ATP敏感钾离子通道开放剂对缺血心肌的保护作用
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摘要
心脏外科中，很多病人手术矫正畸形成功，但术后发生低心排综合征，严重者导致死亡，病理检查发现心肌坏死。许多报道证明心脏术后很多并发症与心肌受损害的程度有密切关系。目前需要矫正的心脏畸形或病变日趋复杂，为使心脏在乎术后不受严重的损害，手术中如何保护好心肌以减少缺血缺氧所造成的损害是心脏外科一项必须解决的课题。高钾停搏液虽可起到良好的心肌保护，但临床上遇到复杂的心脏畸形、术前存在心功能不全及心肌继发性病变等使术后严重低心排甚至导致病人死亡。缺血再灌注损伤机制目前仍不清楚。因此有必要对高钾停搏液进行研究，使其心肌保护的效果更趋完美。近年来ATP敏感钾离子通道开放剂(potassium channel openers)(PCOs)的特殊电生理作用引起许多学者的重视，成为目前心肌保护的热点。本研究采用离体心脏灌流、电镜、单细胞收缩、细胞内钙瞬态、内质网钙释放等功能检测方法在组织、细胞和分子水平对未成熟兔及成熟大鼠缺血心肌进行观察，以探讨心肌缺血再灌注损伤的发生机
Ah域乐钾离于通道开放剂对缺血心肌的保护作用 浙江大学博士学位论文
理及nP敏感钾离子通道开放剂（匹那地尔）对。0肌缺血再灌注损伤的保
护作用及有关机制．
第一部分
：ATP敏感钾离子通遣开放剂
对禾成熟兔缺血心肌的保护作用
研究目的：本实验旨在现察及了解ATP敏感性钾通道开放状态对未
成熟兔缺血＊肌的保护作用．
方法：未成熟大耳白幼兔，雌雄不拘，体重35卜4509，年龄卜4周．
戍巴比妥钠（50屿＆g）腹腔麻醉后，快速开胸摘取心脏悬挂于改良
Langendorff灌流装置．王动脉逆灌注 37CKrebs－Henseleit缓冲剃K－H
液）（含95x 0。和SK CO；混合气，灌注压76cmH；0）．将压力感受器带球囊
导管经左房插入左心室，球囊注水，使左。c室舒张末压保持在 10mmHg．
。G尖、左心房、主动脉根部置。G外膜电极，25分达平衡状态转为工作模
型，然后动物随机分为四组．对照组（。。ntrol组）13只，高钾停搏液组
（正组）13只，（卜H液中加氯化钾使钾浓度为16n。1几），匹那鲑d臼坠化
组（pgu（高钾停搏液中加入匹那地尔 5 oped ／L），格列苯脉桔
帮以X＋P＋G组）13只（匹那地尔强化液加格列苯胀10cpc VL）．control
组·r跳稳定后夹闭主动脉不绍H壬何停搏液，K、K＋P、K＋P＋G组主动脉夹闭
后分别给 20一25ml停搏液，每 15分钟重复一次，心脏置于 36 t IC恒温
器中，缺血30分，开放主动脉再灌n 30分．检测指标：lerMIJ
定：采用Pc ah＠生物信号处理系统软件进行来集和分析，计算左·a室发
展压，左心室压力最大上升和下降速度（f dp／dt。ax），左室发展压缺血
2
fP敏感钾离于通道开放剂对缺血心肌的保护作用 浙江大学搏士学位论文
后恢复率．＠冠状动脉流量测定（CF）．＠·q肋酶值测定：测定冠状动脉
漏出液中乳酸脱氢酶（LDH），肌酸磷酸激酶（CK），谷草转氨酶（AST），
采用常规生化法．④·C耿上因跃结构电镜检查：实验完毕，取左室前壁心肌
置入戊二醛液固定后送电镜检查．
气
结果：1．缺血复灌后K＋P组左心室发展压恢复百分率（K）（97．00
沪
士10．00。78．OO士9．co P（0．01）、左心室压力最大上升速度（。Hg／s）
（1262．84土585．11—913．35士163．82尸＜0．05）和左。C室压力最大下
降速度（mmHg／s）（1039．48t506．06 vs769．08ill7．46P＜0．05）明显
高于【组； 2．冠脉流量变化恤l灿in）： 缺血后 k＋P组 CF（26．13 t 8．78）
增加高于二组（17．45．03）与【＋Ped组（15．03．23），尸＜ 0．01．3．
缺血前后心肌酶值变化：缺血复灌工作sk＋I，后心肌酶值以【＋P组较低，
缺血前后差异无显著性意义，而其他三组心肌酶值缺血后均有不同程度升
高．4，u肌超微结构观察control组：线粒体肿胀、峭稀疏、模糊不清，
核肿大，染色质变深边聚明显，肌丝明暗带模糊不清，排列紊乱部分断裂．
二＋P组：线粒体结构完整，岭清晰，染色质无边聚现象，肌丝排列整齐，
明暗带清晰．K组、X＋P＋G组变化相似，部分线粒体肿胀，空泡化，略不
清，部分结构完好．部分染色质变深，边聚现象，部分肌丝排列?
