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.
Received May 25, 2005; de novo received August 4, 2005; revision received October 5, 2005; accepted October 7, 2005.
Ischemic preconditioning (IPC) was first described in Circulation in 1986 as a procedure giving powerful protection against myocardial infarction.1 Despite extensive research, the cellular pathways and mechanisms involved have not been fully clarified,2 nor is it clear where they converge mechanistically.3 Different pathways appear to mediate classic (early-phase) IPC and late IPC.2 Much current attention is focused on activation of the reperfusion injury salvage kinase (RISK) pathway during early reperfusion, whereby the prosurvival kinases phosphatidylinositol 3-kinase (PI3K)-Akt, the extracellular signal–regulated kinase (Erk 1/2), and the downstream kinase mTOR/p70s6 kinase are activated in response to classic IPC and to various pharmacological preconditioning stimuli.4,5 However, activation of this pathway after pharmacological preconditioning by tumor necrosis factor-α (TNF-α) has not been reported.
Clinical Perspective p 3918
TNF-α, when administered in a time- and dose-dependent manner, mimics classic IPC and thus confers cardioprotection, as we have shown in a rat model6,7 and as confirmed by other investigators.8,9 Furthermore, we have shown that classic IPC was abrogated in vitro in TNF-α–knockout mice10 or in vivo in the presence of a soluble TNF-α receptor11 that acts as an inhibitor, strongly suggesting a role for TNF-α in IPC. The complete signaling cascade responsible for the cardioprotective effect of TNF-α has not yet been elucidated, but activation of several triggers, such as free radicals,12 sphingolipids,6 and the mitochondrial ATP-dependent potassium channel,6 could all play an important role. We hypothesized that preconditioning by TNF-α might involve a protective pathway involving the signal transducer activator of transcription-3 (STAT-3) rather than the RISK pathway, our reasoning being as follows.
We have already shown that classic IPC in the heart requires activation of STAT-3 in the trigger phase before the ischemic insult.13 STAT factors are a family of cytoplasmic transcription factors that mediate intracellular signaling initiated at cytokine cell surface receptors (eg, TNF-α receptors) and transmitted to the nucleus. STAT-3 is activated after phosphorylation by Janus kinases or by mitogen-activated protein kinases (MAPKs), which allow STAT-3 to dimerize and translocate to the nucleus (see Stephanou et al14 for a review). An essential role for the Janus kinase/STAT pathway has already been reported before the major ischemic insult in classic and late preconditioning.15–17 However, the role of STAT-3 during the early-reperfusion phase has not specifically been explored though already linked to the trigger phase and found at the end of 2 hours of reperfusion.12 With the current emphasis on the crucial timing of modulators of reperfusion damage being limited to the very early minutes of reperfusion,18,19 our findings extend those of Hattori et al16 Furthermore, those earlier studies did not specifically link TNF-α–induced preconditioning to the activation of STAT-3.
To test the hypothesis that pharmacological preconditioning with TNF-α involves STAT-3 activation at reperfusion rather than the RISK pathway, we used various models (chiefly isolated rat hearts; also isolated mouse cardiomyocytes and C2C12 mouse myotubes). We also investigated whether classic IPC involves this proposed alternative protective pathway that requires STAT-3 activation.
All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85,23; revised 1996).
In Vivo Surgical Procedures
Male Wistar rats (250 to 300 g) were anesthetized with sodium pentobarbital (60 mg/kg IP) and after intubation were ventilated with room air (2.5 mL/stroke) at a rate of 70 strokes per minute. Rats were placed on a custom-made heating block to maintain body temperature throughout the surgical procedure. A left thoracotomy was performed through the fourth intercostal space, and the lung was collapsed with use of a damp swab. The heart was exteriorized for easier manipulation, and a 6-0 silk suture was passed below the left anterior descending coronary artery &2 to 3 mm from its origin.
Control ischemia/reperfusion (I/R) rats (CTL) were subjected to a period of 30 minutes of ischemia followed by 24 hours of reperfusion. In the IPC rats (IPC group), 3 cycles of temporary regional ischemia (3 minutes) followed by a period of reperfusion (5 minutes) were applied before the I/R protocol. In a third group, a single dose of 0.1 μg/kg IV TNF-α was administered 24 minutes before the CTL protocol. The dose of TNF-α used in vivo correlates with the already-known protective dose of 0.5 ng/mL given in the isolated rat heart model.6 Sham-operated rats were subjected to the same procedure as the CTL group, but the coronary artery was not occluded. After completion of these protocols, the chest wall was closed in a series of layers, and the animals were allowed to recover on a heating pad while receiving supplemental O2.
