【作者】 马会杰；

【导师】 张翼；

【作者基本信息】 河北医科大学 ， 生理学， 2010， 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。有研究报道,CIHH对成年大鼠的心脏缺血/再灌注损伤作用与抗钙超载、保持钙稳态有关。但有关CIHH对幼年动物发育心脏钙稳态的作用尚未明了。本研究旨在应用功能学、细胞学、分子生物学及电生理学方法探讨CIHH对幼年发育大鼠钙稳态的作用及其细胞和分子机制。本研究分为四部分:(1)应用Langendorff灌流技术观察CIHH对发育大鼠心脏功能学及梗死面积的保护作用;(2)应用激光共聚焦显微荧光技术,观察CIHH对发育大鼠的心室肌细胞内游离钙浓度的影响;(3)应用分子生物学技术观察CIHH对钙处理蛋白表达的影响;(4)应用全细胞膜片钳方法观察CIHH对发育大鼠心室肌细胞钙离子通道电流和钠钙交换电流的影响,探讨CIHH对钙稳态作用的离子机制。Ⅰ慢性间歇性低压低氧对发育大鼠心脏的保护作用目的:应用Langendorff灌流系统观察CIHH对发育大鼠缺血/再灌注损伤的心脏功能学及梗死面积的影响。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)。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)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)。实验结束后,测定测定其梗死面积。结果:1基础状态下,CIHH大鼠冠脉流量较对照大鼠明显增多,其余心功能参数及梗死面积与对照大鼠无差异。缺血/再灌注导致动物心功能受损、心肌梗死面积增大,但CON与CIHH动物间具有明显差异。CIHH大鼠缺血/再灌注后心脏功能的恢复明显好于对照动物,表现为左心室发展压(LVDP)和左心室最大压力变化速率(±LVdp/dtmax)恢复增加(P<0.05),左心室舒张末压(LVEDP)恢复降低(P<0.05),梗死面积减小(P<0.05)。CIHH42大鼠抗心脏缺血/再灌注损伤作用强于CIHH28大鼠。2 L型钙通道开放剂,Bay K8644 (0.5μM)和肌浆网内钙释放开放剂,ryanodine (10 nM)可取消CIHH的心脏保护作用(P<0.05)。小结: CIHH可增强发育大鼠抗心肌缺血/再灌注损伤的能力,具有明显的心脏保护作用。其保护作用与心肌细胞钙稳态的维持有关,增加外钙内流和内钙释放均可取消其保护作用。Ⅱ慢性间歇性低压低氧对发育大鼠心室肌细胞内游离钙的影响目的:观察CIHH对发育大鼠心室肌细胞内游离钙的影响并探讨其可能机制。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。通过酶解法得到单个心室肌细胞,并用Fluo3-AM于37°C负载1小时。在缺血前、模拟缺血以及复灌过程中用激光共聚焦显微镜扫描记录荧光强度及图像。结果:1正常台氏液中,CON和CIHH组心室肌细胞内游离钙无显著差异(P > 0.05),在5min模拟缺血和10min再灌注后,CON组心室肌细胞内游离钙浓度增加,而在CIHH28和CIHH42组减弱心室肌细胞内游离钙浓度增加幅度减少,与相应CON有显著差异(P<0.05)。2 CIHH28和CIHH42的这一效应可被L型钙通道开放剂Bay K8644,肌浆网钙泵抑制剂thapsigargin和PKC抑制剂chelerythrine chloride取消。小结: CIHH可抑制发育大鼠心室肌细胞内游离钙浓度的增加,这一效应可被Bay K8644, thapsigargin和chelerythrine chloride取消,提示CIHH的心脏保护效应可能与抗钙超载和PKC激活有关。Ⅲ慢性间歇性低压低氧对发育大鼠钙处理蛋白表达的影响目的:用Western blot方法研究CIHH对发育大鼠钙处理蛋白表达的影响。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。组织匀浆后提取肌浆网和肌膜蛋白。用Western blot方法检测I/R时钠钙交换体(NCX1),Ryanodine受体(RyR2)和肌浆网钙泵(SERCA2)蛋白的表达。结果:基础状态下,各组大鼠心肌NCX1, RyR2和SERCA2蛋白表达无明显差异(P>0.05)。在模拟缺血条件下,CON28和CON42大鼠心肌NCX1, RyR2和SERCA2蛋白表达明显减少(P<0.05),而CIHH28和CIHH42大鼠心肌的NCX1, RyR2和SERCA2蛋白表达无明显变化(P>0.05)。小结: CIHH可对抗缺血/再灌注对发育大鼠心肌NCX1、RyR2和SERCA2蛋白表达的抑制,而发挥抗缺血/再灌注钙超载作用。IV慢性间歇性低压低氧对发育大鼠L型钙电流及钠钙交换电流的影响目的:用全细胞膜片钳技术研究CIHH对发育大鼠L型钙电流(ICaL)及钠钙交换电流(INa/Ca)的影响,探讨CIHH心脏保护及抗钙超载的离子机制。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ICaL及INa/Ca,观察CIHH对大鼠心肌细胞ICaL和INa/Ca的影响。结果:1 ICaL1.1在正常细胞外液中,对照组和间歇性低氧组的ICaL峰值电流和I-V曲线无显著差异(P>0.05)。模拟缺血和再灌注时,CON28和CON42组心肌细胞的ICaL峰值电流明显减少(P<0.05),I-V曲线向上移位,而CIHH28和CIHH42组心肌细胞峰值ICaL电流和I-V曲线无明显变化(P>0.05)。1.2各组心肌细胞ICaL的稳态激活、失活后复活曲线在正常细胞外液、模拟缺血和再灌注条件下均无明显变化(P>0.05)。在正常细胞外液中,各组心肌细胞ICaL的稳态失活曲线无明显变化(P>0.05)。模拟缺血和再灌注时,CON组心肌细胞ICaL的稳态失活曲线向左移位(P<0.05),而CIHH组心肌细胞ICaL的稳态失活曲线无显著变化(P>0.05)。2 INa/Ca2.1正常细胞外液中,各组心肌细胞的INa/Ca无显著差异(P>0.05)。在模拟缺血条件下,CON28和CON42组心肌细胞INa/Ca的内向电流和外向电流部分均减少(P<0.05),内向电流的抑制比外向电流更明显。而CIHH28和CIHH42组心肌细胞INa/Ca无显著变化(P>0.05)。2.2模拟缺血条件下,CON28和CON42组心室肌细胞INa/Ca反转电位向负值方向移位(P<0.05),而CIHH28和CIHH42组心室肌细胞INa/Ca反转电位无显著变化(P>0.05)。小结: CIHH对基础状态的发育大鼠心室肌细胞ICaL和INa/Ca无影响,但可明显对抗缺血/再灌注对ICaL和INa/Ca的抑制作用。此作用可能是CIHH抗心肌缺血/再灌注损伤、对抗钙超载的离子机制之一。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 (I/R) injury, reducing infarct size, limiting cardiac ultrastructure 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. It was reported that the cardioprotective effect of CIHH against I/R was related to anti-calcium overload and preservation of calcium homeostasis. However, it was not clear about the role of CIHH on calcium homeostasis and calcium overload in postnatal developing hearts. The objective of the study was to explore the effects of CIHH on calcium homeostasis and underling cellular and molecular mechanisms on postnatal developing rat using functional, cellular, molecular biology and electrophysiological methods.Our study consists of four parts: (1) To observe cardioprotective effects of CIHH on cardiac function and infart size using Langendorff perfusion technique. (2) To examine effects of CIHH on intracellular free calcium concentration in developing rat ventricular myocytes using laser