Cardioplegic arrest during cardiac surgery has traditionally been accomplished through administration of a hyperkalemic cardioplegic solution. However, ventricular dysfunction has been shown to occur after hyperkalemic cardioplegic arrest which may involve alterations in ionic homeostasis and intracellular pH changes. The Langendorff perfusion model and isolated myocyte model of simulated cardioplegic arrest may be useful to determine mechanisms responsible for contractile dysfunction with reperfusion as well as potential strategies to prevent these effects. Adenosine triphosphate (ATP)-sensitive potassium channels(KATP) exist within the myocyte that open in response to reductions in intracellular ATP or ischemia. The protective effects of ischemic preconditioning in regional or global ischemia have been shown to be mediated in part by the KATP channel. Recent studies have also demonstrated that a brief period of KATP channel activation by a potassium channel opener (PCOs) before ischemia improves myocardial function and reduces infarct size with reperfusion. Moreover, KATP channel activation
before hypothermic, hyperkalemic cardioplegic arrest or used for hyperpolarized cardioplegic arrest improves functional recovery of myocardium. Cardioplegic arrest with simultaneous activation of KATP channels preserves myocyte contractile processes and attenuates the accumulation of intracellular calcium. The role of cellular cation homeostasis in ATP-sensitive K+ channel-induced cardioprotection is poorly understood. As a result of the structural, metabolic and ischemic tolerance differences, clinical myocardial protection strategies and cardioplegic solutions that are effective in adult hearts may be less effective in immature infant or neonatal hearts. In this project we set up two kind experimental models to understand the effects and mechanism of KATP channel opener-Pinacidil. Parti :The purpose of this study is to investigate the effectiveness of pinacidil, an opener of ATP sensitive K+ channels, in protecting myocardium of immature rabbits hearts from ischemic reperfusion injury. Part2:The objectives of the this study are threefold: first, to compare the erfects of cardioplegic arrest through the use of a potassium channel opener (PCO)-supplemented cardioplegia with traditional hyperkalemic cardioplegia on myocyte contractile function; second, to measure intracellular calcium transient in the myocyte with reperfusion ; and third to measure the role of the sarcoplasmic reticulum in the myocyte with reperfusion and calculate the duration of calcium transient evoked by caffeine to demonstrate the role of the Na+-Ca2+ exchanger to better define mechanism of myocyte contractile dysfunction.
Part 1
Myocardial protection of immature rabbits with an ATP-sensitive K* channel opener pinacidil.
