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
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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。
Objectives: To investigate the effects of microbutule depolymerization on mitochondria function, energy metabolism and electrophysiological properties of cardiac ventricular myocytes from rats. Also to analyse the possible machanism and to further find out the roles of microbutule depolymerization during hypoxia by applying colchicine, a microtubule depolymerizing agent, to simulate the damage of microtubule underlying hypoxia.
Methods:
1. Cardiomyocytes were isolated from adult rats and were incubated in the presence or absence of colchicines, a depolymerizing agent.
2. To apply 8μM colchicine in the adult rat cardiomyocytes to simulate the microbutule depolymerization underlying hypoxia, which is divided into 2 groups : normal group (N group) and colchicine group (C group).
3. To use immunochemistry to observe the morphyology of microtubule and mitochondria and changing regularity. To apply Western-blot for detecting the content of microtubule protein.
4. To use TMRE for detecting membrane potential inside mitochondria; to apply Western blot for detecting the content of cytochrome C protein; to detect cellular activity by using MTT method. The concentration of lactic acid was measured by using lactic acid detecting kit.
5. The content of ATP、ADP、AMP were measured by high-performance liquid chromatography (HPLC).
6. The possible related cross-talk protein was screened with a bait that is mitochondrial voltage-dependent anion channel (VDAC) in the yeast two hybrid system. Also the screened protein co-locations were studied by using immunochemistry for further bio-information research.
7. The electrophysiological characteristics of cardiac ventricular myocytes. Whole-cell patch clamp was employed to record the membrane capacity, presence of action potentials (AP) and the features of sodium(INa) and calcium(ICa) ion channels at various stages of differentiation. Data acquisition and analysis were performed by pCLAMP 8.1 software (Axon Instruments).
Results:
1. For the normal newborn rat cardiomyocytes, the microtubules were distributed around the nucleus with radial alignment and distinct tube-like structures. However, for normal adult rat cardiomyocytes, some microtubules were distributed around the nucleus while others were lined in parallel along the long axis of the cell. In the group C, the microtubules from newborn rat cardiomyocytes were fully damaged while from adult rat cardiomyocytes damage was along the myo-section direction. The adult results by immunofluorescence showed that the fluorescence intensity was weaker than newborn group. The microtubule line structure was lost and become rough Western-blot results showed that the content of microtubule protein from C group cardiomyocytes was reduced significantly compared with of N group; Compared with newborn rat cardiomyocytes, the adult group was significantly reduced.
2. The mitochondria from the normal adult cardiomyocytes showed ellipse or long-rod like morphology and were distributed along the long axis of the cell with line-tube like distribution, in parallel with cardiomyo-fiber. The mitochondria from C group were distributed randomly.
3. The mitochondrial membrane potential from C group was reduced significantly than N group with very weak fluorescence intensity, while the content of cytochrome C protein in the C group was increased significantly compared to the N group.
4. After microbutule depolymerization, the ATP content from C group was decreased significantly compared with the N group, while the ADP、AMP increased, and the rate of ADP / ATP was increased significantly. The activity of the cell and the content of lactic acid were decreased significantly.
5. DYNL1、PTPRH, the possible related cross-talk proteins were screened in the yeast two hybrid system, and further confirmed they are positive by the yeast two hybrid test. We can conclude from bio-inofrmation analysis results, the VDAC-DYNL1,VDAC-PTPRH are two pairs of interactional molecules which are novel so far and can be new proteins with specific interaction to microtubule. DYNL1 has probable interaction with microtubules.
6. Compared with N group, the resting membrane potential (RMP) for the C group cells stayed stable after adding medication, but action potentials (AP) showed significant changes in terms of AP shape, action potential amplitude (AMP) and action potential duration (APD). For the N group, their AP shape, AMP and APD accorded with each AP of the same cell. However, for C group the above parameters did not remain similar. The peak of AMP was changeable and APD was reduced significantly. For APD20、APD50和APD90, the C group were shortened significantly compared with the N group.
7. After microbutule depolymerization, INa was increased significantly; I/V curves suggested that current intensities for the C group were significantly higher than N group between -50～-20 mV. The two groups Ica currents intensity-voltage curves stayed and almost overlapped with no significant difference. ICa was obvious voltage-dependent and depolarization stayed at around一40mV where ICa was activitated. ICa got its peak at voltage-10mV.
