大蒜素对大鼠心力衰竭模型心室肌细胞迟后除极和触发活动的作用
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摘要
目的观察大蒜素对大鼠心力衰竭模型细胞迟后除极(DAD)、触发活动(TA)和离子流的作用及机制。方法选取20只SD大鼠利用腹主动脉结扎法制备大鼠心力衰竭模型(模型组),另选取10只SD大鼠制作假手术组作为对照。采用酶解法急性分离2组大鼠心室肌细胞。根据实验设计对模型组是否给以大蒜素干预分为心力衰竭组(未给药)及给药组(200.0μmol/L大蒜素)。膜片钳技术观察心力衰竭组、给药组及对照组细胞DAD和TA的发生,并记录相关离子流(I_(ti),I_(Ca,L),I_(NCX))的改变,以Flou-4AM荧光技术检测细胞内钙离子浓度变化。采用SPSS 17.0软件进行统计学分析。组间比较采用ANOVA分析及SNK-q检验。结果 (1)对照组、心力衰竭组及给药组细胞上DAD和TA发生率依次为[13.3%(2/15),0.0%(0/15)]、[60.0%(9/15),26.7%(4/15)]和[40.0%(6/15),13.3%(2/15)]。与对照组比较,心力衰竭组细胞DAD和TA发生率显著增加;与心力衰竭组比较,给药组细胞上DAD和TA发生率显著下降,下降幅度分别为20.0%和13.4%,差异均有统计学意义(n=15,P<0.01)。(2)与对照组比较,心力衰竭组心室肌细胞上I_(Ca,L)、I_(ti)和I_(NCX)电流密度均增加,而与心力衰竭组比较,给药组上述电流均减少,其中I_(ti)内向峰电流密度从(-1.05±0.06)pA/pF降至(-0.53±0.05)pA/pF(n=10,P<0.01),I_(NCX)内向电流密度从(-5.8±0.7)pA/pF降至(-4.2±0.4)pA/pF(n=10,P<0.01)。同时,给药组通过大蒜素加速通道电流的稳态失活过程,降低I_(Ca,L),I_(Ca,L)的峰电流密度由(-17.2±0.9)pA/pF降为(-13.5±1.0)pA/pF(n=15,P<0.01)。(3)与对照组比较,心力衰竭组细胞静息态钙浓度、钙瞬变幅度和胞内钙最高50%的时间等参数显著升高;与心力衰竭组比较,上述参数在给药组细胞中均显著降低(n=15,P<0.01)。心力衰竭组细胞内钙瞬变衰减率与对照组比较显著下降,而应用大蒜素后,得到显著恢复(P<0.05)。结论大蒜素可能通过减少心力衰竭大鼠心室肌细胞内的钙离子,阻滞I_(ti)和I_(NCX)电流,从而降低心力衰竭后DAD和TA的发生。
Objective To investigate the effect of allicin(All) on the delayed afterdepolarization(DAD), triggered activity(TA) and ion currents in the ventricular myocytes of rats with heart failure and the related mechanism. Methods Heart failure models were established in 20 SD rats by abdominal aortic ligation, and sham operation was performed in 10 SD rats as control. Enzymic digestion was used to isolate ventricular myocytes in the 2 groups. According to the experimental design, the model cells were divided into heart failure group(without administration, HF) and administration group(200.0 μmol/L All, HF + All). Patch clamp technique was used to observe the occurrence of DAD and TA and to record the associated changes of ion currents [the transient inward current(I_(ti)), L-type calcium current(I_(Ca,L)) and sodium-calcium exchanger current(I_(NCX))] in the 3 groups. Flou-4 AM fluorescence technique was used to detect the changes of intracellular calcium concentration. SPSS statistics 17.0 was used for statistical analysis. ANOVA and SNK-q test were used for comparison among groups. Results(1)The incidences of DAD and TA in the control group, HF group and HF + All group were [13.3%(2/15), 0.0%(0/15)], [60.0%(9/15), 26.7%(4/15)] and [40.0%(6/15), 13.3%(2/15)] respectively. Compared with the control group, the incidences of DAD and TA in the HF group increased significantly. Compared with the HF group, the incidences of DAD and TA in the HF+All group decreased significantly by 20.0% and 13.4% respectively(n=15, P<0.01).(2) Compared with control group, I_(ti),I_(NCX) and I_(Ca,L) density increased in the HF group, but compared withHF group, they decreased in the HF+All group, in which I_(ti) peak inward current density decreased from(-1.05±0.06)pA/pF to(-0.53±0.05)pA/pF(n=10, P<0.01) and I_(NCX) inward current density from(-5.8±0.7)pA/pF to(-4.2±0.4)pA/pF(n=10, P<0.01). At the same time, All accelerated the steady-state inactivation of channel currents and decreased the peak current density of I_(Ca,L) from(-17.2+0.9) pA/pF to(-13.5+1.0) pA/pF(n=15, P<0.01).