The overall goal of this proposal is to determine how the expression of two genetic isoforms of creatine kinase (MCK and BCK) is regulated in mammalian heart cells. The specific objectives are: (1) to identify cis-acting DNA elements within the mouse MCK gene which are required for tissue- and/or cardiac cell type-specific expression of MCK; (2) to identify cis-acting DNA elements within the MCK gene which are required for various physiological modulations of its expression; (3) to clone the 5'-flanking and first intron portion of the mouse BCK gene, and to identity similar cell type and physiological regulatory elements within it; (4) to identify and begin purification of the trans-acting factors that bind to these DNA regions or that selectively stimulate or repress transcription from the M- and BCK promoter regions; and (5) to derive permanent cell lines which are representative of the various cardiac muscle cell types (atrial, ventricular, and conducting), as well as cardiac progenitor cells. Basic knowledge derived from these studies may be particularly informative with respect to the general problem of tissue-specific gene regulation during development. Our approach will permit direct comparisons between how the expression of two evolutionarily-related genes is regulated within a set of developmentally-related cell types in the cardiac and skeletal muscle cell lineages. In addition, since the creatine kinases play a critical role in the high-energy phosphate shuttle of cardiac muscle cells, and since CK levels appear to change in response to cardiac hypertrophy, these studies should provide information which is relevant to general problems of heart disease. Finally, the availability of permanent cell lines representing different cardiac muscle cell types would be of great value to virtually all studies of heart cell function. Major methodologies include: gene cloning, modification, transfection, DNAase foot-printing, gel retardation, in vitro transcription assays, clonal cell culture, and spontaneous, oncogene-, and viral-induced transformation.