Discussion and conclusion:
1. The distribution direction of microtubule and mitochondria is with one accord in the cardiac ventricular myocytes. After microbutule depolymerization, the distribution of mitochondria becomes random.
2. The activity of cardiomyocytes from adult rats was significantly decreased by microbutule depolymerization which probably resulted from collapsing of energy producing units by colchicine.
3. The cardiomyocytes mitochondria potential was decreased by microbutule depolymerization, which added leakage of cytochrome C, so it suggested that mitochondria VDAC was regulated by microtubule.
4. The glycolysis of cardiomyocytes was inhibited by microbutule depolymerization. Inside the cardiomyocytes, the glycolysis enzyme adhered to microtubules, and was aligned in some proportion and order resulting in fast producing energy effects. After microbutule depolymerization, so termed regularities were damaged, the efficiency of producing energy by glycolysis was inhibited so the energy was reduced and lactic acid was reduced accordingly.
5. DYNL1、PTPRH, the possible related cross-talk proteins were screened with a bait that is mitochondrial voltage-dependent anion channel (VDAC) in the yeast two hybrid system, and further confirmed as positive by the yeast two hybrid test. We can conclude from bio-inofrmation analysis results, the VDAC-DYNL1,VDAC-PTPRH are two pairs of interactional molecules which is novel and can be new proteins with specific interaction to microtubule. DYNL1 is probably a media-protein in the microtubule which has regulating roles to mitochondria VDAC. PTPRH may regulate mitochondria VDAC, where its possible signal conduction path is ERK-MAPK-PTPRH---VDAC.
6. Microbutule depolymerization also results in cardiomyocytes electrophysiological changes, which probably is the basis of arrhythmia resulted from heart rate speeding up so as to add energy consumption. The possible mechanism is that INa was significantly increased by Microbutule depolymerization. Microbutule depolymerization can increase free-αtubulin,βtubulin dimer, which activate GTP signal and further regulates membrane INa. There are no significant changes in ICa-L by Microbutule depolymerization.
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。
Objectives: To investigate the effects of microbutule depolymerization on mitochondria function, energy metabolism and electrophysiological properties of cardiac ventricular myocytes from rats. Also to analyse the possible machanism and to further find out the roles of microbutule depolymerization during hypoxia by applying colchicine, a microtubule depolymerizing agent, to simulate the damage of microtubule underlying hypoxia.
Methods:
1. Cardiomyocytes were isolated from adult rats and were incubated in the presence or absence of colchicines, a depolymerizing agent.
2. To apply 8μM colchicine in the adult rat cardiomyocytes to simulate the microbutule depolymerization underlying hypoxia, which is divided into 2 groups : normal group (N group) and colchicine group (C group).
3. To use immunochemistry to observe the morphyology of microtubule and mitochondria and changing regularity. To apply Western-blot for detecting the content of microtubule protein.
4. To use TMRE for detecting membrane potential inside mitochondria; to apply Western blot for detecting the content of cytochrome C protein; to detect cellular activity by using MTT method. The concentration of lactic acid was measured by using lactic acid detecting kit.
5. The content of ATP、ADP、AMP were measured by high-performance liquid chromatography (HPLC).
6. The possible related cross-talk protein was screened with a bait that is mitochondrial voltage-dependent anion channel (VDAC) in the yeast two hybrid system. Also the screened protein co-locations were studied by using immunochemistry for further bio-information research.
7. The electrophysiological characteristics of cardiac ventricular myocytes. Whole-cell patch clamp was employed to record the membrane capacity, presence of action potentials (AP) and the features of sodium(INa) and calcium(ICa) ion channels at various stages of differentiation. Data acquisition and analysis were performed by pCLAMP 8.1 software (Axon Instruments).
Results:
1. For the normal newborn rat cardiomyocytes, the microtubules were distributed around the nucleus with radial alignment and distinct tube-like structures. However, for normal adult rat cardiomyocytes, some microtubules were distributed around the nucleus while others were lined in parallel along the long axis of the cell. In the group C, the microtubules from newborn rat cardiomyocytes were fully damaged while from adult rat cardiomyocytes damage was along the myo-section direction. The adult results by immunofluorescence showed that the fluorescence intensity was weaker than newborn group. The microtubule line structure was lost and become rough Western-blot results showed that the content of microtubule protein from C group cardiomyocytes was reduced significantly compared with of N group; Compared with newborn rat cardiomyocytes, the adult group was significantly reduced.