(3) Compared with the control group, the resting calcium concentration, transient calcium amplitude and time for intracellular calcium concentrations to reach 50% of maximum value in the HF group were significantly higher, but those parameters were significantly lower in the HF + All group than in the HF group(n=15, P<0.01). The intracellular calcium transient attenuation rate in the HF group was significantly lower than that in the control group, but it recovered significantly after the application of All(P<0.05). Conclusion All may reduce the occurrence of DAD and TA by reducing intracellular calcium and blocking I_(ti) and I_(NCX) currents in ventricular myocytes of rats with heart failure.
Objective To investigate the effect of allicin(All) on the delayed afterdepolarization(DAD), triggered activity(TA) and ion currents in the ventricular myocytes of rats with heart failure and the related mechanism. Methods Heart failure models were established in 20 SD rats by abdominal aortic ligation, and sham operation was performed in 10 SD rats as control. Enzymic digestion was used to isolate ventricular myocytes in the 2 groups. According to the experimental design, the model cells were divided into heart failure group(without administration, HF) and administration group(200.0 μmol/L All, HF + All). Patch clamp technique was used to observe the occurrence of DAD and TA and to record the associated changes of ion currents [the transient inward current(I_(ti)), L-type calcium current(I_(Ca,L)) and sodium-calcium exchanger current(I_(NCX))] in the 3 groups. Flou-4 AM fluorescence technique was used to detect the changes of intracellular calcium concentration. SPSS statistics 17.0 was used for statistical analysis. ANOVA and SNK-q test were used for comparison among groups. Results(1)The incidences of DAD and TA in the control group, HF group and HF + All group were [13.3%(2/15), 0.0%(0/15)], [60.0%(9/15), 26.7%(4/15)] and [40.0%(6/15), 13.3%(2/15)] respectively. Compared with the control group, the incidences of DAD and TA in the HF group increased significantly. Compared with the HF group, the incidences of DAD and TA in the HF+All group decreased significantly by 20.0% and 13.4% respectively(n=15, P<0.01).(2) Compared with control group, I_(ti),I_(NCX) and I_(Ca,L) density increased in the HF group, but compared withHF group, they decreased in the HF+All group, in which I_(ti) peak inward current density decreased from(-1.05±0.06)pA/pF to(-0.53±0.05)pA/pF(n=10, P<0.01) and I_(NCX) inward current density from(-5.8±0.7)pA/pF to(-4.2±0.4)pA/pF(n=10, P<0.01). At the same time, All accelerated the steady-state inactivation of channel currents and decreased the peak current density of I_(Ca,L) from(-17.2+0.9) pA/pF to(-13.5+1.0) pA/pF(n=15, P<0.01).(3) Compared with the control group, the resting calcium concentration, transient calcium amplitude and time for intracellular calcium concentrations to reach 50% of maximum value in the HF group were significantly higher, but those parameters were significantly lower in the HF + All group than in the HF group(n=15, P<0.01). The intracellular calcium transient attenuation rate in the HF group was significantly lower than that in the control group, but it recovered significantly after the application of All(P<0.05). Conclusion All may reduce the occurrence of DAD and TA by reducing intracellular calcium and blocking I_(ti) and I_(NCX) currents in ventricular myocytes of rats with heart failure.
