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Cardiac fibroblasts are important regulators of myocardial structure and function. Their implications in pathological processes such as Ischemia/Reperfusion are well characterized. Cardiac fibroblasts respond to stress by excessive proliferation and secretion of pro-inflammatory cytokines and other factors, e.g. ATP, leading to purinergic receptors activation. P2Y11 receptor (P2Y11R) is an ATP-sensitive GPCR playing an immunomodulatory role in human dendritic cells (DC). We hypothesized that P2Y11R stimulation modulated the pro-inflammatory responses of human cardiac fibroblasts (HCF) to Hypoxia/Reoxygenation (H/R) mainly by acting on their secretome. P2Y11R stimulation in HCF at the onset of reoxygenation significantly limited H/R-induced proliferation (−19%) and pro-inflammatory cytokines and ATP secretion (−44% and −83% respectively). Exposure of DC to HCF secretome increased their expression of CD83, CD25 and CD86, suggesting a switch from immature to mature phenotype. Under LPS stimulation, DC had a pro-inflammatory profile (high IL-12/IL-10 ratio) that was decreased by HCF secretome (−3,8-fold), indicating induction of a tolerogenic profile. Moreover, P2Y11R inhibition in HCF led to high IL-12 secretion in DC, suggesting that the immunomodulatory effect of HCF secretome is P2Y11R-dependant. HCF secretome reduced H/R-induced cardiomyocytes death (−23%) through RISK pathway activation. P2Y11R inhibition in HCF induced a complete loss of HCF secretome protective effect, highlighting the cardioprotective role of P2Y11R. Our data demonstrated paracrine interactions between HCF, cardiomyocytes and DC following H/R, suggesting a key role of HCF in the cellular responses to reperfusion. These results also demonstrated a beneficial role of P2Y11R in HCF during H/R and strongly support the hypothesis that P2Y11R is a modulator of I/R injury. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]