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各种细胞类型的增殖来生长，包括心肌细胞。绵羊的心肌细胞在145天妊娠期的第100天开始逐渐停止分裂并成为双核细胞（终末分化）。一旦心肌细胞最终分化，它们就不能再分裂，但它们保留着巨大的扩张能力。这一成熟步骤大多发生在绵羊出生前。在细胞失去繁殖能力之前，对心肌细胞复制的调控是很重要的，但决定以下问题的机制尚不清楚：1)多少细胞是最佳的，2)细胞何时应该停止分裂。我们发现，以3,3'，5-三碘- l -甲状腺原氨酸（T3）形式存在的甲状腺激素是135日龄绵羊体外胚胎心肌细胞增殖的强大抑制剂。在妊娠后期，皮质醇刺激较弱的甲状腺素（T4）转化为更强的T3；此后，T3水平开始升高。因此，由于这个巧合的时机，T3已成为心肌近期成熟最强大的调节因子的候选。它也可能是在心脏产生最佳数量的心肌细胞之前就终止增殖的罪魁祸首。因此，T3调节对于母体甲状腺素水平超出正常范围的较为常见的情况具有临床意义。为了确定T3在工作肌细胞中调节细胞周期的程度，我们申请探索性资金，在两年内完成两个目标。目的1将确定甲状腺激素在正常生长的妊娠晚期绵羊胎儿（125-130天；足月~145天）中对心肌细胞增殖和心肌细胞成熟的调节程度。将T3给予完整的和去甲状腺的胎羊，以确定低、正常和高T3循环浓度引起的心肌细胞复制的变化，比正常T3峰值早10天左右。使用Ki-67和BrdU作为标记物监测心肌细胞增殖。去碘酶和甲状腺受体的表达水平也将被测量；关键细胞周期蛋白的水平将通过western blot进行分析。目的2将确定T3在体外刺激的替代信号通路的作用，并导致细胞周期活性的抑制。我们将通过测量ERK、p38、JNK、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。所谓的非基因组途径也将被评估。将测量去碘酶和甲状腺受体的表达水平。本研究为确定甲状腺激素是否对未成熟心肌细胞增殖和心肌成熟具有强大的抑制作用提供了机会。一旦完成，这也应该表明经典和非经典信号通路调节T3刺激心肌细胞行为变化的程度。一个积极的结果将导致进一步在动物和人类群体中开展工作。公共卫生相关性：本研究将确定出生前胎儿T4和T3水平在调节工作心肌细胞增殖中的相关性。由于母体甲状腺激素穿过胎盘影响胎儿水平，母体甲状腺疾病可能严重影响心肌细胞禀赋。心肌细胞数量过低可能导致心肌对其在子宫外生活中所执行的工作不利。

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. .