CASQ2和RyR2在二乙酰吗啡致心肌细胞节律异常中的作用
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摘要
目的探讨二乙酰吗啡致心肌细胞节律改变与心肌钙离子通道异常之间的关系,探究肌钙集蛋白2(CASQ2)与兰碱尼受体2(RYR2)因子与钙通道之间的联系,明确其是否参与心肌细胞钙离子通道异常及心律失常的过程。方法取出生3日龄SD大鼠乳鼠的心肌细胞进行体外培养,分为正常对照组(N组)、二乙酰吗啡干预组(H组)、二乙酰吗啡加维拉帕米干预组(H+V组),二乙酰吗啡浓度为10~(-4) mol/L,药物作用时间24 h。在荧光倒置显微镜下,观察心肌细胞形态和收缩频率节律改变;采用逆转录-聚合酶链反应(RT-PCR)和蛋白印迹法(Western blot)检测CASQ2和RyR2因子的mRNA及蛋白表达,观察H组、N组及H+V组上述2个因子表达含量的变化情况。结果 (1)细胞培养:原代心肌细胞形态多样,呈三角形、长梭形和多边形,细胞核圆形或类圆形,大小正常核膜光滑,细胞间通过伪足相互连接,连接成片的细胞搏动节律一致。(2)心肌细胞形态及节律改变:与N组相比,H组和H+V组心肌细胞形态发生一定程度的改变,收缩频率降低,差异有统计学意义(P<0.05)。(3)与N组相比,H组和H+V组中CASQ2和RYR2蛋白和mRNA表达量减少,差异有统计学意义(P<0.05)。与H组相比,H+V组中CASQ2和RYR2蛋白和mRNA表达量减少,差异有统计学意义(P<0.05)。结论二乙酰吗啡可致心肌细胞自发性搏动频率和节律异常,CASQ2和RyR2因子参与二乙酰吗啡致心肌细胞自发性搏动频率和节律异常的过程。关键字:
Objecive To investigate the relationship between diacetylmorphine-induced myocardial cell rhythm changes and abnormal cardiac calcium channels, and to explore the association between troponin 2(CASQ2), lankine receptor 2(RYR2) factors and calcium channels, and to determine whether it is involved in the process of abnormal cardiac calcium channels and arrhythmias. Methods Myocardial cells from neonatal SD rats(3 rd Day) were cultured in vitro, and divided into control group, diacetylmorphine intervention group and diacetylmorphine plus verapamil intervention group. The concentration of diacetylmorphine was 10~(-4) mol/L. The duration time of drug was 24 h. The myocardial cell morphology change and rhythm was observed by fluorescence inversion microscope. The mRNA and protein expression of CASQ2 and RyR2 were detected by RT-PCR and Western blot. Results(1) The shape of myocardial cells of the primary generation were various kinds, including triangular, long spindle and polygonal, etc. Its nucleus was round or nearly round, its size was normal, and its nuclear membrane was smooth. The cell communication was connected to each other by pseudopod. And the pulsatile rhythm of the connected cells were consistent.(2) Myocardial cell morphology and rhythm changes: Compared to group N, the morphology of myocardial cell were changed at a certain extent, and the contraction frequency of myocardial cell was decreased significantly(P<0.05), in group H and group H+V.(3) Compared to group N, the protein and mRNA expressions of CASQ2 and RYR2 were decreased in group H and group H+V, with statistically significant results(P<0.05). Compared with group H, CASQ2 and RYR2 protein and mRNA expressions was decreased significantly(P<0.05) in group H+V. Conclusion Diacetylmorphine could cause spontaneous beat frequency and abnormal rhythm of cardiac myocyte, and CASQ2 and RyR2 factors were involved in the process of spontaneous beat frequency and abnormal rhythm of cardiac myocyte induced by diacetylmorphine.
