Department of Medicine and Cardiology, Heart Center Dresden University Hospital, University of Technology Dresden, P.O. Box 95, Fetscherstr. 76, Dresden 01307, Germany.
Mol Cell Biochem. 2010 Jun;339(1-2):135-47. doi: 10.1007/s11010-009-0377-x. Epub 2010 Jan 7.
Post-conditioning by repetitive cycles of reperfusion/ischemia after prolonged ischemia protects the heart from infarction. The objectives of this study were: Are kinases (PI3-kinase, mTOR, and GSK-3beta) involved in the signaling pathway of post-conditioning? Does post-conditioning result in a diminished necrosis or apoptosis? In open chest rats the infarct size was determined after 30 min of regional ischemia and 30 min of reperfusion using propidium iodide and microspheres. Post-conditioning was performed by three cycles of 30 s reperfusion and reocclusion each, immediately upon reperfusion. PI3-kinase and mTOR were blocked using wortmannin (0.6 mg/kg) or rapamycin (0.25 mg/kg), respectively. The phosphorylation of GSK-3beta and p70S6K was determined with phospho-specific antibodies. TUNEL staining and detection of apoptosis-inducing factor (AIF) were used for the determination of apoptosis. Control hearts had an infarct size of 49 +/- 3%, while post-conditioning significantly reduced it to 29 +/- 3% (P < 0.01). Wortmannin as well as rapamycin completely blocked the infarct size reduction of post-conditioning (51 +/- 2% and 54 +/- 5%, respectively). Western blot analysis revealed that post-conditioning increased the phosphorylation of GSK-3beta by 2.3 times (P < 0.01), and this increase could be blocked by wortmannin, a PI3-kinase inhibitor. Although rapamycin blocked the infarct size reduction, phosphorylation of p70S6K was not increased in post-conditioned hearts. After 2 h of reperfusion, the post-conditioned hearts had significantly fewer TUNEL-positive nuclei (35 %) compared to control hearts (53%; P < 0.001). AIF was equally reduced in post-conditioned rat hearts (P < 0.05 vs. control). Infarct size reduction by ischemic post-conditioning of the in vivo rat heart is PI3-kinase dependent and involves mTOR. Furthermore, GSK-3beta, which is thought to be a regulator of the mPTP, is part of the signaling pathway of post-conditioning. Finally, apoptosis was inhibited by post-conditioning, which was shown by two independent methods. The role of apoptosis and/or autophagy in post-conditioning has to be further elucidated to find therapeutic targets to protect the heart from the consequences of acute myocardial infarction.
缺血后适应通过重复再灌注/缺血循环对长时间缺血的心脏具有保护作用。本研究的目的是：激酶（PI3-激酶、mTOR 和 GSK-3β）是否参与后适应的信号通路？后适应是否导致坏死或凋亡减少？在开胸大鼠中，使用碘化丙啶和微球在 30 分钟局部缺血和 30 分钟再灌注后确定梗死面积。后适应通过三个 30 秒的再灌注和再闭塞循环来实现，即在再灌注时立即进行。PI3-激酶和 mTOR 分别使用 wortmannin（0.6 mg/kg）或 rapamycin（0.25 mg/kg）阻断。用磷酸化特异性抗体测定 GSK-3β和 p70S6K 的磷酸化。TUNEL 染色和凋亡诱导因子（AIF）的检测用于确定凋亡。对照心脏的梗死面积为 49±3%，而后适应可显著降低至 29±3%（P<0.01）。wortmannin 和 rapamycin 完全阻断后适应的梗死面积减少（分别为 51±2%和 54±5%）。Western blot 分析显示，后适应使 GSK-3β的磷酸化增加 2.3 倍（P<0.01），而 PI3-激酶抑制剂 wortmannin 可阻断该增加。尽管 rapamycin 阻断了梗死面积的减少，但后适应心脏中 p70S6K 的磷酸化并未增加。再灌注 2 小时后，后适应心脏的 TUNEL 阳性核数明显少于对照心脏（分别为 35%和 53%；P<0.001）。后适应大鼠心脏的 AIF 同样减少（与对照相比，P<0.05）。体内大鼠心脏缺血后适应的梗死面积减少依赖于 PI3-激酶，并涉及 mTOR。此外，被认为是 mPTP 调节剂的 GSK-3β是后适应信号通路的一部分。最后，通过两种独立的方法证实，后适应抑制了凋亡。在后适应中凋亡和/或自噬的作用有待进一步阐明，以寻找保护心脏免受急性心肌梗死后果的治疗靶点。
