Stochastic Simulation Of Excitation-contraction Coupling

在上一个项目期间，我们集中研究了局部钙释放事件终止的机制。与Eduardo Rios合作，我们设计了钙火花模拟，并将其与从哺乳动物骨骼肌获得的高分辨率火花图像进行了比较。该分析表明，火花钙源在空间上是扩展的，涉及大量钙释放通道（ryanodine receptor, RyRs）的协调开放。我们开发了facfax语言的火花仿真算法，该算法可以用非线性最小二乘法拟合实验记录。这些模拟现在考虑了SR管腔钙局部耗竭的可能性，这被认为是一种火花终止机制。我们对局部SR钙释放的蒙特卡罗模拟表明，Cheng博士的团队发现的钙火花的量子分解可以用局部胞质钙对静息ryr的强烈失活来解释。然而，到目前为止，这种现象的存在还没有得到孤立ryr研究的证实。因此，我们正在修改模拟算法，以考虑个体SR释放末端钙的动态局部消耗和腔内Ca2+对RyR门控的调节。这涉及到对算法进行重大修改，使SR钙成为局部动态变量。然后，我们将确定实验中发现的量子统计是否可以用管腔钙调节机制来解释。在其他研究中，我们模拟了局部自发钙释放事件对起搏器细胞电流的影响。模型显示舒张期钙释放通过钠钙交换电流强烈耦合，SR钙振荡成为调节心率的主要机制。这与我们实验室最近的实验结果一致。我们还分析了生理条件下记录的第一个单通道l型钙电流，发现通道的钙失活机制似乎与我们在兴奋-收缩耦合随机模拟中假设的模式转换机制一致。

SUMMARY OF WORK During the previous project period we have concentrated on the mechanisms involved in termination of local calcium release events. In collaboration with Eduardo Rios, we devised calcium spark simulations that were compared with high-resolution spark images obtained from mammalian skeletal muscle. This analysis showed that spark calcium sources are spatially extended and involve coordiated opening of large numbers of calcium release channels (ryanodine receptors, RyRs). We developed spark simulation algorithms in the FACSIMILE language which can be fitted to experimental records by non-linear least-squares methods. These simulations now take into account the possibility of local depletion of SR lumenal calcium, which has been suggested as a spark-termination mechanism. Our Monte Carlo simulations of local SR calcium release showed that the quantal decomposition of calcium sparks found by Dr. Cheng's group could be explained by very strong inactivation of of resting RyRs by local cytosolic calcium. However, to date, the existence of such a phenomenon is not confirmed by studies on isolated RyRs. We are therefore modifying the simulation algorithm to take into account dynamic local depletion of calcium in individual SR release terminals and regulation of RyR gating by lumenal Ca2+. This involves major alteration of the algorithm to make SR calcium a local dynamical variable. We will then determine whether the quantal statistics found experimentally can be accounted for by the lumenal calcium regulation mechanism. In other studies, we have modeled the effect of local spontaneous calcium release events on currents in pacemaker cells. The modeling shows that diastolic calcium release couples strongly via sodium-calcium exchange current so that SR calcium oscillations become a dominant mechanism in regulating heart rate. This is consistent with recent experimental results from our laboratory. We have also analyzed the first single-channel L-type calcium currents recored under physiological conditions and find that the mechanism of calcium-inactivation of the channel appears to be consistent with the mode-shift mechanism hypothesized in our stochastic simulations of excitation-contraction coupling.