卡维地洛对模拟缺血时豚鼠乳头肌动作电位和心室肌细胞ATP敏感性钾电流的影响

目的　观察卡维地洛对模拟缺血豚鼠乳头肌动作电位和心室肌细胞ATP敏感性钾电流(IK·ATP)的影响 ,探讨卡维地洛对心室肌ATP敏感性钾通道 (KATP)调节及其在抗心肌缺血、抗心律失常中的作用机制。方法　应用标准玻璃微电极技术 ,观察 0 0 3μM、0 3μM和 3 0 μM 3种不同浓度的卡维地洛对模拟缺血时豚鼠心室乳头肌动作电位的影响 ,同时采用全细胞膜片钳方法 ,记录模拟缺血时上述 3个浓度的卡维地洛对豚鼠心室肌细胞IK·ATP的作用。结果　模拟缺血液灌流细胞 ,其中 1 5 1 %(n =5)的细胞KATP通道在 3min内完全开放 ,这些细胞均在KATP通道完全开放后 (1 2 3 0 0± 33 64)s死亡。模拟缺血液加入 0 0 3μM (n =9)、0 3μM (n =9)、3 0 μM (n =1 0 )卡维地洛灌流细胞的各组 ,KATP通道分别于灌流后 (9 0 0± 3 1 6)min、(9 77± 0 86)min和 (1 1 74± 4 41 )min开放。 +40mV时的IK·ATP(pA/pF)分别是 2 5 44± 1 8 48、2 0 1 7± 30 83和 3 95± 2 48,与对照组 32 65± 36 0 2比较 ,0 0 3μM组差异有显著性 (P <0 0 5) ,后两者差异呈高度显著性 (P <0 0 1 ) ;模拟缺血液灌流 5～ 2 0min(n =1 5)均可见动作电位时限 (APD)显著缩短 ,APD90 从正常对照组的 (2 31 0± 1 4 3)ms降至 (1 30 0±1 2 4     

银杏叶提取物对模拟缺血条件下兔心室肌细胞动作电位及ATP敏感性钾通道的影响

研究银杏叶提取物 (EGb)对模拟缺血条件下兔心室肌细胞三磷酸腺苷敏感性钾通道电流 (IKATP)及跨膜动作电位时程 (APD)的影响 ,以探讨其抗缺血性心律失常作用的电生理机制。采用酶解法分离获取兔心室肌细胞 ,将其分为正常对照、持续缺血、缺血预处理以及含EGb液 (15 ,30 ,6 0 ,12 0 μg/L)灌流 4组。用全细胞膜片钳技术 ,记录不同条件下的IKATP和跨膜动作电位。结果 :①与持续缺血组比较 ,缺血预处理以及EGb(12 0 μg/L)可使单个心室肌细胞APD50 、APD90 明显缩短 (n =5 ,P <0 .0 5 )。EGb处理组与缺血预处理组相比较 ,对APD的影响无显著差异 ;②与持续缺血组比较 ,缺血预处理和EGb(12 0 μg/L)均可以使IKATP由 112 4± 15 3pA增至 344 0± 2 0 5和 2 95 9± 12 9pA(n =5 ,P <0 .0 5 ) ,使得IKATPI V曲线抬高 ;③增大的电流均可被Glibenclimide阻断。结论 :EGb可开放细胞膜ATP敏感性钾通道、缩短APD ,产生类似心肌缺血预适应的病理生理过程。     

辛伐他汀对血脂正常兔心肌缺血再灌注后L型钙电流的影响

目的通过研究辛伐他汀预处理对兔心肌缺血再灌注后L型钙离子通道电流(ICa-L)的影响,探讨他汀类药物抗心律失常的细胞学离子机制。方法45只新西兰大耳白兔随机分为3组:缺血再灌注组(I-R组,结扎冠状动脉左前降支30min后再开放120min);辛伐他汀治疗组(他汀组,手术前给予辛伐他汀5mg·kg-1·d-1,3天);假手术对照组(对照组,只开胸不结扎血管)。观察心律失常发生情况。采用酶解法分离缺血部位心室肌外膜单个心室肌细胞,采用全细胞膜片钳技术,记录ICa-L,同时检测各组血脂水平。结果各组动物血脂水平无显著差异。I-R组心律失常发生率较对照组增加,他汀组较I-R组心律失常发生率明显下降。对照组、I-R组和他汀组ICa-L电流密度峰值(0mV)分别为-3.13±1.22pA/pF(n=16),-4.24±0.92pA/pF(n=15)和-3.46±0.85pA/pF(n=13)。I-R组较对照组明显升高(P<0.05),他汀组较I-R组明显下降(P<0.05),他汀组与对照组无差异(P>0.05)。结论缺血再灌注可导致梗死区心室肌细胞I明显增加,辛伐他汀预处理可逆转这种变化。     

