心肌缺血再灌注损伤和维拉帕米预处理对大鼠心功能和心肌细胞内钙荧光强度以及L型钙电流的影响  

Effects of verapamil preconditioning on cardiac function in vitro and intraceilniar free Ca2＋ and L- type calcium current in rat cardiomyocytes post ischemia-reperfusion injury

作　　者：余薇[1] 汪晶晶[2] 甘文云[1] 林国生[1] 黄从新[1] 

机构地区：[1]武汉大学人民医院心内科武汉大学心血管研究所,430060 [2]解放军总医院心内科

出　　处：《中华心血管病杂志》2010年第3期225-229,共5页Chinese Journal of Cardiology

基　　金：基金项目：国家自然科学基金（30600241）

摘　　要：目的探讨心肌缺血再灌注（I／R）损伤和维拉帕米预处理对大鼠心脏功能和心肌细胞内钙荧光强度和L型钙电流（ICa-L）的影响。为临床认识I/R损伤发生机制和防治措施提供实验依据。方法利用Langendorff灌流系统建立大鼠离体心脏I／R模型，选取sD大鼠20只分离心脏。正常组（n=6）的心脏由蠕动泵向心脏泵入37℃正常Tyrode液30min，采集数据完成；I／R组（n=7）的心脏先向心脏泵入37℃正常Tyrode液30min，后停灌30min，再用正常Tyrode液再灌注心脏30min，完成I／R过程；维拉帕米组（n=7）的心脏先用正常Tyrode液灌流心脏10min，待各监测指标稳定后，在正常Tyrode液中加入20μmol／L维拉帕米灌注心脏30min，然后停灌30min，再灌注心脏30min，采集数据。用酶解法分离单个心肌细胞，激光扫描共聚焦显微镜加Fluo-3／AM荧光染色技术和全细胞膜片钳技术分别观察心肌细胞内Ca2＋荧光强度和ICa-L大小。结果大鼠心脏经I／R损伤后，收缩无力，舒张顺应性差，心律失常频繁发生，心功能指标明显减弱。单个心肌细胞内Ca2＋荧光非常强烈（与正常组相比，P〈0．01），细胞产出量明显减少，形态特征发生改变ICa-L记录难度大，要求技术高，在电压钳制方式下，ICa-L的最大电流密度显著减小（与正常组相比，P〈0．01），I-V曲线显著上抬。20μmoL／L维拉帕米预处理可使心脏收缩和舒张顺应性流畅，心律失常发生明显减少，与I／R相比，左心室发展压恢复明显增加，再灌注痉挛度显著降低（均为P〈0．01）。心肌细胞内Ca2＋荧光强度明显减弱（P〈0．01），分出细胞质量好，可供长时间记录ICa-L维拉帕米预处理能明显抑制ICa-L最大电流密度发生显著改变（与正常组相比，P〈0．01；与I／R组相比，P〈0．05），I—V曲线处在中部。结论I／R损伤能引起心功能下降和心律失常发生，其机�Objective To investigate the influences of verapamil preconditioning on cardiac function in vitro and intracellular free Ca2＋ and L-type calcium current （ICa-L） in rat cardiomyocytes post ischemia- reperfusion （I/R） injury. Methods The isolated rat hearts in control group （37 ℃ Tyrode solution perfusion for 30 min, n = 6）, I/R group （ no flow for 30 min followed 30 min reperfusion with 37℃ Tyrode solution, n = 7） and verapamil preconditioning group [ 37℃ Tyrode solution perfusion for 10 min, adding verapamil （20 μmol/L ） to Tyrode solution and perfusion for another 30 min, followed then by 30 min no flow and 30 min reperfusion, n = 7 ] using Langendorff perfusion system. The fluorescence intensity of intracellular Ca2＋ was detected with Fluo-3/AM loading by the laser scanning confocal microscope. The ICa-L was recorded via whole-cell patch clamp technique in enzymatically dissociated single rat ventricular myoeytes. Results As expected, arrhythmias and cardiac dysfunction were shown post I/R injury. The fluorescence intensities of intracellular free Ca2＋ in cardiomyocytes were significantly increased compared with control group （P 〈0. O1 ）. By voltage clamp protocol, peak current densities of/ca_L was significantly reduced and I-V curve significantly elevated. Post I/R injury compared with control group （P 〈 0. 01 ） which could be reversed by Verapamil preconditioning. Verapamil preconditioning also significantly improved diastolic and systolic functions, and reduced the incidence of arrhythmias. Conclusions Myocardial I/R injury might significantly impair heart functions and induce arrhythmias via cellular Ca2＋ overload. Verapamil preconditioning could prevent heart I/R injury and reduce arrhythmias by decreasing influx of ICa-L, thereby stabilizing cardiomyocytes in myocardial stunning and avoiding occurrence of Ca2＋ -induced Ca2＋ release during I/R injury. [

关 键 词：维拉帕米 心肌缺血 再灌注损伤 钙 

分 类 号：R965[医药卫生—药理学]