Stimulation of P2Y11 receptor protects human cardiomyocytes against Hypoxia/Reoxygenation injury and involves PKCε signaling pathway

Sterile inflammation is a key determinant of myocardial reperfusion injuries. It participates in infarct size determination in acute myocardial infarction and graft rejection following heart transplantation. We previously showed that P2Y11 exerted an immunosuppressive role in human dendritic cells, modulated cardiofibroblasts’ response to ischemia/reperfusion in vitro and delayed graft rejection in an allogeneic heterotopic heart transplantation model. We sought to investigate a possible role of P2Y11 in the cellular response of cardiomyocytes to ischemia/reperfusion. We subjected human AC16 cardiomyocytes to 5 h hypoxia/1 h reoxygenation (H/R). P2Y11R (P2Y11 receptor) selective agonist NF546 and/or antagonist NF340 were added at the onset of reoxygenation. Cellular damages were assessed by LDH release, MTT assay and intracellular ATP level; intracellular signaling pathways were explored. The role of P2Y11R in mitochondria-derived ROS production and mitochondrial respiration was investigated. In vitro H/R injuries were significantly reduced by P2Y11R stimulation at reoxygenation. This protection was suppressed with P2Y11R antagonism. P2Y11R stimulation following H2O2-induced oxidative stress reduced mitochondria-derived ROS production and damages through PKCε signaling pathway activation. Our results suggest a novel protective role of P2Y11 in cardiomyocytes against reperfusion injuries. Pharmacological post-conditioning targeting P2Y11R could therefore contribute to improve myocardial ischemia/reperfusion outcomes in acute myocardial infarction and cardiac transplantation.

Myocardial ischemia/reperfusion (I/R) is a pathologic process responsible for myocardial injuries observed in several common clinical situations. It is the central event in the pathophysiology of acute myocardial infarction, resulting in cardiomyocytes death, but also impacts the short-and long-term outcomes in heart transplantation, including chronic rejection.
Ischemia generates cellular stress leading to irreversible tissue injuries 1 . While shortening the ischemic period is mandatory to ensure cell survival and preserve organ function 2 , the bulky reintroduction of oxygen and nutrients associated with reperfusion generates excessive oxidative stress that further increases cellular damages and cardiomyocytes death 3 .

EA4245 Transplantation, Immunologie et Inflammation, Loire Valley Cardiovascular Collaboration & Université de
Tours, Tours, France. 2 Service de Chirurgie Cardiaque, Hôpital Trousseau, Centre Hospitalier Régional Universitaire de Tours, Tours, France. 3 Service de Cardiologie, Hôpital Trousseau, Centre Hospitalier Régional Universitaire de Tours, Tours, France. 4 Department of Pathophysiology -Functional Sciences, "Victor Babes" University of Medicine and Pharmacy, Timisoara, Romania. 5  I/R injuries lead to the plasma membrane rupture and the release of endogenous danger molecules, also known as damage-associated molecular patterns (DAMPs), that bear the capacity to activate the immune system and promote the inflammatory response to I/R. DAMPs include extracellular ATP (eATP), an important immune regulator and a mediator of sterile inflammation 4 . Physiologically, eATP is locally released at low concentrations and plays the role of primary messenger in intercellular communications notably in the vascular system 5 . An important increase in eATP concentration is associated with cell damage and inflammatory processes 6 . eATP activates purinergic receptors (P2Rs), divided into P2X ATP-gated ion channel receptors (P2X1-7) and G protein-coupled P2Y receptors (P2Y1, 2,4,6,[11][12][13][14]. Both subtypes play important roles in immune cell functions, e.g. neutrophil migration 7 , inflammasome activation 8,9 and dendritic cells (DCs) maturation 10 . DCs are sentinels that orchestrate both innate and adaptive immune response in the presence of DAMPs. They are also important immunoprotective regulators during post-myocardial infarction repair process by controlling monocytes/macrophages homeostasis 11 . We previously reported that eATP mediates human DCs maturation toward an immunosuppressive phenotype through P2Y11 receptor (P2Y11R) stimulation and inhibits Th1 polarization 12 .
