Sarin Somers

Ischaemic postconditioning protects against reperfusion injury via the SAFE pathway

AIMS Ischaemic postconditioning (IPostC) is a powerful protective phenomenon that activates prosurvival intrinsic signalling cascades to limit reperfusion injury. We propose that IPostC confers its infarct-sparing effect via activation of the newly described prosurvival Survivor Activating Factor Enhancement (SAFE) pathway, which involves the activation of the cytokine tumour necrosis factor alpha (TNFalpha) and signal transducer and activator of transcription-3 (STAT-3). METHODS AND RESULTS Isolated ischaemic/reperfused hearts from TNF knockout, TNF receptor-1 knockout, TNF receptor-2 knockout, cardiomyocyte-specific STAT-3-deficient mice or their respective wild-type, (TNF-WT) or (STAT-3-WT), were postconditioned by ischaemic episodes (IPostC) or with exogenous TNFalpha (0.5 microg/L) (TNF-PostC) at the onset of reperfusion. IPostC reduced infarct size (IS) in TNF-WT and TNFR1(-/-) hearts (by 33 and 27%, respectively, P < 0.05), whereas hearts from TNF(-/-) or TNFR2(-/-) failed to be postconditioned. TNF-PostC reduced IS by 37% (P < 0.05) in STAT-3-WT hearts but failed to protect cardiac-specific STAT-3(-/-) hearts. Administration of wortmannin, an inhibitor of PI-3 kinase/Akt, or PD98059, an inhibitor of extracellular regulated kinase 1/2 (Erk1/2), during the postconditioning stimulus did not abolish the infarct-sparing effect of TNF-PostC. AG490, an inhibitor of STAT-3, abrogated the protective effect of TNFalpha. Western blot analysis did not demonstrate the involvement of Akt or Erk1/2 in TNF-PostC, whereas STAT-3 phosphorylation was increased in both IPostC and TNF-PostC. CONCLUSION 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.

Pharmacological Preconditioning With Tumor Necrosis Factor-α Activates Signal Transducer and Activator of Transcription-3 at Reperfusion Without Involving Classic Prosurvival Kinases (Akt and Extracellular Signal–Regulated Kinase)

Background— We previously reported that tumor necrosis-factor-α (TNF-α) can mimic classic ischemic preconditioning (IPC) in a dose- and time-dependent manner. Because TNF-α activates the signal transducer and activator of transcription-3 (STAT-3), we hypothesized that TNF-α–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-α (0.5 ng/mL). Western blot analysis confirmed that IPC phosphorylated Akt and Erk 1/2 after 5 minutes of reperfusion (Akt increased by 34±6% and Erk, by 105±28% versus control; P<0.01). 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-α preconditioning did not phosphorylate these kinases (Akt increased by 7±7% and Erk, by 17±14% versus control; P=NS). Neither wortmannin nor PD-98059 inhibited TNF-α–mediated cardioprotection. However, TNF-α and IPC both phosphorylated STAT-3 and the proapoptotic protein Bcl-2 antagonist of cell death (BAD) (STAT-3 increased by 58±17% with TNF-α or by 68±12% with IPC; BAD increased by 75±8% with TNF-α or by 205±20% with IPC; P<0.01 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-α–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-α and classic IPC. This novel prosurvival pathway may have potential therapeutic significance.

Dual activation of STAT-3 and Akt is required during the trigger phase of ischaemic preconditioning

AIMS During preconditioning by tumour necrosis factor-alpha (TNFalpha), activation of the signal transducer and activator of transcription-3 (STAT-3) but not Akt, is essential, whereas ischaemic cardiac preconditioning (IPC) requires both STAT-3 and Akt at the time of reperfusion. However, it is not known whether the same signalling pattern occurs during the preconditioning stimulus (trigger phase) and whether links exist between STAT-3 and Akt. Hence, our hypothesis is that concomitant activation or co-interaction between these two key signals is required during the trigger phase for IPC. Conversely, we proposed that there would be no such interaction when preconditioning was induced by TNFalpha (TNF-PC). METHODS AND RESULTS Cardiomyocytes, isolated from adult wild-type (WT) and cardiac-specific STAT-3 knockout (KO) mice, were exposed to simulated ischaemia (SI) reperfusion. Cells were preconditioned either by 30 min SI or by 30 min TNFalpha (0.5 ng/mL) in the presence or absence of AG490 (100 nM) or wortmannin (100 nM) to inhibit STAT-3 or Akt, respectively. Cell viability was evaluated by trypan blue, and phosphorylation levels of STAT-3 and Akt were measured by Western blot analysis. Similar experiments were conducted in isolated rat hearts subjected to an ischaemia-reperfusion insult. Both preconditioning stimuli failed to protect KO cardiomyocytes, and addition of AG490 abolished preconditioning in WT cardiomyocytes or isolated hearts. Wortmannin abolished the protection afforded by IPC, but did not affect TNF-PC in both models. Western blot analysis demonstrated that added wortmannin during IPC stimulus decreased STAT-3 phosphorylation while, conversely, AG490 reduced Akt phosphorylation. CONCLUSION 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.

