Thyroid regulation of cardiomyocyte maturation

描述（由申请人提供）：在胎羊中，145天妊娠期的第100天左右，心肌细胞逐渐停止分裂并成为双核（终末分化）。一旦心肌细胞最终分化，它们就不能再分裂，但它们仍保持着显著的扩大能力。这一成熟步骤的标志是绵羊的双核发育，主要发生在出生前。我们发现3,3'，5-三碘- l -甲状腺原氨酸（T3）在体外135天的绵羊心肌细胞中是一种强大的增殖抑制剂。似乎皮质醇刺激了短期内较弱的甲状腺素（T4）向较强的T3的转化，导致T3水平上升，从而抑制了心肌细胞的增殖。由于其时间的巧合，T3已成为心肌成熟最强大的调节因子的主要候选。它也可能在心脏产生最佳数量的心肌细胞之前就终止增殖。因此，T3调节对于母体甲状腺素水平超出正常范围时较为常见的疾病具有临床意义。我们申请资金来研究甲状腺激素在调节绵羊胎儿心肌成熟中的作用。目的1：确定T3在体内抑制胎儿心肌细胞增殖和促进双核/终分化的程度。假设：T3会抑制完整胎羊心肌细胞的增殖速率，增加终末分化速率，刺激心肌细胞成熟。目的2：在体外胎儿心肌细胞暴露于T3后，确定关键信号蛋白（MAPK和PI3K通路）的发育表达和时间激活。假设：MAPK和PI3K均被T3激活，但增殖受MAPK及其与p21的相互作用调控。我们将通过测量ERK、AKT、mTOR和p70S6K以及关键细胞周期蛋白的激活水平来评估MAPK和PI3K信号级联在T3影响下调节增殖中的重要性。“非基因组”途径也将被评估。目的3：测定体内T3浓度升高时胎儿心肌对右心室收缩负荷的不适应程度。假设：T3治疗抑制了心肌细胞对右心室（RV）收缩负荷的正常增殖反应，进一步刺激了心肌细胞的双核和成熟速度。目的4：确定在子宫内暴露于高T3的胎儿的早期产后过渡期间维持心肌细胞生长和成熟的程度。假设：即使IGF-1水平升高，正常的出生后T3激增也会阻止被抑制的心肌细胞数量在出生后立即再生。本研究将确定甲状腺激素在出生前调节心肌成熟中的作用。一旦完成，研究将表明经典和非经典信号通路在多大程度上调节出生前T3刺激心肌细胞行为的变化。

DESCRIPTION (provided by applicant): In fetal sheep, cardiomyocytes gradually cease dividing and become binucleated (terminal differentiation) at ~100 days of a 145 day gestational period. Once cardiac myocytes terminally differentiate they can no longer divide but they retain a remarkable capacity to enlarge. The hallmark of this maturation step is binucleation in sheep which occurs mostly before birth. We discovered that 3,3',5-tri-iodo-L-thyronine (T3), is a powerful inhibitor of proliferation in 135 day ovine cardiomyocytes in vitro. It appears that cortisol stimulates the conversion of the less potent thyroxine (T4) to the more potent T3 near term, causing T3 levels to rise-putting the brakes on cardiomyocyte proliferation. Because of its coincidental timing, T3 has become a primary candidate for being the most powerful regulator of the maturation of the myocardium. It may also terminate proliferation long before the heart has generated its optimal number of cardiomyocytes. Thus, T3 regulation has clinical relevance for the relatively common disease conditions when maternal thyroxine levels are outside the normal range. We request funds to study the role of thyroid hormone in regulating the maturation of the fetal myocardium in sheep. Aim 1: Determine the degree to which T3 suppresses proliferation and promotes binucleation/terminal differentiation of fetal cardiac myocytes in vivo. Hypothesis: T3 will depress the rates of proliferation of intact fetal ovine cardiomyocytes, increase the rate of terminal differentiation and stimulate cardiomyocyte maturation. Aim 2: Determine the developmental expression and temporal activation of key signaling proteins (MAPK & PI3K pathways) following exposure to T3 in fetal cardiac myocytes in vitro. Hypothesis: Both MAPK and PI3K are activated by T3, but proliferation is regulated by MAPK and its interaction with p21. The importance of the MAPK and PI3K signaling cascades in regulating proliferation under the influence of T3 will be evaluated by measuring the activation levels of ERK, AKT, mTOR and p70S6K, as well as key cell cycle proteins. "Non-genomic" pathways will also be evaluated. Aim 3: Determine the degree to which the fetal myocardium mal-adapts to right ventricular systolic load when T3 concentrations are elevated in vivo. Hypothesis: T3 treatment suppresses the normal proliferative response of cardiomyocytes to right ventricular (RV) systolic load and further stimulates the rate of binucleation and maturation of cardiomyocytes. Aim 4: Determine the degree to which cardiomyocyte growth and maturation are maintained during the early postnatal transition in fetuses that have been exposed to high T3 in utero. Hypothesis: The normal postnatal T3 surge will prevent suppressed cardiomyocyte numbers to regenerate during the immediate postnatal period, even in the presence of elevated levels of IGF-1. This study will determine the role of thyroid hormone in regulating the maturation of the myocardium before birth. Once completed the studies will indicate the degrees to which classical and non- classical signaling pathways regulate the T3 stimulated changes in cardiomyocyte behavior before birth. PUBLIC HEALTH RELEVANCE: This study will determine the relevance of fetal T3 levels in regulating the proliferation of working cardiomyocytes before birth. Because maternal thyroid hormones cross the placenta and influence fetal levels, maternal thyroid disease may seriously affect heart cardiomyocyte endowment. Low cardiomyocyte numbers could lead to a myocardium that is disadvantaged for the work it will perform in extrauterine life.