摘要（一）

甲状腺激素水平对肥厚型梗阻性心肌病患者心功能及预后的预测作用

背景：甲状腺激素在心血管系统病理生理学中起着至关重要的调节作用，既往研究发现甲状腺功能异常，尤其是甲状腺功能减退在重症心血管疾病中高达10%-15%，而且在疾病进展和预后中起到重要预测作用。既往有针对心力衰竭的队列研究，但纳入疾病人群具有较大异质性，针对肥厚型梗阻性心肌病单一病因的随访观察研究较少，此研究旨在探索甲状腺激素在肥厚型梗阻性心肌病患者中对心脏功能、心血管危险因素以及预后的预测价值。

方法：研究对象是2009年10月1日至2014年12月31日期间连续收住于中国医学科学院阜外医院的956名肥厚型梗阻性心肌病患者，其中756名患者完成甲状腺功能评估符合入排标准纳入本研究。根据游离三碘甲状腺原氨酸（free triiodothyronine, FT3）水平，将患者均分为三组，FT3<2.81 pg/mL（n=247），2.81 pg/mL ≤FT3≤3.11 pg/mL（n=250），3.12 pg/mL≤FT3≤4.09 pg/mL（n=259）。多元线性回归分析研究甲状腺激素与心功能指标的相关性，logistic回归分析研究甲状腺激素与房颤发生风险的相关性。研究主要终点是全因死亡和心脏移植复合终点。采用Kaplan-Meier法绘制生存曲线，单因素和多因素Cox回归分析用于比较组间主要终点事件的差异。

结果：在相关性分析中，FT3与左心室射血分数呈显著正相关(r=0.109, P=0.003)，FT 4与室间隔厚度呈负相关（r=-0.083, P=0.023），相对于TSH 0.55-2.49 mIU/L，TSH水平升高组TSH 2.50-9.99 mIU/L组（OR 1.977, 95% CI 1.115-3.506, P=0.020）和TSH ≥10.00 mIU/L组（OR 4.301, 95% CI 1.059-17.476, P=0.041）房颤发生风险均显著升高。患者中位随访时间为44个月，随访率为93.7%，期间共有45例患者（6.0%）达到终点事件（全因死亡或心脏移植），其中3组的发生率分别为13.4%，3.6%和1.2%。单因素Cox回归分析发现FT3是全因死亡和心脏移植复合终点的强有力的预测因素（HR 0.111; 95%CI: 0.065, 0.189;P<0.001）。在调整其他危险因素后，FT3仍是主要终点事件的独立的预测因素（HR 0.216; 95% CI: 0.083, 0.559; P=0.002）。与3.12 pg/mL≤FT3≤4.09 pg/mL组患者相比，FT3<2.81 pg/mL组的患者发生主要终点事件的风险显著升高（HR 4.918; 95% CI: 1.076, 22.485; P=0.040）。ROC曲线分析发现即使FT3在正常下限即增加患者全因死亡风险，分析提示FT3的最佳界值为2.73 pg/mL (FT3正常值为1.79-4.09 pg/mL)。

结论：FT3水平与肥厚梗阻性心肌病患者心功能显著相关，并且可作为肥厚梗阻性心肌病患者不良结局（全因死亡和心脏移植）的独立预测因子，TSH升高增加患者房颤发生风险。研究结果提示肥厚梗阻性心肌病患者监测甲状腺功能是有必要的，对于纠正甲状腺功能异常状态能否带来临床获益需要临床试验进一步研究明确。

关键词：游离三碘甲状腺原氨酸，肥厚型梗阻性心肌病，心脏功能，预后

摘要（二）

甲状腺激素对心肌梗死大鼠的心肌保护作用及机制研究

背景：越来越多临床证据显示甲状腺功能异常在心血管疾病中不良预后的作用，对心血管疾病补充三碘甲状腺原氨酸（T3）获益的证据正在逐渐积累，而对机制的研究尚需进一步深入探索。β-受体阻滞剂是心肌梗死和心力衰竭的治疗基石，目前尚缺乏甲状腺激素与心肌梗死和心力衰竭标准治疗药物的直接对比研究。另一方面，由于甲状腺激素与交感神经肾上腺素能系统在机体生理功能等存在多方面的协同交互作用，但研究多集中在产热、生长发育和代谢方面，在心肌梗死和心力衰竭治疗方面的交互对话机制缺乏研究。