Is red wine a SAFE sip away from cardioprotection? Mechanisms involved in resveratrol‐ and melatonin‐induced cardioprotection

Abstract:  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.

HDL protects against ischemia reperfusion injury by preserving mitochondrial integrity.

OBJECTIVE High density lipoproteins (HDL) protect against ischemia reperfusion injury (IRI). However the precise mechanisms are not clearly understood. The novel intrinsic prosurvival signaling pathway named survivor activating factor enhancement (SAFE) path involves the activation of tumor necrosis factor (TNF) alpha and signal transducer and activator of transcription 3 (STAT3). SAFE plays a crucial role in cardioprotection against IRI. We propose that HDL protect against IRI via activation of the SAFE pathway and modulation of the mitochondrial permeability transition pore (mPTP) opening. METHODS AND RESULTS Isolated mouse hearts were subjected to global ischemia (35 min) followed by reperfusion (45 min). HDL were given during the first 7 min of reperfusion. In control hearts, the post-reperfusion infarct size was 41.3 ± 2.3%. Addition of HDL during reperfusion reduced the infarct size in a dose-dependent manner (HDL 200 μg protein/ml: 25.5 ± 1.6%, p < 0.001 vs. control). This protective effect was absent in TNF deficient mice (TNF-KO) or cardiomyocyte-STAT3 deficient mice (STAT3-KO). Similarly, HDL, given as a preconditioning stimulus, improved cell survival and inhibited mPTP opening in isolated cardiomyocytes subjected to simulated ischemia. These protective responses were inhibited in cardiomyocytes from TNF-KO and STAT3-KO mice. CONCLUSION Our data demonstrate that HDL protect against IRI by inhibition of mPTP opening, an effect mediated via activation of the SAFE pathway.

The PGE2 – Stat3 interaction in doxorubicin-induced myocardial apoptosis

AIMS Both cyclooxygenase-2 (COX-2) and the transcription factor signal transducer and activator of transcription 3 (Stat3) are involved in adaptive growth and survival of cardiomyocytes. In ventricular cardiomyocytes, prostaglandin E(2) (PGE(2)), a major COX-2 product, leads to adaptive growth via Stat3 activation, but whether this transcription factor acts as a signalling molecule in PGE(2)-induced cell survival is unknown. Therefore, the purpose of this study was to determine whether PGE(2) counteracts cardiac apoptosis induced by doxorubicin (DOX), and if so, whether Stat3 plays a critical role in this cardioprotective effect. METHODS AND RESULTS Neonatal rat ventricular cardiomyocytes were incubated with DOX (0.5 microM) and/or PGE(2) (1 microM). Apoptosis was assessed by determining caspase3 activation and apoptotic DNA fragmentation. The role of Stat3 was evaluated in vitro and in vivo by transfecting cardiomyocytes with siRNA targeting rat Stat3 and by using cardiomyocyte-restricted Stat3 knockout (Stat3 KO) mice, respectively. Incubation of ventricular cardiomyocytes with PGE(2) led to a time-dependent decrease in the DOX-induced caspase3 activation, reaching a maximal inhibition of 70 +/- 5% after 4 h. Similarly, PGE(2) inhibited DOX-induced DNA fragmentation by 58 +/- 5% after 24 h. This antiapoptotic action of PGE(2) was strongly reduced by the ERK1/2 inhibitor, U0126, whereas the p38 MAP kinase inhibitor, SB203580, had no effect. Depleting Stat3 expression by 50-60% in isolated ventricular cardiomyocytes markedly reduced the protective effect of PGE(2) on DOX-induced caspase3 activation and DNA fragmentation. Likewise, the stable PGE(2) analogue, 16,16-dimethyl-PGE(2), was unable to counteract cardiac apoptosis induced by DOX in Stat3 KO mice. CONCLUSION Our results demonstrate that PGE(2) prevents myocardial apoptosis induced by DOX. This protection requires the activation of the prosurvival pathways of Stat3 and ERK1/2.

