抑制CaMKII-AMPK信号通路优化缺血心肌糖和脂肪酸代谢的机制研究

Optimizing cardiac energy substrate metabolism is currently a research focus for treating ischemic heart disease. Ca2+/calmodulin–dependent protein kinase II (CaMKII) has been shown to increase the transport of glucose into the cell via the glucose transporters (GLUT) 4 in the skeletal muscle. Importantly, CaMKII lies the upstream of AMP-activated protein kinase (AMPK) in regulating free fatty acids (FA) uptake and oxidation in the skeletal muscle. However, the functional role of CaMKII in regulating heart glucose and FA metabolism has not been established. Our pilot studies showed that CaMKII and AMPK expression are increased in the heart of patients with terminal ischemic heart disease who received heart transplantation in our hospital. Our further gene-chip results indicated that transgenic inhibition of cardiac CaMKII (AC3-I) in the heart down-regulates the gene expression of AMPK and other enzymes of FA metabolism after myocardial infarction (MI). Based on these observations, we hypothesized that targeting inhibition of CaMKII in the heart may inhibit glycolysis and down-regulate the AMPK signal pathway, and as a result reducing FA uptake and oxidation to protect the heart against ischemic damage. Thus, this study is designed to demonstrate the predicted phosphorylation sites of AMPK by CaMKII according to the GPS 3.0 software. We'll also check the predicted phosphorylation sites of FAT/CD36, ACC and MCD by AMPK. To test our hypothesis, We'll investigate the involvement of CaMKII-AMPK signal path-way in cardiac glucose and FA uptake and oxidation. We'll also use cultured neonatal mouse myocytes to further study the mechanisms of inhibition of CaMKII in regulating glucose and FA metabolism under the condition of simulated myocardial ischemia. We believe that this study will provide evidence for a basic understanding of CaMKII in regulating glucose and FA metabolism in ischemic heart disease and for finding potentially promising approach in treating ischemic heart disease.

优化缺血性心脏病的能量代谢是当前的研究热点。研究表明骨骼肌的Ca2+/钙调蛋白依赖性蛋白激酶II(CaMKII)的激活可促进葡萄糖的转运和糖酵解，并作用于腺苷酸活化蛋白激酶(AMPK)而促进脂肪酸（FA）代谢，但有关心脏的CaMKII是否具有类似作用未见报道。我们的预实验发现：在转基因心脏特异性CaMKII抑制小鼠，基因芯片揭示心肌梗死后AMPK基因下降，部分FA代谢酶基因发生改变；而且终末期缺血性心脏病患者心肌中的CaMKII和AMPK表达增加。据此我们提出假说：通过抑制心脏CaMKII-AMPK信号通路可抑制糖酵解，并降低FA的摄取和氧化而增加葡萄糖的有氧氧化。本课题拟从整体、器官、细胞和分子水平研究CaMKII-AMPK信号通路在缺血性心脏病糖和FA代谢中的作用及其机制，阐明心脏CaMKII与AMPK、AMPK与下游信号分子的相互作用，从而为寻找缺血性心脏病新的代谢治疗靶点提供依据。

AMP激活激酶（AMPK）信号通路在心肌缺血期间被激活，并促进心肌脂肪酸（FA）的摄取和氧化。而多功能Ca2+/钙调素依赖性蛋白激酶II（CaMKII）也可在心肌缺血时被激活，但两者在心肌FA代谢中的作用及其联系尚不清楚。在本课题，我们通过研究心脏CaMKII对AMPK乙酰辅酶A羧化酶（ACC）、丙二酰辅酶A脱羧酶（MCD）和分化的FA反式酶簇36（FAT/CD36）以及心脏FA摄取和氧化的影响，探讨了CaMKII在心肌缺血期间对FA代谢的影响。此外，我们测试了CaMKII是否和AMPK存在增加蛋白相互作用。我们的研究结果表明晚期缺血性心脏病患者的心脏中CaMKII、AMPK和ACC的磷酸化均显著增加，MCD和FAT/CD36的表达增加。具有CaMKII基因心肌抑制作用的AC3-I小鼠心脏AMPK基因表达减少。在心肌梗死（MI）后AC3-I小心脏中，AMPK-ACC磷酸化、MCD和FAT/CD36水平、心脏FA摄取和FA氧化均显著降低。值得注意的是，我们证明了心脏中CaMKII与AMPKα1和α2亚基存在相互作用。此外，AC3-I小鼠心肌梗死后2周心肌肥厚和细胞凋亡明显减少。以上发现揭示了抑制CaMKII可通过与AMPK信号相互作用而抑制FA代谢，这可能是缺血性心脏病的一种新的机制，抑制CaMKII可能是治疗缺血性心脏病的新的靶点。