Stochastic Simulation Of Excitation-contraction Coupling

在本项目期间，我们集中研究了肌浆网腔内钙耗竭作为终止局部CICR(钙火花)的机制的可能作用。与和平成合作，我们开发了一种方法来监测心肌细胞自发CICR波通过期间的肌浆网钙离子。我们开发了这些波的线性化数学模型，该模型包含了SR腔室内的扩散，但可解析地求解。模型模拟与实验记录的比较表明，与细胞器所占体积分数的预期速率相比，SR内的钙扩散必须受到显著限制，这与局部SR钙耗竭在火花终止中的可能作用是一致的。我们完成了将局部SR耗尽纳入到我们的激发-收缩耦合的蒙特卡罗模型中。该模型表明，局域耗尽可以产生稳定的宏观EC耦合，但在所要求的条件下，郑博士的研究小组所看到的火花幅度的“量子”结构应该是不能观察到的。 与拉什大学的Eduardo Rios合作，我们开发了一种新的方法来估计火花下的局部钙释放通量，方法是将火花的时空过程全局拟合到反应扩散模型，并纳入显微镜光学的影响，这被证明是至关重要的。正在进行的结果似乎表明，心肌中的火花钙流动，可能也包括在骨骼肌中，有一个三角形的衰减时间过程，这与多通道释放事件的局部耗竭或进行性失活相一致。用肌膜L型通道电流产生的“小火花”对模型进行刻度表明，这些触发火花的事件本质上也可能是多通道的。为了研究这些L类事件涉及的通道数和幅度问题，我们进一步提高了膜片钳系统的信噪比，并在生理条件下记录了大量L类单通道数据，这是其他任何组都没有做过的。

SUMMARY OF WORK During this project period we concentrated on the possible role of SR lumenal calcium depletion as a mechanism contributing to the termination of local CICR (calcium sparks). In collaboration with Peace Cheng, we developed a method to monitor SR lumeanl calcium during the passage of spontaneous CICR waves in cardiac myocytes. We developed a linearized mathematical model of these waves which incorporates diffusion within the SR lumenal compartment but is analytically solvable. Comparison of model simulations with experimental records indicates that calcium diffusion within the SR must be significantly restricted compared to the rate that would be expected on the basis of the volume fraction occupied by that organelle, compatible with a possible role for local SR calcium depletion in spark termination. We completed the incorporation of local SR depletion into our Monte Carlo model of excitation-contraction coupling. The model showed that local depletion can give rise to stable macroscopic EC coupling, but under the required conditions the "quantal" structure of spark amplitude seen by Dr. Cheng's group ought not to be observable. In collaboration with Eduardo Rios at Rush University, we developed a novel method to estimate local calcium release flux underlying sparks by globally fitting the space-time course of the spark to a reaction diffusion model, incorporating the effects of microscope optics, which prove to be crucial. Results, which are in progress, appear to show that spark calcium flux in cardiac muscle, and possibly also in skeletal muscle, has a triangular decaying time course, which is compatible with local depletion or progressive inactivation of a multi-channel release event. Calibration of the model using "sparklets" generated by sarcolemmal L-type channel currents indicates that these events, which are the trigger of sparks, may also by multi-channel in nature. In order to study the question of the amplitude and number of channels involved in these L-type events, we have further improved the signal-to-noise ratio of our patch-clamp system and are in the process of recording a large body of L-type single-channel data under physiologic conditions, which has not been done by any other group.