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 ( TNF α ) and the signal transducer and activator of transcription 3 ( STAT 3). Toll‐like receptor 4 ( TLR 4) plays a crucial role in myocardial ischemia/reperfusion injury and activates TNF α . In this study, we investigated whether melatonin may target TLR 4 to activate the SAFE pathway. Isolated hearts from rats or mice were subjected to ischemia/reperfusion injury. Melatonin (75 ng/L) and/or TAK 242 (a specific inhibitor of TLR 4 signaling, 500 n m ) were administered to the rat hearts before the induction of ischemia. Pre‐ischemic myocardial STAT 3 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 TAK 242 (49.2 ± 6.5%). Melatonin administered alone increased the pre‐ischemic activation of mitochondrial STAT 3, and this effect was attenuated with TAK 242. Furthermore, stimulation of TLR 4 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 TNF α receptor 2‐knockout mice or cardiomyocyte‐specific STAT 3‐deficient mice ( P < 0.001). Taken together, these data suggest that cardioprotection induced by melatonin is mediated by TLR 4 to activate the SAFE pathway.