Nishizawa J, Nakai A, Higashi T, Tanabe M, Nomoto S, Matsuda K, Ban T, Nagata K
Department of Cardiovascular Surgery, Faculty of Medicine, Kyoto University, Japan.
Circulation. 1996 Nov 1;94(9):2185-92. doi: 10.1161/01.cir.94.9.2185.
The myocardial protective role of heat shock protein (HSP) has been demonstrated, and there has been increasing interest in stress response in the heart. We examined the DNA-binding activity of heat shock transcription factor (HSF), by which the transcription of heat shock genes is mainly regulated, during heat shock or ischemia/reperfusion in isolated rat heart.
Rat hearts were isolated and perfused with Krebs-Henseleit buffer by the Langendorff method. Whole-cell extracts were prepared for gel mobility shift assay using oligonucleotides containing the heat shock element, which is present upstream of all heat shock genes. Induction of mRNAs for HSP70, HSP90, and GRP78 (glucose-regulated protein) was examined by Northern blot analysis. Although the activation of HSF during global ischemia was weak and rapidly attenuated, postischemic reperfusion induced a significant activation of HSF. In addition, although HSP70 mRNA was hardly induced during ischemia, its burst induction was detected during postischemic reperfusion. Supershift assays using specific antisera for HSF1 and HSF2 revealed that ischemia/reperfusion as well as heat shock induced the activation of HSF1 in hearts. Although the expression of HSP70 mRNA during heat shock was more vigorous than the expression during ischemia/reperfusion, the induction of HSP90 mRNA in postischemic reperfusion was significantly greater than that in heat shock.
Our findings demonstrated that reperfusion causes a significant activation of HSF1 in ischemia-reperfused heart. The striking contrast between the induction of HSP70 mRNA and that of HSP90 mRNA suggests the presence of regulatory mechanisms other than HSF.
热休克蛋白（HSP）的心肌保护作用已得到证实，并且人们对心脏中的应激反应越来越感兴趣。我们研究了在离体大鼠心脏热休克或缺血/再灌注期间，热休克转录因子（HSF）的DNA结合活性，热休克基因的转录主要受该因子调控。
采用Langendorff法分离大鼠心脏，并用Krebs-Henseleit缓冲液灌注。制备全细胞提取物用于凝胶迁移率变动分析，使用含有热休克元件的寡核苷酸，该元件存在于所有热休克基因的上游。通过Northern印迹分析检测HSP70、HSP90和GRP78（葡萄糖调节蛋白）mRNA的诱导情况。虽然在全心缺血期间HSF的激活较弱且迅速减弱，但缺血后再灌注诱导了HSF的显著激活。此外，虽然在缺血期间几乎未诱导HSP70 mRNA，但在缺血后再灌注期间检测到其爆发性诱导。使用针对HSF1和HSF2的特异性抗血清进行的超迁移分析表明，缺血/再灌注以及热休克均可诱导心脏中HSF1的激活。虽然热休克期间HSP70 mRNA的表达比缺血/再灌注期间更活跃，但缺血后再灌注中HSP90 mRNA的诱导明显大于热休克期间。
我们的研究结果表明，再灌注可导致缺血再灌注心脏中HSF1的显著激活。HSP70 mRNA和HSP90 mRNA诱导之间的显著差异表明存在HSF以外的调节机制。