After 24 hours of reperfusion, the animals were anesthetized as described before, and the chest wall was reopened. The coronary artery was occluded with the suture that had been left in place from the previous surgery. A 7.5% patent blue-violet dye was administered intravenously to reveal the zone at risk. The heart was subsequently removed and stored at −80°C. Hearts were then frozen, cut into slices, and incubated in sodium phosphate buffer containing 1% wt/vol triphenyltetrazolium chloride for 15 minutes to visualize the unstained, infarcted region. Infarct and risk-zone areas were determined by planimetry, and infarct size was expressed as a percentage of the risk zone.
Isolated, Perfused Rat Hearts
Hearts from adult, male Long-Evans rats (250 to 300 g) were excised rapidly and perfused retrogradely by the Langendorff technique. All hearts were subjected to 30 minutes of regional standard ischemia by occlusion of the left coronary artery and 120 minutes of reperfusion as described previously.12 Hearts were preconditioned with either low-dose TNF-α (0.5 ng/mL) that was given for 7 minutes followed by a 10-minute wash-out period before standard ischemia (TNF group) or with 2 cycles of 5 minutes of global I/R (IPC group) before standard ischemia. The PI3K inhibitor wortmannin (100 nmol/L), the MAPK/Erk 1/2 kinase (MEK-1/2) inhibitor PD-98059 (10 μmol/L), the mTOR-kinase inhibitor rapamycin (0.5 nmol/L), and the STAT-3 inhibitor AG 490 (100 nmol/L) were given for the first 15 minutes of reperfusion. The concentrations used for wortmannin, PD-98059, or rapamycin have been shown to respectively inhibit PI3K/Akt, Er k1/2, or p70s6 kinase in the isolated, perfused rat heart.18,20 The concentration used for AG 490 corresponds to the IC50 of the drug, and this concentration has previously been shown to inhibit phosphorylation of STAT-3 in cell culture models.21
For infarct size measurement, the coronary artery was reoccluded at the end of the reperfusion period, and a solution of 2.5% Evans blue was perfused to delineate the area at risk. Hearts were then frozen, and the infarcted area was stained with a solution of triphenyltetrazolium chloride as described earlier.
Isolated Mouse Cardiomyocytes
Cardiomyocytes from wild-type mice (30 to 35 g) were isolated according to the modified method of Zhou et al, as described previously.13 Cells were exposed to simulated ischemia for 26 hours in modified Esumi buffer followed by 2 hours of “reperfusion” in normoxic KSLMS (Kawasoto, Sato, Le, McClure, Sato).13 Cardiomyocytes were preconditioned by either low-dose TNF-α (0.5 ng/mL) that was given for 30 minutes followed by 1 hour of washout with normoxic buffer before the index simulated ischemia or exposing the cells to 1% O2 in Esumi buffer at pH 6.4 for 30 minutes, followed by 1 hour of washout with normoxic buffer. The PI3K inhibitor wortmannin (100 nmol/L), the MEK-1/2 inhibitor PD-98059 (10 μmol/L), the mTOR kinase inhibitor rapamycin (0.5 nmol/L), or the STAT-3 inhibitor AG 490 (100 nmol/L) was given during the 2 hours of reperfusion. Cell viability was quantified by exclusion of trypan blue and rod-shaped morphology, and a total of 150 cells or more was counted per sample. The initial yield of the isolation procedure gave between 50% and 60% viable cells.
C2C12 Myotube Preconditioning Protocols
C2C12 myoblasts were obtained from the American Type Culture Collection (Manassas, Va) and were differentiated as previously described.22 Preconditioning was achieved by supplementing the normoxic buffer with TNF-α (0.5 ng/mL) for 30 minutes. After 1 hour of drug-free incubation, cells underwent 7 hours of simulated ischemia followed by 1 hour of reperfusion. Cell viability was analyzed by the uptake of propidium iodide on flow cytometry in a population of 104 cells, as previously described.22
Mitochondrial Function in C2C12 Myotubes
Inner mitochondrial membrane potential determination at reperfusion was performed on C2C12 myotubes by use of the potentiometric dye, JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide, 1 μg/mL; Molecular Probes) and fluorescence-activated cell sorting analysis as described previously.22 Whole-cell respiration was measured at reperfusion with a Clark-type electrode with an integrated temperature control unit (Oxytherm, 37°C).22 State 2 respiration was expressed as nanomoles O2 per minute per 106 viable cells.