P2Y11R is also present in cardiac and endothelial cells 13 . It was shown to be present in cardiofibroblasts, which don't only play the role of support cells but also bear the capacity to modulate the local inflammatory response following I/R in a paracrine manner. Previous data from our group showed that cardiofibroblasts can exert a cardioprotective role in this context, notably when P2Y11R is stimulated 14 . In addition, we demonstrated that P2Y11R activation, in an in vivo murine model of heterotopic heart transplantation, could delay graft rejection through an attenuation of the local immuno-inflammatory response 15 , emphasizing a critical role of P2Y11 in the I/R-induced inflammatory response.
There are growing evidences that P2Y11R stimulation could have a protective role in myocardial I/R: Balogh et al. reported a positive inotropic effects of ATP in murine cardiomyocytes via P2Y11-like receptor signaling 16 ; using a Langendorff rat heart model, Djerada et al. showed effective cardioprotection against I/R with extracellular NAAD pharmacological pre-conditioning involving P2Y11R-like 17 . Last, a P2Y11R polymorphism (Ala-87-Thr) was associated with an increase in both level of C-reactive protein and risk of myocardial infarction 18 . Thus, we hypothesized that pharmacological post-conditioning targeting the modulation of Gq/Gs protein-coupled P2Y11R may directly reduce I/R injuries. This could translate into significant improvements in post-myocardial infarction and post-transplantation outcomes, in addition to its already demonstrated effect on the post I/R inflammatory balance.
In this study, we report P2Y11R as a novel pharmacological post-conditioning target for cardioprotection against I/R through its modulation of oxidative stress in a human cardiomyocytes cell line.

P2Y11R stimulation reduces H2O2-induced mitochondrial ROS production and activates
PKCε signaling pathway, but has no impact on mitochondrial respiration. AC16 cells were subjected to H 2 O 2 (30 min) and mitochondrial ROS production was evaluated by flow cytometry using MitoSOX ™ dye. Oxidative stress significantly increased MitoSOX ™ fluorescence (MFI from 459. www.nature.com/scientificreports www.nature.com/scientificreports/ n = 6) (Fig. 5a,b). Again, co-treatment with NF340 abolished this effect, suggesting a downstream regulation of PKCε signaling pathway by P2Y11R stimulation that may play a key role in this cardioprotective effect.
We finally investigated the impact of P2Y11R on mitochondrial oxygen consumption. Modulation of P2Y11R with its specific agonist/antagonist had no effect on mitochondrial respiration following H/R, whatever the state (data not shown), suggesting that the protective effect of P2Y11R activation cannot be explained by a specific effect on the mitochondrial respiratory chain and oxygen consumption mechanisms.

Discussion
In the present study, we demonstrated a novel cardioprotective role of P2Y11. Pharmacological post-conditioning with selective stimulation of P2Y11R rescued AC16 cardiomyocytes viability after H/R.
We propose AC16 human cardiomyocytes as a new relevant in vitro model to study H/R injuries. Oxygen-nutrients deprivation and resupply decreased AC16 cells viability, increased LDH-assessed necrosis and impaired mitochondrial respiration. Suffering cardiac cells during I/R and particularly cardiomyocytes activate multiple cell death pathways leading to the release of danger signals or DAMPs, among which extracellular ATP [19][20][21][22] has been identified as an important modulator of immune system through P2Rs signaling 4,6,23 . We and others previously reported that eATP through a P2Y11R-dependent signaling induced DCs maturation towards a tolerogeneic phenotype leading to modulation of effector T cells activation by the inhibition of Th1 response 10,12,24,25 . We demonstrated here a cardioprotective post-conditioning-like effect of eATP against H/R injuries. The cardioprotective effects of the hydrolysed form of ATP is now well admitted 26 . Adenosine receptors were associated to the cardioprotective effect of ischemic preconditioning 27 and they showed I/R injuries reduction properties when www.nature.com/scientificreports www.nature.com/scientificreports/ activated at reperfusion 28 . The use of an adenosine receptor antagonist here strongly suggests that the beneficial effect of eATP mainly involved P2R signaling.
