【作者】 董芳；

【导师】 许彦芳；

【作者基本信息】 河北医科大学 ， 药理学， 2007， 硕士

【摘要】 心肌肥厚、心衰是高血压、缺血性心肌病、心瓣膜病等许多心血管疾病的常见合并症,心肌肥厚、心衰常常伴发心律失常,特别是心衰期心源性猝死(SCD)的发生率大大增加。近年来,尽管在心衰的药物治疗上已取得很大的进展,但心衰病人的死亡率仍然较高。故揭示心肌肥厚、心衰病理过程中心律失常产生的分子机制,寻找有效的防治措施,是心血管领域研究的重要课题。迄今,已在多种心肌肥厚、心衰实验动物模型观察到瞬时外向钾电流(Ito)的下调。Ito是心室肌细胞复极化早期的主要电流成分,它是由α亚单位(Kv4.2、Kv4.3)和β亚单位(KChIP2)构成的离子通道形成。Ito密度的减小可来自通道动力学的改变和/或通道蛋白表达的减少,从而造成病理性动作电位(APD)延长。动作电位延长使心脏易发生早后除极产生触发活动及复极的离散度增大是心衰心律失常产生的重要原因。我们前期的实验表明,尽管心肌代偿性肥厚与心衰阶段均有小鼠乳头肌动作电位的延长,但两个阶段延长存在不同形式,前者以早期复极延迟为主,后者不仅有早期、而且存在晚期复极的延迟。提示心肌肥厚、心衰发展过程中电生理改变有随时间而变化的特征,而此种电生理进行性改变是否也伴有相应Ito离子通道的表达变化目前尚不清楚。本实验拟在已建立的小鼠心肌肥厚、心衰模型上,观察不同病理发展阶段小鼠心室肌内膜下(subEndocardial, Endo)、外膜下(subepicardial,Epi)心肌细胞Ito通道蛋白Kv4.2、KChIP2表达随时间变化的特点,并通过应用calcineurin(CaN)的特异性抑制剂环孢素A(CsA),探讨其病理性改变可能的细胞内信号传导通路,从分子生物学角度为心肌肥厚、心衰的电生理重构及机制提供实验依据。一、心肌肥厚和心衰不同时期小鼠左心室游离壁内、外膜下心肌细胞Kv4.2、KChIP2的表达变化目的:观察在压力超负荷性心肌肥厚和心衰发展过程中,小鼠左心室游离壁内膜、外膜下心肌细胞Kv4.2、KChIP2蛋白在不同时间点的表达变化情况。方法:(1)动物模型制备:将小鼠深麻醉后开胸,暴露主动脉弓,部分分离:用外径0.4mm针头与主动脉弓平行结扎,抽出针头,使主动脉弓狭窄65%-70%。术后左心室后负荷增加,血液流出受阻,造成压力超负荷性心肌肥厚和心衰的动物模型;观察动物的一般状况并测定心指数和血流动力学各参数(LVSP、±dp/dt)。(2)用酶消化的方法急性分离小鼠左心室外膜、内膜心肌细胞,采用细胞间接免疫荧光技术,通过激光共聚焦显微镜观察Kv4.2、KChIP2在心内膜、心外膜下心肌细胞上的分布。(3)分别于术后2周、5周、13周提取左心室游离壁内膜、外膜下心肌细胞膜蛋白,采用Western blot技术检测Kv4.2、KChIP2的表达量,并用GAPDH来标准化上样蛋白量。手术结扎(Band)组与同时期假手术(Sham)组对照进行比较。结果:(1)小鼠主动脉弓不完全狭窄术后,2-7周心质量指数和血流动力学各参数呈进行性上升,该时期为代偿性心肌肥厚阶段;8-13周LVSP、±dp/dt呈下降趋势,该时期为失代偿性心力衰竭阶段。(2)细胞免疫荧光观察显示:Kv4.2和KChIP2在内、外膜下心肌细胞上均有表达,且心外膜下心肌细胞Kv4.2荧光强度较内膜高,KChIP2在内、外膜上荧光强度较一致。(3)同时期Sham组内膜、外膜下心肌细胞Kv4.2表达量存在明显差异(P<0.01),心外膜下细胞表达高于心内膜(外膜/内膜=1.82),而不同时间点(2周、5周、13周)内膜及外膜间Kv4.2表达量无显著性差异(P>0.05);与同时期的Sham组相比, Band组内、外膜下心肌细胞Kv4.2表达均降低(P<0.05), 2周、5周、13周外膜下心肌细胞Kv4.2分别降低25.37%、41.33%、62.67%,内膜下心肌细胞分别降低26.32%、41.46%、66.67%;Band组2周与5周外膜下心肌细胞之间、内膜下细胞之间Kv4.2表达量无显著性差异(P>0.05),而Band 13周组内、外膜下心肌细胞Kv4.2与Band2周和5周相比呈显著性下降(P<0.05);同时期Band组内、外膜下心肌细胞Kv4.2表达量仍存在明显差异(外膜/内膜=1.91)(P<0.01)。(4)不同时间点(2周、5周、13周)Sham组内膜和外膜间KChIP2表达量没有显著性差异(P>0.05);与同时期的Sham组相比,Band 2周、5周组内、外膜下心肌细胞KChIP2表达量没有显著性差异,而Band13周组内、外膜下心肌细胞KChIP2较Sham组明显减少(P<0.05),外、内膜降幅分别为48.08%、61.11%。结论:(1)Kv4.2蛋白表达减少是造成肥厚期Ito密度降低的主要分子机制。(2)Kv4.2、KChIP2蛋白的共同下调构成了心衰时Ito下调的分子基础;KChIP2蛋白在内、外膜下心肌的非同步下调可能是心衰期病理性跨壁电异质性增大的原因。二、Calcineurin信号通路在小鼠失代偿性心衰期心室肌内膜、外膜下心肌细胞Kv4.2、KChIP2表达减少中的作用目的:本实验通过应用Calcineurin的特异性抑制剂cyclosporin A(CsA)阻断Cai2+/CaM-calcineurin-NFAT信号通路,评价该信号通路在失代偿性心衰期时Kv4.2、KChIP2表达减少中发挥的作用,为寻找可能的抗心律失常药提供实验基础。方法:在小鼠压力超负荷性心肌肥厚、心衰模型上,于术后9周起皮下注射CsA,25mg/Kg,2次/天,连续两周,提取左心室游离壁内膜、外膜下心肌细胞膜蛋白, Western blot检测Kv4.2、KChIP2的表达量。实验分为:Band11w+CsA组、Band11w+Veh组,为进一步说明CsA对正常和病理状态下心肌作用的选择性,Sham11周组中设与Band11周同期的治疗组和对照组,即Sham11w+CsA组、Sham11w+Veh组。同时监测各组内、外膜下心肌细胞Calcineurin活性、心质量指数,并对各组心肌组织进行组织病理学观察。结果:(1)Sham+CsA组和Sham+Veh组间、Band+CsA组和Band+Veh组间动物外观上无明显差异。Band11w+CsA组死亡率约为8%,较同时期Band11w+Veh组无显著性差异,Sham组动物无死亡。各组小鼠的体重无显著性差异(P>0.05)。心质量指数:Band11w+CsA组同Band11w+Veh相比心指数降低(P<0.01),但没有减少到Sham11w+Veh组水平。