Development of the System for Measurement of Intramyocardial Ion Dynamics in Isolated, Perfused Hearts

心肌内离子动力学的测量对于心脏生理学和病理生理学的研究具有重要意义。在细胞内离子浓度的测量中，使用荧光染料的方法具有时间分辨率高的优点。然而，将荧光染料应用于分离的、灌流的心脏一直是困难的。为了将染料加载到灌流的心脏中，我们开发了使用离子导入技术的系统。在本研究中，我们完成了以下几项工作：1.优化了染料离子导入微电极的制备方法；2.确定了电极的最佳电流条件和最佳位置；3.发展了停止或减弱心脏跳动的方法。首先，降低灌流液中的钙离子浓度，然后增加钾离子浓度或在灌流液中加入BDM。4.在我们的方法中，在心脏上方诱导微电极，从显微镜的顶部进行离子导入。相比之下，紫外线是从显微镜底部进入心脏的。因此，有必要在两次手术之间改变心脏的方向，并导致定向障碍。为了避免这种情况，我们开发了一种保持心脏定位的方法。我们的研究表明，在离体心中进行离子导入是可行的，我们完成了这种方法所需的一些基本技术部分。此外，我们还用荧光染料和核磁共振技术评价了蛋白质对钙离子测量的不同影响。

It is important to measure intramyocardial ion dynamics for the investigation of physiology and pathophysiology of hearts. The measurements of intracellular ion concentration, the method using fluorescent dyes has advantages including high temporal resolution. However, it has been difficult to apply fluorescent dyes into isolated, perfused hearts. To load dyes to perfused hearts, we developed the system using the iontophoresis technique. In this study, we completed the following items ;1.We optimized the method to produce microelectrodes for iontophoresis of dyes.2.We determined the optimal conditions for the current and the optinal positioning of the electrode.3.We developed the method to stop or attenuate the beating of the hearts. First, the calcium concentration in the perfusate was decreased, and then, potasium concentration was increased or BDM was added to the perfusate.4.In our method, microelectrode was induced above the heart, and iontophoresis was performed from the top of the stage of microscope. In contrast, the ultraviolet beam was introduced to the heart from the bottom of the microscope. Thus, it was necessary to change the orientation of the heart between the procedures, and caused the disorientation. To avoid this, we developed the method to keep the orientation of hearts.Our study indicates the feasibility of the iontophoresis in isolated, perfused hearts, and we accomplished some basic part of the techniques required for this approach. Nevertheless, there are still many remaining problems to make this technique more practical.In addition, we also evaluated the different effects of proteins on Ca^<2+> ion measurements using fluorescent dye or NMR technique.