Stochastic Simulation Of Excitation-contraction Coupling

工作总结在本项目期间，我们集中研究SR管腔钙耗竭作为促进局部CICR（钙火花）终止的机制的可能作用。与Peace Cheng合作，我们开发了一种方法来监测心肌细胞中自发CICR波通过期间SR Lumeanl钙。我们开发了一个线性化的数学模型，这些波，其中包括SR内腔室内的扩散，但解析可解。模型模拟与实验记录的比较表明，SR内的钙扩散必须显着限制的速率相比，将被预期的基础上所占的体积分数的细胞器，兼容的火花终止中的局部SR钙耗尽的可能作用。我们完成了纳入本地SR耗尽到我们的Monte Carlo模型的激发-收缩耦合。该模型表明，局部耗尽可以引起稳定的宏观EC耦合，但在所需的条件下，“量子”结构的火花振幅所看到的郑博士的小组应该是不可观察的。 与拉什大学的Eduardo Rios合作，我们开发了一种新方法，通过将火花的时空过程全局拟合到反应扩散模型来估计火花背后的局部钙释放通量，并结合显微镜光学的影响，这被证明是至关重要的。正在进行的结果似乎表明，心肌中的火花钙通量，也可能在骨骼肌中，具有三角形衰减时间过程，这与多通道释放事件的局部耗尽或渐进失活是相容的。使用由肌膜L型通道电流产生的“火花”校准模型表明，这些事件，这是火花的触发，也可能是多通道的性质。为了研究这些L型事件中涉及的通道的幅度和数量的问题，我们进一步提高了我们的膜片钳系统的信噪比，并且正在记录生理条件下的大量L型单通道数据，这是任何其他小组都没有完成的。

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.