对KATP通道介导缺血预适应心肌再灌注损伤的研究

目的探讨优降糖对KATP通道介导心肌缺血预适应作用的影响。方法将44只大鼠随机分为心肌缺血预适应组(IPC组)、优降糖组(GLI组)、优降糖 IPC组(G-P组)和对照组(C组)。心肌缺血预适应由3次10min缺血/10min再灌注组成。所有大鼠均接受30min缺血/60min再灌注。梗死范围由饱和曲利本蓝和红四氮唑蓝染色判定,并以坏死区占缺血区的百分率表示。Ⅱ导联记录心脏室性心律失常。结果IPC能显著缩小缺血/再灌注后的心肌梗死范围,且这种作用能被KATP通道阻滞剂优降糖完全取消。IPC可减少缺血/再灌注所致的室性心律失常的发生,但这种保护作用不能被优降糖所阻断。结论优降糖对KATP介导IPC的心肌保护作用有影响。     

硫化氢对大鼠心房肌细胞三磷酸腺苷敏感性钾通道电流的影响

目的观察内源性及外源性硫化氢(H2S)对大鼠离体心房肌细胞三磷酸腺苷(ATP)敏感性钾通道(KATP)外向电流的影响,以探讨H2S对心房肌细胞的作用。方法对大鼠离体心脏采用胶原酶酶解法得到单个心房肌细胞,采用膜片钳全细胞技术记录H2S生成酶——胱硫醚-γ-裂解酶的不可逆抑制剂DL-propargylglycine(PPG)用药前、用药后5,10,15,20,25 min及不同浓度外源性H2S的供体硫氢化钠(NaHS)干预前后的KATP电流。结果经200μmol/L PPG干预后KATP峰电流密度(+70 mV)显著减小(6.906 6±1.902 9 pA/pF vs 3.924 4±0.988 5 pA/pF,P<0.01),且具有时间依赖性。经9.375,18.75,37.5,75,150μmol/L NaHS干预后KATP峰电流密度呈浓度依赖性增大,至150μmol/L时峰电流密度明显增大(6.5974±1.1527 pA/pF vs 10.463 1±2.329 7 pA/pF,P<0.01)。结论内源性及外源性H2S均可以开放大鼠离体心房肌KATP通道,使KATP电流增加。     

模拟缺血-再灌注对窦房结细胞起搏离子流的影响及K_(ATP)通道开放剂的干预作用

探讨模拟缺血 再灌注对窦房结细胞起搏离子流 (If)的影响及KATP通道开放剂Pinacidil的干预效果。分离乳鼠窦房结细胞 ,纯化培养 2天后进行实验。随机分为对照组、模拟缺血 再灌注组 (I/R)、KATP通道开放剂Pinacidil干预组 (P +I/R)及KATP通道阻断剂 5 HD干预组 (5 HD +P +I/R及 5 HD +I/R)。采用常规全细胞膜片钳技术及多导管灌流系统 ,测定各组细胞If 密度 ,并绘制If 激活曲线。结果 :①每个窦房结细胞均可记录到If 电流 ,在相同指令电压下 ,I/R组窦房结细胞If 密度值较对照组明显增加 (P <0 .0 1) ;而P +I/R组则较I/R组显著减小 (P <0 .0 1) ;5 HD +P +I/R及 5 HD +I/R两组又较P +I/R组明显增加 (P <0 .0 1) ,但与I/R组比较无显著差异。②与对照组比较 ,I/R组窦房结细胞的If 激活曲线发生右移 ,半数最大激活电压由 - 10 8.0± 12 .4mV变为 - 89.5± 7.2mV(P <0 .0 1) ;P +I/R组窦房结细胞If 激活曲线较I/R组左移 ,半数最大激活电压为 - 99.5± 10 .8mV(P <0 .0 5 ) ;KATP通道阻断剂 5 HD可阻断Pinacidil对If 激活曲线的影响。结论 :KATP通道开放剂Pinacidil可对抗模拟缺血 再灌注对窦房结细胞If 的影响 ,此有利于维持模拟缺血 再灌注时窦房结细胞离子稳态和电生理活动的相对稳定     

