Control of mitochondrial metabolism by metabolic stress and hypoxia

生物体工作量的增加需要对新陈代谢进行调整。在细胞水平上，这是通过诱导线粒体生物发生来增加产生ATP的能力来实现的。然而，当氧气需求与适当的供应不匹配时，增加的氧气消耗可能导致缺氧。这很可能发生在缺血性疾病患者身上。细胞缺氧可增加氧自由基（ROS）的形成，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，缺血性心脏必须通过泵送来抵抗血管阻力增加；肺水肿试图通过离子泵送来清除肺泡水肿液）暴露于相反的刺激下，一种刺激会增加线粒体活性，另一种则试图阻止这种刺激。临床（和我们的初步结果显示），缺氧似乎主导了代谢能力的增加，这表明心脏冬眠和心肌收缩力下降。然而，在缺氧条件下，超越线粒体生物发生信号的机制尚不清楚。了解这些机制可以更好地了解患者的临床情况，并可能改善治疗。因此，在这个项目中，我们想要验证一个假设，即缺氧可以阻止线粒体活性的增加，从而保护细胞免受ROS的潜在损伤，我们想要阐明相关的信号通路。实验将在H10细胞上进行，H10细胞是用于研究心肌细胞信号通路的模型细胞。细胞将暴露于AICAR，它在细胞内模拟AMP浓度的增加，这表明ATP周转率增加，即ATP需求增加。作为缺血的一个重要方面，细胞也会暴露于缺氧中，模拟氧气供应的损害。主要读数将是线粒体耗氧量，线粒体膜电位和活性氧产生。RT-PCR和Western blot检测AMP-kinase - PGC1轴和电子传递链主要蛋白，可提示线粒体生物发生的改变。缺氧诱导因子（HIF）诱导的减少机制将包括bniip3依赖性的有丝分裂，丙酮酸脱氢酶（PDH）激酶（PDK）依赖性的PDH失活。在用可用的腺病毒转染系统引入shRNA沉默HIF-1alpha后，将研究与hif无关的机制。所有需要的技术都在我们的实验室建立。

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.