EXCITATION-CONTRACTION IN ISOLATED CARDIAC CELLS

离体心肌细胞的兴奋-收缩

• 批准号: 3821449
• 负责人: M C CAPOGROSSI
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3821449
• 关键词: calcium; electrophysiology; fluorescent dye /probe; heart cell; heart contraction; heart metabolism; mature animal; membrane potentials; myocardium; sarcoplasmic reticulum; single cell analysis

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.

我们分离了成年大鼠、家兔和 豚鼠 这些制剂被用于研究 收缩、电生理和生化特性 在各种不同的条件下。 我们已经开发出一种系统 用于在单个心肌细胞中同时测量 胞浆游离钙、细胞长度和膜的变化 电流/电压，具有高时间分辨率。 系统使用 荧光探针indo-1监测细胞内游离钙 短暂的 Indo-1荧光通过具有以下的落射照射激发： 350 ± 10 nm光的10微秒闪烁， 重复频率高达200 Hz。 印度排放物被收集， 成对的光电倍增管测量刺激光谱 411 ± 20 nm和481 ± 25 nM的窗口 优化收集的光强度和 胞浆游离钙敏感性 荧光发射 由一对快速积分器样本收集， 在VAX控制下定制设计的与保持电路 11/730计算机，用于计算 两个波长作为细胞溶质游离钙的量度， 时间精度优于20微秒。 细胞长度为 通过光学成像仪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 需要毫秒级时间分辨率时使用数组）。 的 膜电位可以与贴片同时监测 选举人 我们的初步结果显示， 完整大鼠钙依赖性收缩和舒张特性， 豚鼠和家兔心肌保留率高， 大多数存活单个肌细胞的保真度 从这些物种的心肌中分离出来， 因此，肌细胞是研究钙依赖性 心脏的兴奋收缩机制 因此 单个肌细胞的可用性和一种装置， 同时测量膜电流、胞浆钙 和功能将允许机械研究的方面， 兴奋-收缩偶联，迄今为止还没有被 适合直接研究。 我们已经开始了一些初步的 这种实验。