MECHANICAL TRANSDUCTION BY CARDIOCYTES

心脏病伴心律失常是西方人的主要死因 世界上，有数据表明，机电反馈发挥着 在这些心律失常的发展过程中起着重要作用。这其中的一个来源 转导可能是已确定的机械敏感离子通道 在一些心肌细胞中。这些经络有不同的药理作用 从传统的电压敏感通道中分离出来，并了解其 特性可能会导致新疗法和治疗方法的发展 探员们。 拟议中的实验将阐明机械敏感的行为 心肌细胞中单通道和全细胞水平的通道 使用从大鼠和小鸡身上急性分离的心肌细胞。目前，只有 其中一篇论文是关于拉伸心肌细胞的电压钳实验。 因此，几乎没有关于这些基本问题的信息。 因为电流的存在，它们的大小是拉伸的函数， 它们的离子选择性、通道或转运体的数量类型 参与这种电流，它们的药理，以及它们的可变性 遍及心脏的不同区域。虽然单声道记录 机械敏感离子通道是由心脏细胞制成的，它是 在膜片钳条件下，未知哪个通道(如果有)可见 在生理上是活跃的。 将开发以最小的损害拉伸单元格的技术 安装在压电式机械手上的探头。平均应变和均匀度 将从细胞长度、膜标记和肌节测量 长度使用视频显微镜。连接到细胞上的膜片钳吸管 固定吸量管附近会跟随电池的运动，避免局部 紧张。电流的特征是渗透性(电导和 选择性)、选通(使用稳态和瞬时长度变化 和电压)、对药剂的反应、温度和 相对于解剖起源的可变性。与拉伸相关的更改 在电流钳条件下，动作电位将被记录下来。 牵张效应与细胞内钙离子变化的相关性 在此期间，将使用钙离子指示剂进行荧光显微镜检查 定义的延伸协议。 单通道膜片钳研究将被用来表征 细胞皮质区的应力与通道的耦合 激活。刺激将包括暂态和稳态机械 压力、电压和药理作用。测量面片几何图形的步骤 以及表征刺激所必需的机械性能， 一种压力伺服和视频显微镜相结合的贴片 将结合拟合例程来估计平均补片应力和 紧张。

Heart disease with arrhythmia is the primary cause of death in the Western world and there is data to suggest that mechanoelectric feedback plays a role in the development of these arrhythmias. One source of this transduction may be mechanosensitive ion channels that have been identified in a number of cardiocytes. These channels have a different pharmacology from traditional voltage sensitive channels, and understanding their properties may lead tot he development of new therapies and therapeutic agents. The proposed experiments will clarify the behavior of mechanosensitive channels in heart cells at the level of single channels and whole cells using acutely isolated cardiocytes from rats and chicks. At present, only one paper exists on voltage clamp experiments on stretched cardiac cells. Consequently there is almost no information on such fundamental questions as the existence of currents, their magnitude as a function of stretch, their ionic selectivity, the number types of channels or transporters involved in such currents, their pharmacology,a nd their variability throughout different regions of the heart. Although single channel records of mechanosensitive ion channels have been made from heart cells, it is unknown which, if any, of the channels seen under patch clamp conditions are physiologically active. Techniques will be developed to stretch cells with minimum damage using probes attached to piezoelectric manipulators. Mean strain and uniformity will be measured from both cell length, membrane markers and from sarcomere length using video microscopy. A patch clamp pipette attached tot he cell near the fixed pipette will follow the motion of the cell to avoid local strain. The currents will be characterized by permeation (conductance and selectivity), gating (using steady state and transient changes in length and voltage), response to pharmacological agents, temperature and variability with respect to anatomical origin. Stretch dependent changes in the action potential will be recorded under current clamp conditions. To correlate the effects of stretch with changes in intracellular Ca2+ levels, fluorescence microscopy with Ca2+ indicators will be done during defined stretch protocols. Single channel patch clamp studies will be used to characterize the coupling of stress in the cortical region of the cells to channel activation. Stimuli will include transient and steady state mechanical stress, voltage and pharmacological agents. To measure the patch geometry and mechanical properties that are necessary to characterize the stimulus, a pressure servo and video microscopy of the patch combined with image fitting routines will be combined to estimate the mean patch stress and strain.