缺氧早期心肌细胞微管结构变化对糖酵解的影响及其机制的研究
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摘要
目的
缺氧是许多疾病过程中重要的病理生理现象之一,缺氧诱导因子(HIF)-1α在缺氧细胞的能量代谢中有着重要作用。本研究旨在明确缺氧早期心肌细胞微管结构破坏是否通过调控HIF-1α来影响细胞糖酵解供能。
材料和方法
1、建立体外培养新生大鼠心肌细胞模型。分别采用常氧、缺氧、以及常氧和缺氧下微管解聚剂和不同浓度微管稳定剂处理心肌细胞;建立心肌细胞高表达微管相关蛋白4和RNA干扰后低表达α-微管蛋白模型。
2、激光扫描共聚焦显微镜观察培养新生大鼠心肌细胞α-微管结构和含量变化,胎盘蓝染色观察细胞存活率,CCK法观察细胞活力,化学比色法和高效液相色谱法分别观察心肌细胞糖酵解关键酶(PK、HK和PFK)活性、肌酸激酶(CK)、乳酸生成,LDH漏出及ATP/ADP生成。
3、免疫印记法及激光共聚焦显微镜观察心肌细胞微管结构改变后HIF-1α蛋白含量和细胞内分布变化,实时定量PCR法检测心肌细胞HIF-1αmRNA表达变化。
结果
1、缺氧后早期,体外培养新生大鼠心肌细胞微管网状结构破坏,聚合态α-微管蛋白含量减少,心肌细胞的活力降低,死亡率升高;稳定微管网状结构可以使细胞存活率和活性升高。
2、缺氧后早期,体外培养新生大鼠心肌细胞微管网状结构破坏引起糖酵解关键酶(PK、HK和PFK)活性降低,代谢终产物乳酸生成减少,细胞ATP生成减少;而稳定微管网状结构可以在缺氧早期一段时间内升高PK、HK和PFK的活性,促进细胞能量生成。
3、缺氧后早期,体外培养新生大鼠心肌细胞微管网状结构破坏使HIF-1α蛋白表达及入核表达均减少。微管稳定剂和高表达微管相关蛋白4可稳定缺氧心肌细胞微管网状结构,上调HIF-1α蛋白含量及入核表达;微管解聚剂和下调微管蛋白表达则可加重缺氧心肌细胞微管结构破坏,HIF-1α蛋白含量及入核表达减少更明显。而且HIF-1α蛋白表达的增加发生在转录后水平。
结论
微管结构变化通过调节HIF-1α可影响缺氧心肌细胞早期糖酵解。稳定微管结构可促进HIF-1α入核表达并提高HIF-1α蛋白含量,提高厌氧糖酵解关键酶活性和能量生成,表明微管结构变化通过调节HIF-1α影响缺氧心肌细胞早期糖酵解,这为临床改善缺氧早期细胞能量代谢提供了潜在的治疗靶点。
Aims Hypoxia is one of the important pathophysiolgical phenomena in many disease processes,and hypoxia inducible factor(HIF)-1αis a key regulator of anaerobic energy metabolism in hypoxic cells. The present study is designed to investigate that cytoskeleton change affects glycolysis under hypoxia and breakdown of microtubular structures influences glycolysis in early hypoxic cardiomyocytes through regulating HIF-1αactivity and distribution in cells.
Materials and methods
1. To establish neonatal rat cardiomyocytes cultured model. Neonatal rat cardiomyocytes were cultured and treated with normoxia, hypoxia, a microtubule stabilizing agent or a microtubule depolymerizing agent which were used to establish the model for high expression of microtubule-associated protein 4 and the model for RNA interference-caused low expression of microtubulin.
2. The microtubular structural changes and intracellular distribution of HIF-1αprotein were observed under laser confocal scanning microscopy. The cell survival was determined by Trypan blue stain. The activity of key glycolytic enzymes, creatine kinase, lactic acid, viability and energy production of cardiomyocyte were determined by colorimetry and high-performance liquid chromatography.
3. The content of HIF-1αprotein following microtubular structural change was examined by Western blotting, and HIF-1αmRNA expression was determined by real-time PCR assay.
Results
1. During the earlier time of hypoxia, microtubular structures were broken in cardiomyocytes. Then the viability of cardiomyocytes and myocardial cell survival decreased. The stablilization of microtubule enhanced the viability of cardiomyocytes and myocardial cell survival.
2. Disorganization of microtubular structures inhibited the activity of key glycolytic enzymes, and contents of lactic acid and ATP decreased during the earlier time of hypoxia. The microtubule stabilizing agent stabilized the reticular microtubular structures in hypoxic cardiomyocytes, increased the activity of key glycolytic enzymes, ameliorated cell energy supply and viability,
3. During the earlier time of hypoxia, disorganization of microtubule structures inhibited the expression of HIF-1αand HIF-1αendonuclear aggregation. The microtubule stabilizing agent and high expression of microtubule-associated protein 4 upregulated HIF-1αprotein expression and endonuclear aggregation. In contrast, the microtubule -depolymerizing agent or knock-down of microtubulin expression aggravated breakdown of microtubular structures of hypoxic cardiomyocytes, further decreased HIF-1αprotein contents and endonuclear aggregation.
Conclusions Microtubular structural changes influence glycolysis of early hypoxic cardiomyocytes by regulating HIF-1αactivity. Stabilizing microtubular structures increases endonuclear and total HIF-1αexpression, the activity of key glycolytic enzymes and energy supply. These findings provide potential therapeutic targets for ameliorating cell energy metabolism during early hypoxia.
