Przyklenk K, Sussman M A, Simkhovich B Z, Kloner R A
Heart Institute, Hospital of the Good Samaritan, Los Angeles, CA 90017, USA.
Circulation. 1995 Sep 15;92(6):1546-57. doi: 10.1161/01.cir.92.6.1546.
Brief episodes of ischemia protect or "precondition" the heart and reduce the size of infarcts caused by subsequent sustained coronary artery occlusion, yet the mechanisms responsible for this cardioprotection remain unresolved. We tested the theory that translocation of protein kinase C (PKC) to the myocyte membranes, initiated in response to brief preconditioning ischemia and manifest during the initial minutes of the sustained occlusion, mediates this phenomenon by attempting to (1) blunt the cardioprotective effects of preconditioning by administration of the PKC inhibitors H-7 and polymyxin B, (2) visualize by fluorescence staining and confocal microscopy changes in the amount or location of PKC, and (3) quantify by incorporation of 32P into PKC-specific peptide changes in the subcellular distribution of PKC in preconditioned versus control hearts.
In the first three limbs of this study, anesthetized open-chest dogs underwent four 5-minute episodes of preconditioning ischemia or a comparable control period before 1 hour of sustained occlusion and 4 to 5 hours of reperfusion. Collateral blood flow was assessed with radioactive microspheres; area at risk (AR) was delineated by injection of blue dye; and the area of necrosis (AN) was measured by tetrazolium staining. AN/AR was smaller in preconditioned versus control dogs that received no treatment (6 +/- 2% versus 19 +/- 3%, P < .01), H-7 (2 +/- 2% versus 14 +/- 5%, P < .02), or polymyxin B (10 +/- 3% versus 29 +/- 5%, P < .01) during the preconditioning or control period. Additional dogs underwent four 5-minute episodes of ischemia, with biopsies obtained at baseline and after the first and fourth occlusions. Frozen sections were stained with a fluorescent probe for active PKC and viewed with confocal microscopy. No differences in the intensity or distribution of fluorescence staining were observed after brief ischemia compared with baseline. Finally, myocardial samples were obtained from dogs subjected to four 5-minute episodes of preconditioning ischemia and time-matched sham-operated controls. Incorporation of 32P into PKC-specific peptide revealed no quantitative difference in the subcellular distribution of PKC between control and preconditioned cohorts.
H-7 and polymyxin B did not blunt the reduction in infant size achieved with ischemic preconditioning. Neither fluorescence staining and confocal microscopy nor biochemical quantification revealed evidence of preconditioning-induced translocation of PKC to the cell membranes. These results fail to support the hypothesis that translocation of PKC, triggered by preconditioning ischemia, is an important mechanism for the reduction in infarct size seen with preconditioning in the dog model.
短暂的缺血发作可保护心脏或使其“预处理”，并减小随后持续性冠状动脉闭塞所导致的梗死面积，然而这种心脏保护作用的机制仍未明确。我们对以下理论进行了验证：蛋白激酶C（PKC）因短暂的预处理缺血而转位至心肌细胞膜，并在持续性闭塞的最初几分钟内表现出来，通过以下方式介导这一现象：（1）给予PKC抑制剂H-7和多粘菌素B以削弱预处理的心脏保护作用；（2）通过荧光染色和共聚焦显微镜观察PKC数量或位置的变化；（3）通过将32P掺入PKC特异性肽来量化预处理心脏与对照心脏中PKC亚细胞分布的变化。
在本研究的前三个部分中，麻醉开胸犬在经历1小时持续性闭塞和4至5小时再灌注之前，先接受4次5分钟的预处理缺血发作或一段类似的对照期。用放射性微球评估侧支血流；通过注射蓝色染料勾勒出危险区域（AR）；通过四氮唑染色测量坏死面积（AN）。在预处理或对照期未接受治疗的预处理犬与对照犬相比，AN/AR较小（6±2%对19±3%，P<.01），接受H-7治疗的犬（2±2%对14±5%，P<.02），以及接受多粘菌素B治疗的犬（10±3%对29±5%，P<.01）。另外的犬接受4次5分钟的缺血发作，在基线以及第一次和第四次闭塞后获取活检组织。冰冻切片用活性PKC的荧光探针染色，并通过共聚焦显微镜观察。与基线相比，短暂缺血后荧光染色的强度或分布未观察到差异。最后，从经历4次5分钟预处理缺血发作的犬以及时间匹配的假手术对照犬获取心肌样本。将32P掺入PKC特异性肽显示，对照组和预处理组之间PKC的亚细胞分布没有定量差异。
H-7和多粘菌素B并未削弱缺血预处理所实现的梗死面积减小。荧光染色和共聚焦显微镜检查以及生化定量均未显示预处理诱导PKC转位至细胞膜的证据。这些结果未能支持以下假设：预处理缺血触发的PKC转位是犬模型中预处理所见梗死面积减小的重要机制。
