【作者】 张浩；

【导师】 张翼； 周兆年；

【作者基本信息】 河北医科大学 ， 生理学， 2009， 博士

【摘要】 大量研究表明,慢性间歇性低压低氧(chronic intermittent hypobaric hypoxia, CIHH)对成年动物具有明显的心脏保护作用,可有效对抗急性缺血/再灌注或急性缺氧/复氧对心肌的损伤、减少心肌梗死面积、限制心肌超微结构的损伤,以及抗心律失常。其机制可能涉及心肌抗氧化能力增强、心肌毛细血管密度及冠脉血流量增加、热休克蛋白表达增加、NO产生增加、抗心肌细胞凋亡、稳定线粒体及钙处理功能、延长心肌动作电位时程及有效不应期等。但有关CIHH对幼年动物发育心脏的作用尚未明了。本研究旨在应用功能学及电生理学方法探讨CIHH对幼年发育大鼠的抗心律失常作用,及其电生理学机制。本研究分为四部分:(1)应用模拟高原低氧处理方法制备发育大鼠间歇性低氧心脏保护动物模型;(2)应用在体冠脉结扎方法诱发心律失常,观察CIHH对发育大鼠的抗心律失常作用;(3)应用细胞内记录方法观察CIHH对发育大鼠心室乳头肌动作电位的影响,探讨CIHH抗心律失常作用的电生理学机制;(4)应用全细胞膜片钳方法观察CIHH对发育大鼠心室肌细胞钾离子通道电流的影响,探讨CIHH抗心律失常的离子机制。Ⅰ发育大鼠慢性间歇性低压低氧心脏保护模型的制备目的:通过应用不同间歇性低氧方式,制备发育大鼠CIHH心脏保护模型。方法:雄性新生SD大鼠按年龄及体重相匹配分成三组:间歇性低压低氧3000米组(CIHH3000)、间歇性低压低氧5000米组(CIHH5000)和对照组(CON)。间歇性低氧处理动物置于低压氧舱,分别接受28天、42天和56天模拟高原3000米(PB = 525 mmHg, PO2 = 108.8 mmHg, 5小时/天)或5000米(PB = 404 mmHg, PO2 = 84 mmHg, 6小时/天)的减压低氧处理。应用langendorff离体心脏灌流技术描记大鼠离体心脏左室功能,给予30分钟全心缺血和60分钟复灌处理。分别记录大鼠心脏缺血前及复灌后不同时间的左室发展压(LVDP)、左室舒张末压(LVEDP)、最大压力变化速率(±LV dp/dt)等心功能参数和冠脉流量(CF)。并测定冠脉流出液的乳酸脱氢酶(LDH)活性。实验结束后,分别测量全心、左心室及右心室重量。结果:(1) CIHH3000组大鼠体重增长与对照组无明显差异;CIHH5000组大鼠体重增长明显慢于对照组及CIHH3000组大鼠(P < 0.01)。(2)对照组28天、42天及56天的大鼠在基础状态及缺血/复灌后心功能分别相比无显著差异(P > 0.05)。(3) CIHH3000组大鼠表现明显的心脏保护效应。与对照组相比较,基础状态下灌脉流量明显增加,在心脏停灌/再灌注60 min时,心功能恢复增强(P < 0.05)、冠状动脉流量增多(P < 0.05)、LDH活性降低(P < 0.05);心脏重量与对照组大鼠无差异;CIHH 42天处理的大鼠心功能恢复明显好于CIHH 28天处理的大鼠(P < 0.05)。(4) CIHH5000组大鼠表现出明显的心脏损伤效应,与对照组相比,心功能的恢复降低(P < 0.05),冠脉流量减少(P < 0.05),复灌过程中LDH活性明显升高(P < 0.05)。右心室重量明显高于对照组大鼠(P < 0.05)。小结:适度的CIHH可保护发育大鼠心脏对抗缺血/再灌注损伤,其保护作用与冠脉流量增加有关;间歇性低氧方式影响其心脏保护作用。Ⅱ慢性间歇性低压低氧对发育大鼠缺血性心律失常的影响目的:利用在体结扎冠脉诱发心律失常的方法,研究CIHH对发育大鼠的抗缺血性心律失常作用。方法:雄性新生SD大鼠,根据年龄及体重相匹配分成四组:28天间歇性低压低氧组(CIHH28)、42天间歇性低压低氧组(CIHH42)和28天对照组(CON28)、42天对照组(CON42)。CIHH动物置于低压氧舱,分别接受28天和42天模拟高原3000米(PB = 525 mmHg, PO2 = 108.8 mmHg, 5小时/天)的减压低氧处理。动物麻醉下,描记动脉血压和心电图。通过在体结扎冠状动脉左前降支造成急性心肌缺血,观察室性早搏(VE)、室性心动过速(VT)及心室纤颤(VF)的发生情况。心律失常评分(arrhythmia score, AC)根据Johnston标准。结果:(1)冠脉阻断后引起的心律失常主要集中在3~8分钟。CON28组与CIHH28组动物相比较,AC无明显差别,分别为0.25±0.11和0.20±0.15(P > 0.05);(2) CON42组与CIHH42组大鼠的AC明显不同。CON42组的AC(2.13±0.74)明显高于CIHH42组(0.38±0.24)大鼠(P < 0.05)。(3)经CIHH处理后,CIHH28组与CIHH42组动物的AC无明显差别,分别为0.20±0.15, 0.38±0.24(P > 0.05)。小结:本研究结果显示CIHH对发育大鼠具有明显的抗心律失常作用,其作用与动物年龄密切相关。发育大鼠缺血性心律失常的发生在一定范围随年龄而增加。Ⅲ慢性间歇性低压低氧对发育大鼠心室乳头肌动作电位的影响目的:利用CIHH动物模型和记录乳头肌动作电位(AP)的方法,研究CIHH对发育大鼠乳头肌AP的影响。方法:雄性新生SD大鼠,按年龄及体重相匹配分成四组:28天间歇性低压低氧组(CIHH28)、42天间歇性低压低氧组(CIHH42)和28天对照组(CON28)、42天对照组(CON42)。CIHH动物置于低压氧舱,分别接受28天和42天模拟高原3000米(PB = 525 mmHg, PO2 = 108.8 mmHg, 5小时/天)的减压低氧处理。以台氏液及模拟缺血液灌流左室乳头肌标本,利用细胞内微电极技术描记动物乳头肌AP以及缺血前后AP的变化。结果:(1)在台氏液灌流的基础状态下,CON动物与CIHH动物相比,AP各参数无明显差异(P > 0.05)。(2)以模拟缺血液灌流乳头肌标本5分钟后, CON42组与CIHH42组动物乳头肌AP的APA、OS、Vmax及APD90均明显降低(P < 0.05)。但CIHH42组AP的APD90的缩短程度明显小于CON42组(P < 0.05);模拟缺血液灌流时,CON28与CIHH28组动物APD缩短无明显差别(P > 0.05)。(3)在台氏液灌流的基础状态下,CON28组与CON42组大鼠AP相比较无明显差异(P > 0.05)。以模拟缺血液灌流乳头肌标本5分钟后,CON42组大鼠APD90的缩短程度明显大于CON28组大鼠(P < 0.05)。(4)在台氏液灌流的基础状态下,CIHH28组与CIHH42组大鼠AP相比较无明显差异(P > 0.05)。以模拟缺血液灌流乳头肌标本5分钟后,两组大鼠AP缩短的程度相比无明显差异(P > 0.05)。