EXCITATION-CONTRACTION COUPLING IN ANOXIC MYOCYTES

在第一个项目期间，我们已经定义了一个详细的序列， 缺氧对单个心肌细胞功能和离子的影响 和复氧，最终导致细胞中的氧矛盾， 在长时间的能量耗尽后恢复了氧气。 我们已经证明 心肌细胞的舒张动力学明显异常， 短暂缺氧，并表明这种异常是在肌丝 水平 我们开发了一种实时测量 心肌细胞线粒体游离钙，并证明， 在能量消耗期间上升，其水平可预测细胞 复氧存活率。 在下一个阶段，我们将集中精力 在3个方面：（1）研究复氧后机制 肌丝异常，特别是寻找磷酸化 肌丝蛋白的调节位点，无机磷酸盐的作用 在信号通路中，酪氨酸可能参与 激酶。(2)为了进一步确定诱发 复氧过度挛缩，我们将（a）试图分离的变化， 细胞溶质和线粒体钙，以确定哪一个（如果有的话）是 因果关系和（B）确定僵硬期间Na+进入的机制，观察 特别是电压依赖性Na通道参与的证据 要么小瓶小窗口电流激活耗尽的 静息电位或异常失活。(3)因为马克 种属对长时间缺氧反应的差异 肌细胞水平，我们将分离人心室肌细胞 肌内膜活检和移植心脏，并表征反应 缺氧/复氧。

During the first project period we have defined a detailed sequence of functional and ionic changes in single cardiac myocytes exposed to anoxia and reoxygenation, culminating in the oxygen paradox in cells which are reoxygenated after prolonged energy depletion. We have demonstrated marked abnormality of relaxation kinetics in myocytes reoxygenated after brief anoxia, and shown that this abnormality is at the myofilament level. We have developed a method for realtime measurement of mitochondrial free calcium in cardiac myocytes, and demonstrated that it rises during energy depletion and its level is predictive of cell survival at reoxygenation. During the next period we will concentrate on 3 areas: (1) We will pursue the mechanism of post- reoxygenation myofilament abnormality, looking particularly for phosphorylation of regulatory sites on myofilament proteins, the role of inorganic phosphate in the signaling pathway, and the possible participation of tyrosine kinases. (2) To define further the conditions predisposing to reoxygenation hypercontracture, we will (a) try to dissociate changes in cytosolic and mitochondrial calcium to determine which (if either) is causal and (b) Determine the mechanism of Na+ entry during rigor, looking particularly for evidence of involvement of voltage-dependent Na channels either vial small window currents activated by deplorization of the resting potential or by abnormal inactivation. (3) Because of marked species differences in the response to prolonged anoxia at the single myocyte level, we will isolate human ventricular myocytes from endomyocardial biopsies and explant hearts and characterize the response to anoxia/reoxygenation in these cells.