引文
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[7] 王瑞,王萍,徐斌,等.大蒜素对自发性高血压大鼠肠系膜动脉平滑肌细胞钙电流的作用[J].心脏杂志,2014,26(4):378-383.DOI:10.13191/j.chj.2014.0037.Wang R,Wang P,Xu B,et al.Effects of garlicin on L-type calcium current of mesenteric artery smooth muscle cells in spontaneously hypertensive rats[J].Chin Heart J,2014,26(4):378-383.DOI:10.13191/j.chj.2014.0037.
[8] 徐叔云,卞如濂,陈修.药理实验方法学[M].北京:人民卫生出版社,1991:675.Xu SY,Bian RL,Chen X.Pharmacological Experimental Metho-dology[M].Beijing:People′s Medical Publishing House,1991:675.
[9] Chen YJ,Chen YC,Yeh HI,et al.Electrophysiology and arrhythmogenic activity of single cardiomyocytes from canine superior vena cava[J].Circulation,2002,105(22):2679-2685.
[10] Patton KK,Hellkamp AS,Lee KL,et al.Unexpected deviation in circadian variation of ventricular arrhythmias:the SCD-HeFT (Sudden Cardiac Death in Heart Failure Trial)[J].J Am Coll Cardiol,2014,63(24):2702-2708.DOI:10.1016/j.jacc.2013.11.072.
[11] Khoo CY,Allen JC,Chia SY,et al.Mortality outcome and predictive risk factors for death in patients with heart failure and reduced ejection fraction who declined implantable cardioverter defibrillator implantation in Singapore[J].J Arrhythm,2018,34(5):536-540.DOI:10.1002/joa3.12106.
[12] Liu MB,Ko CY,Song Z,et al.A dynamical threshold for cardiac delayed afterdepolarization-mediated triggered activity[J].Biophys J,2016,111(11):2523-2533.DOI:10.1016/j.bpj.2016.10.009.
[13] Hegyi B,Horváth B,Váczi K,et al.Ca2+-activated Cl- current is antiarrhythmic by reducing both spatial and temporal heterogeneity of cardiac repolarization[J].J Mol Cell Cardiol,2017,109:27-37.DOI:10.1016/j.yjmcc.2017.06.014.
[14] Watanabe Y,Kimura J.Inhibitory effect of azimilide on Na+/Ca2+ exchange current in Guinea-pig cardiac myocytes[J].J Pharmacol Sci,2010,114(1):111-114.
[15] Xu B,Fu Y,Liu L,et al.Effect of α-allocryptopine on delayed afterdepolarizations and triggered activities in mice cardiomyocytes treated with isoproterenol[J].Evid Based Complement Alternat Med,2015,2015:634172.DOI:10.1155/2015/634172.
[16] Ko CY,Liu MB,Song Z,et al.Multiscale determinants of delayed afterdepolarization amplitude in cardiac tissue[J].Biophys J,2017,112(9):1949-1961.DOI:10.1016/j.bpj.2017.03.006.
[17] Liu MB,de Lange E,Garfinkel A,et al.Delayed afterdepolarizations generate both triggers and a vulnerable substrate promoting reentry in cardiac tissue[J].Heart Rhythm,2015,12(10):2115-2124.DOI:10.1016/j.hrthm.2015.06.019.
[2] Molina CE,Abu-Taha IH,Wang Q,et al.Profibrotic,electrical,and calcium-handling remodeling of the atria in heart failure patients with and without atrial fibrillation[J].Front Physiol,2018,9:1383.DOI:10.3389/fphys.2018.01383.
[3] Xie Y,Sato D,Garfinkel A,et al.So little source,so much sink:requirements for afterdepolarizations to propagate in tissue[J].Biophys J,2010,99(5):1408-1415.DOI:10.1016/j.bpj.2010.06.042.
[4] 柯俊,李泱,陈锋,等.大蒜素对心力衰竭兔心室肌细胞钙电流的作用[J].中华急诊医学杂志,2018,27(7):729-734.DOI:10.3760/cma.j.issn.1671-0282.2018.07.005.Ke J,Li Y,Chen F,et al.The effect of allicin on the ventricular myocytes of rabbit with heart failure[J].Chin J Emerg Med,2018,27(7):729-734.DOI:10.3760/cma.j.issn.1671-0282.2018.07.005.
