Is thyroid hormone a key regulator of fetal cardiomyocyte proliferation?

描述(申请人提供)：发育中的胎儿心脏主要通过包括心肌细胞在内的所有细胞类型的增殖而生长。在绵羊中，心肌细胞逐渐停止分裂并成为双核(终末分化)，从怀孕100天开始，妊娠期为145天。一旦心肌细胞终末分化，它们就不能再分裂了，但它们保留了巨大的放大能力。这一成熟阶段大多发生在绵羊出生之前。在细胞失去生殖能力之前，对心肌细胞复制的调控是重要的，但决定：1)多少细胞是最佳的，以及2)细胞何时应该停止分裂的机制尚不清楚。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对体外培养的135日龄绵羊胚胎心肌细胞的增殖有很强的抑制作用。在妊娠晚期，皮质醇刺激较弱的甲状腺素(T4)转化为较强的T3；此后，T3水平开始上升。因此，由于这一巧合的时机，T3已成为心肌近期成熟最强大的调节器的候选者。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，当母亲的甲状腺激素水平超出正常范围时，T3调节对于相对常见的情况具有临床意义。为了确定T3在多大程度上调节工作心肌细胞的细胞周期，我们要求探索性资金在两年内实现两个目标。目的1测定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控程度。给正常和切除甲状腺的胎羊注射T3，以确定在比正常T3峰值早10天的日龄时，低、正常和高T3循环浓度引起的心肌细胞复制的变化。将使用Ki-67和BrdU作为标志物来监测心肌细胞的增殖。脱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蛋白质印迹进行分析。目的2将确定可供选择的信号通路在体外被T3刺激并导致抑制细胞周期活性的作用。将通过检测ERK、p38、JNK、AKT、mTOR和p70S6K的激活水平以及关键的细胞周期蛋白来评估MAPK和PI3K信号通路在T3影响下调控细胞增殖的重要性。所谓的非基因组途径也将得到评估。将测量脱碘酶和甲状腺受体的表达水平。这项研究提供了确定甲状腺激素是否是未成熟心肌细胞增殖和心肌成熟的有力抑制因素的机会。一旦完成，这也应该表明经典和非经典信号通路调节T3刺激的心肌细胞行为变化的程度。一个积极的结果将导致动物和人类种群的进一步工作。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通过胎盘影响胎儿水平，母体甲状腺疾病可能会严重影响心肌细胞的功能。低心肌细胞数可能导致心肌在宫外生命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. .