Objective: As a result of the structural, metabolic and ischemic tolerance differences, clinical myocardial protection strategies and cardioplegic solutions that are effective in adult hearts may be less effective in immature infant or neonatal hearts. The purpose of this study is to investigate the effectiveness of pinacidil, an opener of ATP sensitive K+ channels, in protecting myocardium of immature rabbits hearts from ischemic reperfusion injury. Methods On modified Langendorff apparatus heart underwent 30 minutes of global normothermic ischemia followed by 30 minutes of reperfusion. 52 isolated hearts of 3-4 weeks old immature rabbits were divided into four groups randomly. During ischemia, three different cardioplegic solutions were administered intermittently by infusion every 15 minutes (20-25ml every time in all groups). Control group (n=13); 16K-cardioplegia group(K group): K-H solution with potassium (16 mmol/L)(n=13); PCO/16K-cardioplegia group (K+P group): K-H solution with potassium (16mmol/L) and pinacidil (50|imol/L)(n=13); PCO/ PCO antagonist/16K-cardioplegia group (K+P+G group): K-H solution with potassium (16mmol/L), pinacidil (50umol/L) and glibenclamide (10jimol/L)(n=13). The pre-ischemic and pos
Ah域乐钾离于通道开放剂对缺血心肌的保护作用 浙江大学博士学位论文
理及nP敏感钾离子通道开放剂（匹那地尔）对。0肌缺血再灌注损伤的保
护作用及有关机制．
第一部分
：ATP敏感钾离子通遣开放剂
对禾成熟兔缺血心肌的保护作用
研究目的：本实验旨在现察及了解ATP敏感性钾通道开放状态对未
成熟兔缺血＊肌的保护作用．
方法：未成熟大耳白幼兔，雌雄不拘，体重35卜4509，年龄卜4周．
戍巴比妥钠（50屿＆g）腹腔麻醉后，快速开胸摘取心脏悬挂于改良
Langendorff灌流装置．王动脉逆灌注 37CKrebs－Henseleit缓冲剃K－H
液）（含95x 0。和SK CO；混合气，灌注压76cmH；0）．将压力感受器带球囊
导管经左房插入左心室，球囊注水，使左。c室舒张末压保持在 10mmHg．
。G尖、左心房、主动脉根部置。G外膜电极，25分达平衡状态转为工作模
型，然后动物随机分为四组．对照组（。。ntrol组）13只，高钾停搏液组
（正组）13只，（卜H液中加氯化钾使钾浓度为16n。1几），匹那鲑d臼坠化
组（pgu（高钾停搏液中加入匹那地尔 5 oped ／L），格列苯脉桔
帮以X＋P＋G组）13只（匹那地尔强化液加格列苯胀10cpc VL）．control
组·r跳稳定后夹闭主动脉不绍H壬何停搏液，K、K＋P、K＋P＋G组主动脉夹闭
后分别给 20一25ml停搏液，每 15分钟重复一次，心脏置于 36 t IC恒温
器中，缺血30分，开放主动脉再灌n 30分．检测指标：lerMIJ
定：采用Pc ah＠生物信号处理系统软件进行来集和分析，计算左·a室发
展压，左心室压力最大上升和下降速度（f dp／dt。ax），左室发展压缺血
2
fP敏感钾离于通道开放剂对缺血心肌的保护作用 浙江大学搏士学位论文
后恢复率．＠冠状动脉流量测定（CF）．＠·q肋酶值测定：测定冠状动脉
漏出液中乳酸脱氢酶（LDH），肌酸磷酸激酶（CK），谷草转氨酶（AST），
采用常规生化法．④·C耿上因跃结构电镜检查：实验完毕，取左室前壁心肌
置入戊二醛液固定后送电镜检查．
气
结果：1．缺血复灌后K＋P组左心室发展压恢复百分率（K）（97．00
沪
士10．00。78．OO士9．co P（0．01）、左心室压力最大上升速度（。Hg／s）
（1262．84土585．11—913．35士163．82尸＜0．05）和左。C室压力最大下
降速度（mmHg／s）（1039．48t506．06 vs769．08ill7．46P＜0．05）明显
高于【组； 2．冠脉流量变化恤l灿in）： 缺血后 k＋P组 CF（26．13 t 8．78）
增加高于二组（17．45．03）与【＋Ped组（15．03．23），尸＜ 0．01．3．
缺血前后心肌酶值变化：缺血复灌工作sk＋I，后心肌酶值以【＋P组较低，
缺血前后差异无显著性意义，而其他三组心肌酶值缺血后均有不同程度升
高．4，u肌超微结构观察control组：线粒体肿胀、峭稀疏、模糊不清，
核肿大，染色质变深边聚明显，肌丝明暗带模糊不清，排列紊乱部分断裂．
二＋P组：线粒体结构完整，岭清晰，染色质无边聚现象，肌丝排列整齐，
明暗带清晰．K组、X＋P＋G组变化相似，部分线粒体肿胀，空泡化，略不
清，部分结构完好．部分染色质变深，边聚现象，部分肌丝排列?