Western Blot Analysis
CTL or preconditioned rat hearts were subjected to 30 minutes of regional ischemia and 5 minutes of reperfusion. At the end of reperfusion, the ventricular tissue at risk was excised and freeze-clamped in LN2 before being stored at −80°C. Cytosolic and nuclear proteins were extracted as previously published.23 Phosphorylated states of Akt (phospho-Akt; Ser 473), Erk 1/2 (phospho-Erk; Thr 202/204), STAT-3 (phospho-STAT-3; Ser 727 or Tyr 705), and the proapoptotic protein Bcl-2 antagonist of cell death (BAD) (phospho-BAD; Ser 136) and total levels of Akt, Erk, STAT-3, BAD, and β-actin were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis with antibodies from Cell Signaling Technology. Equal loading was verified with Ponceau staining or β-actin, and levels of phosphorylated proteins were normalized to their total protein levels performed in the same samples and under the same conditions but on a separate membrane. Relative densitometry was determined with use of a computerized software package.
Data are presented as mean±SEM. Comparisons between multiple groups were performed by 1-way ANOVA followed by the Student-Newman-Keuls post hoc test (Graph Pad Instat). A value of P<0.05 was considered statistically significant.
TNF-α Can Precondition the Heart Subjected to an I/R Insult In Vivo
To ensure that exogenous TNF-α could mimic IPC in vivo in a similar manner as in the isolated rat heart, hearts were preconditioned with either an IPC stimulus or a single injection of TNF-α. Ischemic CTL hearts had an infarct size of 37.8±5.8% (calculated as a percentage of the risk zone; Figure 1). Although the area at risk did not differ among the various groups (data not shown), both IPC and pharmacological preconditioning with TNF-α reduced infarct size compared with the ischemic CTL group (P<0.01).
Inhibition of Akt and Erk Activation at Reperfusion Abrogates the Protection Induced by IPC but Not by TNF-α
To investigate the role of the RISK pathway, we measured infarct size as the percentage of area at risk in hearts perfused with an inhibitor of PI3K (wortmannin), an inhibitor of MEK 1/2 (PD-98059), or an inhibitor of mTOR kinase (rapamycin) during the first 15 minutes of reperfusion. Ischemic CTL hearts had an infarct size of 31.1±2.8% (calculated as a percentage of the risk zone; Figure 2). This confirms previous data from our laboratories.6,24 Both IPC and pharmacological preconditioning with TNF-α reduced infarct size compared with the ischemic CTL group (P<0.001; Figure 2). When wortmannin, PD-98059, or rapamycin was given during early reperfusion, the protective effect of IPC was abrogated. In contrast, these inhibitors did not alter the protection afforded by TNF-α preconditioning.
Similarly, both IPC and TNF-α preconditioning in isolated cardiomyocytes subjected to simulated I/R improved cell viability (P<0.001; Table 1). Addition of wortmannin, PD-98059, or rapamycin during reperfusion abrogated the protective effect of IPC but did not affect preconditioning with TNF-α.
IPC but Not TNF-α Preconditioning Activates Phosphorylation of Akt and Erk at the Time of Reperfusion
We examined whether Akt and Erk were phosphorylated during early reperfusion in IPC and pharmacological preconditioning with TNF-α. As shown in Figure 3, IPC increased both Akt (in arbitrary units [AU] from 100.0±3.0 in ischemic CTL to 134.0±5.8 in IPC; P<0.01) and Erk phosphorylation (from 100.0±11.3 AU in ischemic CTL to 204.7±28.4 AU in IPC; P<0.01) after 5 minutes of reperfusion. By contrast, TNF-α did not affect phosphorylation of these 2 kinases (Akt, 107.4±6.7 AU; Erk, 117±14.2 AU). Similar results were obtained after 15 minutes of reperfusion (data not shown).
Inhibition of STAT-3 Activation at the Time of Reperfusion Abrogates the Protection Induced by Both IPC and TNF-α Preconditioning
To investigate the role of STAT-3 at the time of reperfusion in IPC and pharmacological preconditioning with TNF-α, we administrated an inhibitor of STAT-3, AG 490. Treatment with AG 490 during the reperfusion period abrogated the protective effect of IPC and TNF-α preconditioning in both the isolated rat heart model (Figure 4) and isolated cardiomyocytes (Table 1). Western blot analysis after 5 minutes of reperfusion showed an increase in nuclear and cytosolic phosphorylation of STAT-3 in both IPC and TNF-α preconditioning (Figure 5A). However, this increase was lost when AG 490 was perfused at reperfusion (Figure 5B).
