Thyroid regulation of cardiomyocyte maturation

描述(申请人提供)：在胚胎绵羊中，心肌细胞逐渐停止分裂，并在145天的妊娠期中的~100天变成双核(终末分化)。一旦心肌细胞终末分化，它们就不能再分裂了，但它们保留了非凡的放大能力。这一成熟步骤的标志是绵羊的双核，这主要发生在出生之前。我们发现3，3‘，5-三碘-L-甲状腺原氨酸(T3)对体外培养的135日龄绵羊心肌细胞有很强的增殖抑制作用。皮质醇似乎刺激较弱的甲状腺素(T4)在短期内转化为较强的T3，导致T3水平上升-抑制心肌细胞的增殖。由于其巧合的时机，T3已成为心肌成熟最强大的调节剂的主要候选者。它也可能在心脏产生最佳数量的心肌细胞之前很久就终止增殖。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疾病状况具有临床意义。我们请求资金研究甲状腺激素在调节绵羊胚胎心肌成熟中的作用。目的1：确定T3在体内抑制胎儿心肌细胞增殖和促进双核/终末分化的程度。假设：T3可抑制完整胎羊心肌细胞的增殖率，提高终末分化率，促进心肌细胞成熟。目的：研究T3对体外培养的胎儿心肌细胞发育过程中关键信号蛋白(MAPK和PI3K信号通路)的表达和激活的影响。假设：MAPK和PI3K均由T3激活，但其增殖受MAPK及其与p21相互作用的调控。将通过检测ERK、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。“非基因组”途径也将被评估。目的3：测定体内T3浓度升高时，胎儿心肌对右室收缩负荷的不良适应程度。假设：T3治疗抑制了心肌细胞对右室收缩负荷的正常增殖反应，并进一步刺激了心肌细胞的双核和成熟率。目的4：测定宫内暴露于高T_3的胎儿在出生后早期心肌细胞生长和成熟的维持程度。假设：正常的出生后T3激增将阻止被抑制的心肌细胞数量在出生后立即再生，即使在IGF-1水平升高的情况下也是如此。这项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，这些研究将指出经典和非经典信号通路在多大程度上调节T3刺激的出生前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.