DEVELOPMENT OF CARDIAC EXCITATION/CONTRACTION COUPLING

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Despite many recent advances in the general understanding of Ca2+ handling during cardiac excitation-contraction (EC) coupling, little is known regarding the functional assembly of the various molecular components of EC coupling during the course of embryological development. This is critical information considering that many drugs used in the treatment of heart disease affect Ca2+ handling in some way and, in the pregnant mother, these are likely to affect the fetus differently. Secondly, it is likely that the normal development of these mechanisms is altered in congenital heart disease. In this proposal, the experiments are designed to test how functional aspects of cardiac EC coupling develop in parallel with cardiac junctional complexes and expression of specific proteins in embryonic heart. Junctional complexes are the structural manifestations of EC coupling which have recently been reported in detail in the chick embryo with electron and confocal microscopy. It is anticipated that the time course of development of EC coupling will parallel the appearance and expression of the key protein components. The approaches that will be used include patch clamping, measurements of Ca2+ transients, fluorescence confocal microscopy, and Western blotting. These studies will be carried out in the chick embryo which is the most studied model for embryological development of most cellular systems including EC coupling. Parallel experiments will be conducted in the developing mouse heart for comparison with a mammalian species. The Specific Aims are: 1) to determine the normal development of Ca-induced-Ca-release from the sarcoplasmic reticulum; 2) to determine the normal development of the mechanisms which remove Ca2+ from the cytoplasm; and 3) to determine the relative expression and assembly of the key proteins in cardiac EC coupling. The long term objective of this work is to understand the development of the critical mechanisms which regulate cytosolic Ca2+ on a beat-to-beat basis from the earliest heart tube to the mature four- chambered heart.