缺血再灌注期间不同时相氟比洛芬酯处理对乳鼠心肌细胞钠电流的影响

目的：研究缺血再灌注（ischemia/reperfusion,I/R）期间不同时相（缺血时、缺血后再灌时）氟比洛芬酯处殚对乳鼠心肌细胞钠电流的影响。方法：实验分为四组：1组（对照组）为体外培养乳鼠心室肌细胞;Ⅱ组（I/R组）为相同的细胞,缺血3小时,再灌注1小时;III组（氟比洛芬酯缺血时处理组）在I/R缺血时相加入氟比洛芬哺（0.15μM）;IV组（氟比洛芬酯缺血后再灌注时处理组）在I/R再灌注时相加入氟比洛芬酯（0．15μM）。采用膜片钳全细胞模式分别记录并比较四组的钠电流。结果：和I组相比,II组存每一指令电压上都增大钠电流;在测试电压为-20mV的条件下,钠电流峰值电流密度从-375．47±70．31（pA/pF）增至-557．11±12786（pA/pF）（n=5,P〈f0.05）;稳态激活半激活电压（V1/2）分别为-4281±1．21mV和-3149±2．40mV（n=5,P〈005）;稳态欠活半激活电压（V1/2）分别为-7424±5．89mV和-68．70±6．26mV（n=5,P〈005）;通道失活后恢复T分别为2569±19．47ms和7．534±3．24ms（n=5,P〈0．05）。III、IV组钠电流峰值电流密度从-375．47±70．31（pA/pF）分别降至-208．80±121．44和-278．91±188．26（pA/pF）（n=5,P〈0．05）;通道稳念激活V1/2分别为-40．89±9．81mV和-39．49±3．70mV（n=5,P〉O05）;稳态失活V1/2分别为74．45±5．02mY和-75494±3．32mV（n=5,P〉0．05）;通道失活后恢复T分别为24．98±16．41ms和26．30±11．82ms（n=5,P〉005）。III、IV组之间各指标间的差异无统计学意义。结论：I/R处土型可以增大钠电流的峰值电流,并使电压电流曲线下移;减慢通道的时间依赖性激活,促进通道的电爪依赖性失活,失活后恢复变快。而在I/R缺血和再灌注两个时相使用氟比洛芬酯均能有效阻制钠通道的这螳改变。     

长期应用左旋精氨酸对慢性低氧肺动脉平滑肌细胞钾通道的作用

目的　探讨长期应用左旋精氨酸 (L Arg)对慢性低氧大鼠肺动脉平滑肌细胞 (PASMC)膜钾通道的作用 ,为慢性低氧性肺动脉高压的防治手段提供理论依据。方法　雄性Wistar大鼠 18只 ,每组 6只 ,随机分为三组 :生理盐水对照组 (A组 )、慢性低氧组 (B组 )和慢性低氧 +L Arg组 (C组 )。单个大鼠的PASMC获得采用急性酶分离法 (胶原酶Ⅰ型和木瓜蛋白酶 )。用全细胞膜片钳技术测定三组PASMC的静息膜电位 (Em)、电压门控钾通道 (Kv通道 )电流和钙激活钾通道 (KCa通道 )电流。结果　 (1)B组大鼠PASMC的静息膜电位为 (- 31± 8)mV ,A组为 (- 4 2± 5 )mV ,两组比较差异有显著性 (P <0 0 1)。C组大鼠PASMC的静息膜电位为 (- 39± 4 )mV ,与B组比较差异有显著性 (P <0 0 5 )。(2 )对Kv通道 (在 +5 0mV电压刺激时峰值电流 ) :B组大鼠PASMC的峰值电流为 (6 2± 5 )pA/pF ,A组为 (12 1± 9)pA/pF ,两组比较差异也有显著性 (P <0 0 1)。C组大鼠PASMC的峰值电流为 (95± 3)pA/pF ,与B组比较差异也有显著性 (P <0 0 0 1)。 (3)对KCa通道 (+5 0mV电压刺激时的峰值电流 ) :B组大鼠PASMC的峰值电流为 (74 7± 4 1)pA/pF ,A组为 (5 3 6± 5 9)pA/pF ,两组比较差异有显著性 (P <0 0 5 )。应用L Arg后 ,C组大鼠PASMC的峰值电     

兔急性心肌梗死后二月心室肌细胞钠离子通道活性的变化

研究急性心肌梗死 (AMI)后心室肌细胞钠离子通道活性的变化。采用结扎兔冠状动脉左前降支的方法建立AMI动物模型 ,应用膜片钳全细胞记录方法 ,观察AMI后 2个月心外膜梗死区心肌细胞钠通道电流 (INa)的变化。结果 :①正常对照组INa电流密度峰值 (去极化电位 - 30mV时 )为 45 .5± 5 .33pA/pF(n =12 ) ,心肌梗死 (简称心梗 )组为 16 .4± 4.43pA/pF(n =13) ,心梗组较对照组明显下降 ,P <0 .0 1。心梗组INa电流 电压关系曲线较对照组明显下移。②心梗组INa失活曲线较对照组明显左移 (即向超级化方向移动 ) ,对照组半数失活电压 (V0 .5)为 - 76 .2± 5 .3mV(n =5 ) ,心梗组V0 .5为 - 82 .4± 5 .6mV(n =12 ) ,P <0 .0 5。③心梗组钠通道灭活后恢复时程较对照组减慢 ,恢复曲线下移。结论 :AMI可导致梗死区心室肌细胞INa下降、钠通道动力学发生变化 ,引起心肌传导速度下降和不应性延长 ,此可能是导致AMI后出现折返性室性心律失常的原因。     

