014–2015) from University ‘‘Politecnica delle Marche’’. The authors wish to thank Gerardo Galeazzi, Franco Pettinari and Carlo Alfredo Violet for their excellent technical assistance. References Adameova, A., Carnicka, S., Rajtik, T., Szobi, A., Nemcekova, M., Svec, P., Ravingerova, T., 2012. Upregulation of CaMKIIdelta during ischaemia-reperfusion is associated with reperfusion-induced arrhythmias and mechanical dysfunction of the rat heart: involvement of sarcolemmal Ca2+-cycling proteins. Can. J. Physiol. Pharm. 90, 1127–1134. Aronsen, J.M., Swift, F., Sejersted, O.M., 2013. Cardiac sodium transport and excitationcontraction coupling. J. Mol. Cell Cardiol. 61, 11–19. Barrientos, G., Bose, D.D., Feng, W., Padilla, I., Pessah, I.N., 2009. The Na+/Ca2+ exchange inhibitor 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate (KB-R7943) also blocks ryanodine receptors type 1 (RyR1) and type 2 (RyR2) channels. Mol. Pharmacol. 76, 560–568. Bell, R.M., Mocanu, M.M., Yellon, D.M., 2011. Retrograde heart perfusion: the Langendorff technique of isolated heart perfusion. J. Mol. Cell Cardiol. 50, 940–950. Birinyi, P., Toth, A., Jona, I., Acsai, K., Almassy, J., Nagy, N., Prorok, J., Gherasim, I., Papp, Z., Hertelendi, Z., Szentandrassy, N., Banyasz, T., Fulop, F., Papp, J.G., Varro, A., Nanasi, P.P., Magyar, J., 2008. The Na+/Ca2+ exchange blocker SEA0400 fails to enhance cytosolic Ca2+ transient and contractility in canine ventricular cardiomyocytes. Cardiovasc. Res. 78, 476–484. Bohl, S., Lygate, C.A., Barnes, H., Medway, D., Stork, L.A., Schulz-Menger, J., Neubauer, S., Schneider, J.E., 2009. Advanced methods for quantification of infarct size in mice using three-dimensional high-field late gadolinium enhancement MRI. Am. J. Physiol. Heart Circ. Physiol. 296, H1200–H1208. Bouwman, R.A., Salic, K., Padding, F.G., Eringa, E.C., van Beek-Harmsen, B.J., Matsuda, T., Baba, A., Musters, R.J., Paulus, W.J., de Lange, J.J., Boer, C., 2006. Cardioprotection via activation of protein kinase C-delta depends on modulation of the reverse mode of the Na+/Ca2+ exchanger. Circulation 114, I226–I232. Bulvik, B.E., Berenshtein, E., Meyron-Holtz, E.G., Konijn, A.M., Chevion, M., 2012. Cardiac protection by preconditioning is generated via an iron-signal created by

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P. Castaldo et al.

Orrenius, S., Zhivotovsky, B., Nicotera, P., 2003. Regulation of cell death: the calciumapoptosis link. Nat. Rev. Mol. Cell Biol. 4, 552–565. Palfi, A., Toth, A., Kulcsar, G., Hanto, K., Deres, P., Bartha, E., Halmosi, R., Szabados, E., Czopf, L., Kalai, T., Hideg, K., Sumegi, B., Toth, K., 2005. The role of Akt and mitogen-activated protein kinase systems in the protective effect of poly(ADP-ribose) polymerase inhibition in Langendorff perfused and in isoproterenol-damaged rat hearts. J. Pharm. Exp. Ther. 315, 273–282. Parhamifar, L., Andersen, H., Moghimi, S.M., 2013. Lactate dehydrogenase assay for assessment of polycation cytotoxicity. Methods Mol. Biol. 948, 13–22. Penna, C., Mancardi, D., Raimondo, S., Geuna, S., Pagliaro, P., 2008. The paradigm of postconditioning to protect the heart. J. Cell Mol. Med 12, 435–458. Przyklenk, K., Hata, K., Kloner, R.A., 1997. Is calcium a mediator of infarct size reduction with preconditioning in canine myocardium? Circulation 96, 1305–1312. Quinn, F.R., Currie, S., Duncan, A.M., Miller, S., Sayeed, R., Cobbe, S.M., Smith, G.L., 2003. Myocardial infarction causes increased expression but decreased activity of the myocardial Na+-Ca2+ exchanger in the rabbit. J. Physiol. 