confocal microscope system. (3) To investigate the effect of chronic intermittent hypobaric hypoxia on the expression of calcium handling protein in developing rats using molecular biology technique. (4) To examine of effects of CIHH on calcium currents and Na+/Ca2+ exchange currents in ventricular myocytes of developing rat by using whole-cell patch clamp techenique, and to explore the ionic mechanism of CIHH cardiac protection against IR- induced injury and calcium overload.I The cardiac protective effect of chronic intermittent hypobaric hypoxia on developing rat heartsObjective: to observe the effect of CIHH on cardiac function and infart size during ischemia/reperfusion in developing rat hearts.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. The isolated hearts were perfused in the langendorff apparatus, undergoing 30min global ischemia and 60min reperfusion(I/R). 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) were recorded, respectively. The infart size was measured at the end of the experiment.Results:1 Under the basic condition,the coronary flow (CF) in CIHH rats was significant higher than that in CON rats(P>0.05), while other parameters of cardiac function and infart size were not changed significantly(P<0.05). The recovery of cardiac function from IR in CIHH rats was much better than that in CON rats, including the increase of LVDP and±LVdP/dt, the decrease of LVEDP (P<0.05). Also, the myocardial infart size of was significantly smaller in CIHH rats than that in CON rats(P<0.05).2 The cardiac protection of CIHH rats was abolished by Bay K8644 (a L-type calcium channel agonist, 0.5μM) or ryanodine (a SR ryanodine receptor agonist, 10 nM) pretreatment (P<0.05). Conclusion: CIHH has significant cardiac protection, enhancing the resistance against ishchemia/reperfusion injury in developing rat heart, which might be related with preservation of calcium hoeostasis of cardiomyocytes during I/R. The cardiac protection could be abolished by increase of calcium influx via L-type calcium channel and release from SR in cardiomyocytes.ⅡEffects of chronic intermittent hypobaric hypoxia on intracellular free calcium concentration in developing rat ventricular myocytesObjective: To observe the effects of CIHH on intracellular free calcium concentration ([Ca2+]i) in ventricular myocytes of developing rat and the possible mechanisms involved.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. Isolated ventricular myocytes were were obtained from ventricles by enzymatic dissociation method and incubated with Fluo3-AM solution at 37°C for 60 min. Fluorescence intensity was recorded and images were scanned using laser confocal microscope system during pre-ischemia, simulated ischemia and reperfusion.Results:1 There was no difference of [Ca2+]i of ventricular myocytes under normal Tyrode’s solution between CON and CIHH rats. [Ca2+]i in ventricular myocytes was increased after 5 min simulated ischemia and 10 min reperfusion, but the increasing of [Ca2+]i was blunted in CIHH28 and CIHH42 rats (P<0.05 ).2 The effect of CIHH28 and CIHH42 on [Ca2+]i in ventricular myocytes during I/R was abolished by Bay K8644 (a L type calcium channel agonist), thapsigargin (a inhibitor of Ca2+ ATPase in SR), and chelerythrine chloride (a PKC inhibitor) (P<0.05).Conclusion: CIHH can inhibite the elevation of [Ca2+]i during I/R in developing rat ventricular myocytes. The the effect of CIHH on [Ca2+]i during I/R can be abolished by Bay K8644, thapsigargin, and chelerythrine chloride, suggesting the cardioprotective effect of CIHH is related with anti-calcium overload effect and PKC activation.III Effect of chronic intermittent hypobaric hypoxia on the expression of calcium handling proteins in developing ratsObjective: To investigate the effect of CIHH on the expression of calcium handling proteins in developing rats using Western blot 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 