氯通道在缺血再灌注心肌舒张期钙超载形成中的作用与机制

Intracellular calcium overload is a major factor in eliciting cell death during myocardial ischemia/reperfusion injury. However, the mechansim behind calcium overload has not been well elucidated. Many chloride channels such as voltage-dependent, PKA activated (CFTR), and calcium-activated chloride are present in the heart. We and others have previously demonstrated that NPPB, the chloride channel blocker, attenuated the calcium overload-induced heart injury, therefore, the aim of this study is to investigate which subtype of chloride channels is involved in the development of calcium overload, and whether activation of sodium/calcium exchanger mediates this effect. In the first series experimental protocol, siRNA technique and channel blockers are used to decrease the expression or block the opening of chloride channels, and the ischemia/reperfusion-induced cell death and arrhythmias are evaluated. Secondly, the effects of chloride channels on the intracellular calcium level during myocardial ischemia/reperfusion are measured using Fura-2, and its effects on calcium overload-induced injury are studied in the cultured myocytes and isolated rat hearts by measuring the activity of calpain, a calcium-dependent protease. Finally, the effects of chloride channels on the activity of sodium/calcium exchanger, calcium pump in sarcolemmal membrane and SR are also measured, and the effects of calcium overload on chloride current are also studied. The results from this project would help us to understand the role for chloride channels in calcium overload in the ischemia/reperfused heart. It may also provide some clues to improve the treatments of heart injury.

胞内钙超载是缺血再灌注心肌损伤的重要原因，其形成机制并不十分明确。心肌存在电压依赖、囊性纤维化跨膜电导调节体和钙激活型氯通道，现认为，氯通道激活主要影响膜电位。然而，我们发现，氯通道抑制剂及氯离子替代可拮抗钙超载造成的钙依赖蛋白酶calpain的活化与细胞死亡。据此我们的科学问题是：氯通道是否参与缺血再灌注心肌钙超载形成？参与的亚型有哪些？其作用是否与已知参与钙超载形成的钠-钙交换体有关？本项目拟采用siRNA、阻断剂干预、基因敲除及钙荧光强度测定等方法，在细胞和离体心脏水平探讨：氯通道在缺血再灌注和急性钙超载（无钙液与有钙液接替灌注）所引发心肌损伤中的作用，对舒张期胞内钙水平和calpain活化的影响，对质膜钠-钙交换体与钙泵，以及肌浆网钙泵的影响，同时观察钙超载对不同种类氯电流的影响。完成此课题有助于阐明氯通道在缺血再灌注心肌钙超载形成中的作用与分子机制，为临床心肌保护提供新思路。

临床资料发现，动脉搭桥手术后发生房颤的患者伴随血清Cl-浓度的升高。基础研究表明，氯通道阻断剂NPPB和DIDS减轻缺氧-复氧心肌损伤。但氯通道引起心肌损伤的分子机制并不十分清楚。本课题从钙超载角度探讨氯通道引起心肌损伤的机制。在离体灌注大鼠心脏，建立全心缺血（45 min）/再灌注（2 h）模型，或无钙液（3 min）、有钙液（30 min）接替灌注的钙反常模型（造成胞内钙超载，心肌大面积死亡），并采用氯通道阻断剂NPPB和DIDS，以及含90 mM Cl-的KH液灌注减少Cl-跨膜转运。实验观察到，三种处理方式显著减轻缺血再灌注、钙反常造成的心肌损伤，减弱钙超载损伤分子钙蛋白酶的活性，减少钙蛋白酶底物分子a-fodrin的水解。钙/钙调素依赖的蛋白激酶（CaMKII）是心肌胞内钙超载的重要分子，在钙反常模型中可见，Ryanodine和thapsigargin（分别抑制肌浆网钙释放通道和钙泵）并未减轻钙反常引发的心肌损伤，但CaMKII抑制剂KN-62显著减弱钙蛋白酶的活性，减少膜骨架蛋白的降解，保护膜的完整性。谷氨酸的阴离子特性可竞争性拮抗Cl-跨膜转运，灌流液中添加谷氨酸显著增加其胞内水平，减少钙反常所致的心肌坏死与凋亡，保护心功能，同时降低心肌组织钙蛋白酶活性。谷氨酸转运体抑制剂DL-TBOA减弱这些现象。这些结果提示：（1）氯通道引起的心肌损伤作用与加重胞内钙超载，激活损伤分子钙蛋白酶有关；（2）CaMKII可不依赖肌浆网钙上调钙蛋白酶活性，进而引起心肌损伤；也提示，氯通道的损伤作用可不依赖肌浆网钙；（3）肌膜存在谷氨酸转运体，添加谷氨酸主要通过增加胞内浓度实现心肌保护作用，其竞争性拮抗Cl-跨膜转运的作用不可排除。