兔冠状动脉结扎1小时缺血区心室肌细胞瞬间外向钾通道的变化

为探讨在急性心肌梗死 (AMI)早期瞬间外向钾通道的变化及其在室性心律失常发生中的作用 ,以开胸冠状动脉 (简称冠脉 )结扎法制备兔急性心肌缺血模型 ,1h后处死动物分离心室肌细胞 ,采用全细胞膜片钳记录技术观察缺血区心外膜心室肌细胞瞬间外向钾通道电流 (Ito)的变化 ,以正常心肌Ito为对照。结果 :急性冠脉结扎 1h兔缺血区心室肌细胞Ito受到抑制 ,电流密度$C电压关系曲线下移 ,测试电压 + 60mV时的Ito电流密度对比显示 :对照组为 1 7.39± 5 .2 4pA/pF (n =1 2 ) ,冠脉结扎 1h组为 7.75± 3.1 1pA/pF (n =1 0 ) ,与对照组相比下降了 5 7% ,P <0 .0 0 1 ;其失活曲线左移 ,半数最大失活电压 (V1 /2 )对照组为 - 35 .2± 5 .3mV(n =1 2 ) ,冠脉结扎 1h组为 - 5 5 .1± 5 .6mV(n =1 0 ) ,与对照组比较失活速度加快 ,P <0 .0 1 ;冠脉结扎后 1h组Ito恢复明显减慢 ,恢复时程延长 ,P <0 .0 5。结论 :冠脉结扎后 1h缺血区心室肌细胞瞬间外向钾通道受抑制 ,影响动作电位复极 ,容易诱发 2相折返 ,可能为AMI后室性心律失常发生的机制之一。     

Inhibiting mitochondrial permeability transition pore opening: a new paradigm for myocardial preconditioning?

OBJECTIVE: We propose that ischemic preconditioning (IPC) and mitochondrial K(ATP) channel activation protect the myocardium by inhibiting mitochondrial permeability transition pore (MPTP) opening at reperfusion. METHODS: Isolated rat hearts were subjected to 35 min ischemia/120 min reperfusion and assigned to the following groups: (1) control; (2) IPC of 2x5 min each of preceding global ischemia; (3,4,5) 0.2 micromol/l cyclosporin A (CsA, which inhibits MPTP opening), 5 micromol/l FK506 (which inhibits the phosphatase calcineurin without inhibiting MPTP opening), or 20 micromol/l atractyloside (Atr, a MPTP opener) given at reperfusion; (6,7) pre-treatment with 30 micromol/l diazoxide (Diaz, a mitochondrial K(ATP) channel opener) or 200 nmol/l 2 chloro-N(6)-cyclopentyl-adenosine (CCPA, an adenosine A1 receptor agonist); (8) IPC+Atr; (9) Diaz+Atr; (10) CCPA+Atr. The effect of mitochondrial K(ATP) channel activation on calcium-induced MPTP opening in isolated calcein-loaded mitochondria was also assessed. RESULTS: IPC, CsA when given at reperfusion, and pre-treatment with diazoxide or CCPA all limited infarct size (19.9+/-2.6% in IPC; 24.6+/-1.9% in CsA, 18.0+/-1.7% in Diaz, 20.4+/-3.3% in CCPA vs. 44.7+/-2.0% in control, P<0.0001). Opening the MPTP with atractyloside at reperfusion abolished this cardio-protective effect (47.7+/-1.8% in IPC+Atr, 42.3+/-3.2% in Diaz+Atr, 51.2+/-1.6% in CCPA+Atr). Atractyloside and FK506, given at reperfusion, did not influence infarct size (45.7+/-2.1% in Atr and 43.1+/-3.6% in FK506 vs. 44.7+/-2.0% in control, P=NS). Diazoxide (30 micromol/l) was shown to reduce calcium-induced MPTP opening by 52.5+/-8.0% in calcein-loaded mitochondria. 5-Hydroxydecanoic acid (100 micromol/l) was able to abolish the cardio-protective effects of both diazoxide and IPC. CONCLUSION: One interpretation of these data is that IPC and mitochondrial K(ATP) channel activation may protect the myocardium by inhibiting MPTP opening at reperfusion.     

