Thyroid hormone exerts marked influences on cardiac function and a prolonged cardiac diastole occurs in hypothyroidism. The duration of diastolic relaxation is closely linked to the activity of the sarcoplasmic endoplasmic reticulum Ca2+ ATPase pump (SERCA2) and the gene coding for SERCA2 shows marked T3-induced increases in transcription. The promoter for this gene contains three differently configured thyroid response elements (TRE) as recently shown by us. TRE1 at NT-481 to -458 is a direct repeat spaced by four nucleotides (DR +4); TRE2 at NT - 310 to -298 is an everted repeat or inverted palindrome spaced by four nucleotides (IP+4); TRE3 at NT -219 to -194 is an inverted palindrome plus six nucleotides (IP+6). We would like to delineate in the first aim how the three TREs of the SERCA2 gene contribute in a cooperative manner and individually to the regulation of SERCA2 gene transcription under in vivo and in vitro conditions. Transient transfection assays and in vivo studies using intraventricular wall injection and transgenic mice will be used to evaluate the contribution of TREs functioning individually or in combination in the context of the SERCA2 promoter. Our preliminary results indicate that a muscle-specific transcription factor myocyte enhancer factor 2 (MEF2) a member of the MADS transcription factor family interacts in a synergistic manner with thyroid hormone receptors in the transcriptional regulation of the SERCA2 gene. In the second aim, we will determine the mechanisms which govern the interaction between the MEF2 and the thyroid receptor (T3R). In addition, we will explore the interaction between the T3R with other transcription factors. In the third aim, mechanisms leading to modifications of normal T3 action as it occurs in cardiac ventricular myocytes will be examined. Specifically, modifications of T3 actions exerted by T3-beta receptor mutants obtained from patients with a generalized thyroid hormone resistance syndrome will be explored by transient transfection studies and in transgenic mice expressing these receptors at high levels in their heart. These investigations will clarify the mechanisms which underlie T3-induced changes in cardiac contractility and contribute to knowledge related to cardiac myocyte-type specific and gene-specific mechanisms of T3 action under in vitro and in vivo conditions.
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