小结:我们的研究显示, CIHH处理不影响基础状态下发育大鼠乳头肌的AP。但随年龄增长,CIHH可显著延缓APD90在缺血时的缩短,稳定膜的复极化。Ⅳ慢性间歇性低压低氧对发育大鼠心室肌细胞钾通道的影响目的:通过全细胞膜片钳方法观察CIHH对发育大鼠心室肌细胞钾离子通道电流的影响,探讨CIHH抗心律失常的离子机制。方法:雄性新生SD大鼠,按年龄及体重相匹配分成四组:28天间歇性低压低氧组(CIHH28)、42天间歇性低压低氧组(CIHH42)和28天对照组(CON28)、42天对照组(CON42)。CIHH动物置于低压氧舱,分别接受28天和42天模拟高原3000米(PB = 525 mmHg, PO2 = 108.8 mmHg, 5小时/天)的减压低氧处理。通过酶解法得到单个心肌细胞,利用全细胞膜片钳技术记录瞬时外向钾电流(Ito),延迟整流钾电流(IK)和内向整流钾电流,观察CON和CIHH大鼠心肌细胞在模拟缺血液灌流前后上述钾通道电流的变化。结果:(1) CON28组与CIHH28组大鼠心室肌细胞IK密度在模拟缺血液前后无明显变化(P > 0.05); CON42组与CIHH42组大鼠心室肌细胞IK密度在模拟缺血前相比无差异(P > 0.05),但在模拟缺血灌流5分钟后,CON42组大鼠心室肌细胞IK密度较CIHH42组明显增大(P < 0.05)。(2) CON28组与CIHH28组大鼠心室肌细胞IK1密度在模拟缺血液前后无明显变化(P > 0.05);CON42组与CIHH42组大鼠心室肌细胞IK1密度在模拟缺血前相比无差异(P > 0.05),但在模拟缺血灌流5分钟后,CON42组心室肌细胞IK1密度较CIHH42组明显增大(P < 0.05)。(3) CON与CIHH大鼠心室肌细胞Ito密度在模拟缺血前后相比无明显差别(P > 0.05)。在模拟缺血时,心室肌细胞Ito密度降低,稳态激活和失活曲线向负电压方向移位而且复活时间常数延长(P < 0.05)。小结:研究结果显示CIHH对生后发育大鼠心脏复极化电流产生复杂的影响。CIHH不影响基础状态下的IK、IK1和Ito,但CIHH可显著减轻IK,IK1在模拟缺血状态下的增强。更多还原

【Abstract】 A lot of researches have demonstrated that chronic intermittent hypobaric hypoxia (CIHH) confers significant cardioprotetive effects on adult heart, such as enchancing the resistance of heart against acute hypoxia/re-oxygen or acute ischemia/reperfusion injury, reducing infarct size, limiting cardiac ultrastruc- ture damage and anti-arrhythmia. The candidate mechanisms involved enhancement of antioxidation, increase of myocardium capillary desity and coronary flow, overexpression of heat shock protein, increase of NO production, anti-apoptosis, stabilization of mitochondria and function of handling calcium, prolongation of action potential duration and effective refractory period. However, it was not clear about the role of CIHH on postnatal developing hearts. The objective of the study was to explore the anti-arrhythmic effects of CIHH and underling electrophysiological mechanism on postnatal developing rat using functional and electro- physiological methods。Our study consists of four parts: (1) To make an animal model of CIHH cardioprotection through exposing the developing rat to simulated high altitude hypoxia. (2) To examine anti-arrhythmic effects of CIHH on developing rat via analysis of arrhythmia induced by ligation of coronary artery in vivo. (3) To examine effects of CIHH on action potential in papillary muscle from developing rat through intracellular recording, and to explore electrophysiological mechanism of CIHH against arrhythmias. (4) To examine of effects of CIHH on potassium currents in ventricular myocytes of developing rat by using whole-cell patch clamp techenique, and to explore the ionic mechanism of CIHH against arrhythmias. I Preparation of animal mode for cardioprotection of CIHH in developing ratObjective: to make animal model for cardioprotection of CIHH in developing rat by using different modes of CIHH.Methods: Age- and body weight- matched postnatal male rats were