553, 229–242. Reuter, H., Henderson, S.A., Han, T., Matsuda, T., Baba, A., Ross, R.S., Goldhaber, J.I., Philipson, K.D., 2002. Knockout mice for pharmacological screening: testing the specificity of Na+-Ca2+ exchange inhibitors. Circ. Res. 91, 90–92. Sanada, S., Komuro, I., Kitakaze, M., 2011. Pathophysiology of myocardial reperfusion injury: preconditioning, postconditioning, and translational aspects of protective measures. Am. J. Physiol. Heart Circ. Physiol. 301, H1723–H1741. Seki, S., Taniguchi, M., Takeda, H., Nagai, M., Taniguchi, I., Mochizuki, S., 2002. Inhibition by KB-r7943 of the reverse mode of the Na+/Ca2+ exchanger reduces Ca2+ overload in ischemic-reperfused rat hearts. Circ. J. 66, 390–396. Shintani, Y., Node, K., Asanuma, H., Sanada, S., Takashima, S., Asano, Y., Liao, Y., Fujita, M., Hirata, A., Shinozaki, Y., Fukushima, T., Nagamachi, Y., Okuda, H., Kim, J., Tomoike, H., Hori, M., Kitakaze, M., 2004. Opening of Ca2+-activated K+ channels is involved in ischemic preconditioning in canine hearts. J. Mol. Cell Cardiol. 37, 1213–1218. Sipido, K.R., Volders, P.G., de Groot, S.H., Verdonck, F., Van de Werf, F., Wellens, H.J., Vos, M.A., 2000. Enhanced Ca(2+) release and Na/Ca exchange activity in hypertrophied canine ventricular myocytes: potential link between contractile adaptation and arrhythmogenesis. Circulation 102, 2137–2144. Song, C.L., Liu, B., Diao, H.Y., Shi, Y.F., Zhang, J.C., Li, Y.X., Liu, N., Yu, Y.P., Wang, G., Wang, J.P., Li, Q., 2016. Down-regulation of microRNA-320 suppresses cardiomyocyte apoptosis and protects against myocardial ischemia and reperfusion injury by targeting IGF-1. Oncotarget. Studer, R., Reinecke, H., Bilger, J., Eschenhagen, T., Bohm, M., Hasenfuss, G., Just, H., Holtz, J., Drexler, H., 1994. Gene expression of the cardiac Na(+)-Ca2+ exchanger in end-stage human heart failure. Circ. Res. 75, 443–453. Vinten-Johansen, J., Shi, W., 2011. Perconditioning and postconditioning: current knowledge, knowledge gaps, barriers to adoption, and future directions. J. Cardiovasc. Pharmacol. Ther. 16, 260–266. Xu, W., Liu, Y., Wang, S., McDonald, T., Van Eyk, J.E., Sidor, A., O'Rourke, B., 2002. Cytoprotective role of Ca2+- activated K+ channels in the cardiac inner mitochondrial membrane. Science 298, 1029–1033. Yang, E., Korsmeyer, S.J., 1996. Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood 88, 386–401. Yang, X., Cohen, M.V., Downey, J.M., 2010. Mechanism of cardioprotection by early ischemic preconditioning. Cardiovasc Drugs Ther. 24, 225–234. Yellon, D.M., Hausenloy, D.J., 2007. Myocardial reperfusion injury. N. Engl. J. Med 357, 1121–1135. Zhang, J.Y., Cheng, K., Lai, D., Kong, L.H., Shen, M., Yi, F., Liu, B., Wu, F., Zhou, J.J., 2015. Cardiac sodium/calcium exchanger preconditioning promotes anti-arrhythmic and cardioprotective effects through mitochondrial calcium-activated potassium channel. Int J. Clin. Exp. Pathol. 8, 10239–10249. Zlatkovic, J., Filipovic, D., 2012. Bax and B-cell-lymphoma 2 mediate proapoptotic signaling following chronic isolation stress in rat brain. Neuroscience 223, 238–245.