(2)组织病理学观察:光镜下可见Sham11w+Veh、Sham11w+CsA组心肌纤维结构清晰,形态完整,排列均匀整齐,横纹清楚,细胞大小均一,仅有少量的胶原纤维;Band11w+Veh组心肌纤维染色变浅,心肌细胞体积明显增大,排列紊乱,横纹不清或消失,细胞水肿,细胞核淡染或不清楚,局部心肌溶解,胞浆疏松化;Band11w+CsA组较Band11w+Veh组心肌纤维体积增大不明显,排列较整齐,细胞水肿减轻,病变程度明显好转。(3)各组左室游离壁内膜下心肌细胞的CaN活性高于外膜(P<0.01);与Sham11w+Veh组相比,Band11w+Veh组内、外膜下心肌细胞CaN活性均显著性升高(P<0.01),内膜细胞升高幅度约为109.89%,外膜细胞升高约84.41%;CsA治疗后,Band11w+CsA组同Band11w+Veh组相比内、外膜下心肌细胞CaN活性均降低(P<0.05),且完全恢复到Sham11w+Veh水平(P>0.05)。(4)CsA治疗后,Band11w+CsA同Band11w+Veh组相比内、外膜下心肌细胞Kv4.2表达均增加(P<0.05),但没有完全恢复到Sham11w+Veh组水平;CsA对内、外膜下心肌细胞KChIP2表达无影响,同Sham11w+Veh组相比,Band11w+CsA组、Band11w+Veh组内、外膜下心肌细胞KChIP2表达均减少(P<0.05);(5)CsA对假手术组动物的一般状况、心质量指数、CaN活性及内、外膜下心肌细胞Kv4.2、KChIP2表达均无影响。结论:(1)CsA可改善心肌组织病理学形态改变程度并逆转心衰期内、外膜下心室肌细胞calcineurin活性升高。(2)calcineurin信号通路参与失代偿性心衰状态下心室内、外膜下心肌细胞Ito通道蛋白Kv4.2的下调改变,但不参与KChIP2的下调。更多还原

【Abstract】 Cardiac hypertrophy and failure is the common complication for such diseases as hypertension, ischemic heart disease, and heart valve disease, etc. Cardiac hypertrophy and failure often accompany with arrhythmias. Heart failure in particular is associated with a significant increase in the risk of sudden cardiac death (SCD). Despite considerable progresses have been made in the treatment of heart failure in recent years, the mortality of patients with heart failure remains high. Therefore, it is of significance to elucidate the molecular mechanism underlying the arrhythmias companying cardiac hypertrophy and failure and seek for the effective medication.Up to now, the reduction of transient outward potassium current (Ito) density has been observed in many experimental animal models with cardiac hypertrophy and failure. Ito plays an important role in cardiac early repolarization. The Ito channel is composed ofαsubunits (Kv4.2, Kv4.3) andβsubunit (KChIP2). The reduction of Ito results from the changes of the kinetics properties and/or the molecular expression of the channel and contributes to the prolongation of action potential duration (APD), which is believed to predispose the heart to afterdepolarization and reentrant arrhythmias.In our previous study we found a progressing prolongation of APD in mouse papillary muscle as the development of cardiac hypertrophy and the failure in a mouse cardiac pressure overload model. The electrical remodeling in this model is a dynamic process and perhaps depends on the degree and the state of ventricular functional compensation. It is known that Ito is main component for the repolarization in mouse myocardium. However, little is known about the changes ofαandβsubunits of the ion channel and underlying mechanism during the pathological process of cardiac hypertrophy and failure.In this study, we quantified the titation of Kv4.2 and KChIP2 proteins in subepicardial and subendocardial ventricular myocardium either in hypertrophied or failing hearts in the mouse cardiac pressure over-loaded model. By treating animals with calcineurin inhibitor cyclosporin A(CsA), We evaluated the possible role of calcineurin pathway in