方法：12周龄的200-224g雌性Sprague-Dawley大鼠左冠状动脉前降支结扎制作心肌梗死模型，存活的心肌梗死大鼠随机分组至心肌梗死安慰剂治疗组（MI+Veh，n=11）、心肌梗死T3治疗组（MI+T3，n=11）和心肌梗死美托洛尔治疗组（MI+Met，n=11），假手术组大鼠开胸前降支穿线但不结扎作为正常对照组（Sham, n=8）。T3和美托洛尔溶解在饮用水给予大鼠治疗，其剂量分别为5µg/kg/d和100mg/kg/d。治疗8周后，检测各组大鼠心脏超声、血流动力学、心律失常诱发实验、血管舒张功能以及分子生物学等实验。

结果：相比心肌梗死安慰剂治疗组，T3和美托洛尔治疗明显改善了心脏的收缩功能（左室缩短分数LVFS，21.37±2.58%, 21.14±3.71% vs. 17.88±1.23%, T3, Met vs. Veh）。T3和美托洛尔分别降低快速性房性心律失常诱发率达到87.5%和62.5%。总体两者治疗效果类似，但在以下指标与心肌梗死安慰剂组相比，T3具有显著统计学差异，而美托洛尔虽有改善但未达到统计学效果，比如诱发的心律失常持续时间、左房直径、左房纤维化水平、血管对内皮依赖性舒张剂乙酰胆碱（ACh）的舒张反应性等。定量qPCR array结果显示T3和美托洛尔可以显著降低炎症因子和氧化应激有关基因的表达水平，改善离子通道和心脏关键收缩蛋白的基因表达水平。更重要的是，甲状腺激素和美托洛尔可部分或完全恢复甲状腺激素和β-肾上腺素能受体信号通路的基因表达。

结论：T3在心肌梗死治疗方面的效应与美托洛尔相似，可能与其降低炎症因子和氧化应激有关基因的表达水平，改善离子通道和心脏关键收缩蛋白的基因表达水平相关。本研究发现T3可以上调心肌梗死中下调的β-受体，可能与其改善心肌梗死后心脏功能和心室重塑相关，提示甲状腺激素信号通路可能与β-肾上腺素能受体信号通路存在有益的交互对话机制，这对于进一步改善心肌梗死和心力衰竭的治疗具有重要意义，尤其对于临床不能耐受β-受体阻滞剂的患者。

关键词：三碘甲状腺原氨酸，美托洛尔，心肌梗死，心脏功能

摘要（三）

甲状腺激素通过调节血管平滑肌细胞PKG/VASP信号通路促进血管舒张的研究

背景：既往研究发现甲状腺激素具有促进血管舒张的作用，通过内皮细胞（EC）的内皮依赖性血管舒张作用比较清楚，近些年发现血管平滑肌细胞（VSMC）也是甲状腺激素作用的重要靶点，其也可通过非内皮依赖性机制促进血管舒张，但具体机制尚不清楚。近年研究发现环鸟苷酸（cGMP）依赖的蛋白激酶G（PKG）的激活在一氧化氮（NO）引起的血管舒张中起重要作用。我们前期研究发现三碘甲状腺原氨酸（T3）具有促进血管舒张、改善内皮功能的作用，但具体机制尚待进一步研究。因此，我们假设T3作用于内皮细胞促进NO释放，然后通过血管平滑肌细胞内的NO/cGMP/PKG信号通路促进血管舒张。

方法：人主动脉内皮细胞（HAEC）和血管平滑肌细胞分别接受T3短时间（2分钟到60分钟）和长期（24小时）的治疗。用NO荧光探针（DAF-FM  DA）检测内皮细胞内NO的释放量。分离健康成年雄性Sprague-Dawley大鼠胸主动脉环并在离体条件下接受T3治疗20分钟，然后利用DMT 620M离体微血管张力测定系统观察其对内皮依赖性血管舒张剂乙酰胆碱（ACh）和非内皮依赖性血管舒张剂硝普钠（SNP）的累积剂量反应曲线。用Western Blot方法检测内皮细胞和血管平滑肌细胞在T3作用下相关信号通路蛋白的表达量。

