Control of mitochondrial metabolism by metabolic stress and hypoxia

通过代谢应激和缺氧控制线粒体代谢

• 批准号: 253342100
• 负责人: Professor Dr. Heimo Mairbäurl
• 金额: --
• 依托单位: Abteilung Innere Medizin VII: Sportmedizin
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/253342100?language=en
• 关键词: Control; mitochondrial; metabolism; metabolic; stress

An increased work load of an organism requires adjustments of metabolism. At the cellular Level this is achieved by inducing mitochondrial biogenesis to increase the capacity to produce ATP. However, the increased oxygen consumption might result in hypoxia when the oxygen demand is not matched by an appropriate supply. This is likely to happen in patients with ischemic diseases. Cellular hypoxia might increase the formation of oxygen radicals (ROS) leading to cell damage. Thus, a major adaptation to hypoxia is a decrease in mitochondrial activity in order to prevent the damage. Cells with increased metabolic demand but inadequate oxygen supply (typical clinical situations are e.g. an ischemic heart that has to pump against an increased vascular resistance; an edematous lung trying to remove alveolar edema fluid by ion pumping) are therefore exposed to opposing stimuli, one that increases mitochondrial activity, and a second trying to prevent that. Clinically (and shown by our preliminary results), hypoxia seems to dominate over increased metabolic capacity as indicated by the hibernating heart and decreased myocardial contractility. However, the mechanisms overriding the signals increasing mitochondrial biogenesis in hypoxia are poorly understood. Knowledge of these mechanisms provides a better understanding of a clinical situation of a patient and might result in an improved treatment. Therefore, in this project we want to test the hypothesis that hypoxia prevents an increase in mitochondrial activity in order to protect cells from potential damage by ROS, and we want to elucidate the involved signaling pathways.Experiments will be performed on H10 cells, a model cell used for studying cardiomyocyte signaling pathways. Cells will be exposed to AICAR, which inside the cell mimics an increased concentration of AMP, which is indicative of an increased ATP-turnover, i.e. an increased ATP demand. Cells will also be exposed to hypoxia simulating the impairment of oxygen supply as one significant aspect of ischemia. Major readouts will be mitochondrial oxygen consumption, mitochondrial membrane potential, and ROS production. Measurement of the AMP-kinase - PGC1 - axis and major proteins of the electron transfer chain by RT-PCR and Western blot of will indicate altered mitochondrial biogenesis. Hypoxia-inducible factor (HIF) induced decrease mechanisms studied will include BNIP3 dependent mitophagy, pyruvate dehydrogenase (PDH)-kinase (PDK) dependent inactivation of PDH. HIF-independent mechanisms will be studied after silencing HIF-1alpha with shRNA introduced with an available adenoviral transfection system. All required techniques are established in our laboratory.

生物体工作负荷的增加需要新陈代谢的调整。在细胞水平，这是通过诱导线粒体生物合成来增加产生ATP的能力来实现的。然而，当氧气需求量与适当的供应量不匹配时，增加的氧气消耗可能导致缺氧。这可能发生在缺血性疾病患者中。细胞缺氧可增加氧自由基（ROS）的生成，导致细胞损伤。因此，对缺氧的主要适应是线粒体活性的降低，以防止损伤。因此，代谢需求增加但氧气供应不足的细胞（典型的临床情况是例如必须对抗增加的血管阻力进行泵送的缺血性心脏;试图通过离子泵送去除肺泡水肿液的水肿肺）暴露于相反的刺激，一种刺激增加线粒体活性，另一种刺激试图阻止线粒体活性。在临床上（我们的初步结果显示），缺氧似乎占主导地位的代谢能力增加所示的冬眠的心脏和心肌收缩力下降。然而，在缺氧中增加线粒体生物合成的信号的机制知之甚少。这些机制的知识提供了对患者临床情况的更好理解，并可能导致改善的治疗。因此，本课题以研究心肌细胞信号通路的模型细胞H10细胞为对象，对缺氧抑制线粒体活性的增加，从而保护细胞免受ROS的潜在损伤的假说进行验证，并对其中的信号通路进行解析。细胞将暴露于AICAR，AICAR在细胞内模拟增加的AMP浓度，这指示增加的ATP周转，即增加的ATP需求。细胞还将暴露于缺氧，模拟作为缺血的一个重要方面的氧供应受损。主要读数将是线粒体耗氧量、线粒体膜电位和ROS产生。通过RT-PCR和Western blot测量AMP-激酶-PGC 1-轴和电子传递链的主要蛋白质，将表明线粒体生物发生的改变。缺氧诱导因子（HIF）诱导的减少机制研究将包括BNIP 3依赖性线粒体自噬，丙酮酸脱氢酶（PDH）-激酶（PDK）依赖性PDH失活。HIF-1 α沉默后，将研究与现有的腺病毒转染系统引入的shRNA HIF-1 α的非依赖性机制。所有所需的技术都在我们的实验室中建立。