the regulation of Kv4.2 and KChIP2 protein levels in heart failure.Part 1. Changes of Kv4.2 and KChIP2 protein level in subepicardial and subendocardial ventricular myocardium during the development of pressure over-loaded mouse cardiac hypertrophy and failureObjective: To understand the molecular mechanism of ion channel remodeling in the pathological condition by determining Kv4.2 and KChIP2 protein level in subepicardial and subendocardial ventricular myocardium in hypertrophied or failing hearts.Methods: (1) Mouse model of cardiac pressure overload: Transverse aorta was banded by using microsurgical techniques to create cardiac pressure overload, as described by others. Briefly, Kunming male mice, 4-5 weeks old (provided by Experimental Animal Center of Hebei Province), were anesthetized with ketamine (25mg/kg, intraperitoneal injection). The mice were orally intubated and ventilated (Harvard Apparatus). The chest cavity was opened in the second intercostal space and transverse aortic banding was performed by tying a nylon suture against a 27-gauge needle to produce an aortic narrowing 0.4 mm in diameter when the needle was removed. The procedure resulted in a reproducible transverse aortic banding of 65-70%. Age-matched mice were subjected to a sham operation in which the aortic arch was visualized but not banded. Mice were then maintained for 13 weeks after operation. Hemodynamic variables in some TAB or sham-operated mice were measured by placing a catheter in the isolated right carotid artery, which was advanced as far as the aorta and left ventricle. Cardiac mass index was also assessed and compared to the sham-operated animals at the different time after operation. (2) Immunofluorescence staining: Immunofluorescence labeling was performed in isolated subepicardial and subendocardial ventricular myocytes. The cells were treated with anti-Kv4.2 or KChIP2 antibody. Immunofluorescence labeling for confocal microscopy were done by treatment with fluorescein isothiocyanate-conjugated goat anti-rabbit antibody. Immunofluorescence-labeled samples were examined using a confocal laser scanning microscopy. Identical settings were used for all the specimens. (3) Western blot analysis: Immunoblots were performed using membrane fraction from mouse subepicardial and subendocardial ventricular myocardium at week 2, 5, 13 after operation. Anti-Kv4.2 and anti-KChIP2 were used as primary antibodies. Anti-GAPDH antibody was used as an internal loading control. Quantification of the signals was performed by densitometry. The protein bands were normalized to the GAPDH band in each sample. The value was then averaged from all the different sets of experiments.Results: (1) A mouse pressure over-loaded cardiac hypertrophy and failure model was established by aorta banding. The results showed that first 7 week hearts in banded mice were manifested the progressive increase