Molecular mechanism of ischemic preconditioning

采用α-肌球蛋白重链启动子制备心脏特异性过表达MEK5的转基因小鼠。MEK5调控BMK1活性，northern blotting证实转基因基因在心脏中成功表达。小鼠心脏兴奋，用Langendorf法逆行灌注克雷布斯缓冲液。全脑缺血后连续监测左室压及其衍生物（dP/dt）。在过表达MEK5的转基因小鼠心脏中，缺血后左心室压和dP/dt的恢复比野生型小鼠更快。这些数据提示MEK5-BMK1通路可能对心肌缺血具有心脏保护作用，并参与缺血预处理的分子机制。蛋白激酶C （PKC）是缺血预处理和心肌肥厚的关键信号分子。二酰基甘油激酶（DGK）是一种控制细胞二酰基甘油水平的酶，可能作为PKC活性的内源性调节剂。缺氧未改变新生儿心肌细胞DGK基因表达水平。内皮素-1增加心肌细胞DGK的表达。AP1是PKC下游的转录因子。荧光素酶报告基因法测定AP1 dna结合活性。内皮素-1增加AP-1 DNA结合活性。转染DGK后，内皮素-1不能以基因剂量依赖的方式增加API的dna结合活性。这些数据表明DGK可能通过控制细胞二酰基甘油水平来调节PKC活性。DGK可能作为gaq偶联受体信号的内源性调节剂，调节缺血预处理和心肌肥厚。

Transgenic mice with cardiac specific overexpression of MEK5 were generated using α-myosin heavy chain promoter. MEK5 regulates BMK1 activity, and successful expression of transgene in the heart was confirmed by Nothern blotting. The mouse heart was excited, and coronary arteries were perfused retrogradely with Krebs buffer by the Langendorf method. Left ventricular pressure and its derivatives (dP/dt) were continuously monitored after global ischemia. In transgenic mouse hearts overexpressing MEK5, recovery of left ventricular pressure and dP/dt after ischemia was more prompt than in wild type mouse hearts. These data suggest that MEK5-BMK1 pathway may act as cardio-protective against myocardial ischemia, and be involved in molecular mechanisms of ischemic preconditioning.Protein kinase C (PKC) is a key signaling molecule for both ischemic preconditioning and cardiac hypertrophy. Diacylglycerol kinase (DGK) is an enzyme responsible for controlling cellular levels of diacylglycerol, and possibly may work as an endogenous regulator of PKC activity. Hypoxia did not change levels of DGK gene expression in neonatal cardiomyocytes. Endothelin-1 increased DGK expression in cardiomyocytes. AP1 is a transcription factor downstream of PKC. AP1 DNA-binding activity was measured by luciferase reporter gene assay. AP-1 DNA binding activity was increased by endothelin-1. After transfection of DGK, endothelin-1 failed to increase API DNA-binding activity in a gene-dose dependent manner. These data suggest that DGK regulates PKC activity possible via controlling cellular diacylglycerol levels. DGK may work as an endogenous regulator of Gaq-coupled receptor signaling and modulate ischemic preconditioning and cardiac hypertrophy.