The developmental program of cardiac myocytes has been the subject of several sophisticated studies. As these cells progress through embryonic, fetal, neonatal, and adult phases their contractile phenotypes can change significantly. Investigations of mammalian cardiac development , however, are seriously restrained by the lack of a suitable culture system for studying the differentiation of heart cells in vitro. Neoplasias are rarely associated with the heart, so that tumor cell lines derived from cardiac tissues are not available. Embryonic and fetal cardiocytes, while viable in culture for extended periods, do not proliferate appreciably and thus represent a limited source of biomaterials. In addition, variability occurs in the developmental expression of contractile proteins in the hearts of different mammalian species, making it difficult to determine the developmental program of human cardiac myocytes from observations made in other animal systems. And, obviously, experiments that require the sacrifice of experimental animals cannot be performed with human hearts. This proposal is concerned with the generation of a novel system for culturing cells from autopsy samples of human fetal heart. Rather than culturing cardiocytes, however, the proposed experiments will focus on culturing cardioblasts. The cardioblast is defined as an undifferentiated cardiac myocyte, which has the capacity to proliferate, but is committed to the cardiocytes lineage. Initial experiments indicate that cardioblasts emerge in mitogen-rich medium from cultures of human fetal heart cells, and, when changed to mitogen-poor medium, differentiate into more mature cardiocytes. The experiments in this proposal will detail the culture parameters associated with the proliferation and subsequent differentiation of cardioblast cells. In addition, procedures to immortalize the existing human cardioblast strain into a permanent cell line will be performed. With existing and future strains of human cardioblasts, several developmental studies will be possible. Those chosen for this proposal will characterize, at the transcriptional and post-transcriptional levels, alterations in the expression of a variety of cloned genes during the differentiation program of these cells. These will include probes for contractile proteins, developmental proteins (proto-oncogenes), and Atrial Natriuretic Factor. Experiments are also proposed to observe the effects of co-cultured neuronal cells on the differentiation program of human cardioblasts. Finally, human cardioblasts will be used to develop monoclonal antibodies reactive with cell surface markers of myocardial development. Surface molecules identified by these monoclonal antibodies may be associated with specific populations of cardiac myocytes, and defects in their expression may be pathogenic.