Li, D.F., Tian, J., Guo, X., Huang, L.M., Xu, Y., Wang, C.C., Wang, J.F., Ren, A.J., Yuan, W.J., Lin, L., 2012. Induction of microRNA-24 by HIF-1 protects against ischemic injury in rat cardiomyocytes. Physiol. Res. / Acad. Sci. Bohemoslov. 61, 555–565. Li, P.C., Yang, Y.C., Hwang, G.Y., Kao, L.S., Lin, C.Y., 2014. Inhibition of reverse-mode sodium-calcium exchanger activity and apoptosis by levosimendan in human cardiomyocyte progenitor cell-derived cardiomyocytes after anoxia and reoxygenation. PloS One 9, e85909. Li, S.Z., Wu, F., Wang, B., Wei, G.Z., Jin, Z.X., Zang, Y.M., Zhou, J.J., Wong, T.M., 2007. Role of reverse mode Na+/Ca2+ exchanger in the cardioprotection of metabolic inhibition preconditioning in rat ventricular myocytes. Eur. J. Pharmacol. 561, 14–22. Lytton, J., 2007. Na+/Ca2+ exchangers: three mammalian gene families control Ca2+ transport. Biochem. J. 406, 365–382. Magi, S., Arcangeli, S., Castaldo, P., Nasti, A.A., Berrino, L., Piegari, E., Bernardini, R., Amoroso, S., Lariccia, V., 2013. Glutamate-induced ATP synthesis: relationship between plasma membrane Na+/Ca2+ exchanger and excitatory amino acid transporters in brain and heart cell models. Mol. Pharm. 84, 603–614. Magi, S., Lariccia, V., Castaldo, P., Arcangeli, S., Nasti, A.A., Giordano, A., Amoroso, S., 2012. Physical and functional interaction of NCX1 and EAAC1 transporters leading to glutamate-enhanced ATP production in brain mitochondria. PloS One 7, e34015. Magi, S., Nasti, A.A., Gratteri, S., Castaldo, P., Bompadre, S., Amoroso, S., Lariccia, V., 2015. Gram-negative endotoxin lipopolysaccharide induces cardiac hypertrophy: detrimental role of Na(+)-Ca(2+) exchanger. Eur. J. Pharm. 746, 31–40. Marber, M.S., Latchman, D.S., Walker, J.M., Yellon, D.M., 1993. Cardiac stress protein elevation 24h after brief ischemia or heat stress is associated with resistance to myocardial infarction. Circulation 88, 1264–1272. Matsumoto, T., Miura, T., Miki, T., Nishino, Y., Nakamura, Y., Shimamoto, K., 2003. Does enhanced expression of the Na+-Ca2+ exchanger increase myocardial vulnerability to ischemia/reperfusion injury in rabbit hearts? Mol. Cell Biochem 248, 141–147. Meldrum, D.R., Cleveland, J.C., Jr., Sheridan, B.C., Rowland, R.T., Banerjee, A., Harken, A.H., 1996. Cardiac preconditioning with calcium: clinically accessible myocardial protection. J. Thorac. Cardiovasc Surg. 112, 778–786. Miyawaki, H., Ashraf, M., 1997. Ca2+ as a mediator of ischemic preconditioning. Circ. Res. 80, 790–799. Murphy, K.M., Ranganathan, V., Farnsworth, M.L., Kavallaris, M., Lock, R.B., 2000. Bcl2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells. Cell Death Differ. 7, 102–111. Murry, C.E., Jennings, R.B., Reimer, K.A., 1986. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74, 1124–1136. Namekata, I., Shimada, H., Kawanishi, T., Tanaka, H., Shigenobu, K., 2006. Reduction by SEA0400 of myocardial ischemia-induced cytoplasmic and mitochondrial Ca2+ overload. Eur. J. Pharmacol. 543, 108–115. Niu, C.F., Watanabe, Y., Ono, K., Iwamoto, T., Yamashita, K., Satoh, H., Urushida, T., Hayashi, H., Kimura, J., 2007. Characterization of SN-6, a novel Na+/Ca2+ exchange inhibitor in guinea pig cardiac ventricular myocytes. Eur. J. Pharmacol. 573, 161–169. O'Connell, T.D., Rodrigo, M.C., Simpson, P.C., 2007. Isolation and culture of adult mouse cardiac myocytes. Methods Mol. Biol. 357, 271–296. Ohnuma, Y., Miura, T., Miki, T., Tanno, M., Kuno, A., Tsuchida, A., Shimamoto, K., 2002. Opening of mitochondrial K(ATP) channel occurs downstream of PKC-epsilon activation in the mechanism of preconditioning. Am. J. Physiol. Heart Circ. Physiol. 283, H440–H447. Ohtsuka, M., Takano, H., Suzuki, M., Zou, Y., Akazawa, H., Tamagawa, M., Wakimoto, K., Nakaya, H., Komuro, I., 2004. Role of Na+-Ca2+ exchanger in myocardial ischemia/reperfusion injury: evaluation using a heterozygous Na+-Ca2+ exchanger knockout mouse model. Biochem. Biophys. Res. Commun. 314, 849–853. Oltvai, Z.N., Milliman, C.L., Korsmeyer, S.J., 1993. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74, 609–619.

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