The overall aim of the proposed research is to investigate the mechanism by which the release of calcium ions (Ca2+) from the sarcoplasmic reticulum (SR) of mammalian cardiac muscle is controlled, or 'graded', by surface membrane Ca2+-current. Previous work has shown that Ca2+ entering cardiac cells via L-type Ca2+-channels does activate release of Ca2+ from the SR, that the amount released is in rough proportion to the influx of Ca2+ (i.e., it is 'graded') and that membrane voltage has no direct role in this. Furthermore, the release of Ca2+ from the SR is characterized by 'high gain', in that as much as ten times more Ca2+ is released from the SR than enters the cell via the L-type Ca2+-channels. The major question remaining concerns how control is achieved, given the fact that Ca2+ released from the SR (presumably in the proximity of both a surface membrane Ca2+-channel and an activating site) might activate uncontrolled further release, in a positive feedback loop.
The Specific Aims of the research are: 1) Construct a mathematical model of the entry, diffusion, release and binding of Ca2+ in a representative section of a cardiac sarcomere, with sufficient spatial resolution to consider the possible local accumulation of Ca2+ near co-associated Ca2+-channels in the t- tubule and SR membranes. 2) Calculate the afflux of Ca2+ from SR of intact cells during E-C coupling, considering the existence of spatial inhomogeneity of [Ca2+]i. 3) Test the hypothesis that control of potential positive feedback on SR Ca2+-release is achieved through a Ca2+-dependent negative feedback on SR Ca2+-release. 4) Test the hypothesis that control (gradation) is achieved through the voltage- dependent kinetics of single L-type Ca2+-channels, the stochastic nature of Ca2+-release through SR Ca2+-channels, and the characteristics of accumulation and dissipation of Ca2+ in the t-SR junction at a site that activates SR Ca2+-release.
Specific Aim 1 will be achieved through the use of a supercomputer to solve the system of partial differential equations that describe Ca2+ diffusion and binding. This will allow consideration of new detailed data on the ultrastructure of the t-tubule SR junction and of the stochastic properties of SR and sarcolemmal (SL) Ca2+-channels. The model will serve to generate explicit, experimentally testable predictions of complex hypotheses and also serve as a framework for evaluating certain phenomena that cannot be observed directly. Experimentally, [Ca2+]i and whole-cell Ca2+-current will be measured in single, voltage-clamped, guinea-pig ventricular myocytes perfused internally with fluorescent Ca2+-indicators. Unitary Ca2+-currents will be measured using cell-attached patch. [Ca2+]i will be manipulated through the use of intracellular buffers and through flash photolysis of caged Ca2+-chelators. The research addresses the fundamental physiological question remaining in the study of excitation-contraction coupling in mammalian heart muscle.
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