Mitochondrial K(ATP) channel opening is important during index ischemia and following myocardial reperfusion in ischemic preconditioned rat hearts

We have previously demonstrated that K(ATP)channel openers administered just prior to and throughout reperfusion induce cardioprotection in the blood-perfused canine heart. However, a recent report suggests that the mitochondrial K(ATP)channel is only a trigger of ischemic preconditioning (IPC). These recent data are, however, in contrast to most previous investigations that suggested that activation of the mitochondrial K(ATP)channel is an important downstream mediator of cardioprotection. Therefore, we examined the role of the mitochondrial K(ATP)channel as a downstream mediator of IPC in a rat model by administering the selective mitochondrial K(ATP)channel antagonist, 5-hydroxydecanoate (5-HD), at several points during IPC. Infarct size (IS) was determined by tetrazolium chloride staining and expressed as a percent of the area at risk (AAR). Control animals had an IS/AAR of 58.4+/-0.6 and IS/AAR was reduced to 6.2+/-1.7 following IPC. 5-HD (10 mg/kg), attenuated cardioprotection when administered either 5 min prior to the IPC stimulus (40.4+/-1.4), during the reperfusion phase of the IPC stimulus (39.7+/-5.9), or 5 min prior to reperfusion during prolonged ischemia (34.3+/-6.9). Additionally, when 5-HD was administered at 5 mg/kg during the reperfusion phase of index ischemia plus 5 min prior to IPC or plus during the reperfusion phase of IPC, cardioprotection was also attenuated (36.3+/-5.5 and 43.8+/-6.9, respectively). These data suggest that activation of the mitochondrial K(ATP) channel is an important downstream regulator of myocardial protection with effects lasting into the reperfusion period following prolonged ischemia.     

Effect of classic preconditioning and diazoxide on endothelial function and O2- and NO generation in the post-ischemic guinea-pig heart

OBJECTIVES: A hypothesis was tested that a reaction product between superoxide (O2-) and nitric oxide (NO) mediates post-ischemic coronary endothelial dysfunction that ischemic preconditioning (IPC) protects the endothelium by preventing post-ischemic cardiac O2- and/or NO formation, and that the opening of the mitochondrial ATP-dependent potassium channel (mKATP) plays a role in the mechanism of IPC. METHODS: Langendorff-perfused guinea-pig hearts were subjected either to 30 min global ischemia/30 min reperfusion (IR) or were preconditioned prior to IR with three cycles of either 5 min ischemia/5 min reperfusion or 5 min infusion/5 min wash-out of mKATP opener, diazoxide (0.5 microM). Coronary flow responses to acetylcholine (ACh) and nitroprusside were used as measures of endothelium-dependent and -independent vascular function, respectively. Myocardial outflow of O2- and NO, and functional recoveries were followed during reperfusion. RESULTS: IR impaired the ACh response by approximately 60% and augmented cardiac O2- and NO outflow. Superoxide dismutase (150 U/ml) and NO synthase inhibitor, l-NMMA (100 microM) inhibited the burst of O2- and NO, respectively, and afforded partial preservation of the ACh response in IR hearts. NO scavenger, oxyhemoglobin (25 microM), afforded similar endothelial protection. IPC and diazoxide preconditioning attenuated post-ischemic burst of O2-, but not of NO, and afforded a complete endothelial protection. Diazoxide given after 30-min ischemia increased the O2- burst and was not protective. The effects of IPC and diazoxide preconditioning were not affected by HMR-1098 (25 microM), a selective blocker of plasmalemmal KATP, and were abolished by glibenclamide (0.6 microM) and 5-hydroxydecanoate (100 microM), a nonselective and selective mK(ATP) blocker, respectively. 5-Hydroxydecanoate produced similar effects, whether it was given as a continuous treatment or was washed out prior to IR. CONCLUSION: The results suggest that in guinea-pig heart: (i) a reaction product between O2- and NO mediates the post-ischemic endothelial dysfunction; (ii) the mK(ATP) opening serves as a trigger of the IPC and diazoxide protection; and (iii) the mK(ATP) opening protects the endothelium in the mechanism that involves the attenuation of the O2- burst at reperfusion.     

