Thyroid hormone exerts marked influences on cardiac contractility and some of these changes result from alterations in specific cardiac proteins. A prolonged cardiac diastole occurs in hypothyroidism and the speed of diastolic relaxation of the heart is closely linked to the activity of the sarcoplasmic reticulum (SR) Ca++ ATPase pump. To gain further insight into the molecular mechanisms which are responsible for T3-induced increases in diastolic relaxation speed, we have demonstrated that T3 increases the transcription of the slow/cardiac isoform of the SR Ca++ ATPase (SR Ca2) gene. To define mechanisms of T3 action in further detail, we will determine the location, number, and nucleotide sequence of thyroid hormone response elements (TRE). We will delineate such TREs by the ability of deletion mutants of the SR Ca2 gene regulatory region to transmit T3- induced transactivation of a CAT reporter gene and by specific binding and footprinting assays. Preliminary results point to the existence of TREs in separate regions of the SR Ca2 gene regulatory region. The putative TREs are different from each other and are divergent from currently described TREs. Results in vivo and in cell culture show that marked differences in the level of SR Ca2 gene expression occur in different myocytic and non- myocytic cell types. In addition, T3 increases expression of the SR Ca2 gene in certain tissues and cells and decreases its expression in other muscle or cell types. To define factors which influence T3-induced SR Ca2 gene expression in a cell-type specific context, we will identify myocyte specific cis-acting sequences in the SR Ca2 gene regulatory region using functional transfection assays and binding and footprinting assays. Nucleotide sequences presenting perfect or imperfect motifs of myocyte- specific cis-acting elements occur in the SR Ca2 regulatory region but it is currently unclear if they are functional in cardiac myocytes or striated muscle cells. After such elements have been shown to be functional their interaction with TREs will be investigated in further detail. Our results indicate that nuclear extracts of neonatal myocytes contain proteins which markedly enhance the binding of T3 receptors (T3R) to TREs. We will determine if cell-type specific and TRE-specific of T3R enhancer binding proteins occur in myocyte nuclear extracts using ABCD assay, gel morbidity shift assay, and a protein cross-linking approach. In addition, we will determine if T3R binding enhancer proteins contact specific regions of TREs or adjacent DNA. Identification of specific nucleotide sequences to which such proteins attach will allow for fractionation and purification of these proteins. Investigation of the mechanism by which T3 alters transcription of the SR Ca2 gene will elucidate one of the causes which underlie T3- induced changes in cardiac contractility and will contribute to knowledge related to cell-type and gene-type specific mechanisms of T3 action.
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