Proliferating cardiac myoblasts are observed in situ only during a short developmental period in early embryogenesis, after which all of the muscle cells of the myocardium - even the proliferating cells - bear contractile elements and are partially differentiated. Since the partially differentiated cells possess only a limited capacity for mitotic growth, replication of cardiac myocytes stops soon after birth, leaving the myocardium with no significant regenerative ability. The components of the embryonic extracellular matrix in which cardiac myoblasts proliferate and differentiate are quite different from those of fetal or adult heart, and their composition and architecture are not well understood. One reason is the limited amount of material available for analysis at such an early developmental period. To fully understand the induction of cardiac myogenesis, a better understanding of the cellular and extracellular matrix components of the embryonic heart is required. The purpose of this proposal is to recreate in culture the earliest events of cardiac organogenesis observed in the embryo. Based on in situ observations, such culture systems should consist of three cellular components: cardiac myoblasts, endodermal cells, and embryonic endocardial cells. The extracellular matrix components should include the basal lamina between the endoderm and the cardiac myoblast and cardiac jelly found between the embryonic endocardium and the cardiac myoblasts. In this proposal, culture equivalents for all three cellular components will be characterized. Co- culture experiments are proposed that will examine the inductive interactions between these three embryonic cell types, and the mechanisms that lead to the elaboration of the embryonic cardiac matrices. Defined embryonic, fetal and adult extracellular matrices then will be assembled in vitro, and their effects on the phenotypes of cardiac myoblasts and myocytes will be elucidated. By comparing the cellular and extracellular components of the embryonic and adult heart, insights should be gained into the forces that stifle regeneration of the myocardium.
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