and 42days, respectively. The heart tissues were homogenated and SR and cell sarcolemmal membrane protein were extracted. The protein expressions of ryanodine receptors (RyR2), Ca2+ ATPase in the sarcoplasmic reticulum (SERCA2) and Na+/Ca2+ exchanger (NCX1) in the sarcolemmal membrane were assessed during I/R using Western blot method.Results:The protein expression of NCX1,RyR2, and SERCA2 were decreased markedly at 30 min of ischemia (I30), 5 min (R5) and 60 min (R60) of reperfusion in CON rats (P<0.05), but the inhibition of NCX1, RyR2, and SERCA2 expression during I/R were significantly attenuated in CIHH rats compared with CON rats (P<0.05).Conclusion: CIHH can effectively antagonize the depression of the protein expression of NCX1, RyR2 and SERCA2 in myocardium during I/R, suggesting that CIHH may maintain Ca2+ homeostasis and suppress I/R-induced cytosolic Ca2+ overload by reserving the protein expression of NCX1, RyR2 and SERCA2.IV Effect of chronic intermittent hypobaric hypoxia on L-type Ca2+ currents and Na+-Ca2+ exchange currents in cardiomyocytes of developing ratObjective: To explore the effect of CIHH on L-type Ca2+ currents (ICaL) and Na+-Ca2+ exchange currents (INa/Ca) in ventricular cardiomyocyte of developing rat by using whole-cell patch clamp technique, and the ionic mechanism for CIHH cardioprotection and anti-calcium overload.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 put into a hypobaric chamber to get CIHH mimicking 3000-m altitude (PB = 525 mmHg, PO2 = 108.8 mmHg, 5 hrs/day) for 28days and 42days, respectively. Isolated myocytes were obtained from ventricles by enzymatic dissociation method. ICaL and Ni2+ sensitive INa/Ca were recorded before and during simulated ischemia as well as reperfusion in CON and CIHH groups.Results:1 ICaL1.1 There were no significant differences of peak current and current-voltage relationship curve of ICaL between CON and CIHH cardiomyocytes under normal extracullar solution (P>0.05). The peak amplitude of ICaL was decreased and I-V relationship curve was shifted upward during simulated ischemia and reperfusion in CON28 and CON42 cardiomyocytes (P<0.05). However, the peak amplitude and I–V relationship of ICaL in CIHH28 and CIHH42 cardiomyocytes was not changed significantly during simulated ischemia and reperfusion (P>0.05).1.2 There were no significant differences of steady-state activation curve and steady-state inactivation curve of ICaL under normal extracullar solution in all cardiomyocytes (P>0.05). The steady-state inactivation curve of ICaL was not changed significantly under normal extracullar solution condition in all cardiomyocytes (P>0.05), but was shifted to the left in CON28 and CON42 (P<0.05), not in CIHH28 and CIHH42 (P>0.05), cardiomyocytes during simulated ischemia and reperfusion.2 INa/Ca2.1 There was no significant difference of Ni2+ sensitive Na+-Ca2+ exchange current (INa/Ca) between all cardiomyocytes(P>0.05). During simulated ischemia and reperfusion, INa/Ca in CON28 and CON42 cardiomyocytes was decreased(P<0.05), the decreasing of inward current part in INa/Ca was more significant than that of outward current part. But INa/Ca in CIHH28 and CIHH42 cardiomyocytes was not changed signifcantly during simulated ischemia (P>0.05).2.2 The reversal potential of INa/Ca in CON28 and CON42 cardiomyocytes was shifted toward negative potential during simulated ischemia (P<0.05), but the reversal potential of INa/Ca in CIHH28 and CIHH42 cardiomyocytes was changed significantly during simulated ischemia(P>0.05).Conclusion: CIHH has no effect on ICaL and INa/Ca of ventricular myocytes in developing rat under basic condition, but effectively antagonizes the inhibition of ICaL and INa/Ca in ventricular myocytes during simulated ischemia and reperfusion, which might be one of ionic mechanism of CIHH cardiac protection against I/R injury and anti-calcium overload.更多还原