银杏酮酯预处理改善心肌缺血再灌注损伤的钙稳态调节机制

Sarcoplasmic Ca2+ overload is a critical mechanism of ischemia reperfusion (I/R) injury at the cardiomyocyte level.Recently, we showed that Ginkgo biloba extract 50(GBE50) has a significant preventive effect on heart function decline induced by the lesion of myocardial ischemia-reperfusion. Although calcium handling dysfunction is known to impair cardiac calcium homeostasis under I/R conditions, the status of calcium handling proteins and ion channels during I/R in the hearts pretreated by GBE 50 is unknown..In the project, after animal studies (Langendorf perfusion surgery), the effects of GBE50 on the protein expression of calcium handling proteins within myocardial tissue of rats, including Cav1.2(α1c subunit of L-type calcium channel), ryanodine receptor(RyR2),inositol 1,4,5-trisphosphate 2 (IP3R2),sarco(endo)plasmic reticulum Ca2+-ATPase(SERCA),Na+/Ca2+ exchange (NCX),protein kinase A (PKA) and protein kinase C(PKC), will be investigated during I/R and Ca2+ handling in the hearts of rats subjected to myocardial ischemia reperfusion injury will be determined. The effects of GBE50 on ICa-L, IK-ATP and INa/Ca of single ventricular myocytes during I/R will be recorded by whole-cell patch clamp technique in voltage clamp mode. By using spectro?uorometry to measure intracellular Ca2+ concentration ([Ca2+]i) and its responses to electrical stimulation and caffeine in myocytes from the left ventricle of rats, the effects of GBE50 on the uptake of Ca2+ by SERCA and extrusion by Na+/Ca2+ exchange (NCX) during I/R ,with or without inhibitors of PKA or PKC, will be determined..These studies will identify the role of GBE50 on altered handling of Ca2+ by SR and sarcolemmal membrane and proteins damaged during I/R and potentially lead GBE50 to clinical therapies for myocardial ischemia reperfusion. Our project will provide a new insight into the understanding of the intrinsic defensive mechanism and demonstrate the importance of preserving SR and calcium handling protein function in the cardiac protection afforded by GBE50.

心肌细胞内钙超载是心肌缺血再灌注（I/R）损伤导致心肌细胞损伤的最终共同通路。我们前期工作发现银杏酮酯（GBE50）能预防实验性心肌I/R损伤。本课题拟在离体水平观察GBE50预处理对I/R大鼠心肌与钙转运相关的L型钙通道的α1c亚单位，雷诺定受体2 型，三磷酸肌醇受体2型，肌浆网钙ATP 酶(SERCA)，钠钙交换蛋白（NCX）及NCX磷酸化调节通路中蛋白激酶PKA和PKC表达的作用；在细胞水平研究GBE50对I/R心肌细胞L型钙通道、ATP敏感钾通道和钠钙交换等离子通道的影响；观察在有/无蛋白激酶抑制剂时I/R心肌细胞内钙瞬变（E[Ca2+]i和C[Ca2+]i）的变化，探讨GBE50对I/R心肌细胞SERCA再摄取和NCX移除Ca2+功能的影响及其是否通过PKA和/或PKC起作用。旨在明确GBE50影响I/R心肌细胞钙转运的作用靶点，阐明GBE50改善心肌I/R损伤的钙稳态调节机制。

心肌细胞内钙超载是心肌缺血再灌注（I/R）损伤导致心肌细胞损伤的最终共同通路。我们前期工作发现银杏酮酯（GBE50）可改善在体急性心肌缺血大鼠心功能和减轻心脏形态学损伤的抗心肌缺血效应与GBE50减轻心肌缺血时钠钙交换体（NCX）的上调有关。本课题先在离体水平观察到GBE50通过开放缺血时ATP敏感的钾通道，减轻再灌时逆向钠钙交换改善心脏缺血再灌后的心功能；在细胞水平通过膜片钳实验发现GBE50减轻缺血再灌时钠钙交换的外向电流即逆向钠钙交换，但对L型钙电流无影响，且细胞内免疫荧光结果并未显示钠钙交换和Cav 1.2蛋白表达的差异；心肌细胞内钙瞬变（E[Ca2+]i和C[Ca2+]i）的测量结果显示I/R使咖啡因诱导的钙瞬变幅度降低，衰变延迟，提示I/R导致肌浆网钙含量减少，钠钙交换活性减弱，使钙从胞浆排出的速度减慢，GBE50可逆转I/R导致的咖啡因诱导的钙瞬变幅度即肌浆网钙含量降低，及促进其衰变，增加钠钙交换的活性，加快钙从胞浆排出。以上结果说明GBE50作用 I/R心肌细胞钙转运的靶点位于钠钙交换体，GBE50通过加强顺向钠钙交换向胞外移除Ca2+功能，抑制逆向钠钙交换，改善心肌I/R损伤导致的钙超载。本课题阐明银杏酮酯预处理改善心肌缺血再灌注损伤的钙稳态调节的内源性保护机制，为心肌缺血再灌注损伤提供新型药物和靶点，丰富心肌缺血预适应和药物预适应的内涵，为其今后运用于临床提供理论依据。