Cardiac contractile function is determined by the expression of distinct set of tissue-specific genes. These include, myofibrilla isoforms, cell surface receptors, as well as lineage-specific enzymes and structural proteins. The tissue specific expression of these genes is controlled at the level of transcription. However, relatively little is understood about the molecular mechanisms that control cardiac-specific transcription. Because of its restricted pattern of expression, we have used the murine slow/cardiac troponin C (cTnC) gene as a model system to define the molecular mechanisms that regulate gene expression in cardiac myocytes. We have shown both in vitro, and by the direct injection of plasmid DNA into the adult rat heart in vivo, that cTnC gene expression in cardiac myocytes is controlled by a cardiac-specific promoter/enhancer located in the immediate 5' flanking region of the gene (bp-124-+1). This transcriptional enhancer contains previously undescribed nuclear protein binding sites that bind novel cardiac-specific nuclear protein complexes. The demonstration of cardiac-specific transacting factors suggests that a distinct set of cardiac-specific transcription factor may play a central role in cardiac myocyte-specific gene expression. The proposed studies are designed to elucidate the molecular mechanism that control cTnC gene expression during murine development in vivo with particular attention placed on examining the role of these novel cardiac- specific nuclear protein complexes. Specifically, we propose to i) examine the tissue-and developmental-specificity of the cTnC 5' flanking promoter/enhancer in transgenic mice, ii) examine how protein-protein interactions between factors that bind to the cTnC enhancer and promoter control the transcriptional activation of the cTnC gene in cardiac myocytes, and iii) biochemically characterize and clone the transcription factors that regulate cTnC gene expression, with particular emphasis on purification of cardiac-specific nuclear protein complexes. Elucidation of the transcriptional program that controls cTnC gene expression in cardiac myocytes should fundamentally increase our understanding of the molecular mechanisms that control gene expression in the heart. The proposed studies are relevant both to normal cardiac development and to the molecular pathogenesis of cardiac hypertrophy.
