微管对心肌细胞线粒体功能及能量代谢与电生理影响研究

发布时间：2018-01-02 22:07

本文关键词：微管对心肌细胞线粒体功能及能量代谢与电生理影响研究　出处：《第三军医大学》2008年博士论文　论文类型：学位论文

【摘要】： 我们对“休克心”发生机理的长期研究中发现,在心肌缺血缺氧早期,微管即发生显著破坏,而微管在缺氧引起的一系列效应中所起的作用及对缺氧所致的心肌细胞的能量代谢障碍的影响及其发生机制目前罕见报道。作为细胞能量供应核心细胞器的线粒体,在缺氧条件下细胞状态的改变中起至关重要的作用。线粒体是心肌细胞缺氧损害的核心靶细胞器,线粒体损害是“休克心”及全身性缺氧损害的最关键环节。线粒体在细胞缺氧性损害中起重要作用,不仅因其是细胞生物反应过程重要的能量供应者,他们还能通过其膜上的通透性转换孔(mPTP)直接参与启动细胞的坏死和凋亡。我们以往的研究发现,mPTP的开放引起线粒体通透性转换(MPT),导致线粒体基质膨胀、外膜破裂,凋亡信号分子从内外膜间释放,引起细胞的坏死和凋亡;mPTP的开放导致线粒体的不可逆损伤是缺氧性心肌细胞损害的关键环节。而微管与线粒体之间有密切的联系。近年来,对细胞骨架的深入研究表明,微管除了作为胞内的刚性物质,具有锚定亚细胞结构如线粒体、高尔基体、细胞核等而对细胞起稳定性作用外,还参与调节信号转导、核转录及蛋白质合成等。细胞骨架除了对线粒体胞内定位及分布起一定作用外,还可能参与线粒体呼吸功能的调节过程。我们前期在乳鼠心肌细胞缺氧的研究中发现,缺氧条件下微管破坏可导致线粒体通透性转换孔MPTP的持续开放,进而使线粒体呼吸功能下降,提示微管对乳鼠心肌细胞线粒体具有重要的调节作用,而这一具体调控环节尚不清楚。由于VDAC是MPTP在线粒体外膜上的通道蛋白,故我们推测,微管可能是通过某种未知机制对VDAC的开放状态产生影响。通过上述途径改变,微管影响了细胞的产能,进一步使得心肌细胞的功能产生各种不同的影响。作为心肌细胞,在功能研究中,最重要的是其电生理活性,与能量代谢息息相关,破坏微管后,大鼠成体心肌细胞的电生理活性改变,罕见报道。在心脏缺氧的实验研究中,大鼠成体心肌细胞与乳鼠心肌细胞相比,存在显著的差异,由于成体心肌细胞已经分化成熟,其自我修复能力远较乳鼠细胞差,其对缺氧的代偿能力也较差。在活性及功能研究方面,成体细胞更加接近整体水平,其结果更具有说服力。对原代培养的成体心肌细胞而言,容易受外界各种环境影响,故培养的难度较大,但对缺氧及各种实验施加因素更为敏感。另外原代培养的成体心肌细胞背景更加一致,试验数据稳定、可信度高。在其结构、功能方面则区别更大,其线粒体的分布更加具有规律,各项电生理及收缩功能发育完善,有利于深入的机理研究。基于上述特点,本研究采用大鼠成体心肌细胞作为研究对象,并利用一定浓度的秋水仙碱固定时间处理细胞,模拟缺氧条件下的微管解聚状态,作为单一实验处理因素,观察大鼠成体心肌细胞的各种指标变化,以分析单纯微管解聚所引起的各种变化。本研究假设:微管破坏可能通过改变线粒体的亚细胞定位;通过某中间微管相互作用蛋白分子对VDAC进行调控,使VDAC开放增加,线粒体活性下降,两条途径加重心肌细胞能量代谢障碍,进而使其功能产生改变,细胞膜电生理活性下降。研究目的应用微管解聚剂模拟缺氧条件下微管的破坏,研究微管解聚对心肌细胞线粒体功能及能量代谢与电生理的影响,分析其可能机制,深入探讨微管解聚在缺氧过程中的作用。材料和方法 1、成年大鼠心肌细胞培养 2、利用8μM微管解聚剂秋水仙碱(colchicine)作用于大鼠成体心肌细胞,模拟缺氧引发的微管解聚状态。实验分组为正常对照组(N组)及紫杉醇微管解聚组(C组)。 3、利用免疫细胞化学染色观察N组、C组心肌细胞聚合态微管、线粒体形态及分布变化规律,Western blot法检测各组心肌细胞聚合态微管蛋白含量变化。 4、利用四甲基罗丹明乙酯(TMRE)检测线粒体内膜电位;使用免疫印记法检测胞浆中细胞色素C含量变化;运用MTT法测定细胞活性;运用乳酸测定试剂盒检测心肌细胞内乳酸浓度。 5、高效液相色谱测定心肌细胞中ATP、ADP、AMP含量。 6、利用酵母双杂交实验系统,以VDAC为诱饵在肝细胞文库中筛选可能与其有相互作用的蛋白,对实验结果进行酵母回转验证,并对筛选出的蛋白进行免疫组化细胞共定位研究;进一步实验结果进行生物信息学研究。 7、应用膜片钳技术全细胞纪录方法,纪录心肌细胞膜电容、动作电位、钠电流(INa)、钙电流(ICa),并应用Axon公司的pClamp8.1软件中的Clampit进行数据分析。主要结果 1、正常乳鼠心肌细胞微管围绕核周呈放射状排列,微管管状结构清晰。正常大鼠成体心肌细胞微管部分围绕核周排列,其他呈线性沿细胞长轴方向平行排列。C组乳鼠心肌细胞细胞微管结构遭受破坏,大鼠成体心肌细胞微管沿肌小节纵轴方向规律排列破坏,表现为免疫荧光强度减弱,微管结构的连续性丧失,变得粗糙且不光滑,微管结构不清晰,且呈特征性卷曲状结构。