Cardioplegic arrest during cardiac surgery has traditionally been accomplished through administration of a hyperkalemic cardioplegic solution. However, ventricular dysfunction has been shown to occur after hyperkalemic cardioplegic arrest which may involve alterations in ionic homeostasis and intracellular pH changes. The Langendorff perfusion model and isolated myocyte model of simulated cardioplegic arrest may be useful to determine mechanisms responsible for contractile dysfunction with reperfusion as well as potential strategies to prevent these effects. Adenosine triphosphate (ATP)-sensitive potassium channels(KATP) exist within the myocyte that open in response to reductions in intracellular ATP or ischemia. The protective effects of ischemic preconditioning in regional or global ischemia have been shown to be mediated in part by the KATP channel. Recent studies have also demonstrated that a brief period of KATP channel activation by a potassium channel opener (PCOs) before ischemia improves myocardial function and reduces infarct size with reperfusion. Moreover, KATP channel activation
before hypothermic, hyperkalemic cardioplegic arrest or used for hyperpolarized cardioplegic arrest improves functional recovery of myocardium. Cardioplegic arrest with simultaneous activation of KATP channels preserves myocyte contractile processes and attenuates the accumulation of intracellular calcium. The role of cellular cation homeostasis in ATP-sensitive K+ channel-induced cardioprotection is poorly understood. As a result of the structural, metabolic and ischemic tolerance differences, clinical myocardial protection strategies and cardioplegic solutions that are effective in adult hearts may be less effective in immature infant or neonatal hearts. In this project we set up two kind experimental models to understand the effects and mechanism of KATP channel opener-Pinacidil. Parti :The purpose of this study is to investigate the effectiveness of pinacidil, an opener of ATP sensitive K+ channels, in protecting myocardium of immature rabbits hearts from ischemic reperfusion injury. Part2:The objectives of the this study are threefold: first, to compare the erfects of cardioplegic arrest through the use of a potassium channel opener (PCO)-supplemented cardioplegia with traditional hyperkalemic cardioplegia on myocyte contractile function; second, to measure intracellular calcium transient in the myocyte with reperfusion ; and third to measure the role of the sarcoplasmic reticulum in the myocyte with reperfusion and calculate the duration of calcium transient evoked by caffeine to demonstrate the role of the Na+-Ca2+ exchanger to better define mechanism of myocyte contractile dysfunction.
Part 1
Myocardial protection of immature rabbits with an ATP-sensitive K* channel opener pinacidil.
Objective: As a result of the structural, metabolic and ischemic tolerance differences, clinical myocardial protection strategies and cardioplegic solutions that are effective in adult hearts may be less effective in immature infant or neonatal hearts. The purpose of this study is to investigate the effectiveness of pinacidil, an opener of ATP sensitive K+ channels, in protecting myocardium of immature rabbits hearts from ischemic reperfusion injury. Methods On modified Langendorff apparatus heart underwent 30 minutes of global normothermic ischemia followed by 30 minutes of reperfusion. 52 isolated hearts of 3-4 weeks old immature rabbits were divided into four groups randomly. During ischemia, three different cardioplegic solutions were administered intermittently by infusion every 15 minutes (20-25ml every time in all groups). Control group (n=13); 16K-cardioplegia group(K group): K-H solution with potassium (16 mmol/L)(n=13); PCO/16K-cardioplegia group (K+P group): K-H solution with potassium (16mmol/L) and pinacidil (50|imol/L)(n=13); PCO/ PCO antagonist/16K-cardioplegia group (K+P+G group): K-H solution with potassium (16mmol/L), pinacidil (50umol/L) and glibenclamide (10jimol/L)(n=13). The pre-ischemic and pos
引文
1. Feng J, Li H, Rosenkranz ER. Pinacidil pretreatment extends ischemia tolerance of neonatal rabbit hearts. J Surg Res. 2000 May 15;90:131-137.
