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长时间保存的急性分离心肌细胞代谢产物影响L-型Ca2+通道电流

岳志杰, 圣娟娟, 谢满江, 余志斌^*

第四军医大学航空航天医学教育部重点实验室，航空航天生理教研室，西安 710032

摘要

采取较小容量保存缓冲液保存急性分离的心肌细胞，进行L-型Ca2+通道特性观测时，在细胞分离6 h后，L-型Ca2+通道电流峰值变异性明显增大，其原因尚不清楚。为获取更多的实验数据，且确保数据的准确与稳定性，本研究旨在观测小容量保存缓冲液保存体系pH的变化及其对心肌细胞形态、线粒体功能与L-型Ca2+通道特性的影响。将急性分离的心肌细胞分别保存于100 mL大容量保存液和20 mL小容量保存液中。结果显示，在10 h保存过程中，100 mL大容量保存液组pH保持不变；而20 mL小容量保存液组，其溶液pH从7.20降至6.95，导致轻度酸中毒。20 mL小容量保存液组酸中毒不仅使异常形态的长杆状心肌细胞比例增加(保存时间≥6 h)，而且使心肌细胞线粒体膜电位逐步降低(保存时间≥8 h)，线粒体NADPH自发荧光由蓝色转向绿色(保存时间≥8 h)，提示能量代谢受阻。由于这些变化，保存10 h时，心肌细胞L-型Ca2+通道电流峰值呈显著性降低，峰值电流钳制电位从+10 mV向0 mV偏移。上述结果提示长时间保存急性分离心肌细胞，宜采用较大容量的缓冲液体系，或者采取心肌细胞形态与NADPH蓝色荧光两种方法相结合，选择线粒体功能良好的细胞，进行L-型Ca2+通道特性观测。

关键词： 心肌细胞; 酸中毒; 线粒体膜电位; L-型Ca2+通道

分类号：R337.2

[Metabolites of long-time preserved-acutely isolated rat cardiomyocytes affect L-type Ca2+ channel current.] [Article in Chinese]

YUE Zhi-Jie, Sheng Juan-Juan, Xie Man-Jiang, YU Zhi-Bin^*

Key Laboratory of Aerospace Medicine, Ministry of Education； Department of Aerospace Physiology, the Fourth Military Medical University, Xi’an 710032, China

Abstract

The variability of peak current of L-type calcium channel (I(Ca,L)) shows an increase in cardiomyocytes after 6 h of preservation when the acutely isolated cardiomyocytes are preserved in a small volume buffer solution. The mechanism of the increased variability of I(Ca,L) is not clear. In order to obtain more accurately and stably experimental data of I(Ca,L), the aim of this study was to observe the pH changes of preservation buffer solution with acutely isolated rat cardiomyocytes, and the effects of pH changes on the shape of cardiomyocytes, the function of mitochondria and the gating property of L-type calcium channel. The results indicated that the pH was kept stable in 100 mL buffer solution, but was decreased from 7.20 to 6.95 in 20 mL buffer solution during 10 h of cardiomyocyte preservation. Therefore, 100 mL or 20 mL preservation solution was used as a normal control or acidotic group, respectively. The ratio of abnormal to normal rod-shaped cardiomyocytes increased in the acidotic group after 6 h of preservation. The acidosis induced a reduction in mitochondrial membrane potential indicated by JC-1 fluorescent probe after 8 h of cardiomyocyte preservation. The acidosis also shifted the autofluorescence of NADPH from blue to green after 8 h of cardiomyocyte preservation. The above changes in mitochondrial function induced a significant decrease in the peak I(Ca,L) and a shift in the clamped voltage at peak I(Ca,L) from +10 mV to 0 mV, after 10 h of cardiomyocyte preservation. These results suggest that the best way to preserve acutely isolated cardiomyocytes is to use a larger volume buffer system. In order to get stable peak I(Ca,L), we need to not only select a normal shape of cardiomyocyte at a bright field but also a blue fluorescent myocyte at an ultraviolet excitation.

Key words: Cardiomyocyte; acidosis; mitochondrial membrane potential; L-type calcium channel

收稿日期：2012-05-12　　录用日期：2012-06-30

通讯作者：余志斌　　E-mail: yuzhib@fmmu.edu.cn

引用本文：

岳志杰, 圣娟娟, 谢满江, 余志斌. 长时间保存的急性分离心肌细胞代谢产物影响L-型Ca2+通道电流[J]. 生理学报 2013; 65 (1): 83-88.

YUE Zhi-Jie, Sheng Juan-Juan, Xie Man-Jiang, YU Zhi-Bin. [Metabolites of long-time preserved-acutely isolated rat cardiomyocytes affect L-type Ca2+ channel current.] [Article in Chinese]. Acta Physiol Sin 2013; 65 (1): 83-88 (in Chinese with English abstract).