Role of toll-like receptor 4 in melatonin-induced cardioprotection.

Melatonin protects the heart against myocardial ischemia/reperfusion injury via the activation of the survivor activating factor enhancement (SAFE) pathway which involves tumor necrosis factor alpha (TNFα) and the signal transducer and activator of transcription 3 (STAT3). Toll-like receptor 4 (TLR4) plays a crucial role in myocardial ischemia/reperfusion injury and activates TNFα. In this study, we investigated whether melatonin may target TLR4 to activate the SAFE pathway. Isolated hearts from rats or mice were subjected to ischemia/reperfusion injury. Melatonin (75 ng/L) and/or TAK242 (a specific inhibitor of TLR4 signaling, 500 nm) were administered to the rat hearts before the induction of ischemia. Pre-ischemic myocardial STAT3 was evaluated by Western blotting. Lipopolysaccharide (LPS, a stimulator of TLR4) was administered to wild type, TNFα receptor 2 knockout or cardiomyocyte-specific STAT3-deficient mice (2.8 mg/kg, i.p) 45 min before the heart isolation. Myocardial infarct size was measured as an endpoint. Compared to the control, administration of melatonin reduced myocardial infarct size (34.7 ± 2.8% versus 62.6 ± 2.7%, P < 0.01). This protective effect was abolished in the presence of TAK242 (49.2 ± 6.5%). Melatonin administered alone increased the pre-ischemic activation of mitochondrial STAT3, and this effect was attenuated with TAK242. Furthermore, stimulation of TLR4 with LPS pretreatment to mice reduced myocardial infarct size of the hearts isolated from wild-type animals but failed to protect the hearts isolated from TNFα receptor 2-knockout mice or cardiomyocyte-specific STAT3-deficient mice (P < 0.001). Taken together, these data suggest that cardioprotection induced by melatonin is mediated by TLR4 to activate the SAFE pathway.

Role of melatonin, melatonin receptors and STAT3 in the cardioprotective effect of chronic and moderate consumption of red wine.

We have recently discovered that melatonin, given acutely and directly to the isolated heart at the concentration found in wine, confers cardioprotection against ischemia-reperfusion (I/R). However, whether the presence of melatonin in wine contributes to the cardioprotective effect of chronic and moderate consumption of wine and its signalling mechanisms of protection are unknown. We therefore used both in vivo and in vitro models of I/R to investigate whether the presence of melatonin in red wine may contribute to the cardioprotective effect of chronic and moderate consumption of red wine. Wistar rats and C57black6 mice (WT) received drinking water supplemented daily with a moderate amount of red wine or melatonin given at the concentration found in the red wine. Rats were also pretreated with luzindole, a specific inhibitor of melatonin receptors 1 and 2 (2.3 mg/kg/day, intraperitoneally) or prazosin, a specific inhibitor of melatonin receptor type 3 (2.5 mg/kg/day, intraperitoneally). After 14 days, hearts were subjected to I/R in vivo or ex vivo. Red wine reduced the infarct size in both rats and WT mice (p < 0.001). Luzindole did not affect wine-induced cardioprotection, while prazosin reduced the infarct sparing effect of red wine (p < 0.05). Furthermore, red wine or melatonin failed to protect tumor necrosis factor alpha (TNF) receptor 2 knockout or cardiomyocyte specific signal transducer and activator of transcription 3 (STAT3) deficient mice (n.s. vs. control). Our novel findings suggest that the presence of melatonin in red wine contributes to the cardioprotective effect of chronic and moderate consumption of red wine against lethal I/R injuries. This effect is most likely mediated, at least in part, via melatonin receptor 3 and the activation of TNF and STAT3, both key players of the prosurvival and well described SAFE pathway.