WB结果显示:C组心肌细胞聚合态微管蛋白含量较N组明显减少;成体心肌细胞减少程度较乳鼠有显著增加。 2、正常成体心肌细胞线粒体呈椭圆或长杆状,沿细胞长轴分布,与各肌束间呈线性均匀分布。大鼠成体心肌细胞微管呈线性管状分布,与心肌纤维方向平行,VDAC显示的线粒体呈颗粒装分布,其分布方向与微管相同,并重叠其上,提示成体心肌细胞线粒体沿微管分布。C组线粒体的分布散乱,失去规律性。 3、C组线粒体内膜电位较N组明显降低,表现为线粒体荧光强度减弱;C组心肌细胞胞浆中细胞色素C含量较正常对照明显增高。 4、微管解聚后心肌细胞与正常对照相比ATP含量下降、ADP、AMP含量上升,ADP / ATP明显升高,能荷下降;细胞活性明显降低;心肌细胞内乳酸含量下降。 5、应用酵母双杂交技术,在人肝脏文库中筛选出VDAC的可能相互作用蛋白为DYNL1、PTPRH,经酵母回转验证结果为阳性。生物信息学分析结果提示,本实验研究发现VDAC-DYNL1,VDAC-PTPRH的两对相互作用分子,目前未见报道,为新的可能存在的相互作用蛋白,DYNL1与微管有明确的相互作用。 6、与N组相比较,C组静息电位(rest potential, RP)无显著变化;而连续动作电位(action potential, AP)的形状发生显著改变,N组心肌细胞连续AP形态一致,动作电位振幅(action potential amplitude,AMP)峰值一致,复极化时动作电位持续时间(action potential duration,APD)APD时长一致,C组AP形态不稳定,AMP峰值大小不一,APD时长明显减小。微管解聚组APD20、APD50和APD90较对照组明显缩短。 7、微管解聚后INa电流显著增加;I/V曲线结果提示微管解聚组电流密度在-50～-20 mV的电压范围内均明显高于正常对照组。两组Ica电流密度一电压曲线均一致,几乎重叠,微管解聚组与对照组组间无明显差别。ICa有明显的电压依赖性,去极化电压正于一40mV时ICa被激活,去极化电压至-10mV时ICa最大。讨论与结论 1、微管与线粒体在成体心肌细胞内分布方向一致。微管解聚后心肌细胞线粒体的排列分布规律紊乱。 2、微管解聚使大鼠成体心肌细胞活性显著下降,推测为微管解聚使细胞内能量生成单元崩解,降低了心肌细胞的能量供应。 3、微管解聚使心肌细胞线粒体膜电位降低,细胞色素C漏出增加,表明微管对线粒体VDAC存在调控作用。 4、微管解聚抑制心肌细胞糖酵解。心肌细胞内糖酵解酶依附于微管,按照一定比例及次序排列,构成最佳的快速产能效应。微管解聚后,这种规律排列遭到严重破坏,糖酵解产能效率受到抑制,故能量生成减少,相应乳酸生成减少。 5、应用酵母双杂交技术,在人肝脏文库中筛选出VDAC的可能相互作用蛋白为DYNL1、PTPRH,经酵母回转验证结果为阳性。生物信息学分析结果提示,本实验研究发现VDAC-DYNL1,VDAC-PTPRH的两对相互作用分子,目前未见报道,为新的可能存在的相互作用蛋白,DYNL1与微管有明确的相互作用。DYNL1可能为微管对线粒体VDAC进行调控作用的中间蛋白。PTPRH可能对线粒体VDAC具有调控作用,其信号传导途径可能为ERK-MAPK-PTPRH---VDAC 6、微管解聚使心肌细胞电生理发生改变,可能导致心律加快增加能量消耗及诱发心律失常。其机理可能为微管解聚使INa电流显著增加所致。微管解聚可使游离态αtubulin,βtubulin二聚体增加,使得GTP信号激活,进而对细胞膜INa产生调节。微管解聚对L-钙通道电流(ICa-L)无显著影响,但由于APD缩短,总钙离子内流减少,使心肌收缩力下降。
[Abstract]:We found that the "long term study on pathogenesis of cardiac shock in early myocardial ischemia and hypoxia, which is significant microtubule damage, and the effect of energy metabolism plays a series of effects of microtubules in hypoxia induced in vitro and on myocardial cell induced by hypoxia and the vigorous system is rarely reported. As the cell energy the supply of core organelles mitochondria play a vital role in cells under hypoxic conditions change. Mitochondria