Cardiac preconditioning with sphingosine-1-phosphate requires activation of signal transducer and activator of transcription-3.

Summary Aims Sphingosine-1-phosphate (S1P) is a cardioprotective agent. Signal transducer and activator of transcription 3 (STAT-3) is a key mediator of many cardioprotective agents. We aimed to explore whether STAT-3 is a key mediator in S1P-induced preconditioning. Methods Langendorff-perfused hearts from Wistar rats and wild-type or cardiomyocyte-specific STAT-3 knockout mice were pre-treated with S1P (10 nmol/l), with or without the STAT-3 pathway inhibitor AG490, before an ischaemia–reperfusion insult. Triphenyltetrazolium chloride and Evans blue staining were used for the determination of infarct size. Western blot analysis was carried out on the S1P pre-treated hearts for detection of cytosolic, nuclear and mitochondrial phosphorylated and total STAT-3 proteins. Results Pre-treatment with S1P decreased the infarct size in isolated rat (5 ± 3% vs control 26 ± 8%, p < 0.01) and wild-type mouse hearts (13 ± 1% vs control 33 ± 3%, p < 0.05). This protective effect was abolished in the rat hearts pre-treated with AG490 (30 ± 10%, p = ns vs control) and in the hearts from STAT-3 knockout mice (35 ± 4% vs control 30 ± 3%, p = ns). Levels of phosphorylated STAT-3 were significantly increased in both the nuclear (p < 0.05 vs control) and mitochondrial (p < 0.05 vs control) fractions in the S1P pre-treated hearts, but remained unchanged in the cytosolic fraction (p = ns vs control). Conclusion These novel results demonstrate that pharmacological preconditioning with S1P in the isolated heart is mediated by activation of mitochondrial and nuclear STAT-3, therefore suggesting that S1P may be a novel therapeutic target to modulate mitochondrial and nuclear function in cardiovascular disease in order to protect the heart against ischaemia–reperfusion.

Cardiac Postconditioning: An Additional Therapy to Limit Cell Death Following Myocardial Infarction

Following acute myocardial infarction (AMI), early reperfusion therapy with thrombolytic therapy or primary percutaneous coronary intervention therapy (PCI) is the best way to salvage the heart by limiting the infarct size and preserving the left ventricular function. The early survival benefits of reperfusion are probably sustained lifelong and after 20 years, the survival rate of 27% in patients treated with conventional therapy is increased to 37% in patients treated with reperfusion therapy (thrombolytics and/or PCI) (van Domburg et al. 2005).

TNFα-induced cardioprotection is independent of the activation of the prosurvival kinase Erk

Introduction : Tumour Necrosis Factor alpha (TNFα) contributes to cardiac dysfunction following ischaemia-reperfusion. Nonetheless, brief exposure to exogenous low doses of TNFα can mimic ischaemic preconditioning and thus be cardio-protective. The extracellular signal-regulated kinase (Erk) has been implicated in the protection against ischaemia-reperfusion and is known to be activated by TNFα. However, whether Erk activation contributes to TNFα-induced cardio-protection remains unknown. Methods : PD 98059 (10µM), an Erk inhibitor, was used to evaluate the role of this prosurvival kinase with respect to infarct size (expressed as a percentage of the area at risk) in isolated rat hearts subjected to ischaemic preconditioning (IPC) or TNFα preconditioning (TNFαP). Western blot analyses were used to determine the degree of Erk phosphorylation after the application of the preconditioning stimulus. Results : Pre-treatment of the hearts with 0.5ng/ml of TNFα (for 7min) or 2 cycles of 5min ischaemia-reperfusion prior to 30min regional index ischaemia and 2h of reperfusion reduced the infarct size by 76% and 88%, respectively, versus control. Western blot analysis of isolated rat hearts revealed that IPC but not TNFαP activated phosphorylation of Erk (+54% for IPC vs. control). Co-administration of PD 98059 during the preconditioning stimulus did not influence the infarct size. Conclusion : These findings confirm that activation of TNFα may be considered as a novel therapeutic approach against ischaemic heart disease and that its effect is independent of the activation of the classic prosurvival factor Erk.