PKC isoform inhibition in cardiac ischemia/reperfusion

描述（由申请人提供）：心肌缺血后再灌注（MI/R）导致心脏收缩功能障碍。再灌注损伤的特征在于：1）内皮源性一氧化氮（NO）的基础释放减少（内皮功能障碍）; 2）多形核白细胞（PMN）-内皮相互作用增强; 3）PMN浸润到心肌中。缺血后冠状动脉内皮的蛋白激酶C（PKC）抑制剂保留了基础内皮NO释放并抑制随后的PMN粘附和迁移到缺血后心脏组织中。对于特定的PKC亚型，特别是PKC β II和zeta，在MI/R中介导冠状动脉内皮功能障碍和PMN活化的作用知之甚少。亚型特异性PKC抑制剂有可能成为临床MI/R治疗（如冠状动脉血管成形术和心脏移植）的高选择性治疗工具。目前的项目将测试抑制PKC β II和/或PKC zeta将证明在MI/R环境中具有心脏保护作用的假设。将通过这些特定目的来检验该假设：1）在存在和不存在PKC β II肽抑制剂、PKC ζ肽抑制剂以及PKC β II和ζ抑制剂的组合的情况下，将在分离的灌注大鼠心脏中测量左心室发展压力（LVDP）和LVDP的最大速率（+dP/dt max）。将PKC抑制剂在血浆中稀释，并在再灌注期间与PMN共输注到心脏中; 2）在存在/不存在PKC β II抑制剂和/或PKC ζ抑制剂的情况下测量大鼠主动脉内皮的基础NO释放; 3）当用甲酰基-Met-Leu-Phe（fMLP）或佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）刺激时，将测量来自大鼠PMN的超氧化物释放。4）当在存在/不存在PKC β II抑制剂和/或PKC ζ抑制剂的情况下用fMLP或PMA致敏时，测量PMN趋化性; 5）PMN冠状血管粘连，将在存在/不存在PKC抑制剂的情况下评价缺血后心脏组织中的浸润和内皮粘附分子表达。与对照组相比，PKC β II和/或zeta抑制剂可显著改善LVDP、+dP/dt max和内皮基础NO释放，减少PMN超氧化物释放、趋化性、浸润和内皮粘附分子表达。

DESCRIPTION (provided by applicant): Myocardial ischemia followed by reperfusion (MI/R) results in cardiac contractile dysfunction. Reperfusion injury is characterized by: 1) a decrease in the basal release of endothelium-derived nitric oxide (NO) (endothelial dysfunction); 2) enhanced polymorphonuclear leukocyte (PMN)-endothelium interaction; 3) PMN infiltration into the myocardium. Protein kinase C (PKC) inhibition of the post-ischemic coronary endothelium preserves basal endothelial NO release and inhibits the subsequent PMN adherence and transmigration into post-ischemic cardiac tissue. The role of specific PKC isoforms, particularly, PKC beta II and zeta, mediating coronary endothelial dysfunction and PMN activation in MI/R is poorly understood. Isoform specific PKC inhibitors have the potential to be highly selective therapeutic tools in the treatment of clinical MI/R such as coronary angioplasty and in heart transplantation. The current project will test the hypothesis that inhibiting PKC beta II and/or PKC zeta will prove to be cardioprotective in the setting of MI/R. The hypothesis will be tested by these specific aims: 1) Left ventricular developed pressure (LVDP) and the maximal rate of LVDP (+dP/dt max) will be measured in the isolated perfused rat heart in the presence and absence of the PKC beta II peptide inhibitor, a PKC zeta peptide inhibitor, and the combination of PKC beta II and zeta inhibitors. The PKC inhibitors will be diluted in plasma and co-infused with PMNs into the heart during reperfusion; 2) Basal NO release from rat aortic endothelium will be measured in the presence / absence of the PKC beta II inhibitor and/or the PKC zeta inhibitor; 3) Superoxide release from rat PMNs will be measured when stimulated with formyI-Met-Leu-Phe (fMLP) or phorbol-12-myristate-13-acetate (PMA) in the presence / absence of the PKC beta II inhibitor and/or the PKC zeta inhibitor; 4) PMN chemotaxis will be measured when primed with fMLP or PMA in the presence / absence of the PKC beta II inhibitor and/or the PKC zeta inhibitor; 5) PMN coronary vascular adherence, infiltration and endothelial adhesion molecule expression in post-ischemic cardiac tissue will be evaluated in the presence / absence of the PKC inhibitors. It is anticipated that PKC beta II and/or zeta inhibition will significantly improve LVDP, +dP/dt max and endothelial basal NO release, decrease PMN superoxide release, chemotaxis, infiltration and endothelial adhesion molecule expression compared to controls.