ALCAT1介导心肌肥厚的病理进程及调控线粒体氧化应激和自噬的机制

发布时间：2018-06-21 14:03

本文选题：心磷脂 + 心磷脂乙酰转移酶　；参考：《中南大学》2012年博士论文

【摘要】：氧化应激和线粒体的受损是引起心肌功能不全和心衰的重要原因,但其机制仍未被完全研究和阐述清楚。心磷脂(CL)是参与氧化磷酸化和维持心脏功能所需的一种线粒体的磷脂,其支链结构长度的改变,酰基不饱和度的异常和脂质过氧化程度的增加与心功能障碍有关。心磷脂乙酰转移酶(ALCAT1)是一种的重要的CL酰基转移酶,它对CL的支链结构有重构性的修饰作用,由于ALCAT1对CL的病理重构是很多心脏病的普遍现象,而且ALCAT1本身可被氧化应激和年龄相关的代谢性疾病所诱导而表达上调,因此我们研究在心肌肥厚疾病中,ALCAT1对心脏病理发展的可能作用和影响机制。在小鼠模型中,考察ALCAT1的敲缺对T4引起的心肌肥厚程度,心肌功能受损的程度,心室纤维化的程度,线粒体的损伤,和PINK1相关的线粒体自噬程度的影响。在H9C2细胞株中,我们考察ALCAT1高表达对氧化应激和脂质过氧化水平,mtDNA的拷贝数,细胞骨架蛋白的重构和细胞形态的影响。在MEF细胞上,考察ALCAT1的敲除对氧化应激引起的线粒体损伤和碎片化程度的影响。整个研究结果,确定了具有对CL重构作用的ALCAT1在心肌肥厚和心脏功能不全的病理进程中有重要的介导作用,其机制与氧化应激,线粒体的氧化损伤,自噬体系的调控等有关,整个研究结果表明了ALCAT1在心脏病源学研究中的意义。本课题主要研究结果如下： 1. ALCTA1的敲除减轻了甲状腺激素诱导的小鼠心肌肥厚的表型,包括小鼠的心脏和体重比值,左心室厚度和心肌功能,心肌细胞的容积,心室纤维化程度,心肌细胞肥厚和纤维化的标志物mRNA水平,和组织脂质过氧化程度。 2.从蛋白水平确证了ALCTA1在小鼠心肌肥厚的病理组织中表达上调。 3. ALCTA1的敲除减轻了甲状腺激素诱导后的心肌组织中的线粒体损伤程度。 4. ALCTA1的敲除增加了PINK1和P62在小鼠心肌中的表达水平 5. ALCTA1的敲除不会影响短期甲状腺激素处理对心肌中Akt通路的激活程度,但会在一定程度上减轻或逆转长期甲状腺激素处理对心肌组织中Akt通路的抑制程度。 6.构建和筛选出稳定高表达Vector和ALCTA1的H9C2细胞株。 7.在H9C2细胞株上,ALCTA1的高表达增加了细胞的容积和增殖速度,阻断了细胞分化能力。 8. ALCTA1的高表达增加了H9C2线粒体氧自由基的产生速度,以及氧化应激诱导的脂质过氧化程度和mtDNA的氧化缺失。 9. ALCTA1的过表达和敲除改变了骨架蛋白在H9C2和MEF细胞内的分布。 10.ALCTA1介导了H9C2和MEF细胞中的线粒体网络结构在碎片化和连续管状结构的转换,ALCTA1的敲除可以减轻氧化应激引起的线粒体碎片化程度。 11.ALCTA1的高表达减少了VDAC在H9C2中的表达,ALCTA1的敲除增加了VDAC在MEF的表达。 12.ALCTA1的敲除减缓了MEF的细胞衰老进程。 13.ALCTA1引起的氧化应激诱导了H9C2的胰岛素信号抵抗。 14.ALCTA1的高表达阻断了甲状腺激素对Akt通路的磷酸化激活。总之,本课题在甲亢诱导的心肌肥厚模型中,考察ALCAT1的敲除对心肌肥厚表型的影响,从分子机制和信号传导通路的调控等方面进行机制的探讨。在H9C2和MEF细胞水平上研究ALCAT1对细胞形态,增殖,分化能力,氧自由基产生,以及氧化应激诱导的脂质过氧化度和线粒体缺失,线粒体网络结构和自噬,骨架蛋白重构和胰岛素信号通路的影响。我们首次发现ALCAT1在甲状腺激素诱导的心肌肥厚病理进程中的调控作用,并发现其机制跟氧化应激,线粒体的融合和分裂,线粒体氧化损伤和自噬体系,以及病理状态下Akt信号通路的上调有关。研究结果为其它心脏疾病的分子病理机制研究提供了新思路,为临床的心肌肥厚和心衰等疾病的药物治疗提供了新的靶标。
[Abstract]:Oxidative stress and damage to mitochondria are important causes of myocardial dysfunction and heart failure, but their mechanisms are still not fully studied and elaborated. Cardiolipin (CL) is a mitochondrial phospholipid required to participate in oxidative phosphorylation and maintenance of heart function, changes in the length of its branched chain structure, abnormality of acyl unsaturation, and lipid peroxy. The increase is associated with cardiac dysfunction. Cardiolipin acetyltransferase (ALCAT1) is an important CL acyl transferase, which has a reconstructive modification to the branched structure of CL. Because ALCAT1's pathological remodeling of CL is a common phenomenon in many heart diseases, and ALCAT1 itself can be oxidative stress and metabolic disease related to age. We study the possible role and mechanism of ALCAT1 in cardiac histopathological development in cardiac hypertrophy. In the mouse model, we examine the degree of myocardial hypertrophy caused by ALCAT1's knockout, the extent of myocardial dysfunction, the degree of ventricular fibrosis, the damage of the mitochondria, and the lines associated with the PINK1. The effect of autophagy. In the H9C2 cell strain, we examined the effect of ALCAT1 high expression on oxidative stress and lipid peroxidation, the number of copies of mtDNA, the remodeling of cytoskeleton protein and the effect of cell morphology. On MEF cells, the effects of ALCAT1 knockout on the damage and fragmentation of linear particles caused by oxidative stress were investigated. As a result, the role of ALCAT1 in the pathological process of cardiac hypertrophy and cardiac dysfunction was identified, and the mechanism was related to oxidative stress, oxidative damage of mitochondria, and regulation of autophagy. The whole research results showed the significance of ALCAT1 in the study of cardiac pathogeny.