2. The mitochondria from the normal adult cardiomyocytes showed ellipse or long-rod like morphology and were distributed along the long axis of the cell with line-tube like distribution, in parallel with cardiomyo-fiber. The mitochondria from C group were distributed randomly.
3. The mitochondrial membrane potential from C group was reduced significantly than N group with very weak fluorescence intensity, while the content of cytochrome C protein in the C group was increased significantly compared to the N group.
4. After microbutule depolymerization, the ATP content from C group was decreased significantly compared with the N group, while the ADP、AMP increased, and the rate of ADP / ATP was increased significantly. The activity of the cell and the content of lactic acid were decreased significantly.
5. DYNL1、PTPRH, the possible related cross-talk proteins were screened in the yeast two hybrid system, and further confirmed they are positive by the yeast two hybrid test. We can conclude from bio-inofrmation analysis results, the VDAC-DYNL1,VDAC-PTPRH are two pairs of interactional molecules which are novel so far and can be new proteins with specific interaction to microtubule. DYNL1 has probable interaction with microtubules.
6. Compared with N group, the resting membrane potential (RMP) for the C group cells stayed stable after adding medication, but action potentials (AP) showed significant changes in terms of AP shape, action potential amplitude (AMP) and action potential duration (APD). For the N group, their AP shape, AMP and APD accorded with each AP of the same cell. However, for C group the above parameters did not remain similar. The peak of AMP was changeable and APD was reduced significantly. For APD20、APD50和APD90, the C group were shortened significantly compared with the N group.
7. After microbutule depolymerization, INa was increased significantly; I/V curves suggested that current intensities for the C group were significantly higher than N group between -50～-20 mV. The two groups Ica currents intensity-voltage curves stayed and almost overlapped with no significant difference. ICa was obvious voltage-dependent and depolarization stayed at around一40mV where ICa was activitated. ICa got its peak at voltage-10mV.
Discussion and conclusion:
1. The distribution direction of microtubule and mitochondria is with one accord in the cardiac ventricular myocytes. After microbutule depolymerization, the distribution of mitochondria becomes random.
2. The activity of cardiomyocytes from adult rats was significantly decreased by microbutule depolymerization which probably resulted from collapsing of energy producing units by colchicine.
3. The cardiomyocytes mitochondria potential was decreased by microbutule depolymerization, which added leakage of cytochrome C, so it suggested that mitochondria VDAC was regulated by microtubule.
4. The glycolysis of cardiomyocytes was inhibited by microbutule depolymerization. Inside the cardiomyocytes, the glycolysis enzyme adhered to microtubules, and was aligned in some proportion and order resulting in fast producing energy effects. After microbutule depolymerization, so termed regularities were damaged, the efficiency of producing energy by glycolysis was inhibited so the energy was reduced and lactic acid was reduced accordingly.
5. DYNL1、PTPRH, the possible related cross-talk proteins were screened with a bait that is mitochondrial voltage-dependent anion channel (VDAC) in the yeast two hybrid system, and further confirmed as positive by the yeast two hybrid test. We can conclude from bio-inofrmation analysis results, the VDAC-DYNL1,VDAC-PTPRH are two pairs of interactional molecules which is novel and can be new proteins with specific interaction to microtubule. DYNL1 is probably a media-protein in the microtubule which has regulating roles to mitochondria VDAC. PTPRH may regulate mitochondria VDAC, where its possible signal conduction path is ERK-MAPK-PTPRH---VDAC.
6. Microbutule depolymerization also results in cardiomyocytes electrophysiological changes, which probably is the basis of arrhythmia resulted from heart rate speeding up so as to add energy consumption. The possible mechanism is that INa was significantly increased by Microbutule depolymerization. Microbutule depolymerization can increase free-αtubulin,βtubulin dimer, which activate GTP signal and further regulates membrane INa. There are no significant changes in ICa-L by Microbutule depolymerization.
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