2. Lin RU, Zhu XK, Zhang ZW, etal. Myocardial protection of immature rabbits with an ATP-sensitive K~+ channel opener pinacidil. Chin Med J. 2001;114:1184-1188.
3. Takashi E, Wang Y, Ashraf M. Activation of mitochondrial K(ATP)channel elicits late preconditioning against myocardial infarction via protein kinase C signaling pathway. Circ Res. 1999 Dec 3-17;85:1146-1153.
4. Ducko CT, Stephenson ER Jr, Jayawant AM, etal. Potassium channel openers: are they effective as pretreatment or additives to cardioplegia? Ann Thorac Surg 2000 May;69:1363-1368
5. Toyoda Y, Levitsky S, McCully JD. Opening of mitochondrial ATP-sensitive potassium channels enhances cardioplegic protection. Ann Thorac Surg 2001 Apr;71:1281-1288; discussion 1288-1289.
6. Lawton JS, Harrington GC, Allen CT, etal. Myocardial protection with pinacidil cardioplegia in the blood-perfused heart. Ann Thorac Surg 1996 Jun;61:1680-1688.
7．黄杰，姚震，高尚志．心脏停搏液对不成熟心肌保护的实验研究进展．中华胸心血管外科杂志．1991；7：51-54．
8．黄杰，高尚志，程邦昌，等．心脏停搏液对未成熟心肌的保护作用．中华胸心血管外科杂志．1998；14：45-47．
9. Auchampach JA, Maruyama M, Cavero I, et al. Phormacological evidence for a role of ATP-dependent potassium channels in myocardial stunning. Circulation. 1992;86:311-319.
10. higcmatsu S, Sato T, Abe T, et al. Pharmacological evidence for persistent activation of ATP-sensitive K~+ channels in early phase of reperfusion and its protective role against myocardial stunning. Circulation. 1995;92:2266-2275.
11． Menasche P, Mouas C, Grousset C. Is potassium channel opening an effective form of preconditioning before cardioplegia? Ann Thorac Surg. 1996;61:1764-1768．
12． Cohen NM, Wise RM, Wechsler AS, et al. Elective cardial arrest with a hyperpolarizing adenosine triphosphate-sensitive potassium channel opener. J Thorac Cardiovasc Surg. 1993;106:317-328．
13． Steven YW, Menachem F, Robert GJ, et al.Adenosine triphosphate-sensitive K channels mediate postcardioplegia coronary hyperemia. J Thorac Cardiovasc Surg. 1995;110:1073-1082．
14． Mark J, Edward RS, Ralph JD, et al. Advantages of continuous hyperpolarized arrest with pinacidil over St. thomas' hospital solution during prolonged ischemia. J Thorac Cardiovasc Surg. 1998;116:131-138．
15． Kevelaitis E, Oubenaissa A, Peynet J,etal. Preconditioning by mitochondrial ATP-sensitive potassium channel openers: An effective approach for improving the preservation of heart transplants. Circulation 1999 9; 100(19 Suppl):Ⅱ345-350．
16． Ghosh S, Standen NB,Galinanes M.Evidence for mitochondrial KATP channels as effectors of human myopcardial preconditioning. Cardiovasc Res. 2000;45:934-940．
17． Holmuhamedov EL, Wang L, Terzic A. ATP-sensitive K+channel openers prevent Ca2+ overload in rat cardiac mitochondria. J Physiol. 1999;519: Pt 2347-2360．
18． Wang S,Cone J,Liu Y. Dual roles of mitochongrial K(ATP) channels in diazoxide-mediated ptrotection in isolated rabbit hearts. Am J Physiol Heart Circ Physiol. 2001;280: H246-255．
19． B. Hugh Dorman, Latha H, Robert B. H etal . Preservation of Myocyte Contractile Function After Hypothermic Cardioplegic Arrest by Activation of ATP-Sensitive Potassium Channels. Circulation. 1997;96:2376-2384．
2. Lin RU, Zhu XK, Zhang ZW, etal. Myocardial protection of immature rabbits with an ATP-sensitive K~+ channel opener pinacidil. Chin Med J. 2001;114:1184-1188.
