Suleman Naushaad, Somers Sarin, Smith Robert, Opie Lionel H, Lecour Sandrine C
Hatter Cardiovascular Research Institute, Department of Medicine, Faculty of Health Sciences, University of Cape Town, Private Bag 3, 7935 Observatory, South Africa.
Cardiovasc Res. 2008 Jul 1;79(1):127-33. doi: 10.1093/cvr/cvn067. Epub 2008 Mar 13.
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).
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.
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.
在肿瘤坏死因子-α（TNFα）预处理过程中，信号转导及转录激活因子3（STAT-3）而非Akt的激活至关重要，而缺血性心脏预处理（IPC）在再灌注时需要STAT-3和Akt两者。然而，尚不清楚在预处理刺激（触发阶段）期间是否出现相同的信号传导模式，以及STAT-3和Akt之间是否存在联系。因此，我们的假设是，在IPC的触发阶段需要这两个关键信号的同时激活或共同相互作用。相反，我们提出，当由TNFα诱导预处理（TNF-PC）时不会有这种相互作用。
从成年野生型（WT）和心脏特异性STAT-3基因敲除（KO）小鼠分离的心肌细胞，暴露于模拟缺血（SI）再灌注。细胞分别通过30分钟SI或30分钟TNFα（0.5 ng/mL）进行预处理，存在或不存在AG490（100 nM）或渥曼青霉素（100 nM）以分别抑制STAT-3或Akt。通过台盼蓝评估细胞活力，并通过蛋白质印迹分析测量STAT-3和Akt的磷酸化水平。在遭受缺血-再灌注损伤的离体大鼠心脏中进行了类似实验。两种预处理刺激均未能保护KO心肌细胞，并且添加AG490消除了WT心肌细胞或离体心脏中的预处理。渥曼青霉素消除了IPC提供的保护，但在两种模型中均不影响TNF-PC。蛋白质印迹分析表明，在IPC刺激期间添加渥曼青霉素会降低STAT-3磷酸化，相反，AG490会降低Akt磷酸化。
在TNF-PC期间，STAT-3的激活可以独立于Akt实现。相反，在IPC刺激期间，两种促生存信号分子级联协同作用，因此抑制STAT-3的激活也会抑制Akt的激活反之亦然。
