从钙离子调控改变探讨兔肾素血管紧张素系统阻断剂逆转甲状腺素促心肌肥厚的机制

目的 研究甲状腺素诱导的肥厚心肌细胞内钙调控变化及其机制,探讨肾素血管紧张素系统(RAS)阻断剂逆转甲状腺素促心肌肥厚的机制.方法 腹腔注射左旋甲状腺素(L-Thy)建立兔甲状腺素性心肌肥厚模型,经胸二维超声心动图测定室间隔厚度(IVS)、左心室内径(LV)及左心室后壁厚度(LVPW)后收集心肌标本,光镜及透射电镜下观察心肌细胞结构改变,Fluo3/AM荷载共聚焦显微镜检测细胞内钙离子浓度,逆转录聚合酶链半定量检测L型Ca2+通道(LCC)、兰尼碱受体(RyR)、肌浆网钙泵(SERCA)mRNA表达,对硝基苯磷酸酯(P-nitrophenyl phosphate,P-NPP)法检测SERCA活性,免疫组织化学法检测IP3R蛋白表达.结果 甲状腺素可诱导心肌肥厚、心肌细胞结构改变及纤维组织增生,甲状腺素组细胞内钙离子浓度明显高于对照组,分别为(576.2±41.7)nmol/L比(314.6±35.6)nmol/L(P<0.01);甲状腺素组RyR mRNA、SERCA mRNA及IP3R蛋白较对照组表达上调,分别为1.19±0.21比0.73±0.15(P<0.01)、1.01±0.08比0.76±0.09(P<0.01)、65.3±13.7比47.9±10.2(P<0.01);LCC mRNA表达和SERCA活力降低,分别为0.48±0.11比0.75±0.16(P<0.01)、(0.062±0.013)μmol·min-1·g-1比(0.133±0.022)μmol·min-1·g-1(P<0.01).咪达普利和缬沙坦干预可显著抑制甲状腺素诱导的心肌细胞肥厚,降低细胞内钙离子浓度,咪达普利组和缬沙坦组细胞内钙离子浓度分别为(376.4±32.5)nmol/L和(392.6±41.2)nmol/L均显著低于甲状腺素组(576.2±41.7)nmol/L(P<0.01);咪达普利和缬沙坦干预后LCC mRNA表达高于甲状腺素组,分别为0.68±0.14比0.48±0.11(P<0.01)、0.64±0.13比0.48±0.11(P<0.01);咪达普利和缬沙坦干预增加SERCA的活力,分别为(0.115±0.019)μmol·min-1·g-1比甲状腺素组(0.062±0.013)μmol·min-1·g-1(P<0.01)、(0.109±0.015)μmol·min-1·g-1比甲状腺素组(0.052±0.013)μmol·min-1·g-1(P<0.01),但对RyR mRNA、SERCA mRNA及IP3R蛋白表达无影响.结论 RAS和细胞内钙超载可能参与甲状腺索性心肌肥厚的发病机制,SERCA活力降低可能是导致甲状腺素性肥厚心肌细胞内钙超载的重要因素.咪达普利和缬沙坦可以改善L-Thy诱导的心肌重构和细胞内钙超载并对SERCA活力具有保护作用.

Renin-agiotensin system blocking agents reverse the myocardial hypertrophy in experimental hyperthyroid cardiomyopathy via altering intracellular calcium handling

Objective To explore the mechanisms of myocardial hypertrophy induced by Levothyroxine (L-Thy). Methods A rabbit model of hyperthyroidism was established by daily intraperitoneal injections of L-Thy (45 μg/kg per day) for 28 days. New Zealand rabbits were randomly divided into four groups (n = 10 each): control group, L-Thy group (L-Thy alone), imidapril group (L-Thy + 0. 5 mg/kg imidapril), and valsartan group (L-Thy + 8 mg/kg valsartan). All rabbits were treated for 4 weeks. At the end of the treatments, all rabbits underwent echocardiography and IVS, LV and LVPW thickness were measured. Ventricular tissues were then collected. Cardic hypertrophy index, cardiomyocytediameter, structural and ultrastructural changes were obtained. Ventricular myocytes were isolated by enzymatic digestion method and intracellular Ca2+ concentration was determined with the fluoresent Ca2+ indicator Fluo3/AM and laser scanning confocal microscopy. Activity of Sarco/Endoplasmic reticulum Ca2+ Tpase(SERCA) was evaluated with P-NPP method, Mrna expression of L-typo Ca2+ channel (LCC), ryanodine receptor (RyR) ,and SERCA was semi-quantified with RT-PCR. Protein of IP3R was localized by immunostaining and semi-quantified with pathological image analytic system. Results Compared with control group, rabbits treated with L-Thy displayed remarkable myocardial hypertrophy and morphological changes in both structure and ultrastructure levels. Increased intracellular Ca2+ concentration (576. 2±41.7) nmol/L vs. (314. 6±35. 6) nmol/L,P <0. 01 ] and decreased SERCA activity (0. 062±0. 013) μmol · min-1 g-1 vs. (0. 133±0. 022) μmol · min-1 · g-1 ,P <0. 01 ] were detected in L-Thy treated rabbits. RT-PCR analysis and (or) immunohistochemistry revealed decreased Mrna expression of LCC Mrna (0. 48±0. 11 vs. 0. 75±0. 16,P < 0. 01 ) and increased RyR Mrna ( 1.19±0. 21 vs. 0. 73±0. 15, P < 0. 01 ), SERCA Mrna ( 1.01±0. 08 vs. 0.76±0. 09, P < 0. 01 ) and IP3R protein (65.3±13.7 vs. 47. 9±10. 2, P < 0. 01 ) expression in L-Thy treated rabbits. Both imidapril and valsartan could significantly attenuate cardiomyocyte hypertrophy and structural remodeling induced by L-Thy. Compared with L-Thy group, decreased intracellular Ca2+ concentration ( 376. 4±32. 5 ) nmol/L vs. ( 576. 2±41.7 ) nmol/L, P < 0. 01 and (392. 6±41.2) nmol/L vs. (576. 2±41.7) nmol/L, P <0. 01, respectively], and increased LCC Mrna (0.68±0.14 vs. 0.48±0.11,P <0.01; 0.64±0.13 vs. 0.48±0.11,P <0.01, respectively) and SERCA activity (0. 115±0. 019) μmol · min-1 · g-1 vs. (0. 062±0. 013) μmol · min-1 · g-1, P <0. 01 ; (0. 109±0. 015) μmol · min-1· g-1 vs. (0. 062±0. 013) μmol · min-1 · g-1,P <0. 01, respectively] were found in both imidapril and valsartan treated rabbits, but expression of RyR, SERCA and IP3R remained unchanged.Conclusion Intracellular Ca2+ overload may play important roles in myocardial hypertrophy induced by L-Thy. Imidapril and valsartan may exert beneficial effects on hyperthyroid myocardial hypertrophy via altering intracellular calcium handling.