结果：T3治疗的胸主动脉对ACh的内皮依赖性血管舒张反应性和SNP的非内皮依赖性血管舒张反应性显著增强。在PKG抑制剂作用下，T3的促血管舒张作用明显减弱。T3促进了内皮细胞蛋白激酶B（protein kinase B, Akt）在Ser 473位点和内皮型一氧化氮合酶（endothelial nitric synthetase, eNOS）在Ser 1177位点的磷酸化，从而促进了NO的生成释放。T3在血管平滑肌细胞通过PKG信号通路促进了血管扩张剂刺激磷蛋白（VASP）Ser 239位点的磷酸化，此磷酸化作用在PKG抑制剂作用下显著减弱。此外，离体血管以及血管平滑肌细胞接受T3直接作用下，PKG蛋白水平较对照组显著升高，而mRNA水平无显著差异。

结论：研究发现T3促血管舒张作用需要内皮细胞和血管平滑肌细胞共同参与的一个整体协调过程，在内皮细胞通过Akt/ eNOS信号通路促进NO释放，NO弥散至邻近的血管平滑肌细胞，通过PKG/VASP信号通路促进血管舒张作用。此机制的进一步明确为T3在心血管疾病中的转化应用提供了新的证据，为心血管疾病的治疗提供了新的治疗靶点。

关键词：三碘甲状腺原氨酸，血管平滑肌细胞，蛋白激酶G，血管扩张剂刺激磷蛋白，血管舒张

Abstract 1

Free triiodothyronine level correlates with cardiac function and long-term prognosis in patients with hypertrophic obstructive cardiomyopathy

Background: Thyroid hormone (TH) acts as a fundamental regulator in cardiovascular homeostasis under both and physiological pathological conditions. Even though subtle deficit of free triiodothyronine (FT3), the active form of TH, can lead to a series of disorders in the cardiovascular system and contributes to worse prognosis. We aimed to examine whether FT3 could be an independent predictor of adverse events and further to determine the optimal cutoff value of FT3 in predicting adverse events in patients with hypertrophic obstructive cardiomyopathy (HOCM).

Methods: The original cohort consisted of 965 consecutive patients with HOCM who were admitted to our institute from October 1, 2009 to December 31, 2014, and 756 patients with complete thyroid function evaluations were finally enrolled into this analysis. Patients were equally divided into three groups based on FT3 levels: tertile 1 (<2.81 pg/mL, n=247), tertile 2 (2.81 to 3.11 pg/mL, n=250), tertile 3 (3.12 to 4.09 pg/mL, n=259). Multiple linear analysis was used to examine the correlation of TH and cardiac functional parameter assessed by echocardiography. Logistic regression analysis was used to determine the association between TH and atrial fibrillation (AF). The primary endpoints of this study were composite of all-cause mortality and cardiac transplantation. Univariate and multiple Cox regression analysis were used to assess the prognostic value of TH. A receiver operating characteristic curve was performed to determine the optimal cutoff value of FT3 for all-cause mortality and cardiac transplantation.

Results: In correlation analysis, FT3 showed significantly positive correlation with left ventricular ejection fraction (r=0.109, P=0.003). The risk of AF significantly increased as TSH elevated. After a median follow-up of 44 months, a total of 45 (6.0%) endpoints (all-cause mortality or cardiac transplantation) occurred with rates of 13.4%, 3.6% and 1.2% in tertiles 1, 2 and 3, respectively. Univariate Cox analysis established FT3 as a predictor of endpoint (HR 0.111; 95%CI: 0.065, 0.189; P <0.001). After adjustment for traditional risk factors, the prognostic value of FT3 level was still significant (HR 0.216; 95% CI: 0.083, 0.559; P =0.002). Compared with patients in tertile 3, those in tertile 1 were at a much higher risk of endpoint (HR 4.918; 95% CI: 1.076, 22.485; P =0.040). The sensitivity and specificity were 79% and 73%, respectively, for FT3 cutoff value of 2.73 pg/mL, with an area under the curve of 0.79.

Conclusions: FT3 correlated with cardiac function and could serve as an independent predictor of all-cause mortality and cardiac transplantation in patients with HOCM. Higher circulating FT3 (normal lower of FT3 in our institute is 1.79 pg/mL) may be necessary to maintain cardiac tissue euthyroid under HOCM condition. These findings support the need for close monitoring of thyroid function in HOCM and suggest that maintaining serum TH levels within normal range may be considered a therapeutic target which needs to be confirmed in the future studies.

