Is thyroid hormone a key regulator of fetal cardiomyocyte proliferation?

描述（由申请人提供）：发育中的胎儿心脏主要通过所有细胞类型（包括心肌细胞）的增殖生长。在绵羊中，从145天妊娠期的100天开始，心肌细胞逐渐停止分裂并变成双核（终末分化）。一旦心肌细胞终末分化，它们就不能再分裂，但它们保留了巨大的扩大能力。这一成熟步骤主要发生在绵羊出生前。在细胞失去生殖能力之前，对心肌细胞复制的调节是重要的，但是决定：1）多少细胞是最佳的，2）细胞何时应该停止分裂的机制尚不清楚。我们发现甲状腺激素以3，3 ′，5-三碘-L-甲状腺原氨酸（T_3）的形式存在，对135天龄绵羊胎儿心肌细胞的体外增殖有明显的抑制作用。在妊娠晚期，皮质醇刺激较弱的甲状腺素（T4）转化为更强的T3;此后，T3水平开始增加。因此，由于这种巧合的时机，T3已成为心肌近期成熟的最强大调节剂的候选者。它也可能是在心脏产生最佳数量的心肌细胞之前很久就终止增殖的罪魁祸首。因此，当母体甲状腺素水平超出正常范围时，T3调节对于相对常见的病症具有临床相关性。为了确定T3调节工作肌细胞细胞周期的程度，我们要求探索性资金在两年内实现两个目标。目的1：研究甲状腺激素对正常妊娠晚期（125-130天;足月~145天）绵羊胎儿心肌细胞增殖和成熟的调节作用。将对完整和甲状腺切除的胎羊给予T3，以确定在比正常T3峰早约10天的年龄时由低、正常和高T3循环浓度引起的心肌细胞复制变化。将使用Ki-67和BrdU作为标志物监测心肌细胞增殖。还将测量脱碘酶和甲状腺受体的表达水平;将通过蛋白质印迹法分析关键细胞周期蛋白的水平。目的二是研究T3在体外刺激细胞周期中的作用。通过测量ERK、p38、JNK、AKT、mTOR和p70 S6 K以及关键细胞周期蛋白的活化水平，评价MAPK和PI 3 K信号级联在T3影响下调节增殖的重要性。也将评估所谓的非基因组途径。将测量脱碘酶和甲状腺受体的表达水平。这项研究提供了一个机会，以确定是否甲状腺激素是一个强大的抑制增殖的未成熟的心肌细胞和成熟的心肌。一旦完成，这也应该表明经典和非经典信号通路调节T3刺激的心肌细胞行为变化的程度。一个积极的结果将导致进一步的工作在动物和人类群体。公共卫生相关性：这项研究将确定胎儿T4和T3水平在出生前调节工作心肌细胞增殖的相关性。由于母体甲状腺激素穿过胎盘并影响胎儿水平，母体甲状腺疾病可能严重影响心脏心肌细胞的禀赋。心肌细胞数量少可能导致心肌在子宫外生活中的工作处于不利地位。.

DESCRIPTION (provided by applicant): The developing fetal heart grows primarily by proliferation of all cell types, including cardiomyocytes. In sheep, cardiomyocytes gradually cease dividing and become binucleated (terminal differentiation) beginning at 100 days gestation of a 145 day gestational period. Once cardiac myocytes terminally differentiate they can no longer divide but they retain an enormous capacity to enlarge. This maturation step occurs mostly before birth in sheep. The regulation of cardiomyocyte replication, before the cells lose their generative capacity, is important, but the mechanisms that determine: 1) how many cells are optimal and 2) when a cell should stop dividing, are not known. We discovered that thyroid hormone, in the form of 3,3',5-tri-iodo-L-thyronine (T3), is a powerful inhibitor of proliferation in cardiomyocytes from135 day old sheep fetuses in vitro. In late gestation, cortisol stimulates the conversion of the less potent thyroxine (T4) to the more potent T3; thereafter, T3 levels begin to increase. Thus, because of this coincidental timing, T3 has become a candidate for the most powerful regulator of the near term maturation of the myocardium. It may also be a culprit in terminating proliferation long before the heart has generated its optimal number of cardiomyocytes. Thus, T3 regulation has clinical relevance for the relatively common conditions when maternal thyroxine levels are outside the normal range. In order to determine the degree to which T3 regulates the cell cycle in working myocytes, we request exploratory funds to carry out two aims over two years. Aim 1 will determine the degree to which thyroid hormone regulates cardiomyocyte proliferation and maturation of cardiomyocytes in normally growing late gestation sheep fetuses (125-130 days; term ~145 days). T3 will be administered to both intact and thyroidectomized fetal sheep to determine the changes in cardiomyocyte replication caused by low, normal, and high T3 circulating concentrations at an age ~10 days earlier than the normal T3 surge. Cardiomyocyte proliferation will be monitored using Ki-67 and BrdU as markers. Expression levels of deiodinases and thyroid receptors will also be measured; levels of key cell cycle proteins will be analyzed by western blot. Aim 2 will determine the roles of alternative signaling pathways that are stimulated by T3 in vitro and lead to inhibition of cell cycle activity. 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, p38, JNK, AKT, mTOR and p70S6K, as well as key cell cycle proteins. So-called non-genomic pathways will also be evaluated. Expression levels of deiodinases and thyroid receptors will be measured. This study offers the opportunity to determine whether thyroid hormone is a powerful suppressant of proliferation in the immature cardiac myocyte and on the maturation of the myocardium. Once completed this should also indicate the degrees to which classical and non-classical signaling pathways regulate the T3 stimulated changes in cardiomyocyte behavior. A positive outcome will lead to further work in animal and human populations. PUBLIC HEALTH RELEVANCE: This study will determine the relevance of fetal T4 and 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. .