The main results of this study are as follows:
1. ALCTA1 knockout alleviates the phenotype of thyroid hormone induced cardiac hypertrophy in mice, including the heart and weight ratio of mice, left ventricular thickness and myocardial function, myocardial cell volume, ventricular fibrosis, myocardial cell hypertrophy and fibrosis markers mRNA level, and tissue lipid peroxidation.
2. protein level confirmed that ALCTA1 was upregulated in the histopathological tissue of mouse cardiac hypertrophy.
3. ALCTA1 knockout alleviated mitochondrial damage in thyroid tissue induced by thyroid hormone.
The knockout of 4. ALCTA1 increased the expression level of PINK1 and P62 in mouse myocardium.
5. ALCTA1 knockout does not affect the activation of the short-term thyroid hormone treatment on the Akt pathway in the myocardium, but to a certain extent alleviates or reverses the inhibition of the long-term thyroid hormone treatment on the Akt pathway in the myocardium.
6. to construct and screen H9C2 cell lines with stable expression of Vector and ALCTA1.
7. on the H9C2 cell line, the high expression of ALCTA1 increased cell volume and proliferation rate, and blocked cell differentiation ability.
The high expression of 8. ALCTA1 increased the production rate of H9C2 mitochondrial oxygen free radicals, as well as the degree of oxidative stress induced lipid peroxidation and the oxidative depletion of mtDNA.
Overexpression and knockout of 9. ALCTA1 changed the distribution of skeleton proteins in H9C2 and MEF cells.
10.ALCTA1 mediated transformation of mitochondrial network structure in H9C2 and MEF cells in fragmentation and continuous tubular structure. ALCTA1 knockout can reduce the degree of mitochondrial fragmentation caused by oxidative stress.
The high expression of 11.ALCTA1 reduced the expression of VDAC in H9C2, and the knockout of ALCTA1 increased the expression of VDAC in MEF.
Knockout of 12.ALCTA1 slowed down the aging process of MEF cells.
Oxidative stress induced by 13.ALCTA1 induces insulin resistance in H9C2.
The high expression of 14.ALCTA1 blocked the phosphorylation of Akt pathway by thyroid hormone.
In conclusion, in the hyperthyroidism induced myocardial hypertrophy model, the effect of ALCAT1 knockout on the myocardial hypertrophy phenotype was investigated, the mechanism of molecular mechanism and the regulation of signal transduction pathway were investigated. The morphology, proliferation, differentiation, oxygen free radical production and oxidative stress of ALCAT1 were studied at the level of H9C2 and MEF cells. Induced lipid peroxidation and mitochondrial deletion, mitochondrial network structure and autophagy, cytoskeleton remodeling and insulin signaling pathways. We first discovered the regulatory role of ALCAT1 in the pathological process of thyroid hormone induced cardiac hypertrophy, and found its mechanism with oxygen stress, mitochondrial fusion and division, mitochondrial oxygen. The damage and autophagy system, as well as the up regulation of the Akt signaling pathway in the pathological state, provide new ideas for the molecular pathological mechanism of other heart diseases, and provide a new target for the clinical drug therapy of cardiac hypertrophy and heart failure.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

【共引文献】