divided into three groups: 3000m chronic intermittent hypoxia (CIHH3000) group, 5000m chronic intermittent hypoxia (CIHH5000) group and control (CON) group. For CIHH groups, neonatal rats with the maternals were exposed to a hypobaric chamber, enduring CIHH mimicking 3000 m altitude (PB = 525 mmHg, PO2 = 108.8 mmHg, 5 hrs/day) or 5000 m altitude (PB = 404 mmHg, PO2 = 84 mmHg, 6 hrs/day) for 28, 42 and 56days, respectively. The isolated hearts were perfused in the langendorff apparatus, undergoing 30min global ischemia and 60min reperfusion. Parameters of cardiac function including left ventricular developing pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), maximal velocity of left ventricular pressure (LVdp/dtmax), coronary flow (CF) and lactate dehydrogenase (LDH) activity were recorded respectively. The total heart weight, right ventricular weight and left ventricular plus inter-ventricular septum weight were measured at the end of the experiment.Results:(1) There was no difference of body weight gaining between CIHH3000 and CON rats. The gain of body weight in CIHH5000 rats was much lower than that in CIHH3000 and CON (P < 0.01).(2) There were no significant differences of cardiac function among 28-day, 42-day and 56-day of control rats under basic condition and during ischemia/reperfusion.(3) Compared with CON, CF in CIHH rats was increased under basic condition, the recovery of cardiac function in CIHH3000 rats was enhanced at 60 min after ischemia/reperfusion (P < 0.05), coronary flow was increased (P < 0.05), and LDH activity was decreased (P < 0.05), which means a cardioprotective effect occurred. There was no significant difference of heart weight between CIHH3000 and CON. In addition, cardiac function restored better in CIHH3000 rat after 42 days CIHH than that after 28 days CIHH (P < 0.05).(4) Compared with CON, the recovery of cardiac function in CIHH5000 rats was lower (P < 0.05), coronary flow was decreased (P < 0.05), and LDH activity was increased (P < 0.05). Right ventricular weight was higher in CIHH5000 than that in CON rats (P < 0.05).Conclusion: The result of the study suggests that moderate CIHH can protect developing rat heart against ischemia/reperfusion injury, which was affected by mode of CIHH exposure and related with CF increase.ⅡEffects of CIHH on ischemic arrhythmia in developing ratsObjective: To examine anti-arrhythmic effects of CIHH on developing rat via analysis of arrhythmia induced by ligation of coronary artery in vivo.Methods: In this part of experiment, age- and body weight-matched postnatal male rats were divided into four groups: 28-day CIHH group (CIHH28), 42-day CIHH group (CIHH42), 28-day control group (CON28) and 42-day control group (CON42). For CIHH group, neonatal rats with the maternals were exposed to a hypobaric chamber, enduring CIHH mimicking 3000 m altitude (PB = 525 mmHg, PO2 = 108.8 