Inhibition of STAT-3 Activation at the Time of Reperfusion Results in Loss of Phosphorylation of BAD
To explore the downstream targets activated by STAT-3 at the time of reperfusion, we investigated the role of the proapoptotic agent BAD. Western blot analysis after 5 minutes of reperfusion showed increased cytosolic phosphorylation of BAD in TNF-α and to a greater extent in IPC compared with the ischemic CTL group (Figure 6A). However, this relative increase in phosphorylation was lost when AG 490 was perfused at the time of reperfusion (Figure 6B).
IPC and TNF-α Improved Mitochondrial Function in C2C12 Myotubes Subjected to Simulated I/R
A simulated I/R insult performed in C2C12 myotubes reduced cell viability, decreased the inner mitochondrial membrane potential, and increased state 2 respiration when compared with the normoxic group (P<0.001; Table 2). IPC and TNF-α preconditioning before the simulated I/R insult prevented cell death and partially restored O2 consumption and the inner mitochondrial membrane potential at reperfusion (IPC and TNF; P=NS versus normoxic).
In different models and animal species, our novel data show, apparently for the first time, that cardioprotection at reperfusion by TNF-α is independent of activation of the prosurvival kinases Akt and Erk 1/2 (the RISK pathway).4 More importantly, we have shown the existence of an alternative protective pathway that involves activation of the transcription factor STAT-3 at the time of reperfusion in response to both TNF-α and IPC. The main data leading to these conclusions are as follows: (1) TNF-α–induced preconditioning did not phosphorylate the kinases Akt and Erk during reperfusion; (2) specific inhibitors of these kinases did not affect cardioprotection by TNF-α; (3) TNF-α and classic IPC phosphorylated STAT-3 during early reperfusion; and (4) addition of the STAT-3 inhibitor AG 490 abolished the protection afforded by both TNF-α and IPC as preconditioning stimuli.
During ischemia,25 in heart failure,26 or after coronary microembolization,27 myocardial and/or serum TNF-α levels increase and are causally involved in contractile dysfunction. In contrast, in vitro experiments conducted in rats or in mouse hearts have shown that exogenous TNF-α could also confer cardioprotection against an I/R insult in a time- and dose-dependent manner.6,10 However, these in vitro models represent limitations in that they do not take into account the intracoronary activation of leukocytes, which may result in an opposite effect of TNF-α in the setting of myocardial ischemia. In the present study, we have confirmed that TNF-α could mimic IPC in vivo to a similar extent as in the isolated, rat heart model. Interestingly, the protective dose of TNF-α (0.1 μg/kg) used in vivo is within the range (1) of the dose used to mimic IPC in the isolated, rat heart model (0.5 ng/mL) and (2) of the amount of TNF-α endogenously released in the isolated heart after an I/R insult.28
The concept of multiple pathways in preconditioning has already been suggested in relation to the trigger phase.2 Using an isolated rat heart model with global I/R, we found that sphingolipids are involved with TNF-α–induced preconditioning but not necessarily with IPC.6 In contrast, activation of tyrosine kinase is required in IPC29,30 but not necessarily in pharmacological preconditioning.31 During the early phase of reperfusion, it is now well established that IPC activates the RISK pathway.32,33 In addition, many pharmacological agents, such as adenosine agonists, bradykinin, atorvastatin, urocortin, insulin, and cardiotrophin-1, all protect the heart by activating the RISK pathway when given at the beginning of reperfusion.4 However, now for the first time (to our knowledge), we have found that a preconditioning pharmacological agent (TNF-α) could confer cardioprotection independently of activation of the RISK pathway during early reperfusion. By contrast, we found that an alternative and RISK-independent pathway is activated by both classic IPC and TNF-α preconditioning. This novel pathway requires phosphorylation of STAT-3 at the time of reperfusion.
The role of STAT-3 has previously been reported during the trigger phase in both TNF-α preconditioning13 and IPC16,17 but not during the early-reperfusion phase. Because we have already demonstrated that TNF-α acts as a trigger in classic IPC,10,11 we now postulate that IPC activates STAT-3 via TNF-α. However, further experiments will be required to determine whether STAT-3 may be activated in a TNF-α–independent manner in IPC. Our data with different pharmacological inhibitors suggest that Akt, Erk, and STAT-3 are all essential for cardioprotection at reperfusion and that activation of these 3 components is required to reach a postulated threshold of protection in IPC. Inhibiting 1 of these components means that the threshold for protection cannot be reached. As an example, Hausenloy et al20 and our group (vide infra) have shown that inhibiting Akt at the time of reperfusion with wortmannin blocks IPC, even if the activity of Erk is not inhibited. These experiments demonstrate that although Erk is not a component of a linear pathway nor is it downstream of Akt, the protection can be lost if Akt is blocked. Similarly, with TNF-α, activation of Erk and Akt is not essential to reach the threshold for protection by TNF-α. Another possibility would be that STAT-3 is a downstream target of Erk and/or Akt. TNF-α could directly activate STAT-3, whereas IPC would activate STAT-3 via Erk and/or Akt. Further investigations will be required to determine the exact link or possible cross-talk between these different pathways.
