CONTINUAL ELECTRIC-FIELD STIMULATION PRESERVES CONTRACTILE FUNCTION OF ADULT VENTRICULAR MYOCYTES IN PRIMARY CULTURE
CONTINUAL ELECTRIC-FIELD STIMULATION PRESERVES CONTRACTILE FUNCTION OF ADULT VENTRICULAR MYOCYTES IN PRIMARY CULTURE
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DOI：
10.1152/ajpheart.1994.266.1.h341
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发表时间：
1994-01-01
影响因子：
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通讯作者：
KELLY, RA
中科院分区：
文献类型：
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作者：
BERGER, HJ;PRASAD, SK;KELLY, RA
为了更逼真地模拟原代培养中新鲜分离的心肌鳗鱼的机电功能，我们描述了一种以生理频率连续电刺激成年心肌细胞数天的技术。构建可重复使用的塑料盖，以适应标准的一次性175 cm（2）组织培养瓶，并沿每个培养瓶的长轴沿着固定平行的石墨电极，从而产生均匀的电场，导致心室肌细胞的捕获效率为75- 80%。计算机控制的放大器被设计成能够在一定范围的刺激频率（从0.1到7.0 Hz）内同时驱动多个培养瓶，每个培养瓶含有多达4 × 10（6）个肌细胞，每次刺激后电极极性反转，以防止每个电极周围形成pH梯度。与静止的、未受刺激的心肌细胞不同，在3-5 Hz起搏长达72 h的心肌细胞中，收缩幅度、缩短速度和舒张速度没有变化。起搏心肌细胞正常收缩功能的维持需要机械收缩本身，因为起搏心肌细胞由于在培养基中加入2.5 μ M维拉帕米48小时而保持静止，在去除维拉帕米后起搏时也表现出收缩力下降。同样，与未起搏的静止细胞相比，起搏增加了峰值钙电流。因此，在限定培养基中的短期原代培养物中，由连续均匀电场刺激诱导的生理频率下的肌细胞收缩维持了与新鲜分离的成人心室肌细胞中观察到的那些相似的肌细胞表型的一些生物物理参数。
To model with greater fidelity the electromechanical function of freshly isolated heart muscle eels in primary culture, we describe a technique for the continual electrical stimulation of adult myocytes at physiological frequencies for several days. A reusable plastic cover was constructed to fit standard, disposable 175-cm(2) tissue culture flasks and to hold parallel graphite electrodes along the long axis of each flask, which created a uniform electric field that resulted in a capture efficiency of ventricular myocytes of 75-80%. Computer-controlled amplifiers were designed to be capable of driving a number of flasks concurrently, each containing up to 4 x 10(6) myocytes, over a range of stimulation frequencies (from 0.1 to 7.0 Hz) with reversal of electrode polarity after each stimulus to prevent the development of pH gradients around each electrode. Unlike quiescent, unstimulated myocytes, the amplitude of contraction, and velocities of shortening and relaxation did not change in myocytes paced at 3-5 Hz for up to 72 h. The maintenance of normal contractile function in paced myocytes required mechanical contraction per se, since paced myocytes that remained quiescent due to the inclusion of 2.5 mu M verapamil in the culture medium for 48 h also exhibited a decline in contractility when paced after verapamil removal. Similarly, pacing increased peak calcium current compared with quiescent cells that had not been paced. Thus myocyte contraction at physiological frequencies induced by continual uniform electric field stimulation in short-term primary culture in defined medium maintains some biophysical parameters of myocyte phenotype that are similar to those observed in freshly isolated adult ventricular myocytes.