This research seeks to identify and characterize known and unknown growth factors that regulate development of the embryonic heart. Results from this work will contribute to an understanding of the mechanisms of congenital heart disease and will suggest strategies for inducing the regeneration of injured mature cardiac myocytes in the adult. We have already determined that two known growth factor families -- the activins and the fibroblast growth factors (FGFs) -- may have a major role in the cardiogenic process. We have shown that activin comprises approximately 40% of the secretory product of embryonic endoderm, cells in which may induce cardiogenesis, and that activin alone is able to effect cardiogenesis in defined medium. And, using isoform-specific antibodies, we have shown that FGF family members aFGF, FGFK, FGF5 and bFGF are concentrated in the earliest myocardial cells. We have also shown that oligodeoxynucleotide (ODN)-mediated deletion of the bFGF inhibits heart development, and that bFGF, like activin, is sufficient to support heart development in culture. During the renewal years, these findings will be extended by determining the individual and combined function of these factors during heart development. species' homologous probes for isoforms of activin and FGF will be isolated from a cDNA expression library and sequenced. The amount and location of activin and FGF mRNA and protein isoforms will be assessed using mRNA hybridization and immunochemical analyses. The function of each factor will be directly tested by adding/deleting these proteins to/from heart cells during development in culture, and in the embryo. Deletions will be performed by (i) antisense ODN-mediated knockout of mRNA and (ii) antibody neutralization of protein. The hypothesis that FGF induces cardiogenesis by up-regulating serum response factor (SRF) protein, which causes expression of sarcomeric actin genes, will be tested. Myocardial cell membrane receptors for activin and FGF will be characterized by PCR (polymerase chain reaction) amplification and DNA sequencing; the effects of receptor deletion on cardiogenesis will also be determined. Finally, another major goal of this research is to identify and characterize novel, unknown cardiogenic inducer molecules that are secreted by endoderm. Antibodies to an unknown, major 25 kD endoderm-secreted protein that we have identified, as well as minor endodermal proteins, will be prepared for library screening, immunohistochemical mapping and functional analysis during cardiogenesis.
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