in heart mass index and contractile function, which we defined the stage of compensatory hypertrophy. The following was the stage of heart failure, characterized by declining contraction. (2) Immunofluorescence labeling revealed that Kv4.2 and KChIP2 were both intensely observed on cell membrane in subepicardial and subendocardial ventricular myocytes. Kv4.2 protein appeared more strongly expressed in subepicardial myocytes than in subendocardial myocytes, while expression of KChIP2 protein was similar in subepicardial and subendocardial myocytes. (3) Quantification of channel proteins showed that there was a transmural gradient of Kv4.2 protein level in sham operated mice, i.e. higher amount of protein in subepicardium than in subendocardium with a ratio 1.82. The quantity of Kv4.2 protein was significantly decreased both in endocardium and epicardium in banded mice ( P<0.05 ) . Compared to sham-operated animals, amount of the protein in aorta-banded mice was decreased by 25.37%、41.33%、62.67% (P < 0.05) in subepicardium at week 2, 5, 13 after operation respectively, and in endocardial myocytes was respectively decreased 26.32%、41.46%、66.67% (P < 0.05). There were no significant differences in Kv4.2 protein level either in subendocardium or subepicardium between band 2w and band 5w groups. However, Kv4.2 protein level in 13w banded mice was less both in subendocardium and subepicardium than it in band 2w and band 5w ( P<0.05 ) mice. (4) Amount of KChIP2 protein in subepicardium is similar to that in subendocardium in sham-operated mice at week 2, 5, 13 after sham operation. Compared to sham-operated mice, no significant changes of KChIP2 level were observed in band 2w and 5w mice. However, KChIP2 protein was down-regulated at week 13 after operation(P<0.05), and the degree of reduction in subendocardium and subepicardium was 61.11% and 48.08%.Conclusions: (1) It was in the early phase that amount of Kv4.2 protein started to be decreased both in subepicardium and subendocardium in hypertrophied hearts, but KChIP2 expression was not changed in compensated hypertrophy stage. (2) The expression of Kv4.2 protein was kept reduced in heart failure phase. KChIP2 protein level was decreased in failing hearts, and the reduction degree was higher in subendocardium than in subepicardium. The results suggest that the changes of protein expression level are the molecular basis contributing to abnormal repolarization of cardiomyocyte in the pathological condition. Both Kv4.2 and KChIP2 proteins were down-regulated in heart failure, showing the possible reason for the propensity for arrhythmias in this stage.Part 2. The possible role of calcineurin pathway in down- regulation of Kv4.2, KChIP2 protein expression in failing heartsObjective: To evaluate the role of calcineurin signaling pathway in down-regulation of Kv4.2, KChIP2 protein expression in failing heartsMethods: Sham operated (Sham) or aorta banded (Band) mice were randomized to receive CsA (25mg/kg, injected subcutaneously twice daily, North China Pharmaceutical Group Corporation) or vehicle (Veh) for 2 weeks. Treatments were started from 9 weeks after operation, which represented heart failure phases. At the end of treatment, i.e. 11 weeks after operation, the amount of Kv4.2 and KChIP2 proteins from subepicardium and subendocardium were determined by using Western blot technique in CsA or Veh treatment mice, which including following groups: Band 11w + CsA, Band 11w + Veh, Sham 11w + CsA and Sham 11w + Veh. Meantime, calcineurin activity in subepicardium and subendocardium, cardiac mass index and cardiac histopathology were assessed.Results: (1) There were no significant differences in appearances between the Band 11w + CsA group and Band 11w + Veh group, also in Sham 11w + CsA and Sham 11w + Veh groups. The mortality was 8% in Band 11w + CsA group, which was similar to that in Band 11w + Veh mice. There was no mortality in sham-operated mice. The cardiac mass index of Band 11w + CsA mice was significantly decreased compared to Band 11w + Veh group (P<0.01),but not down to the level in Sham 11w + Veh group. (2) Cardiac histopathology: cardiac myocytes from Sham 11w + CsA, Band 11w + CsA and Sham 11w + CsA had well-arranged appearance, with similar sizes and clear transverse lines. There was little collagen between the cells. However, cells from Band 11w + Veh were lightly dyed and had blurry-transverse lines, with bigger size and disorganized appearance. There were a lot of denatured cells with dark-dyed and blurry nucleus. (3) Calcineurin activity was significant higher in subendocardium than in subepicardium (P<0.01) in Sham mice. Compared to Sham 11w + Veh group, calcineurin activity in Band 11w + Veh mice was greatly increased both in subepicardium and subendocardium (P<0.01), and subendocardium with higher activity than subepicardium. CsA could completely revised the increase of calcineurin activity since there was no difference in calcineurin activity between Band 11w + CsA and Sham 11w + Veh. (4) CsA treatment partially up-regulated Kv4.2 protein level both in subepicardium and subendocardium in banded mice. The expression of KChIP2 was no sigificiant differences in epicardium or endocardium between Band 11w + CsA and Band 11w + Veh groups. (5) In sham-operated mice, there was no effect of CsA on general conditions, cardic mass index, calcineurin activity and the expressions of Kv4.2, KChIP2 proteins.Conclusions: Inhibition of calcineurin signaling pathway attenuated the changes of cardiac histopathology and partially reversed the reduction of Kv4.2 protein level in heart failure, but it didn’t affect the expression of KChIP2 proteins. The result suggests that calcineurin pathway may down regulate Ito by inhibiting the expression of Kv4.2 protein.更多还原