缺氧是许多疾病过程中重要的病理生理现象之一,缺氧诱导因子(HIF)-1α在缺氧细胞的能量代谢中有着重要作用。本研究旨在明确缺氧早期心肌细胞微管结构破坏是否通过调控HIF-1α来影响细胞糖酵解供能。
材料和方法
1、建立体外培养新生大鼠心肌细胞模型。分别采用常氧、缺氧、以及常氧和缺氧下微管解聚剂和不同浓度微管稳定剂处理心肌细胞;建立心肌细胞高表达微管相关蛋白4和RNA干扰后低表达α-微管蛋白模型。
2、激光扫描共聚焦显微镜观察培养新生大鼠心肌细胞α-微管结构和含量变化,胎盘蓝染色观察细胞存活率,CCK法观察细胞活力,化学比色法和高效液相色谱法分别观察心肌细胞糖酵解关键酶(PK、HK和PFK)活性、肌酸激酶(CK)、乳酸生成,LDH漏出及ATP/ADP生成。
3、免疫印记法及激光共聚焦显微镜观察心肌细胞微管结构改变后HIF-1α蛋白含量和细胞内分布变化,实时定量PCR法检测心肌细胞HIF-1αmRNA表达变化。
结果
1、缺氧后早期,体外培养新生大鼠心肌细胞微管网状结构破坏,聚合态α-微管蛋白含量减少,心肌细胞的活力降低,死亡率升高;稳定微管网状结构可以使细胞存活率和活性升高。
2、缺氧后早期,体外培养新生大鼠心肌细胞微管网状结构破坏引起糖酵解关键酶(PK、HK和PFK)活性降低,代谢终产物乳酸生成减少,细胞ATP生成减少;而稳定微管网状结构可以在缺氧早期一段时间内升高PK、HK和PFK的活性,促进细胞能量生成。
3、缺氧后早期,体外培养新生大鼠心肌细胞微管网状结构破坏使HIF-1α蛋白表达及入核表达均减少。微管稳定剂和高表达微管相关蛋白4可稳定缺氧心肌细胞微管网状结构,上调HIF-1α蛋白含量及入核表达;微管解聚剂和下调微管蛋白表达则可加重缺氧心肌细胞微管结构破坏,HIF-1α蛋白含量及入核表达减少更明显。而且HIF-1α蛋白表达的增加发生在转录后水平。
结论
微管结构变化通过调节HIF-1α可影响缺氧心肌细胞早期糖酵解。稳定微管结构可促进HIF-1α入核表达并提高HIF-1α蛋白含量,提高厌氧糖酵解关键酶活性和能量生成,表明微管结构变化通过调节HIF-1α影响缺氧心肌细胞早期糖酵解,这为临床改善缺氧早期细胞能量代谢提供了潜在的治疗靶点。
Aims Hypoxia is one of the important pathophysiolgical phenomena in many disease processes,and hypoxia inducible factor(HIF)-1αis a key regulator of anaerobic energy metabolism in hypoxic cells. The present study is designed to investigate that cytoskeleton change affects glycolysis under hypoxia and breakdown of microtubular structures influences glycolysis in early hypoxic cardiomyocytes through regulating HIF-1αactivity and distribution in cells.
Materials and methods
1. To establish neonatal rat cardiomyocytes cultured model. Neonatal rat cardiomyocytes were cultured and treated with normoxia, hypoxia, a microtubule stabilizing agent or a microtubule depolymerizing agent which were used to establish the model for high expression of microtubule-associated protein 4 and the model for RNA interference-caused low expression of microtubulin.
2. The microtubular structural changes and intracellular distribution of HIF-1αprotein were observed under laser confocal scanning microscopy. The cell survival was determined by Trypan blue stain. The activity of key glycolytic enzymes, creatine kinase, lactic acid, viability and energy production of cardiomyocyte were determined by colorimetry and high-performance liquid chromatography.
3. The content of HIF-1αprotein following microtubular structural change was examined by Western blotting, and HIF-1αmRNA expression was determined by real-time PCR assay.
Results
1. During the earlier time of hypoxia, microtubular structures were broken in cardiomyocytes. Then the viability of cardiomyocytes and myocardial cell survival decreased. The stablilization of microtubule enhanced the viability of cardiomyocytes and myocardial cell survival.
2. Disorganization of microtubular structures inhibited the activity of key glycolytic enzymes, and contents of lactic acid and ATP decreased during the earlier time of hypoxia. The microtubule stabilizing agent stabilized the reticular microtubular structures in hypoxic cardiomyocytes, increased the activity of key glycolytic enzymes, ameliorated cell energy supply and viability,
3. During the earlier time of hypoxia, disorganization of microtubule structures inhibited the expression of HIF-1αand HIF-1αendonuclear aggregation. The microtubule stabilizing agent and high expression of microtubule-associated protein 4 upregulated HIF-1αprotein expression and endonuclear aggregation. In contrast, the microtubule -depolymerizing agent or knock-down of microtubulin expression aggravated breakdown of microtubular structures of hypoxic cardiomyocytes, further decreased HIF-1αprotein contents and endonuclear aggregation.
Conclusions Microtubular structural changes influence glycolysis of early hypoxic cardiomyocytes by regulating HIF-1αactivity. Stabilizing microtubular structures increases endonuclear and total HIF-1αexpression, the activity of key glycolytic enzymes and energy supply. These findings provide potential therapeutic targets for ameliorating cell energy metabolism during early hypoxia.
引文
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