This research will be done primarily in Slovak Republic as an extension of NIH grant # ROl HL52620. The overall goal of this proposal is to elucidate the mechanisms of calcium signaling in dyads of mammalian cardiac muscle cells. These structures, representing excitation-contraction coupling units, contain clusters of L-type calcium channels (the excitation unit) in apposition to clusters of ryanodine receptor calcium release channels (the release unit) functionally coupled by calcium ions. The feed-forward calcium signaling steps are important for activation of calcium release by excitation, the feedback calcium signaling steps are important for inactivation of calcium current and calcium release, and their relationship to the topology of channels in the excitation-contraction coupling unit will be determined. To this end, measurements of the kinetics of whole-cell calcium currents, whole-cell calcium transients, and dyadic calcium release events during stimulation and during recovery from inactivation will be carried out. The properties of the excitation-contraction coupling unit, such as calcium channel gating and permeation, the fraction of active calcium channels, and the rate of calcium binding to the ryanodine receptor, will be manipulated. Quantitative analysis of the kinetics of calcium current and calcium release in parallel will be used to estimate parameters of calcium signaling critical for control of gradation and efficiency of excitation-contraction coupling. Computer simulations of experiments on channel clusters that interact by way of calcium signaling will be used to ascertain possible mechanisms and to design experiments with high predictive power. Understanding the regulation of cardiac E-C coupling is important since it might become a strategic site for therapeutic intervention in cardiac muscle dysfunctions. Furthermore, defining mechanisms of calcium-based interaction between channels in co-localized clusters has a broader significance for understanding processes such as synaptic transmission, neuroendocrine secretion and regulation of vascular tone.