Objecive To investigate the relationship between diacetylmorphine-induced myocardial cell rhythm changes and abnormal cardiac calcium channels, and to explore the association between troponin 2(CASQ2), lankine receptor 2(RYR2) factors and calcium channels, and to determine whether it is involved in the process of abnormal cardiac calcium channels and arrhythmias. Methods Myocardial cells from neonatal SD rats(3 rd Day) were cultured in vitro, and divided into control group, diacetylmorphine intervention group and diacetylmorphine plus verapamil intervention group. The concentration of diacetylmorphine was 10~(-4) mol/L. The duration time of drug was 24 h. The myocardial cell morphology change and rhythm was observed by fluorescence inversion microscope. The mRNA and protein expression of CASQ2 and RyR2 were detected by RT-PCR and Western blot. Results(1) The shape of myocardial cells of the primary generation were various kinds, including triangular, long spindle and polygonal, etc. Its nucleus was round or nearly round, its size was normal, and its nuclear membrane was smooth. The cell communication was connected to each other by pseudopod. And the pulsatile rhythm of the connected cells were consistent.(2) Myocardial cell morphology and rhythm changes: Compared to group N, the morphology of myocardial cell were changed at a certain extent, and the contraction frequency of myocardial cell was decreased significantly(P<0.05), in group H and group H+V.(3) Compared to group N, the protein and mRNA expressions of CASQ2 and RYR2 were decreased in group H and group H+V, with statistically significant results(P<0.05). Compared with group H, CASQ2 and RYR2 protein and mRNA expressions was decreased significantly(P<0.05) in group H+V. Conclusion Diacetylmorphine could cause spontaneous beat frequency and abnormal rhythm of cardiac myocyte, and CASQ2 and RyR2 factors were involved in the process of spontaneous beat frequency and abnormal rhythm of cardiac myocyte induced by diacetylmorphine.
引文
[1] PAVLIDIS P, DEFTEREOU T E, KARAKASI M V, et al. Intravenous heroin abuse and acute myocardial infarction: forensic study[J]. Am J Foren Med Path,2016,37(2):1.
[2] MILROY C M,PARAI J L.The histopathology of drugs of abuse[J]. Histopathology,2011,59(4):579-593.
[3] 刘小山.二醋吗啡对大鼠心肌细胞游离Ca2+浓度的作用[J].法医学杂志,2007,23(6):424-427.
[4] CARTER S, PITT S J, COLYER J, et al. Ca2+-dependent phosphorylation of RyR2 can uncouple channel gating from direct cytosolic Ca2+ regulation[J].J Membrane Biol,2011,40(1):21-33.
[5] GALIMBERTI E S,KNOLLMANN B C.ryanodine receptor use-dependent block suppresses Ca2+ waves in permeabilized Casq2-/- and RyR2-R4496C myocytes[J].Biophys J,2012,102(3):99a.
[6] 朱晓丽,王丽,马依彤,等.乳鼠心肌细胞和心肌成纤维细胞体外分离培养方法的优化[J].中国动脉硬化杂志,2015,23(1):90-93.
[7] KESHERWANI V, AGRAWAL S K. Upregulation of RyR2 in hypoxic/reperfusion injury[J].J Neurotrauma,2012,29(6):1255-1265.
[8] 揭娅,朱少华,蒋艳伟,等.大鼠急性心肌缺血后肌浆网RyR2 mRNA表达变化[J].法医学杂志,2008,24(5):327-329,338.
[9] DARKE S, DUFLOU J, TOROK M. The comparative toxicology and major organ pathology of fatal methadone and heroin toxicity cases[J]. Drug Alcohol Depend. 2010,106(1):1-6.
[10] REVELO A E, PAllAVI R, ESPANA-SCHMIDT C, et al. ‘Stoned’ people can get stunned myocardium: A case of heroin withdrawal precipitating Tako-Tsubo cardiomyopathy[J].Int J Cardiol,2013, 168(3):e96-e98.
[11] BUTLER B, RUBIN G, LAWRANCE A, et al. Estimating the risk of fatal arrhythmia in patients in methadone maintenance treatment for heroin addiction[J]. Drug Alcohol Review, 2011, 30(2):173-180.
[12] PHILLIPS K A, EPSTEIN D H, PRESTON K L. Cardiac complications of unwitting co-injection of quinine/quinidine with heroin in an intravenous drug user[J]. J Gen Intern Med, 2012, 27(12):1722-1725.
[13] SANTOS P E, BARCELLOS L C, MILL J G, et al. Ventricular action potential and L-type calcium channel in infarct-induced hypertrophy in rats[J]. J Cardiovasc Electrophysiol, 2010, 6(11):1004-1014.
