The protective effect of the SAFE pathway is shown in IPostC, with the activation of TNFalpha, its receptor type 2, and STAT-3. This signalling cascade is activated independently of the well-known Reperfusion Injury Salvage Kinases (RISK) pathway, which involves the kinases Akt and Erk1/2.

Background-We previously reported that tumor necrosis-factor-␣ (TNF-␣) can mimic classic ischemic preconditioning (IPC) in a dose-and time-dependent manner. Because TNF-␣ activates the signal transducer and activator of transcription-3 (STAT-3), we hypothesized that TNF-␣-induced preconditioning requires phosphorylation of STAT-3 rather than involving the classic prosurvival kinases, Akt and extracellular signal-regulated kinase (Erk) 1/2, during early reperfusion. Methods and Results-Isolated, ischemic/reperfused rat hearts were preconditioned by either IPC or low-dose TNF-␣ (0.5 ng/mL). Western blot analysis confirmed that IPC phosphorylated Akt and Erk 1/2 after 5 minutes of reperfusion (Akt increased by 34Ϯ6% and Erk, by 105Ϯ28% versus control; PϽ0.01). Phosphatidylinositol 3-kinase/Akt inhibition (wortmannin) or mitogen-activated protein kinase-Erk 1/2 kinase inhibition (PD-98059) during early reperfusion abolished the infarct-sparing effect of IPC. In contrast, TNF-␣ preconditioning did not phosphorylate these kinases (Akt increased by 7Ϯ7% and Erk, by 17Ϯ14% versus control; PϭNS). Neither wortmannin nor PD-98059 inhibited TNF-␣-mediated cardioprotection. However, TNF-␣ and IPC both phosphorylated STAT-3 and the proapoptotic protein Bcl-2 antagonist of cell death (BAD) (STAT-3 increased by 58Ϯ17% with TNF-␣ or by 68Ϯ12% with IPC; BAD increased by 75Ϯ8% with TNF-␣ or by 205Ϯ20% with IPC; PϽ0.01 versus control), thereby activating the former and inactivating the latter. The STAT-3 inhibitor AG 490 abolished cardioprotection and BAD phosphorylation with both preconditioning stimuli. Conclusions-Activation of the classic prosurvival kinases (Akt and Erk 1/2) is not essential for TNF-␣-induced preconditioning in the early reperfusion phase. We show the existence of an alternative protective pathway that involves STAT-3 activation specifically at reperfusion in response to both TNF-␣ and classic IPC. This novel prosurvival pathway may have potential therapeutic significance. (Circulation. 2005;112:3911-3918.)

STAT-3 activation could be achieved independent of Akt during TNF-PC. In contrast, during an IPC stimulus, both prosurvival signalling molecule cascades acted in concert so that inhibiting activation of STAT-3 also inhibited that of Akt and vice versa.

Epidemiological studies suggest that regular moderate consumption of red wine confers cardioprotection but the mechanisms involved in this effect remain unclear. Recent studies demonstrate the presence of melatonin in wine. We propose that melatonin, at a concentration found in red wine, confers cardioprotection against ischemia-reperfusion injury. Furthermore, we investigated whether both melatonin and resveratrol protect via the activation of the newly discovered survivor activating factor enhancement (SAFE) prosurvival signaling pathway that involves the activation of tumor necrosis factor alpha (TNFα) and the signal transducer and activator of transcription 3 (STAT3). Isolated perfused male mouse (wild type, TNFα receptor 2 knockout mice, and cardiomyocyte-specific STAT3-deficient mice) or rat hearts (Wistars) were subjected to ischemia-reperfusion. Resveratrol (2.3 mg/L) or melatonin (75 ng/L) was perfused for 15 min with a 10-min washout period prior to an ischemia-reperfusion insult. Infarct size was measured at the end of the protocol, and Western blot analysis was performed to evaluate STAT3 activation prior to the ischemic insult. Both resveratrol and melatonin, at concentrations found in red wine, significantly reduced infarct size compared with control hearts in wild-type mouse hearts (25 ± 3% and 25 ± 3% respectively versus control 69 ± 3%, P < 0.001) but failed to protect in TNF receptor 2 knockout or STAT3-deficient mice. Furthermore, perfusion with either melatonin or resveratrol increased STAT3 phosphorylation prior to ischemia by 79% and 50%, respectively (P < 0.001 versus control). Our data demonstrate that both melatonin and resveratrol, as found in red wine, protect the heart in an experimental model of myocardial infarction via the SAFE pathway.