P2Rs are expressed in different cell types of the cardiovascular system and their involvement in cardiac physiology recently emerged. Besides the positive inotropic effects of ATP, UTP and UDP on cardiomyocytes through P2Y11R, P2Y2R and P2Y6R activation respectively 13,16,29 , there are growing evidence that P2Rs play key roles in the physiopathology of I/R. Wee et al. reported a significant worsening in ischemic tolerance and post-ischemic outcomes due to P2Rs antagonism with suramin in a murine Langendorff model 30 . Moreover, both ATP and UTP released during cardiac ischemia displayed cardioprotective effects in several rodent model studies 30,31 . But because of the lack of selective agonists/antagonists until recently and the differences in pharmacological properties between human and rodent P2Rs, the receptors involved in such effects were not clearly identified. NF546 has been identified by Meis et al. 32 as a relatively selective, non-nucleotide agonist for P2Y11R over other P2YR, with a competitive behaviour toward the nanomolar potency antagonist NF340. Using NF546, we are the first to show that selective P2Y11R stimulation at the onset of reoxygenation protected human AC16 cardiomyocytes from H/R injuries. This is in line with Djerada et al. who reported that rat hearts treated with NAADP before I/R sequence were protected against I/R injuries through P2Y11R activation in a Langendorff model 17 . In the publication of Amisted et al. reporting an increased risk of myocardial infarction associated with a P2Y11R polymorphism in residue 87, the suggested mechanisms were probable impairments in ligand binding and signaling 18 . www.nature.com/scientificreports www.nature.com/scientificreports/ Regarding the molecular mechanisms involved in the cardioprotective effect of P2Y11R, we showed that its stimulation following H 2 O 2 -induced oxidative stress increased cell recovery through reduction of mitochondrial ROS production. Our data suggest that this effect was related to an increased PKCε phosphorylation. This is particularly relevant as PKCε can be activated by phospholipase C/diacylglycerol signaling pathway following Gq protein-coupled receptors stimulation (e.g. by P2Y11R), enabling its importation to mitochondria where it exerts its cardioprotective effect. In line with this, our data suggest that the inhibition of PKCε translocation to mitochondria abolished P2Y11R-mediated cardioprotection following H 2 O 2 -induced oxidative stress, with no additional effect of P2Y11R antagonist. These results support a close association between P2Y11R stimulation and PKCε signaling pathway in transducing cardioprotective signal. In our experiments, we couldn't link PKCε activation to a modulation of mitochondrial oxygen metabolism. Yet, modulation of oxidative phosphorylation is not the sole-described effect of PKCε signaling pathway. PKCε is considered to play a critical part in cardioprotection due to its ability to interact with substantial mitochondrial proteins, e.g. extracellular signal-regulated kinases (ERK) or ATP-sensitive K+ channels thereafter modifying mitochondrial permeability transition 33 .
All our data strongly suggest that P2Y11 may modulate myocardial H/R injuries. Limiting both I/R injuries and oxidative stress through P2Y11R stimulation might indirectly influence the inflammatory response (by decreasing the release of DAMPs and ROS), which could be synergistic with the effects of P2Y11R stimulation on DCs as previously described in vitro 12 and in vivo 15 . Of note, Certal et al. reported an anti-proliferative effect of P2Y11 in cardiac myofibroblasts, suggesting that this receptor may also be an interesting target to modulate cardiac remodeling 34 . We recently reported a similar anti-fibrotic effect of P2Y11R activation in cardiac fibroblasts that also displayed an immunomodulatory and cardioprotective role 14 . A limitation of this study has to do with the characteristic of AC16 cells, which is a proliferating cardiomyocyte cell line. Though they have retained the nuclear and mitochondrial DNA of the primary cardiomyocytes, cellular metabolism and proliferative status are correlates and both could modify response to I/R. As a reminder, we obtained similar data using the well-characterized H9c2 cardiomyocytes cell line 15 . Yet, with regards to this point, the effect of P2Y11 on primary cardiomyocytes remains speculative and needs to be confirmed. Nevertheless, the protective effect of P2Y11R previously reported regarding inhibition of graft rejection on transplanted hearts could be promising as to his role on non-proliferative cardiomyocytes.
The next step to demonstrate the therapeutic relevance of this approach, i.e. modulation of P2Y11R, in the context of myocardial ischemia/reperfusion should be through an in vivo model of acute myocardial infarction, with the additional advantage to validate the concept on non-proliferative cardiomyocytes.
