NIH FIRCA RO3 TW005543/2

Principal Investigator: Sandor Gyorke, Ohio State University, Columbus, OH, USA
International Collaborator: Alexandra Zahradníková, Ivan Zahradník

Duration: May 2005 – April 2008
Coordinating Organization: Ohio State University, Columbus, OH, USA

Annotation:

Excitation-contraction coupling (ECC) in mammalian cardiac muscle relies on calcium signaling across the tubulo – reticular junction. Our central hypothesis is that the specific microarchitecture of the tubulo-reticular junction, consisting of juxtaposed clusters of DHPR and RyR channels, together with their specific calcium-dependent gating properties, convey the unique properties of ECC in cardiac myocytes. We particularize this hypothesis in the concept of the excitation-contraction coupling unit (ECCU). The ECCU is understood as a particular structure, the cardiac dyad, with the whole set of associated ionic channels and other proteins. In the first approximation ECCU consists of the excitation unit (EU) represented by a segment of the plasma membrane with a cluster of DHPR channels, and the release unit (RU) represented by a segment of the SR membrane with a cluster RyR channels). Ion channels are defined by their conductive and gating properties and by their topological disposition and ionic environment. The objective of this proposal is to define the role of changes in the microarchitecture of cardiac cells, i.e., their cellular distribution, morphology and topological properties, in compromising calcium release efficiency during heart failure. To achieve this objective, we will extensively employ several original methods developed at the Slovak site, i.e., the model of calcium current kinetics with release dependent inactivation, the software for simulation of the electrophysiological experiment based on simulation of interacting channel clusters, and the technique for selective induction of calcium release-dependent inactivation of calcium current.

Keywords:

ryanodine receptor, cardiac myocyte, excitation-contraction coupling

Objectives:

This research will be done primarily in Slovak Republic as an extension of NIH grant #HL074045.
The overall goal of this proposal is to elucidate the changes of calcium signaling mechanisms in dyads of mammalian cardiac muscle cells during heart failure. These structures contain clusters of L-type calcium channels (DHPRs) in apposition to clusters of ryanodine receptor calcium release channels functionally coupled by calcium ions. Alterations in calcium signaling during excitation-contraction coupling contribute to the decreased performance of failing hearts. The changes in calcium signaling steps during activation of calcium release by DHPRs and their relationship to the ultrastructural organization of dyads and the topology of channels in the excitation-contraction coupling units will be determined. To this end, measurements of the kinetics of whole-cell calcium currents, whole-cell calcium transients, and dyadic calcium release events (calcium spikes) will be carried out. These measurements will be supplemented by morphological analysis of the volume and surface densities of the tubular system and of cisternal and longitudinal sarcoplasmic reticulum, and of colocalization between calcium channels and calcium release channels, using electron microscopy and confocal microscopy. 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 simulation of experiments will be used to ascertain possible mechanisms and to design experiments with high predictive power. Understanding the abnormalities of cardiac E-C coupling in heart failure is important since it might become a strategic site for therapeutic intervention. Furthermore, defining the relationships between the spatial organization of channel clusters and their calcium signaling has a broader significance for understanding processes such as synaptic transmission, neuroendocrine secretion and regulation of vascular tone.

Publications: