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.
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