Although BAD, BAX, p70s6 kinase, and endothelial NO synthase appear to be the downstream components responsible for mediating the protection associated with activation of the RISK pathway at the time of reperfusion,4 the cell survival components activated by the RISK-independent pathway still remain to be determined. Our work delineates inhibition of the apoptosis-promoting factor BAD as 1 of the components responsible for mediating protection at reperfusion that is associated with the activation of STAT-3. Because BAD is also known to be a downstream component of the RISK pathway,4 our results suggest a convergence of the RISK and RISK-independent pathways at the level of activity of BAD. In addition, phosphorylation of BAD with IPC appears to be greater than with TNF-α, suggesting that phosphorylation of BAD in IPC may result in the combined activation of both pathways. Interestingly, the presence of AG 490 in the IPC group abrogated the cardioprotection but reduced the phosphorylation of BAD to a level similar to that in the cardioprotective TNF-α group. Although Western blot analysis is a semiquantitative technique that is often used to explore the activation of proteins via their phosphorylation levels, it is important to emphasize that phosphorylation of a protein may not always reflect the activation of a protein. An explanation for our apparently inconsistent data for phospho-BAD may be due to sample collection. To perform Western blot analyses, we collected samples from the full transmural thickness of the myocardium at risk (a mixture of necrotic and viable myocardium and the amount of viable tissue may vary from 1 group to another), as previously described by other inverstigators.33,34 Recent data have shown that collecting samples from viable, previously ischemic subepicardium may lead to different results for some protein analyses.35 However, this apparent fundamental inconsistency in our results with phospho-BAD is most likely related to the time point selected to explore the phosphorylation of the different proteins (5 minutes of reperfusion). Western blot analysis on samples collected after 15 minutes of reperfusion showed an equal increase in cytosolic phospho-BAD in TNF-α and IPC groups. Furthermore, the addition of AG 490 at this time point reduced phospho-BAD to a similar extent in TNF-α and IPC groups (data not shown).
Many of the antiapoptotic pathways activated by prosurvival kinase cascades converge on the mitochondria. Juhaszova et al36 have recently shown that preconditioning prevents mitochondrial pore opening by activating Erk and Akt. We have previously reported that pharmacological preconditioning with TNF-α involves the mitochondrial potassium ATP-dependent channel.6 Our present study confirms that TNF-α and IPC both act on mitochondrial function by improving the inner mitochondrial membrane potential and cell respiration. It would be interesting to investigate whether STAT-3 activation, like the RISK pathway, confers protection by preventing the mitochondrial permeability transition pore opening, now postulated to be the end point of IPC.37,38 If so, the end point of both pathways would be the same.
In summary, the present study demonstrates that pharmacological preconditioning induced by TNF-α does not require activation of the RISK pathway at early reperfusion. Moreover, we provide evidence for an alternative protective pathway that involves the activation of STAT-3. Delineating the exact prosurvival components involved in this novel RISK-independent pathway, as well as the possible link between these 2 pathways both involving STAT-3, is of current interest, as it may have potential therapeutic significance in the mitigation of reperfusion-induced cardiac damage.
This work was supported in part by the Hatter Institute Foundation, a CRIG grant from the Wellcome Trust, and the Interuniversity Cape Heart Group of the South African Medical Research Council. S.L. was supported by a Servier Senior Fellowship for Research in Heart Failure and G.D., by the Wellcome Trust. We thank Suzelle Hattingh for technical help and Professors Amanda Lochner and Daan Nel for helpful comments.
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During myocardial ischemia or in heart failure, myocardial and/or serum TNF-α levels are increased and are causally involved in contractile dysfunction. By contrast, low physiological levels of TNF-α could be protective. The present study shows in vitro and in vivo that low doses of TNF-α can mimic IPC and thus confer cardioprotection. We also show that protection from reperfusion damage can be achieved by the activation of a new protective pathway independent of the classic prosurvival kinases (Akt and Erk). This new pathway depends on the activation of STAT-3. Thus, pharmacological activation of STAT-3 at the time of reperfusion may provide a new therapeutic approach to lessen reperfusion damage caused by revascularization for acute myocardial infarction.
Guest Editor for this article was Robert Kloner, MD, PhD.