are the core target organelles of myocardial hypoxia damage, mitochondrial damage is the key link of cardiac shock and systemic hypoxia damage. Mitochondria play an important role in cell hypoxia injury, not only because of its is an important energy supplier cell biological process, they will be able to use the membrane permeability transition pore (mPTP) directly involved in initiating cell necrosis and apoptosis. We Previous studies have found that mPTP caused the opening of mitochondrial permeability transition (MPT), leading to mitochondrial matrix swelling, membrane rupture, apoptotic signal molecules released from the inner and outer membrane, causing necrosis and apoptosis; irreversible injury induced mitochondrial mPTP open is a key link of myocardial hypoxia damage.
But there is a strong relationship between microtubules and mitochondria. In recent years, in-depth study of the cytoskeleton showed that microtubules except as rigid intracellular substances, has anchored subcellular structures such as mitochondria, Golgi, nucleus and the cell stability effect, but also involved in the regulation of signal transduction, transcription and protein synthesis. In addition to the cytoskeletal distribution and localization of intracellular mitochondria play a role, the adjustment process may also be involved in mitochondrial respiratory function. We found in the previous study of hypoxic myocardial cells of neonatal rats in hypoxia micro tube disruption can lead to continuous opening of mitochondrial permeability transition pore MPTP, and mitochondrial respiratory function decline, suggesting that microtubule has an important role in the regulation of mitochondria in rat myocardial cell, and the specific regulation is not clear. Because the VDAC is MPTP in the mitochondrial membrane channel Therefore, we speculate that microtubules may have an effect on the open state of VDAC through some unknown mechanism. Through these pathways, microtubules affect the production of cells, and further affect the function of cardiac myocytes.
