During development, heart muscle cells diversify into a heterogeneous population of cells with distinct contractile and secretory functions. Atrial natriuretic factor (ANF) is secreted predominately from atrial myocytes, and distinct patterns of myosin heavy chain (MHC) isoforms are expressed in atrial, ventricular, and conducting system myocytes at different stages of development. The long-term goal of our work is to understand the genetic mechanisms underlying the formation of cardiac myocytes with diverse secretory and contractile functions. The molecular mechanisms of cardiac secretion and cardiac muscle cell diversification remain largely unknown. Work on other secretory and contractile cells, however, suggests possible mechanisms of heart cell function. Proteins (p38, p65, SV2, and synaptobrevin) have been identified that are common to secretory vesicles of endocrine and neural cells, but the developmental expression patterns and functions of these secretory vesicle proteins in cardiac muscle are not known. Skeletal muscle differentiation has been shown to be regulated by fibroblast growth factor (FGF) and transforming growth factor beta (TGFbeta), as well as by DNA-binding, muscle regulatory proteins such as MyoD1, myogenin, and Myf-5. FGF and TGFbeta are expressed in the heart, but the roles of growth factors in cardiac myocyte development and diversification have not been fully explored, nor have any cardiac muscle-specific regulatory proteins been identified. The proposed experiments address questions about the diversification of secretory and contractile function in developing mammalian cardiac myocytes. The experiments have three specific aims: (i) The patterns of expression of secretory vesicle proteins and ANF secretion pathways will be determined in developing atrial, ventricular, and conducting system myocytes in vitro and in vivo, (ii) The roles of FGF and TGFbeta in the development of ANF secretion and in MHC isoform expression will be determined using cultured cardiac myocytes, and (iii) Homology screening of cDNA libraries and PCR-based homology amplification of cardiac mRNA will be used to isolate cDNAs encoding possible cardiac myocyte-specific, DNA-binding proteins. Methods will include cell culture, ANF secretion assays, immunofluorescence, Northern and Western blotting, cDNA library preparation and screening, and polymerase chain reaction amplification of homologous genes.
| Marazzi, G; Buckley, K M (1993) Accumulation of mRNAs encoding synaptic vesicle-specific proteins precedes neurite extension during early neuronal development. Dev Dyn 197:115-24 |
