EXCITATION-CONTRACTION IN ISOLATED CARDIAC CELLS

我们分离了成年大鼠、兔和 豚鼠。这些制剂正被用于研究 收缩、电生理和生化特征 在各种不同的条件下。我们已经开发了一个系统 用于在单个心肌细胞中同时测量 细胞内游离钙、细胞长度和细胞膜的变化 电流/电压，时间分辨率高。系统使用 用INDO-1荧光探针监测胞浆游离钙 转瞬即逝。INDO-1荧光被外照光激发 350加或减10纳米光的10微秒闪光 重复频率高达200赫兹。印度的排放是由 用于测量刺激光谱的成对光电倍增管 411加或减20 nm和481加或减25 nm的窗口 优化收集光强度和光强之间的权衡 胞浆游离钙敏感性。荧光发射 从每个闪光灯中收集一对快速积分器样本- 在VAX控制下定制设计的与保持电路 11/730计算机，它计算在 两个波长作为细胞内游离钙的量度 时间精度优于20微秒。单元格长度为 通过光学装置从细胞的亮场图像测量 使用视频边缘检测器(或光电二极管)的边缘跟踪方法 数组，当需要毫秒时间分辨率时)。这个 膜电位可与贴片同时监测 选举人。我们的初步结果显示， 正常大鼠钙依赖的收缩和舒张期特性， 豚鼠和兔心肌保持较高的 大多数存活的单个肌细胞的保真度 从这些物种的心肌中分离出来，而这些 因此，心肌细胞是研究钙依赖的有效模型。 心脏的兴奋-收缩机制。因此， 单个心肌细胞的可用性和一种我们可以用来 同时测定膜电流、细胞内钙离子 和函数将允许对以下方面进行机械性研究 到目前为止还没有的兴奋收缩耦合 服从于指导研究。我们已经开始了一些初步的工作 这类实验。

We have dissociated cardiac cells from adult rats, rabbits and guinea pigs. These preparations are being used to study the contractile, electrophysiological, and biochemical characteristics in a variety of different conditions. We have developed a system for simultaneous measurement, in single cardiac myocytes, of changes in cytosolic free calcium, cell length and membrane current/voltage, with high time resolution. The system uses the fluorescent probe indo-1 to monitor cytosolic free calcium transient. Indo-1 fluorescence is excited by epi-illumination with 10 microsecond flashes of 350 plus or minus 10 nm light at repetition rates of up to 200 Hz. Indo emission is collected by paired photomultipliers to measure stimultaneously spectral windows of 411 plus or minus 20 nm and 481 plus or minus 25 nM optimizing the trade -off between collected light intensity and cytosolic free calcium sensitivity. The fluorescence emission from each flash is collected by a pair of fast integrator sample- and-hold circuits of custom design under the control of a VAX 11/730 computer which computes the ratio of indo emission at the two wavelengths as a measure of cytosolic free calcium with a time precision of better than 20 micro-seconds. Cells length is measured from the bright-field image of the cell by an optical edge tracking method using a vedio edge detector (or a photodiode array when millisecond time resolution is required). The membrane potential may be monitored simultaneously with patch electodes. Our initial results have shown the widely divergent, calcium-dependent systolic and diastolic properties of intact rat, guinea pig and rabbit cardiac muscle are retained with a high degree of fidelity in the majority of viable single myocytes isolated from the myocardium of these species, and that these myocytes are thus a valid model of studies of calcium-dependent excitation-contraction mechanisms in the heart. Thus, the availability of single myocytes and an apparatus whereby we can simultaneously measure membrane currents, cytosolic calcium and function will permit mechanistic studies of aspects of excitation-contraction coupling that have heretofore not been amenable to direct study. We have already begun some initial experiments of this sort.