EXCITATION-CONTRACTION COUPLINGY IN ANOXIC MYOCYTES

摘要：缺氧性收缩衰竭的机制和 心肌中的再氧合损伤，将在一个 单个心肌细胞缺氧模型。 单个分离的心室肌细胞， 大鼠、家兔和豚鼠将经受缺氧或分级 缺氧（1-3 ℃）使用层流逆流屏障井，一种新的 主要研究者开发的器械，允许用于 第一次，开放进入细胞进行电生理研究， 深度缺氧 肌细胞将被“全细胞”钳位电压 在缺氧和复氧过程中的贴片吸管技术， 通过视频边缘跟踪同时监测收缩， 使用频闪激发的indo-1荧光的细胞溶质钙瞬变。 肌浆网钙储备将通过快速 将来自微量移液管的咖啡因灌流到负载indo-1的细胞中。的 动作电位变窄的重要性（由于外向电流）， 抑制慢内向钙电流和改变 肌丝敏感性在快速缺氧性收缩衰竭中的作用 将通过测量钙瞬变和收缩来确定 当缺氧外向电流被电压钳克服时的振幅， 用甲苯磺丁脲阻断。反复发作的缺氧性收缩 失败，然后在挛缩前再氧合，将用于 确定心肌“顿抑”是否存在于单细胞水平;如果 因此，将确定钙瞬变的改变是否 和/或肌膜电流起作用。“早”的机制 钙超载，在初步研究中发现在分级期间发生 将研究缺氧，但不研究缺氧。增长的根源在于 缺氧性挛缩期间的细胞溶质钙将通过 改变离子环境，应用咖啡因，兰尼定和缓慢 通道阻滞剂，在收缩（僵直）的indo-1负载的肌细胞中 状态 挛缩后的复氧导致四舍五入（氧 悖论）的肌细胞，随着概率的增加， 僵硬的持续时间 钙积累、钠 积累，肌膜损伤和腺嘌呤核苷酸消耗， 这一过程将通过操纵细胞外和 在僵直和复氧期间的细胞内环境。

ABSTRACT: The mechanisms responsible for hypoxic contractile failure and reoxygenation injury in the myocardium, will be studied in a single-myocyte hypoxia model. Single isolated ventricular myocytes from rats, rabbits and guinea-pigs will be subjected to anoxia or graded hypoxia (1-3 torr) using the Laminar Counterflow Barrier Well, a novel device developed by the Principal Investigator, which permits, for the first time, open access to the cell for electrophysiologic studies during profound hypoxia. Myocytes will be voltage clamped by the "whole cell" patch pipette technique during hypoxia and reoxygenation, with simultaneous monitoring of contraction by video edge tracking, and of cytosolic calcium transients using strobe-excited indo-1 fluorescence. Sarcoplasmic reticulum calcium stores will be estimated by rapid superfusion of caffeine from a micropipette in indo-1 loaded cells. The importance of action potential narrowing (due to outward currents), depression of the slow inward calcium current and alteration of myofilament sensitivity in producing rapid hypoxic contractile failure will be determined by measuring the calcium transient and contraction amplitude when hypoxic outward currents are overcome by voltage clamp and blockade with Tolbutamide. Repeated episodes of hypoxic contractile failure, followed by reoxygenation before contracture, will be used to determine if myocardial "stunning" exists at the single cell level; if so, it will be determined whether alteration of the calcium transient and/or sarcolemmal currents are responsible. The mechanism of "early" calcium overload, which was found in pilot studies to occur during graded hypoxia, but not anoxia, will be studied. The source of the rise in cytosolic calcium during the hypoxic contracture will be determined by varying the ionic milieu and applying caffeine, ryanodine and slow channel blockers, in indo-1 loaded myocytes in the contracted (rigor) state. Reoxygenation after contracture causes rounding up (oxygen paradox) of myocytes, with a probability which increases with the duration of rigor. The contributions of calcium accumulation, sodium accumulation, sarcolemmal injury and adenine nucleotide depletion to this process will be explored by manipulating the extracellular and intracellular environment during rigor and reoxygenation.