[14] ZHANG R Y. Sulfur dioxide derivatives depress L-type calcium channel in rat cardiomyocytes[J].Clin Exp Pharmacol P,2011, 38(7):366-372.
[15] 蒲红伟, 苏丽萍, 王雪梅, 等.海洛因成瘾模型大鼠心肌细胞的动作电位和L型钙通道电流[J].中国组织工程研究,2015,19(18):2843-2848.
[16] HOUSER S R. Role of RyR2 phosphorylation in heart failure and arrhythmias: protein kinase A-mediated hyperphosphorylation of the ryanodine receptor at serine 2808 does not alter cardiac contractility or cause heart failure and arrhythmias[J]. Circ Res, 2014, 114(8):1320-1327.
[17] GRIMM M, LING H, PEREIRA L, et al. CaMKIIδ mediates β-adrenergic effects on RyR2 phosphorylation and SR Ca2+ leak and the pathophysiological response to chronic β-adrenergic stimulation[J]. J Mol Cell Cardiol, 2015, 85:282-291.
[18] BRIGGS L E, TAKEDA M, CUADRA A E, et al. Perinatal loss of Nkx2-5 results in rapid conduction and contraction defects[J]. Circ Res, 2008, 103(6):580.
[19] HWANG H S, HASDEMIR C, LAVER D, et al. Inhibition of cardiac Ca2+ release channels (RyR2) determines efficacy of class I antiarrhythmic drugs in catecholaminergic polymorphic ventricular tachycardia[J]. Circ Arrhythmia Elec, 2011, 4(2):128.
[20] BRUNELLO L, SLABAUGH J L, RADWANSKI P B, et al. Decreased RyR2 refractoriness determines myocardial synchronization of aberrant Ca2+ release in a genetic model of arrhythmia[J]. P Natl Acad Sci Usa, 2013,110(25):10312-10317.
[21] WEN Y, FANG Z, LAN Y. Age-related increase of early afterdepolarization in calsequestrin-2 knock-in mouse cardiomycyte[J]. J Geriatr Cardiol, 2010, 7(4):171-175.
[22] BONCOMPAGNI S, THOMAS M, LOPEZ J R, et al. Null mutations for triadin and junctin reveal triadin anchors and the requirement for triadin-junctin-CASQ complexes in stablizing RyR resting leak[J]. Biophys J, 2012, 102(3):363a-363a.
[23] BONCOMPAGNI S, THOMAS M, LOPEZ J R, et al. Triadin/junctin double null mouse reveals a differential role for triadin and junctin in anchoring CASQ to the jSR and regulating Ca2+ homeostasis[J]. Plos One, 2012, 7(7):e39962.
[24] WATERS S B, DIAK D M, ZUCKERMANN M, et al. Genetic background influences adaptation to cardiac hypertrophy and Ca2+ handling gene expression[J]. Front Physiol, 2013, 4:11.
[25] Knollmann B C. New roles of calsequestrin and triadin in cardiac muscle[J]. J Physiol, 2009,587(13):3081-3087.
[2] MILROY C M,PARAI J L.The histopathology of drugs of abuse[J]. Histopathology,2011,59(4):579-593.
[3] 刘小山.二醋吗啡对大鼠心肌细胞游离Ca2+浓度的作用[J].法医学杂志,2007,23(6):424-427.
[4] CARTER S, PITT S J, COLYER J, et al. Ca2+-dependent phosphorylation of RyR2 can uncouple channel gating from direct cytosolic Ca2+ regulation[J].J Membrane Biol,2011,40(1):21-33.
[5] GALIMBERTI E S,KNOLLMANN B C.ryanodine receptor use-dependent block suppresses Ca2+ waves in permeabilized Casq2-/- and RyR2-R4496C myocytes[J].Biophys J,2012,102(3):99a.
[6] 朱晓丽,王丽,马依彤,等.乳鼠心肌细胞和心肌成纤维细胞体外分离培养方法的优化[J].中国动脉硬化杂志,2015,23(1):90-93.
[7] KESHERWANI V, AGRAWAL S K. Upregulation of RyR2 in hypoxic/reperfusion injury[J].J Neurotrauma,2012,29(6):1255-1265.
[8] 揭娅,朱少华,蒋艳伟,等.大鼠急性心肌缺血后肌浆网RyR2 mRNA表达变化[J].法医学杂志,2008,24(5):327-329,338.
