The overall aim of this research proposal is to elucidate the mechanisms that may be involved in differentiation and development of skeletal and cardiac muscle, and the processes that control adaptation to external stimuli. To accomplish this goal we have selected myosin HC for our initial analysis. This protein is particularly appropriate to study since it is crucial to muscle contraction and it appears in multiple molecular forms. The expression of these isoforms follows a developmental and tissue specific pattern and, furthermore, can be affected by environmental and hormone stimuli. It may be postulated that such regulation is dependent on specific regulatory gene sequences, which are affected by change in chromatin structure and by interactions with effector molecules. In this proposal will approach these problems experimentally, by first delineating the different myosin HC isoforms in skeletal and cardiac muscle, and then by isolating, reconstructing and characterizing their corresponding genes. cDNA and gene probes will then be used to examine in detail the developmental expression of myosin HC in vivo and in muscle tissue culture systems. The effects of external factors such as repetitive stimuli to fast skeletal muscle, of co-culture with neural elements, and of thyroid hormone on myosin HC gene expression will be evaluated. The role of transcription, processing and translational control will be examined. The relationship of the DNA methylation pattern and of chromatin sensitivity to nuclease in myosin HC gene expression will be studied. Using in vitro transcription and DNA-mediated gene transfer experiments, 5 feet flanking sequences essential for transcriptional initiation and for developmental expression will be analyzed. Of note is that cardiac hypertrophy and other pathological states of the heart muscle are associated with alterations in myosin HC expression. Elucidation of the factors that regulate the expression of cardiac muscle genes should provide a basis for further understanding of cardiac pathology and a rationale to develop effective therapy.
