Inhibition of mitochondrial permeability transition pore opening by ischemic preconditioning is probably mediated by reduction of oxidative stress rather than mitochondrial protein phosphorylation.
Inhibition of mitochondrial permeability transition pore opening by ischemic preconditioning is probably mediated by reduction of oxidative stress rather than mitochondrial protein phosphorylation.
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DOI：
10.1161/circresaha.107.167072
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发表时间：
2008-05-09
影响因子：
20.1
通讯作者：
Halestrap, Andrew P.
中科院分区：
文献类型：
--
作者：
Clarke, Samantha J.;Khaliulin, Igor;Das, Manika;Parker, Joanne E.;Heesom, Kate J.;Halestrap, Andrew P.
关键词：
再灌注时线粒体通透性过渡孔（MPTP）开放的抑制对缺血预处理（IP）的心脏保护至关重要。一些研究暗示线粒体蛋白磷酸化在这种作用中。在这里，我们证实从缺血前对照和IP心脏中快速分离的线粒体在钙介导的MPTP开放方面没有显着差异，而在30分钟的全温缺血或3分钟的再灌注后，IP心脏中分离的线粒体在钙介导的MPTP开放方面抑制MPTP开放。使用2D和1D电泳分析密度梯度纯化线粒体中的蛋白质磷酸化，使用Pro-Q Diamond或磷酸化氨基特异性抗体检测磷酸化蛋白。检测到几种磷酸化蛋白，包括电压依赖性阴离子通道异构体1和2，但在缺血前或缺血再灌注期间均未显示出显著的ip介导的变化。Western blotting和2-D荧光差凝胶电泳（DIGE）也没有检测到蛋白激酶C （α， ε或δ亚型），糖原合成酶激酶3β (GSK3β)或Akt在IP后向线粒体的易位。在冷冻夹紧的心脏中，缺血和再灌注后检测到GSK3β、Akt和amp活化蛋白激酶（AMPK）磷酸化的变化，但没有ip介导的与MPTP抑制或心脏保护相关的变化。然而，线粒体蛋白羰基化（氧化应激的替代标志物）的测量表明，缺血结束和再灌注期间线粒体氧化应激的减少可能是ip介导的MPTP抑制的原因。我们还讨论了介导这种效应并在再灌注过程中维持这种效应的信号通路。
Inhibition of mitochondrial permeability transition pore (MPTP) opening at reperfusion is critical for cardioprotection by ischemic preconditioning (IP). Some studies have implicated mitochondrial protein phosphorylation in this effect. Here we confirm that mitochondria rapidly isolated from pre-ischemic control and IP-hearts show no significant difference in calcium-mediated MPTP opening, whereas IP inhibits MPTP opening in mitochondria isolated from IP-hearts following 30 min global normothermic ischemia or 3 min reperfusion. Analysis of protein phosphorylation in density-gradient purified mitochondria was performed using both 2D and 1D electrophoresis with detection of phosphoproteins using Pro-Q Diamond or phospho-amino specific antibodies. Several phosphoproteins were detected, including voltage-dependent anion channels isoforms 1 and 2, but none showed significant IP-mediated changes either before ischemia or during ischemia and reperfusion. Nor did either Western blotting or 2-D fluorescence difference gel electrophoresis (DIGE) detect translocation of protein kinase C (α, ε or δ isoforms), glycogen synthase kinase 3β (GSK3β), or Akt to the mitochondria following IP. In freeze-clamped hearts changes in phosphorylation of GSK3β, Akt and AMP-activated protein kinase (AMPK) were detected following ischemia and reperfusion but no IP-mediated changes correlated with MPTP inhibition or cardioprotection. However, measurement of mitochondrial protein carbonylation, a surrogate marker for oxidative stress, suggested that a reduction in mitochondrial oxidative stress at the end of ischemia and during reperfusion might account for IP-mediated inhibition of MPTP. The signalling pathways mediating this effect and maintaining it during reperfusion are discussed.