成年大鼠心肌细胞高效激光共聚焦显微镜钙成像方法

Efficient Laser Confocal Ca^(2+) Imaging of Adult Rat Cardiac Myocytes

目的建立高效易行的测定心肌细胞钙成像方法。方法 Langendorff灌流获取成年大鼠心室肌细胞。大鼠心肌细胞经Fluo-4染色后分别加入玻璃底的6孔培养板中,连接Ion C-PaceEP刺激器,于激光共聚焦显微镜下测定胞内钙离子浓度的动态变化。结果约80%的细胞跟随所设频率规律的收缩。激光共聚焦显微镜记录到与刺激器频率相同的有规则的细胞钙瞬变。加入咖啡因后钙释放增加,提示此为钙库的钙释放。结论利用新型的刺激装置,结合激光共聚焦显微技术,我们成功地获得了高效易行的成年大鼠心肌细胞Ca2+成像方法。此法也可用于其他实验动物心肌细胞,易于记录细胞内钙离子浓度的动态变化。

Objective To establish an efficient and reliable method to measure the calcium release in cardiac myocytes freshly isolated from adult rodents. Methods Rat hearts were perfused on a modified I_angen- dorff system. Ventricular myocytes were isolated and loaded with Fluo-4 fluorescent Ca2＋ indicator. Cells were added into different wells of a 6 well glass-bottom plate. Next, confocal Ca2＋ imaging was performed with laserscanned confocal microscopy while the plate was connected to stimulator and cells were paced at different frequencies. Results Adult rat ventricular myocytes were quiescently with rod-shape morphology and clear and smooth edges. Approximately 80% of cells responded to pacing. Single cell line scan showed regular Ca^2＋ transients while caffeine （ 10 mM） triggered a drastic release of Ca^2＋ or depletion of the intracellular calcium store. Conclusion We have successfully developed an efficient method for high quality laser confocal Ca2＋ imaging with isolated adult rat cardiac myocytes. This method allows efficient testing of multiple samples within limited times. Moreover, it can be extent to other animal cardiac myocytes in order to record the Ca2＋ handling properties by Ca2＋ imaging.