再灌注损伤抢救激酶对小鼠缺血后适应心肌再灌注损伤中的减轻作用

目的 研究缺血后适应(IPC)对离体小鼠心肌缺血再灌注(I/R)损伤的作用及其影响因素,探讨再灌注损伤抢救激酶在IPC心肌保护中的作用.方法 建立Langendofff小鼠心肌I/R模型,全心缺血30 min后分为6组[(1)对照组,(2)3次IPC组(采取缺血10 s及再灌注10 s的3次IPC周期),(3)6次IPC组(采取缺血10 s及再灌注10 s的6次IPC周期),(4)延迟IPC组(恢复再灌注1 min后进行IPC),(5)IPC+PD98059组,(6)I/R+PD98059组],随后再灌注2 h;观察IPC对心脏血流动力学、心肌酶的释放、心肌超氧化物歧化酶活性和丙二醛的含量、梗死心肌范围的影响以及与细胞外信号调节激酶(ERK1/2)、磷脂酰肌醇3激酶-蛋白激酶B表达水平的关系.结果 与对照组比较,3次IPC组和6次IPC组小鼠心脏血流动力学显著改善,心肌酶释放减少,心肌丙二醛减少、超氧化物歧化酶活性增加,心肌梗死范围减小.6次与3次IPC周期的保护作用相似.而IPC作用在恢复再灌注1 min后消失.3次IPC组和6次iPC组心肌的ERK1/2磷酸化水平显著增高,蛋白激酶B磷酸化水平无明显变化.PD98059显著抑制IPC所致的ERK1/2的磷酸化,并能消除IPC对心肌的保护作用.结论 IPC能有效地减轻离体小鼠心肌缺血再灌注损伤,增加IPC的周期数并没有扩大保护作用,延迟IPC没有产生类似的保护作用.ERK1/2细胞信号途径参与介导IPC对离体心脏缺血再灌注心肌的保护作用.     

Ischemic preconditioning prevents reperfusion heart injury in cardiac hypertrophy by activation of mitochondrial KATP channels

BACKGROUND: Cardiac hypertrophy has been demonstrated to decreases the ATP-sensitive potassium channels (K(ATP)), the major protective mechanism following the energy depletion, a common condition seen during the reperfusion after open heart surgery. In this study we have demonstrated the role of ischemic preconditioning (IP) in preventing the reperfusion injury of the hypertrophied heart by activation of the depleted K(ATP) channels. METHODS: Pressure overload left ventricular hypertrophy was induced in 6 weeks old male Wistar rats by supra renal transverse abdominal aortic constriction and the study was conducted 10-12 weeks later. Hypertrophied rats were subjected to IP protocols by four episodes of 3 min ischemia each being separated by 10 min reperfusion, followed by 30 min of sustained ischemia and 120 min of reperfusion with or without treating the rats with K(ATP) channel antagonists 5-hydroxydecanoic acid (10 mg/kg per i.v.) or glibenclamide (1 mg/kg per i.v.), 10 min before the sustained ischemia. RESULTS: IP resulted in (a) less incidence of ventricular arrhythmias (b) less area of myocardial infarction (9.3% vs. 48.1%, IP to control) (c) less tissue water content (76.5% vs. 94.8%, IP to control) (d) well preserved myocardial ATP content (P<0.001 from control) content and (e) much fewer apoptotic cells (4.7% vs. 13.2%, IP to control). Pre treating the rats with the K(ATP) channel inhibitors before sustained ischemia resulted in inhibition of these protective effects of IP on cardiac hypertrophy. CONCLUSION: The above results, therefore, suggest to us that IP by activation of K(ATP) channels can afford protection against the ischemia-reperfusion injury in the hypertrophied heart.     

Redox signaling triggers protection during the reperfusion rather than the ischemic phase of preconditioning

In ischemic preconditioning (IPC) brief ischemia/reperfusion renders the heart resistant to infarction from any subsequent ischemic insult. Protection results from binding of surface receptors by ligands released during the preconditioning ischemia. The downstream pathway involves redox signaling as IPC will not protect in the presence of a free radical scavenger. To determine when in the IPC protocol the redox signaling occurs, seven groups of isolated rabbit hearts were studied. All hearts underwent 30 min of coronary branch occlusion and 2 h of reperfusion. IPC groups were subjected to 5 min of regional ischemia followed by 10 min of reperfusion prior to the 30-min coronary occlusion. The Control group had only the 30-min occlusion and 2-h reperfusion. In the second group IPC preceded the index coronary occlusion. The third group was also preconditioned, but the free radical scavenger N-2-mercaptopropionyl glycine (MPG 300 microM) was infused during the 10-min reperfusion and therefore was present in the myocardium in the distribution of the snared coronary artery during the entire reperfusion phase and also during the subsequent 30-min ischemia. In another preconditioned group MPG was added to the perfusate before the preconditioning ischemia and therefore was present in the tissue only during the preconditioning ischemia and then was washed out during reperfusion. In the fifth group MPG was added to the perfusate for only the last 5 min of the preconditioning reperfusion and therefore was present in the tissue during the last minutes of the reperfusion phase and the 30 min of ischemia. In an additional group of IPC hearts MPG was infused for only the initial 5 min of the preconditioning reperfusion and then allowed to wash out so that the scavenger was present for only the first half of the reperfusion phase. Infarct and risk zone sizes were measured by triphenyltetrazolium staining and fluorescent microspheres, resp. IPC reduced infarct size from 31.3 +/- 2.7% of the ischemic zone in control hearts to only 8.4 +/- 1.9%. MPG completely blocked IPC's protection in the third (39.4 +/- 2.8%) and sixth (36.1 +/- 7.7%) groups but did not affect its protection in groups 4 (8.1 +/- 1.5%) or 5 (7.8 +/- 1.1%). When deoxygenated buffer was used during IPC's reperfusion phase in the seventh group of hearts, protection was lost and infarct size was increased over that seen in control hearts (74.5 +/- 9.0%). Hence redox signaling occurs during the reperfusion phase of IPC, and the critical component in that reperfusion phase appears to be molecular oxygen.     

Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium

We have previously shown that a brief episode of ischemia slows the rate of ATP depletion during subsequent ischemic episodes. Additionally, intermittent reperfusion may be beneficial to the myocardium by washing out catabolites that have accumulated during ischemia. Thus, we proposed that multiple brief ischemic episodes might actually protect the heart from a subsequent sustained ischemic insult. To test this hypothesis, two sets of experiments were performed. In the first set, one group of dogs (n = 7) was preconditioned with four 5 min circumflex occlusions, each separated by 5 min of reperfusion, followed by a sustained 40 min occlusion. The control group (n = 5) received a single 40 min occlusion. In the second study, an identical preconditioning protocol was followed, and animals (n = 9) then received a sustained 3 hr occlusion. Control animals (n = 7) received a single 3 hr occlusion. Animals were allowed 4 days of reperfusion thereafter. Histologic infarct size then was measured and was related to the major baseline predictors of infarct size, including the anatomic area at risk and collateral blood flow. In the 40 min study, preconditioning with ischemia paradoxically limited infarct size to 25% of that seen in the control group (p less than .001). Collateral blood flows were not significantly different in the two groups. In the 3 hr study, there was no difference between infarct size in the preconditioned and control groups. The protective effect of preconditioning in the 40 min study may have been due to reduced ATP depletion and/or to reduced catabolite accumulation during the sustained occlusion. These results suggest that the multiple anginal episodes that often precede myocardial infarction in man may delay cell death after coronary occlusion, and thereby allow for greater salvage of myocardium through reperfusion therapy.     

Multiple, brief coronary occlusions during early reperfusion protect rabbit hearts by targeting cell signaling pathways.

OBJECTIVES: An in situ model was used to test whether and how multiple occlusions at reperfusion can protect rabbit myocardium. BACKGROUND: Recently it was demonstrated that postconditioning in dogs salvaged ischemic myocardium. METHODS: Control hearts underwent 30-min regional ischemia/3-h reperfusion, whereas in experimental hearts four postconditioning cycles of 30-s occlusion/30-s reperfusion starting 30 s after release of the index coronary occlusion were added in the presence or absence of various cell signaling antagonists. RESULTS: Postconditioning decreased infarction from 35.4 +/- 2.7% of the risk zone in control hearts to 19.8 +/- 1.8% (p < 0.05). Six cycles did not result in greater protection. If postconditioning cycles were begun after 10 min of reperfusion, protection was no longer evident. Either the non-selective K(ATP) channel closer glibenclamide or the putatively selective mitochondrial K(ATP) channel antagonist 5-hydroxydecanoate administered 5 min before reperfusion blocked the protection afforded by postconditioning, indicating involvement of the mitochondrial K(ATP) channel. PD98059, a mitogen-activated protein/extracellular-signal regulated kinase (MEK) 1/2 and therefore extracellular-signal regulated kinase (ERK) inhibitor, and N(omega)-nitro-L-arginine methyl ester, an antagonist of nitric oxide synthase, infused shortly before reperfusion also aborted the protection afforded by postconditioning. Combined ischemic postconditioning and preconditioning resulted in significantly greater protection than either alone. CONCLUSIONS: Multiple, short, regional coronary occlusions immediately after prolonged myocardial ischemia are an effective cardioprotective intervention in the rabbit, and the mechanism of protection involves activation of ERK, production of nitric oxide, and opening of mitochondrial K(ATP) channels. These observations suggest that a similar approach could be applied in the cardiac catheterization laboratory to protect reperfused myocardium after primary angioplasty in patients with acute myocardial infarction.     

