Thyroid Hormone Receptor Function in Cardiac Hypertrophy

描述(申请人提供)：心脏收缩功能的许多方面是由甲状腺激素，特别是三碘甲状腺原氨酸(T3)对心肌细胞特异性基因转录的作用决定的，这种转录是由T3与一个或多个核甲状腺激素受体(TR)结合所介导的。单个受体(trαI，trβ1)及其转录后修饰在确定特定心肌细胞基因转录中的确切作用尚不清楚，其中包括共肌球蛋白重链(α-MHC)。在病理性和生理性肥厚的情况下，心肌表现出许多T3反应基因的表达改变，这些基因可以通过T3治疗部分恢复正常。目前的应用将检验这一假说，即肥厚心脏的T3反应性受损是由于TR亚型的表达和磷酸化改变的结果，这些改变影响核受体功能的方方面面，包括DNA结合、二聚化、与辅助调节因子的相互作用、配体亲和力和转录活性。在前两个特定目标中，我们提出了一种假设，即肥厚的心脏表型是由于核内TRR亚型含量的变化，以及通过从纯化的成年大鼠心室肌细胞中分离核蛋白来评估单个trαI和β1亚型被差异磷酸化的假设。我们的初步数据表明，非T3结合亚型TRα2具有独特的抑制T3诱导的心脏α-MHC和SERCA2基因转录的能力。重组腺病毒策略将在特定的目标3中在培养的心肌细胞中过表达trα2蛋白，并将通过一种新的定量α-MHC异核RNA的方法来测量由此导致的内源性α-MHC基因转录的变化。特定目标4将验证这样的假设，即在肥大的心肌中激活的蛋白激酶C信号通路导致特定的TR亚型的磷酸化，而这反过来又介导了心脏表型的变化，与甲状腺功能减退的心脏非常相似。我们将研究腺病毒介导的单个PKC Delta、epsilon和Zeta同工酶的过表达对特定的TR亚型磷酸化和α-MHC基因转录的影响。这些研究的完成将为TRs在介导肥厚表型中的作用提供新的机制信息，并为T3在充血性心力衰竭中的潜在治疗作用提供理论基础。

DESCRIPTION (provided by applicant): Many aspects of cardiac contractile function are determined by the action of thyroid hormones, specifically triiodothyronine (T3), on myocyte-specific gene transcription that is mediated byT3 binding to one or more nuclear thyroid hormone receptors (TR). The precise role of the individual TRs (TR alpha I,TR beta 1) and their post-transcriptional modification in determining transcription of specific cardiomyocyte genes, including co-myosin heavy chain (alpha-MHC), remains unknown. The myocardium in conditions of pathologic and physiologic hypertrophy exhibits altered expression of many T3-responsive genes, which can be partially normalized by T3 treatment. The present application will test the hypothesis that impaired T3 responsiveness in the hypertrophied heart is the result of altered expression and phosphorylation of TR isoforms which affect all aspects of nuclear receptor function including DNA binding, dimerization, interaction with co-regulators, ligand affinity and transcriptional activity. In the first two specific aims, we propose to test the hypothesis that the hypertrophic cardiac phenotype is a result of changes in nuclear, content of TR isoforms and that the individual TR alpha I and beta 1 isoforms are differentially phosphorylated as assessed by isolation of nuclear proteins from purified adult rat ventricular myocytes. Our preliminary data suggest that the non-T3 binding isoform, TR alpha 2,has the unique ability to repress T3-inducible transcription of cardiac alpha-MHC and SERCA2 genes. A recombinant adenovirus strategy will be used in specific aim 3 to overexpress TR alpha 2 protein in cultured cardiomyocytes,and the resulting changes in transcription of the endogenous alpha-MHC gene will be measured by a novel assay to quantify alpha-MHC heteronuclear RNA. Specific aim 4 will test will the hypothesis that protein kinase C signaling pathways that are activated in the hypertrophic myocardium result in phosphorylation of specific TR isoforms, which in turn mediate changes in cardiac phenotype remarkably similar to that of the hypothyroid heart. We will study the effects of adenoviral-mediated overexpression of individual PKC delta, epsilon, and zeta isoenzymes on specific TR isoform phosphorylation and alpha-MHC gene transcription. Completion of these studies will provide novel mechanistic information regarding the role of TRs in mediating the hypertrophic phenotype, and provide the rationale for the potential therapeutic utility of T3 in the setting of congestive heart failure.