mmHg, 5 hrs/day) for 28days and 42days, respectively. Arterial blood pressure and electrocardiogram (ECG) were recorded under anesthetized state. Acute myocardial ischemia was produced by ligating left anterior descending coronary artery in anesthetized postnatal male rats. Ventricular extrasystole (VE), tachycardia (VT) and fibrillation (VF) were analyzed from ECG. Arrhythmia scores (AC) were used according to Johnston’s Standards.Results:(1) The arrhythmia was mainly occurred between 3 and 8 min after coronary artery occlusion. There was no significant difference in ACs between CON28 (0.25±0.11) and CIHH28 (0.20±0.15) rats (P > 0.05).(2) There was a significant difference of AC between CON42 and CIHH42 rats. The AC was 2.13±0.74 in CON42 rats, much higher than 0.38±0.24 in CIHH42 rats (P < 0.05).(3) In CIHH group, there was no significant difference of AC between CIHH 28 (0.20±0.15) and CIHH42 (0.38±0.24) rats (P > 0.05).Conclusion: The result of the study demonstrates that CIHH has a significant anti-arrhythmic effect in developing rat, which is closely related with animals’age. The incidence of ischemic arrhythmia in developing rat increases along with rat growing in a certain age range.ⅢEffects of CIHH on action potential in ventricular papillary muscle of developing ratObjective: to investigate the effects of CIHH on action potential (AP) in ventricular papillary muscle of developing rats by using CIHH animal model and intracelluar recording method. Methods: In this part of experiment, age- and body weight-matched postnatal male rats were divided into four groups: 28-day CIHH group (CIHH28), 42-day CIHH group (CIHH42), 28-day control group (CON28) and 42-day control group (CON42). For CIHH group, neonatal rats with the maternals were exposed to a hypobaric chamber, enduring CIHH mimicking 3000 m altitude (PB = 525 mmHg, PO2 = 108.8 mmHg, 5 hrs/day) for 28days, 42days, respectively. The left vetricullar papillary muslces were perfused with Tyrodes solution and simulated ischemic solution. AP in papillary muscle was recorded before and during simulated ischemia by using intracellular microelectrode technique.Results:(1) There was no difference in any parameters of basic AP between CON and CIHH rats during Tyrodes solution perfusion (P > 0.05).(2) APA, OS, Vmax and APD90 of AP in papillary muscle were decreased significantly (P < 0.05) after 5minutes of simulated ischemia solution perfusion, but the decreasing of AP90 in CIHH42 rats was much smaller than than in CON42 rats (P < 0.05). There was no significant difference of AP decreasing during simulated ischemia between CON28 and CIHH28 rats (P > 0.05).(3) In CON groups, there was no difference of AP between CON28 and CON42 rats during Tyrodes solution perfusion (P > 0.05). The decreasing of APD90 of AP after 5minutes of simulated ischemia solution perfusion was more severe in CON 42 rats than that in CON28 rats (P < 0.05).