Molecular mechanism of ischemic preconditioning

使用α-肌球蛋白重链启动子产生心脏特异性过表达MEK 5的转基因小鼠。MEK 5调节BMK 1活性，并且转基因在心脏中的成功表达通过Nothing印迹证实。兴奋小鼠心脏，用Langendorf法用Krebs缓冲液逆行灌注冠状动脉。全脑缺血后连续监测左室压力及其导数（dP/dt）。在转基因小鼠心脏过度表达MEK 5，恢复左心室压力和dP/dt缺血后比野生型小鼠心脏更迅速。提示MEK 5-BMK 1通路可能参与了缺血预适应的分子机制，而蛋白激酶C（PKC）是缺血预适应和心肌肥大的关键信号分子。甘油二酯激酶（DGK）是一种负责控制细胞内甘油二酯水平的酶，可能是PKC活性的内源性调节因子。缺氧并没有改变新生心肌细胞DGK基因表达水平。内皮素-1增加心肌细胞DGK的表达。AP 1是PKC下游的转录因子。用荧光素酶报告基因测定法测定AP 1的DNA结合活性。内皮素-1可增加AP-1的DNA结合活性。转染DGK后，内皮素-1未能以基因剂量依赖性方式增加API DNA结合活性。这些数据表明，DGK可能通过控制细胞二酰甘油水平来调节PKC活性。DGK可能作为Gaq偶联受体信号的内源性调节剂，调节缺血预适应和心肌肥厚。

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.