Key words: free triiodothyronine, hypertrophic obstructive cardiomyopathy, heart function, prognosis

Abstract 2

Cardioprotective effects of therapeutic triiodothyronine in treatment of myocardial infarction in rats

Background: It is frequently and consistently observed that low thyroid hormone (TH) function existed in severe cardiovascular diseases including myocardial infarction. Hypothyroidism has been increasingly identified as an independent and potent predictor for adverse prognosis in patients with cardiovascular disorders. Beta blockers are standard therapy for myocardial infarction (MI). Preclinical studies have shown efficacy and safety of TH treatment of cardiovascular disorders. Since THs interact with the sympathoadrenergic system to control several physiological processes, we aimed to compare triiodothyronine (T3) and metoprolol (Met) treatment of rats with MI on pathophysiology and TH-adrenergic signaling.

Methods: Female Sprague-Dawley rats aged 12 weeks underwent left anterior descending coronary artery ligation (MI) or sham surgeries. T3 (5 µg/kg/d) or Met (100 mg/kg/d) were given in drinking water immediately after surgery for 8 weeks. At the terminal of the experiments, the rats were subjected to morphological, functional, and molecular examination.

Results: T3 and Met significantly enhanced left ventricular (LV) contractility (LVFS 21.37±2.58% and 21.14±3.71%, respectively) compared to untreated MI (17.88±1.23%) and decreased the incidence of inducible atrial tachyarrhythmia by 87.5% and 62.5%. Although both treatments showed efficacy, T3 but not Met showed statistically significant improvements compared to MI in arrhythmia duration, left atrial diameter (T3 vs MI 4.33±0.63 vs. 5.65±1.32 mm, P<0.05), fibrosis (6.1±0.6%, 6.6±0.6% vs. 8.2±0.7%, T3, Met vs. MI, respectively) and aortic vasorelaxation responsiveness (pD2 6.97±0.22, 6.83±0.21 vs. 6.66±0.22, T3, Met vs MI, respectively). Quantitative PCR array showed that T3 and Met attenuated expression of genes associated with inflammation and oxidative stress and restored expression of ion channels and contractile proteins. Importantly, thyroid and β-adrenergic signaling genes were favorably regulated by both treatments.

Conclusion: These results support the efficacy of long-term treatment with a physiological dose of T3 comparable to Met and suggest a potential therapeutic alternative beyond standard β-blockade, especially for patients intolerant to treatment with β-blockers after MI.

Key words: triiodothyronine, metoprolol, myocardial infarction, heart function

Abstract 3

Triiodothyronine potentiates vasorelaxation via PKG/VASP signaling in vascular smooth muscle cells

Background: Vascular relaxation caused by Triiodothyronine (T3) involves direct activation of endothelial cells (EC) and vascular smooth muscle cells (VSMC). Activation of protein kinase G (PKG) has risen as a novel contributor to the vasorelaxation mechanism triggered by numerous stimuli. We hypothesize that T3-induced vasorelaxation involves PKG/vasodilator-stimulated phosphoprotein (VASP) signaling pathway in VSMC.

Methods: Human aortic endothelial cells (HAEC) and VSMC were treated with T3 for short (2 to 60 minutes) and long term (24 hours). Nitric oxide (NO) production was measured using DAF-FM DA. For functional studies, adult male Sprague-Dawley rat thoracic aortas were isolated and treated with T3 for 20 minutes and mounted in a wire myograph. Relaxation was measured by a concentration-dependent response to acetylcholine (ACh) and sodium nitroprusside (SNP). Expression of protein targets was determined using western blot.

Results: Aortas stimulated with T3 exhibited augmented sensitivity to ACh and SNP-induced relaxation, endothelium-dependent and endothelium-independent responses, respectively. T3 directly increased vasorelaxation, which was abolished in the presence of a PKG inhibitor. T3 markedly induced phosphorylation of Akt at Ser 473, eNOS at Ser 1177 and consequently increased NO production in EC. Likewise, T3 induced phosphorylation of VASP at serine 239 via the PKG pathway in VSMC.

Conclusion: Our findings have uncovered a PKG/VASP signaling pathway in VSMC as a key molecular mechanism underlying T3-induced vascular relaxation. This study suggests that T3 has a vasoprotective effect, endorsing it as a potential therapeutic approach to treat vascular disease that involves impaired vasodilation.

Key words: triiodothyronine, vascular smooth muscle cell, protein kinase G, vasodilator-stimulated phosphoprotein, vasorelaxation