[5] 但晴,赵颖,吴志娟,等.大蒜素对自发性高血压大鼠心肌Ito重构的影响[J].药学学报,2015,150(1):39-44.DOI:10.16438/j.0513-4870.2015.01.008.Dan Q,Zhao Y,Wu ZJ,et al.Effect of allicin on remodeling of the transient outward potassium current of ventricular myocytes of spontaneously hypertensive rats[J].Acta Pharm Sin,2015,150(1):39-44.DOI:10.16438/j.0513-4870.2015.01.008.
[6] 张建成,林琨,魏芝雄,等.大蒜素对HEK293细胞HERG电流的阻滞作用[J].南方医学大学学报,2015,35(8):1128-1132.DOI:10.3969/j.issn.1673-4254.2015.08.09.Zhang JC,Lin K,Wei ZX,et al.Effects of allicin on rapidly delayed rectifier potassium current in HEK293 cell line[J].South Med Univ,2015,35(8):1128-1132.DOI:10.3969/j.issn.1673-4254.2015.08.09.
[7] 王瑞,王萍,徐斌,等.大蒜素对自发性高血压大鼠肠系膜动脉平滑肌细胞钙电流的作用[J].心脏杂志,2014,26(4):378-383.DOI:10.13191/j.chj.2014.0037.Wang R,Wang P,Xu B,et al.Effects of garlicin on L-type calcium current of mesenteric artery smooth muscle cells in spontaneously hypertensive rats[J].Chin Heart J,2014,26(4):378-383.DOI:10.13191/j.chj.2014.0037.
[8] 徐叔云,卞如濂,陈修.药理实验方法学[M].北京:人民卫生出版社,1991:675.Xu SY,Bian RL,Chen X.Pharmacological Experimental Metho-dology[M].Beijing:People′s Medical Publishing House,1991:675.
[9] Chen YJ,Chen YC,Yeh HI,et al.Electrophysiology and arrhythmogenic activity of single cardiomyocytes from canine superior vena cava[J].Circulation,2002,105(22):2679-2685.
[10] Patton KK,Hellkamp AS,Lee KL,et al.Unexpected deviation in circadian variation of ventricular arrhythmias:the SCD-HeFT (Sudden Cardiac Death in Heart Failure Trial)[J].J Am Coll Cardiol,2014,63(24):2702-2708.DOI:10.1016/j.jacc.2013.11.072.
[11] Khoo CY,Allen JC,Chia SY,et al.Mortality outcome and predictive risk factors for death in patients with heart failure and reduced ejection fraction who declined implantable cardioverter defibrillator implantation in Singapore[J].J Arrhythm,2018,34(5):536-540.DOI:10.1002/joa3.12106.
[12] Liu MB,Ko CY,Song Z,et al.A dynamical threshold for cardiac delayed afterdepolarization-mediated triggered activity[J].Biophys J,2016,111(11):2523-2533.DOI:10.1016/j.bpj.2016.10.009.
[13] Hegyi B,Horváth B,Váczi K,et al.Ca2+-activated Cl- current is antiarrhythmic by reducing both spatial and temporal heterogeneity of cardiac repolarization[J].J Mol Cell Cardiol,2017,109:27-37.DOI:10.1016/j.yjmcc.2017.06.014.
[14] Watanabe Y,Kimura J.Inhibitory effect of azimilide on Na+/Ca2+ exchange current in Guinea-pig cardiac myocytes[J].J Pharmacol Sci,2010,114(1):111-114.
[15] Xu B,Fu Y,Liu L,et al.Effect of α-allocryptopine on delayed afterdepolarizations and triggered activities in mice cardiomyocytes treated with isoproterenol[J].Evid Based Complement Alternat Med,2015,2015:634172.DOI:10.1155/2015/634172.
[16] Ko CY,Liu MB,Song Z,et al.Multiscale determinants of delayed afterdepolarization amplitude in cardiac tissue[J].Biophys J,2017,112(9):1949-1961.DOI:10.1016/j.bpj.2017.03.006.
[17] Liu MB,de Lange E,Garfinkel A,et al.Delayed afterdepolarizations generate both triggers and a vulnerable substrate promoting reentry in cardiac tissue[J].Heart Rhythm,2015,12(10):2115-2124.DOI:10.1016/j.hrthm.2015.06.019.