[9] DARKE S, DUFLOU J, TOROK M. The comparative toxicology and major organ pathology of fatal methadone and heroin toxicity cases[J]. Drug Alcohol Depend. 2010,106(1):1-6.
[10] REVELO A E, PAllAVI R, ESPANA-SCHMIDT C, et al. ‘Stoned’ people can get stunned myocardium: A case of heroin withdrawal precipitating Tako-Tsubo cardiomyopathy[J].Int J Cardiol,2013, 168(3):e96-e98.
[11] BUTLER B, RUBIN G, LAWRANCE A, et al. Estimating the risk of fatal arrhythmia in patients in methadone maintenance treatment for heroin addiction[J]. Drug Alcohol Review, 2011, 30(2):173-180.
[12] PHILLIPS K A, EPSTEIN D H, PRESTON K L. Cardiac complications of unwitting co-injection of quinine/quinidine with heroin in an intravenous drug user[J]. J Gen Intern Med, 2012, 27(12):1722-1725.
[13] SANTOS P E, BARCELLOS L C, MILL J G, et al. Ventricular action potential and L-type calcium channel in infarct-induced hypertrophy in rats[J]. J Cardiovasc Electrophysiol, 2010, 6(11):1004-1014.
[14] ZHANG R Y. Sulfur dioxide derivatives depress L-type calcium channel in rat cardiomyocytes[J].Clin Exp Pharmacol P,2011, 38(7):366-372.
[15] 蒲红伟, 苏丽萍, 王雪梅, 等.海洛因成瘾模型大鼠心肌细胞的动作电位和L型钙通道电流[J].中国组织工程研究,2015,19(18):2843-2848.
[16] HOUSER S R. Role of RyR2 phosphorylation in heart failure and arrhythmias: protein kinase A-mediated hyperphosphorylation of the ryanodine receptor at serine 2808 does not alter cardiac contractility or cause heart failure and arrhythmias[J]. Circ Res, 2014, 114(8):1320-1327.
[17] GRIMM M, LING H, PEREIRA L, et al. CaMKIIδ mediates β-adrenergic effects on RyR2 phosphorylation and SR Ca2+ leak and the pathophysiological response to chronic β-adrenergic stimulation[J]. J Mol Cell Cardiol, 2015, 85:282-291.
[18] BRIGGS L E, TAKEDA M, CUADRA A E, et al. Perinatal loss of Nkx2-5 results in rapid conduction and contraction defects[J]. Circ Res, 2008, 103(6):580.
[19] HWANG H S, HASDEMIR C, LAVER D, et al. Inhibition of cardiac Ca2+ release channels (RyR2) determines efficacy of class I antiarrhythmic drugs in catecholaminergic polymorphic ventricular tachycardia[J]. Circ Arrhythmia Elec, 2011, 4(2):128.
[20] BRUNELLO L, SLABAUGH J L, RADWANSKI P B, et al. Decreased RyR2 refractoriness determines myocardial synchronization of aberrant Ca2+ release in a genetic model of arrhythmia[J]. P Natl Acad Sci Usa, 2013,110(25):10312-10317.
[21] WEN Y, FANG Z, LAN Y. Age-related increase of early afterdepolarization in calsequestrin-2 knock-in mouse cardiomycyte[J]. J Geriatr Cardiol, 2010, 7(4):171-175.
[22] BONCOMPAGNI S, THOMAS M, LOPEZ J R, et al. Null mutations for triadin and junctin reveal triadin anchors and the requirement for triadin-junctin-CASQ complexes in stablizing RyR resting leak[J]. Biophys J, 2012, 102(3):363a-363a.
[23] BONCOMPAGNI S, THOMAS M, LOPEZ J R, et al. Triadin/junctin double null mouse reveals a differential role for triadin and junctin in anchoring CASQ to the jSR and regulating Ca2+ homeostasis[J]. Plos One, 2012, 7(7):e39962.
[24] WATERS S B, DIAK D M, ZUCKERMANN M, et al. Genetic background influences adaptation to cardiac hypertrophy and Ca2+ handling gene expression[J]. Front Physiol, 2013, 4:11.
[25] Knollmann B C. New roles of calsequestrin and triadin in cardiac muscle[J]. J Physiol, 2009,587(13):3081-3087.