As a cardiac muscle cell, the most important function is its electrophysiological activity, which is closely related to the energy metabolism. After destroying the microtubule, the electrophysiological activity of adult rat cardiomyocytes has been rarely reported.
In the experimental study of cardiac hypoxia in rat adult cardiomyocytes and cardiomyocytes were compared, there are significant differences, because adult cardiomyocytes had matured, the self repair ability than cells of neonatal rats, the hypoxia compensatory ability is poor. In the study of activity and function of adult the cell more close to the overall level, the result is more convincing. The myocardial cells into primary cultured, susceptible to external environmental impact, so the training is difficult, but on the hypoxia and various experimental factors applied more sensitive. The background of myocardial cell body into another primary culture more consistent, test data stable and reliable. In the structure, function of the difference is bigger, the distribution of mitochondria is more regular, the electrophysiological and contractile function of development, is conducive to the further study on the mechanism. Based on the above characteristics, the Study on the adult cardiomyocytes of rats as the research object, and using the fixed time of colchicine treated cells in certain concentration, state of microtubule depolymerization under simulated hypoxia, as single treatment factors, observe the changes of various indexes of myocardial cells of rats, changes in microtubule depolymerization caused by simple analysis.
The research hypothesis: the destruction of microtubules by subcellular localization changes of mitochondria; interaction through a middle molecular regulation of VDAC tubulin, VDAC increases, mitochondrial activity decreased, two ways to increase myocardial energy metabolic disorder, and thus the function change, decreased cell membrane electrophysiological activity. The purpose of the study
Microtubule depolymerization agent was used to simulate the destruction of microtubules under anoxic condition. The effects of microtubule depolymerization on mitochondrial function and energy metabolism and electrophysiology of cardiomyocytes were studied, and its possible mechanism was analyzed. The role of microtubule depolymerization in anoxia process was further discussed.
Materials and methods
1, adult rat cardiomyocyte culture
2, we used 8 micron M microtubule depolymerization agent, colchicine (colchicine), to treat adult rat cardiomyocytes, simulating the microtubule depolymerization state induced by hypoxia. The experiment was divided into normal control group (group N) and paclitaxel microtubule depolymerization group (group C).
3, immunocytochemical staining was used to observe the changes in the morphology and distribution of myocardial microtubules and mitochondria in group N and group C, and the changes of aggregated tubulin content in each group were detected by Western blot.
4, the mitochondrial potential was detected by four methyl Luo Danming ethyl ester (TMRE). The content of cytochrome C in cytoplasm was detected by immuno imprinting. Cell activity was detected by MTT assay, and lactate concentration in cardiac muscle cells was detected by lactate test kit.
5, high performance liquid chromatography (HPLC) was used to determine the content of ATP, ADP and AMP in cardiac myocytes.
6, using the yeast two hybrid experiment system, using VDAC as a bait in liver cDNA library screening and interacting proteins by yeast, rotary verification of the experimental results, and the screened protein of cells were co localization; further experimental results in bioinformatics research.
7, patch clamp technique was used to record myocardial cell membrane capacitance, action potential, sodium current (INa) and calcium current (ICa). The data were analyzed by Clampit in Axon's pClamp8.1 software.
Main results
1, the normal rat myocardial cell microtubules around the nucleus are arranged radially, microtubule tubular structure clear. Normal rats into myocardial cells arranged around the perinuclear microtubule body part, the other is linear parallel arranged along the long axis of the cell group.C rat myocardial cell microtubule structure damaged, rat myocardial cell microtubules along the longitudinal axis of the regular arrangement of sarcomere damage, showed decreased fluorescence intensity, continuous loss of microtubules, become rough and not smooth, microtubule structure is not clear, and a characteristic structure of curl.WB showed that myocardial cells of C group of polymeric tubulin content was significantly reduced compared with N group; adult cardiomyocytes was reduced significantly in neonatal rats.
