酒精性心肌病小鼠动物模型的建立及大量饮酒对QTc延长的影响机制
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摘要
目的分析酒精性心肌病(ACM)小鼠动物模型的建立及大量饮酒对校正QT间期(QTc)延长的影响机制。方法取20只C57BL/6小鼠,随机分为对照组和实验组,两组各10只。实验组经长期饮酒和灌胃构建ACM小鼠模型,采用心肌胶原染色、病理和电镜下验证ACM模型是否成功。造模成功后进行心电图检查,并采用小动物超声成像系统对两组小鼠心功能相关参数进行测量;采用酶解法急性分离小鼠心室肌细胞,采用全细胞膜片钳技术电生理学检测心室肌细胞动作电位和各种离子通道,急性检测通道相关动力学。结果建模6个月后,经心电图检查结果显示,相比对照组,实验组小鼠QTc明显延长(P <0. 01);而两组QRS间期、RR间期、PR间期及心率的比较,并无显著差异(P> 0. 05)。经超声心动图检查结果显示,实验组小鼠左心室短轴缩短率(LVFS)和左心室射血分数(LVEF)较对照组显著下降(P <0. 05);而两组室间隔厚度(IVST)和左心室后壁厚度(LVPWd)的比较,并无显著差异(P> 0. 05)。全细胞膜片钳实验结果显示,实验组小鼠心室肌细胞动作电位时程明显延长,其心室肌细胞动作电位复极至90%所需时间较对照组明显延长(P <0. 01)。经心室肌细胞膜片钳实验结果显示,实验组L型钙通道电流密度绝对值明显低于对照组(P <0. 01),但实验组失活动力学检测结果提示半失活电压向负极方向移动,说明较对照组明显失活延迟(P <0. 01),而两组激活动力学检测结果提示无统计学意义(P> 0. 05);实验组心肌细胞钠电流密度与对照组比较,差异无统计学意义(P> 0. 05);两组激活与失活动力学中半失活电位的比较,差异亦无统计学意义(P> 0. 05)。结论长期大量饮酒可使得心室肌钙通道失活延迟,可进一步使得心室肌细胞动作电位延长,这可能是ACM小鼠心电图QTc延长的主要原因之一。
Objective To establish an animal model of alcoholic cardiomyopathy( ACM) in mice and to analyze the effect of heavy drinking on correcting the prolongation of QTc. Methods Twenty C57 BL/6 mice were randomly divided into control group and experimental group with10 mice in each group. The ACM mice in the experimental group were established by long-term drinking and gavage. The success of the ACM model was verified by myocardial collagen staining,pathology and electron microscopy. After the successful establishment of the model,electrocardiogram was examined,and the parameters related to cardiac function were measured by small animal ultrasound imaging system; the ventricular myocytes were isolated by enzymatic hydrolysis,and action potential and ion channels were detected by whole cell patch clamp electrophysiology,and channel-related dynamics were detected. Results After 6 months of modeling,the results of electrocardiogram showed that compared with the control group,the QTc of mice in the experimental group was significantly prolonged( P < 0. 01),while there was no significant difference in QRS interval,RR interval,PR interval and heart rate between the two groups( P > 0. 05). The results of echocardiography showed that the left ventricular fraction shortening( LVFS) and left ventricular ejection fraction( LVEF) in the experimental group were significantly lower than those in the control group( P < 0. 05),while there was no significant difference in the comparisons of interventricular septal thickness( IVST) and left ventricular posterior wall depth( LVPWd) between the two groups( P > 0. 05). The whole-cell patch clamp experiment showed that the action potential duration of ventricular myocytes in the experimental group was significantly prolonged,and the time required for action potential repolarization to 90% of the ventricular myocytes in the experimental group was significantly longer than that in the control group( P < 0. 01). The re-sults of patch clamp experiment on ventricular myocytes showed that the absolute current density of L-type calcium channel in the experimental group was significantly lower than that in the control group( P < 0. 01),but the results of inactivation mechanics in the experimental group indicated that the half-inactivation voltage moved towards the negative pole,indicating that the inactivation delay was significantly longer than that in the control group( P < 0. 01),while the results of activation kinetics in the experimental group showed no statistical significance( P > 0. 05).There was no significant difference in the sodium current density of myocytes between the two groups( P > 0. 05),and there was no significant difference in the semi-inactivation potential of activation and inactivation mechanics between the two groups( P > 0. 05). Conclusion Long-term heavy drinking can delay the inactivation of calcium channels in ventricular myocytes and further prolong the action potential of ventricular myocytes,which may be one of the main reasons for the prolongation of QTc inelectrocardiogram of ACM mice.