In conclusion, our results propose a novel protective role of P2Y11R as a pharmacological post-conditioning target through a reduction of myocardial I/R injuries. This property combined with our previous observations of immunomodulatory and anti-fibrotic effects support the idea that therapeutic interventions aiming at stimulating P2Y11R could provide beneficial effects in the setting of acute myocardial infarction and cardiac transplantation and improve patients' outcomes. Cell culture. AC16 cells were purchased from American Type Culture Collection (ATCC ® , LGC Standards) and cultured in Gibco ® Dulbecco's modified Eagle's medium: Nutrient mixture F-12 (DMEM/F12) supplemented with 10% foetal bovine serum (FBS) (HyClone ™ , GE Healthcare Life Sciences) and Penicillin-Streptomycin (100 U/mL, Gibco ® ) in a humidified incubator at 37 °C with 5% CO 2 .  Oxidative stress was induced by H 2 O 2 (50 µM-1 mM in DMEM) for the indicated time. Then, cells were re-placed in fresh DMEM for 1 h. Treatments were added before, during or after the oxidative stress sequence for the indicated duration.

Cardiomyocytes viability and death assessments. MTT reduction and CellTiter-Glo ® Luminescent
Assay (Promega) measuring intracellular ATP production were used as indicators of cell viability.
Following H/R or oxidative stress sequences, cells were incubated for 1 h with a MTT solution (0.5 mg/mL in DMEM with 5% FBS) at 37 °C in a humidified incubator with 5% CO 2 . The resulting formazan produced by viable mitochondria was solubilized in DMSO for 45 min. Absorbance was recorded at 550 nm using a microplate reader. High resolution respirometry. Oxygen consumption was measured in non-attached AC16 cells resuspended in DMEM without FBS (2.5 × 10 6 cells/ml) using a 2 mL chamber OROBOROS ® Oxygraph 2 K (Oroboros Instruments, Innsbruck, Austria) at 37 °C. Respiration rates were calculated as the time derivative of oxygen concentration measured in the closed respirometer, expressed per million viable cells and corrected by non-mitochondrial oxygen consumption (energy wasting process) measured with antimycin A (2 μM). Oxygen consumption was measured in intact cells following 5 h of hypoxia and 1 h of reoxygenation at basal state, state 4 (non-phosphorylating state) using oligomycin B (10 μg/mL) and ETS (Electron Transfer System) capacity (maximum uncoupled respiration) induced by Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, 0.8 µM). Results were expressed as O 2 flow (pmol.min −1 ) per million cells.
Non-quantitative polymerase chain reaction. Total RNA was extracted from cardiomyocytes using the Illustra RNAspin mini kit (GE Healthcare Life Sciences) and reverse transcribed using the Protoscript ® II Reverse Transcriptase (New England Biolabs) according to manufacturer's instruction. cDNA were subjected to 40 cycles of amplification using OneTaq ® DNA Polymerase (New England Biolabs) and specific primers targeting human P2X1-7 and P2Y1, 2, 4, 6, 11-14 receptors (see Table 1) as previously described 12 with modified Tm.
PCR products were separated on 2% Agarose gels containing 1/10,000 SYBR ® Safe DNA Gel Stain (Invitrogen) and revealed under UV light with a PXi/PXi Touch gel imaging system and UltraSlim blue LED transilluminator (Syngene).
The primary antibodies (see Table 2) were incubated overnight at 4 °C. Membranes were further incubated for 1 h at room temperature with appropriate secondary antibodies. Proteins were detected with Amersham ™ ECL ™ Prime reagent (GE Healthcare Life Sciences) using a PXi/PXi Touch gel imaging system and bands were quantified by densitometric analysis using Fiji software (distributed by ImageJ) 35 . Of note, for each experiment, proteins of every conditions were loaded in the same gel and transferred to a membrane that was then cut in order to incubate appropriate antibodies. Densitometric analyses of bands were calculated as relative to control protein loading (e.g. HSC70 or ß-Actin). Intracellular cAMP quantification. Following a 15 min treatment with NF546 10 µM and/or NF340 10 µM in FBS-free DMEM, cAMP production in AC16 cells was quantified using the luminescent cAMP-Glo ™ Assay kit  Table 2. Antibodies for Western Blot.