3. Takashi E, Wang Y, Ashraf M. Activation of mitochondrial K(ATP)channel elicits late preconditioning against myocardial infarction via protein kinase C signaling pathway. Circ Res. 1999 Dec 3-17;85:1146-1153.
4. Ducko CT, Stephenson ER Jr, Jayawant AM, etal. Potassium channel openers: are they effective as pretreatment or additives to cardioplegia? Ann Thorac Surg 2000 May;69:1363-1368
5. Toyoda Y, Levitsky S, McCully JD. Opening of mitochondrial ATP-sensitive potassium channels enhances cardioplegic protection. Ann Thorac Surg 2001 Apr;71:1281-1288; discussion 1288-1289.
6. Lawton JS, Harrington GC, Allen CT, etal. Myocardial protection with pinacidil cardioplegia in the blood-perfused heart. Ann Thorac Surg 1996 Jun;61:1680-1688.
7．黄杰，姚震，高尚志．心脏停搏液对不成熟心肌保护的实验研究进展．中华胸心血管外科杂志．1991；7：51-54．
8．黄杰，高尚志，程邦昌，等．心脏停搏液对未成熟心肌的保护作用．中华胸心血管外科杂志．1998；14：45-47．
9. Auchampach JA, Maruyama M, Cavero I, et al. Phormacological evidence for a role of ATP-dependent potassium channels in myocardial stunning. Circulation. 1992;86:311-319.
10. higcmatsu S, Sato T, Abe T, et al. Pharmacological evidence for persistent activation of ATP-sensitive K~+ channels in early phase of reperfusion and its protective role against myocardial stunning. Circulation. 1995;92:2266-2275.
11． Menasche P, Mouas C, Grousset C. Is potassium channel opening an effective form of preconditioning before cardioplegia? Ann Thorac Surg. 1996;61:1764-1768．
12． Cohen NM, Wise RM, Wechsler AS, et al. Elective cardial arrest with a hyperpolarizing adenosine triphosphate-sensitive potassium channel opener. J Thorac Cardiovasc Surg. 1993;106:317-328．
13． Steven YW, Menachem F, Robert GJ, et al.Adenosine triphosphate-sensitive K channels mediate postcardioplegia coronary hyperemia. J Thorac Cardiovasc Surg. 1995;110:1073-1082．
14． Mark J, Edward RS, Ralph JD, et al. Advantages of continuous hyperpolarized arrest with pinacidil over St. thomas' hospital solution during prolonged ischemia. J Thorac Cardiovasc Surg. 1998;116:131-138．
15． Kevelaitis E, Oubenaissa A, Peynet J,etal. Preconditioning by mitochondrial ATP-sensitive potassium channel openers: An effective approach for improving the preservation of heart transplants. Circulation 1999 9; 100(19 Suppl):Ⅱ345-350．
16． Ghosh S, Standen NB,Galinanes M.Evidence for mitochondrial KATP channels as effectors of human myopcardial preconditioning. Cardiovasc Res. 2000;45:934-940．
17． Holmuhamedov EL, Wang L, Terzic A. ATP-sensitive K+channel openers prevent Ca2+ overload in rat cardiac mitochondria. J Physiol. 1999;519: Pt 2347-2360．
18． Wang S,Cone J,Liu Y. Dual roles of mitochongrial K(ATP) channels in diazoxide-mediated ptrotection in isolated rabbit hearts. Am J Physiol Heart Circ Physiol. 2001;280: H246-255．
19． B. Hugh Dorman, Latha H, Robert B. H etal . Preservation of Myocyte Contractile Function After Hypothermic Cardioplegic Arrest by Activation of ATP-Sensitive Potassium Channels. Circulation. 1997;96:2376-2384．