(4) In CIHH groups, there was no difference of AP between CIHH28 and CIHH42 rats during Tyrodes solution perfusion (P > 0.05). Also there was no significant difference of AP decreasing after 5minutes of simulated ischemia solution perfusion between CIHH28 and CIHH42 rats (P > 0.05).Conclusion: The result of the study shows that CIHH does not affect basic AP in papillary muscle of developing rats. CIHH, however, can significantly alleviate the decreasing of APD90 induced by simulated ischemia, stabilizing the membrane repolarization during ischemia.ⅣEffect of CIHH on potassium currents in ventricular myocytes of developing ratObjective: to explore the effect of CIHH on cardiac potassium currents in developing rat heart by using whole-cell patch clamp technique, and the ionic mechanism of CIHH against arrhythmias.Methods: In this part of experiment, age- and body weight-matched postnatal male rats were divided into four groups: 28-day CIHH group (CIHH28), 42-day CIHH group (CIHH42), 28-day control group (CON28) and 42-day control group (CON42). For CIHH group, neonatal rats with the maternals were exposed to a hypobaric chamber, enduring CIHH mimicking 3000 m altitude (PB = 525 mmHg, PO2 = 108.8 mmHg, 5 hrs/day) for 28days, 42days, respectively. Isolated myocytes were obtained from ventricles by acute enzymic digestion method. Transient ourward potassium current (Ito), delayed rectifier potassium current (IK), inwardly rectifier potassium (IK1) and ATP-sensitive potassium current (IKATP) from myocytes were recorded before and during simulated ischemic solution perfusion. Results:(1) There was no difference of IK density between CON28 and CIHH28 rats before and during simulated ischemia. There was no difference of IK density between CON42 and CIHH42 rats before simulated ischemia, but IK density was increased significantly after 5minutes of simulated ischemia in CON42 rats compared with CIHH42 rats (P < 0.05).(2) There was no difference of IK1 density between CON28 and CIHH28 rats before and during simulated ischemia. There was no difference of IK1 density between CON42 and CIHH42 rats before simulated ischemia, but IK1 density was increased significantly after 5minutes of simulated ischemia in CON42 rats compared with CIHH42 rats (P < 0.05).(3) There was no difference of Ito density between CON and CIHH groups before or during simulated ischemia (P > 0.05). During simulated ischemia, Ito density was decreased, steady-state activation and inactivation curves of Ito were shifted negatively, and the time constant of recovery was prolonged (P < 0.05).Conclusion: The result of the study shows that CIHH has complicated effects on repolarization currents in cardiomyocytes of developing rats. CIHH has no effects on the basic IK, IK1 and Ito, but can significantly alliviate the increase of IK, IK1 during simulated ischemia.更多还原