Difference in the cardioprotective mechanisms between ischemic preconditioning and pharmacological preconditioning by diazoxide in rat hearts.

BACKGROUND: Recent studies have implicated the opening of mitochondrial K(ATP) (mitoK(ATP)) channels and the production of reactive oxygen species (ROS) in the cardioprotective mechanism of ischemic preconditioning (IPC). METHODS AND RESULTS: The involvement of mitoK(ATP) channels and ROS in the cardioprotective effects of both IPC and the mitoK(ATP) channel opener diazoxide (DZ) was investigated in ischemic/reperfused rat hearts. The effects of IPC and DZ on myocardial high-energy phosphate concentrations and intracellular pH (pH(i)) were also examined using (31)P nuclear magnetic resonance spectroscopy. Although both the mitoK(ATP) channel inhibitor 5-hydroxydecanoate and the antioxidant N-acetylcysteine abolished the postischemic recovery of contractile function by DZ, neither of them inhibited that by IPC. IPC attenuated the decline in pHi during ischemia, but DZ did not (6.28+/-0.04 in IPC, p<0.05, and 6.02+/-0.05 in DZ vs 6.02 +/-0.06 in control hearts). DZ, but not IPC, reduced the decrease in ATP levels during ischemia (ATP levels at 20-min ischemia: 26.3+/-3.4% of initial value in DZ, p<0.05, and 8.1+/-3.0% in IPC vs 15.1+/-1.3% in control hearts). CONCLUSIONS: These results suggest that DZ-induced cardioprotection is related to ROS production and reduced ATP degradation during ischemia, whereas attenuated acidification during ischemia is involved in IPC-induced cardioprotection, which is not mediated through mitoK(ATP) channel opening or ROS production.     

Preconditioning protects endothelium by preventing ET-1-induced activation of NADPH oxidase and xanthine oxidase in post-ischemic heart

The hypothesis was tested that endothelin-1 (ET-1)-induced superoxide (O(2)(-)) generation mediates post-ischemic coronary endothelial injury, that ischemic preconditioning (IPC) affords endothelial protection by preventing post-ischemic ET-1, and thus O(2)(-), generation, and that opening of the mitochondrial ATP-dependent potassium channel (mK(ATP)) triggers the mechanism of IPC. Furthermore, the study was aimed at identifying the source of O(2)(-) mediating the endothelial injury. Langendorff-perfused guinea-pig hearts were subjected either to 30 min ischemia/35 min reperfusion (IR) or were preconditioned prior to IR with three cycles of either 5 min ischemia/5 min reperfusion or 5 min infusion/5 min washout of mK(ATP) opener diazoxide (0.5 mM). Coronary flow responses to acetylcholine (ACh) served as a measure of endothelium-dependent vascular function. Myocardial outflow of ET-1 and O(2)(-) and functional recoveries were followed during reperfusion. NADPH oxidase and xanthine oxidase (XO) activities were measured in cardiac homogenates. IR augmented ET-1 and O(2)(-) outflow and impaired ACh response. All these effects were attenuated or prevented by IPC and diazoxide, and 5-hydroxydecanoate (a selective mK(ATP) blocker) abolished the effects of IPC and diazoxide. Superoxide dismutase and tezosentan (a mixed ET-1-receptor antagonist) mimicked the effects of IPC, although they had no effect on the ET-1 generation. IR augmented also the activity of NADPH oxidase and XO. Apocynin treatment, that resulted in NADPH oxidase inhibition, prevented XO activation and O(2)(-) generation in IR hearts. The inhibition of XO, either by allopurinol or feeding the animals with tungsten-enriched chow, prevented post-ischemic O(2)(-) generation, although these interventions had no effect on the NADPH activity. In addition, the post-ischemic activation of NADPH oxidase and XO, and O(2)(-) generation were prevented by IPC, tezosentan, thenoyltrifluoroacetone (mitochondrial complex II inhibitor), and tempol (cell-membrane permeable O(2)(-) scavenger). In guinea-pig heart: (i) ET-1-induced O(2)(-) generation mediates post-ischemic endothelial dysfunction; (ii) IPC and diazoxide afford endothelial protection by attenuating the ET-1, and thus O(2)(-) generation, and the mK(ATP) opening triggers the protection; (iii) the NADPH oxidase maintains the activity of XO, and the XO-derived O(2)(-) mediates the endothelial injury, and (iv) ET-1 and O(2)(-) (probably of mitochondrial origin) are upstream activators of the NADPH oxidase-XO cascade, and IPC prevents the cascade activation and the endothelial dysfunction by preventing the ET-1 generation.