2, normal myocardial mitochondria of body oval or long rod, distributed along the cell axis, a linear and uniform distribution of each muscle. The rat myocardial cell body into microtubules showed a linear tubular distribution, parallel with myocardial fiber orientation, VDAC display of the mitochondria in the particle loaded distribution, its distribution and the same direction with microtubules. The overlap, suggesting that into myocardial mitochondria along microtubules distribution group.C mitochondria scattered, lost regularity.
3, the mitochondrial potential of C group was significantly lower than that of N group, showing a decrease in mitochondrial fluorescence intensity. The cytochrome C content in cytoplasm of C group was significantly higher than that of normal control group.
4, after the microtubule depolymerization, the content of myocardial cells decreased compared with normal contrast. The content of ADP and AMP increased, ADP / ATP increased significantly, the energy load decreased, cell activity decreased significantly, and lactate content in myocardial cells decreased.
5, using the yeast two hybrid technique in human liver cDNA library, screened for proteins interacting with DYNL1, PTPRH VDAC, after the yeast rotary verification result is positive. Bioinformatics analysis showed that the experimental study found that two of VDAC-DYNL1, the interaction of VDAC-PTPRH molecules, there are no reports, for interacting proteins there are new possibilities, DYNL1 and microtubule interactions clear.
6, compared with the N group, C group (rest potential, RP of resting potential) showed no significant changes; and continuous action potentials (action potential, AP) the shape changed significantly, the myocardial cells of N group AP consistent shape, amplitude of action potential (action potential, amplitude, AMP) peak, repolarization of action potential duration (action potential, duration, APD) APD long C group, AP form is not stable, the peak value of AMP size, APD length was significantly reduced. The microtubule depolymerization groups APD20, APD50 and APD90 was significantly shorter than the control group.
7, INa current increased significantly after microtubule depolymerization; I/V curve showed that group of microtubule depolymerization current density in the control group -50 ~ -20 voltage range in mV were significantly higher than those in group Ica were consistent. Two, current density voltage curves almost overlap, microtubule depolymerization group and the control group had no significant difference between groups.ICa dependent obviously the voltage is a 40mV voltage depolarization when ICa is activated, depolarizing voltage to -10mV ICa.
Discussion and conclusion
1, the distribution of microtubules and mitochondria in the adult cardiomyocytes is the same. After the microtubule depolymerization, the distribution of mitochondria in cardiac myocytes is irregular.
2, microtubule depolymerization significantly reduced the activity of adult rat cardiomyocytes, suggesting that microtubule depolymerization can disintegrate energy generation units and reduce the energy supply of cardiomyocytes.
3, microtubule depolymerization made the mitochondrial membrane potential of myocardial cells decreased and the leakage of cytochrome C increased, indicating the regulation of microtubules on mitochondrial VDAC.
4, microtubule depolymerization inhibition of myocardial cell glycolysis. Intracellular glycolytic enzymes attached to microtubules, arranged according to a certain proportion and order, a capacity for rapid effects. After the regular arrangement of microtubule depolymerization, was severely damaged, the production efficiency of glycolysis is inhibited, so the energy production is reduced, the corresponding reduction of lactic acid.
5, using the yeast two hybrid technique in human liver cDNA library, screened for proteins interacting with DYNL1, PTPRH VDAC, after the yeast rotary verification result is positive. Bioinformatics analysis showed that the experimental study found that two of VDAC-DYNL1, the interaction of VDAC-PTPRH molecules, there are no reports, for interacting proteins there are new possibilities, DYNL1 and microtubule interacting.DYNL1 may regulate microtubule specific effects on mitochondrial VDAC intermediate protein.PTPRH may play a role in the regulation of mitochondrial VDAC and its signal transduction pathway may be ERK-MAPK-PTPRH---VDAC
6, the microtubule depolymerization in cardiomyocytes electrophysiological changes, may lead to increased energy consumption and heart rate induced arrhythmia. The possible mechanism is that microtubule depolymerization was significantly increased by INa current. The microtubule depolymerization free alpha tubulin beta two tubulin dimer increase, making GTP activation signal, and then produce the regulation on cell. INa on L- membrane microtubule depolymerization calcium current (ICa-L) had no significant effect, but due to the shortening of APD, the total decrease of calcium influx, which decreased myocardial contractility.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363

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