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


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.


CONCLUSIONS
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.
Suleman
Naushaad
N
Hatter Cardiovascular Research Institute, Department of Medicine, Faculty of Health Sciences, University of Cape Town, Private Bag 3, 7935 Observatory, South Africa.
Somers
Sarin
S
Smith
Robert
R
Opie
Lionel H
LH
Lecour
Sandrine C
SC
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D019157:Ischemic Preconditioning, Myocardial; D053612:Janus Kinases / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D023421:Models, Animal; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D020318:Rats, Long-Evans; D050796:STAT3 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology*; D020032:Tyrphostins / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D050796:STAT3 Transcription Factor; C494087:Stat3 protein, mouse; D014409:Tumor Necrosis Factor-alpha; D020032:Tyrphostins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide; D019869:Phosphatidylinositol 3-Kinases; D053612:Janus Kinases; D051057:Proto-Oncogene Proteins c-akt; D000077191:Wortmannin