Thyroid regulation of cardiomyocyte maturation

描述（由申请人提供）：在145天妊娠期的约100天，胎羊心肌细胞逐渐停止分裂并变为双核（终末分化）。一旦心肌细胞终末分化，它们就不能再分裂，但它们保留了显著的扩大能力。这个成熟步骤的标志是绵羊的双核化，主要发生在出生前。我们发现3，3 ′，5-三碘-L-甲状腺原氨酸（T_3）对135日龄绵羊心肌细胞的增殖有明显的抑制作用。皮质醇似乎在短期内刺激较弱的甲状腺素（T4）转化为较强的T3，导致T3水平升高，从而抑制心肌细胞增殖。由于其巧合的时机，T3已成为心肌成熟的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，当母体甲状腺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相关性。我们要求基金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/终末分化的程度。假设：T3可抑制完整胎羊心肌细胞的增殖，促进终末分化，促进心肌细胞成熟。目标二：测定T3暴露于体外胎儿心肌细胞后关键信号蛋白（MAPK和PI 3 K通路）的发育表达和时间激活。假设：MAPK和PI 3 K都被T3激活，但增殖受MAPK及其与p21相互作用的调节。通过测量ERK、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。还将评估“非基因组”途径。目的3：确定胎儿心肌在体内T3浓度升高时对右心室收缩负荷的适应不良程度。假设：T3处理抑制心肌细胞对右心室（RV）收缩负荷的正常增殖反应，并进一步刺激心肌细胞的双核化和成熟率。目标4：确定在子宫内暴露于高T3的胎儿在出生后早期过渡期间心肌细胞生长和成熟的维持程度。假设：正常的出生后T3激增将阻止抑制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。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，这些研究将表明经典和非经典信号通路调节出生前T3刺激的心肌细胞行为变化的程度。 公共卫生相关性：这项研究将确定胎儿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): 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.