Objective To establish an animal model of alcoholic cardiomyopathy( ACM) in mice and to analyze the effect of heavy drinking on correcting the prolongation of QTc. Methods Twenty C57 BL/6 mice were randomly divided into control group and experimental group with10 mice in each group. The ACM mice in the experimental group were established by long-term drinking and gavage. The success of the ACM model was verified by myocardial collagen staining,pathology and electron microscopy. After the successful establishment of the model,electrocardiogram was examined,and the parameters related to cardiac function were measured by small animal ultrasound imaging system; the ventricular myocytes were isolated by enzymatic hydrolysis,and action potential and ion channels were detected by whole cell patch clamp electrophysiology,and channel-related dynamics were detected. Results After 6 months of modeling,the results of electrocardiogram showed that compared with the control group,the QTc of mice in the experimental group was significantly prolonged( P < 0. 01),while there was no significant difference in QRS interval,RR interval,PR interval and heart rate between the two groups( P > 0. 05). The results of echocardiography showed that the left ventricular fraction shortening( LVFS) and left ventricular ejection fraction( LVEF) in the experimental group were significantly lower than those in the control group( P < 0. 05),while there was no significant difference in the comparisons of interventricular septal thickness( IVST) and left ventricular posterior wall depth( LVPWd) between the two groups( P > 0. 05). The whole-cell patch clamp experiment showed that the action potential duration of ventricular myocytes in the experimental group was significantly prolonged,and the time required for action potential repolarization to 90% of the ventricular myocytes in the experimental group was significantly longer than that in the control group( P < 0. 01). The re-sults of patch clamp experiment on ventricular myocytes showed that the absolute current density of L-type calcium channel in the experimental group was significantly lower than that in the control group( P < 0. 01),but the results of inactivation mechanics in the experimental group indicated that the half-inactivation voltage moved towards the negative pole,indicating that the inactivation delay was significantly longer than that in the control group( P < 0. 01),while the results of activation kinetics in the experimental group showed no statistical significance( P > 0. 05).There was no significant difference in the sodium current density of myocytes between the two groups( P > 0. 05),and there was no significant difference in the semi-inactivation potential of activation and inactivation mechanics between the two groups( P > 0. 05). Conclusion Long-term heavy drinking can delay the inactivation of calcium channels in ventricular myocytes and further prolong the action potential of ventricular myocytes,which may be one of the main reasons for the prolongation of QTc inelectrocardiogram of ACM mice.
引文
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[13] Landstrom AP,Dobrev D,Wehrens XHT. Calcium signaling and cardiac arrhythmias[J]. Circ Res,2017,120(12):1969-1993.
[14] Rehm J,Shield KD,Roerecke M,et al. Modelling the impact of alcohol consumption on cardiovascular disease mortality for comparative risk assessments:an overview[J]. BMC Public Health,2016,16:363.
[15] de Beaurepaire R. A review of the potential mechanisms of action of baclofen in alcohol use disorder[J]. Front Psychiatry,2018,9:506.
[2] Kryzhanovskii SA,Kolik LG,Tsorin IB,et al. Alcoholic Cardiomyopathy:Translation Model[J]. Bull Exp Biol Med,2017,163(5):627-631.
[3] Liang D,Wu Y,Zhou L,et al. LRP5 controls cardiac QT interval by modulating the metabolic homeostasis of L-type calcium channel[J].Int J Cardiol,2019,275:120-128.
[4] Kanae H,Hamaguchi S,Wakasugi Y,et al. Pathological prolongation of action potential duration as a cause of the reduced alpha-adrenoceptor-mediated negative inotropy in streptozotocin-induced diabetic mice myocardium[J]. J Pharmacol Sci,2017,135(3):131-133.
[5] Sokolova OV. The morphological changes in the myocardial tissue after sudden cardiac death from alcoholic cardiomyopathy[J]. Sud Med Ekspert,2016,59(1):3-6.
[6] Perki9mki J,Hookana E,Kaikkonen K,et al. Blood alcohol in victims of sudden cardiac death in northern Finland[J]. Europace,2016,18(7):1006-1009.
[7]李明,黄京璐,王小广,等.广东地区622例猝死案例的流行病学调查[J].中国法医学杂志,2015,30(1):66-69.
[8] Rehm J,Hasan OSM,Imtiaz S,et al. Quantifying the contribution of alcohol to cardiomyopathy:A systematic review[J]. Alcohol,2017,61:9-15.
[9] Spaziano M,Sawaya FJ,Lefèvre T. Alcohol septal ablation for hypertrophic obstructive cardiomyopathy:Indications,technical aspects,and clinical outcomes[J]. J Invasive Cardiol,2017,29(12):404-410.
[10] Szentandrássy N,Kistamás K,Hegyi B,et al. Contribution of ion currents to beat-to-beat variability of action potential duration in canine ventricular myocytes[J]. Pflugers Arch,2015,467(7):1431-1443.
[11] Pipilas DC,Johnson CN,Webster G,et al. Novel calmodulin mutations associated with congenital long QT syndrome affect calcium current in human cardiomyocytes[J]. Heart Rhythm,2016,13(10):2012-2019.
[12] Boczek NJ,Miller EM,Ye D,et al. Novel timothy syndrome mutation leading to increase in CACNA1C window current[J]. Heart Rhythm,2015,12(1):211-219.
[13] Landstrom AP,Dobrev D,Wehrens XHT. Calcium signaling and cardiac arrhythmias[J]. Circ Res,2017,120(12):1969-1993.
[14] Rehm J,Shield KD,Roerecke M,et al. Modelling the impact of alcohol consumption on cardiovascular disease mortality for comparative risk assessments:an overview[J]. BMC Public Health,2016,16:363.
[15] de Beaurepaire R. A review of the potential mechanisms of action of baclofen in alcohol use disorder[J]. Front Psychiatry,2018,9:506.