Control Of Cellular Energy Metabolism

这些研究的目的是利用现代系统生物学方法更好地了解生物组织的能量代谢。为了实现这一目标，实验室专注于在蛋白质组学、代谢组学、蛋白质结构、翻译后修饰、微创代谢成像信息和光谱学等方面使用筛选方法。这个项目的一个主要假设是，执行氧化磷酸化的多蛋白复合物的活性以某种方式协调，以平衡细胞中ATP的产生率和利用率。这导致了观察到的代谢稳态，即使在工作量发生重大变化时，细胞中做功的势能也保持在接近恒定的水平。在过去的一年里，我们取得了以下主要发现：1)我们扩大了线粒体在完整跳动的心脏中功能的透射光谱学研究，重点关注小鼠心脏模型的缺血再灌注。我们还验证了使用氧化磷酸化复合物III的发色团来提供跳动心脏线粒体膜电位的非侵入性测定。使用这种方法，我们已经建立了线粒体膜电位的动态和线粒体在缺血，收缩和再灌注在各种条件下的整体氧化还原平衡。初步研究结果表明，再灌注时线粒体膜电位的快速恢复可能有助于活性氧的产生以及线粒体基质中Ca的积累。这两种情况都可能导致再灌注阶段的线粒体损伤，目前正在评估区分这些并发症的方法（见下文）。我们正在继续开发一种新的模型，在体内对心脏进行这些光学研究。2)我们完成了在再灌注事件发生前不同程度缺血心脏的全3D高分辨率电子显微镜屏幕。这些研究表明，线粒体网的降解是高度异质性的形态反应，而线粒体网的降解是我们实验室先前描述的心脏能量分布的关键。这些数据提示线粒体网的退化可能是再灌注损伤的早期损伤事件之一。3)与Murphy博士合作，我们开发了一个系统，使用RHOD2作为完整灌注心脏中线粒体游离钙的荧光指示器，允许监测RHOD2的荧光，同时跟踪心脏壁对光的一次和二次过滤。这是通过在同一颗心脏中交替进行白光透射研究和532nm激光荧光研究来完成的。这允许校正初级和次级光学过滤的荧光数据，一直是一个主要问题，在过去的完整组织研究。通过这种方法，我们发现了一个令人惊讶的现象，即当假定的主要钙转运体之一MCU被敲除时，线粒体钙不会增加，但对激素刺激的代谢和功能反应不变。这一结果表明，心脏负荷的适度增加并不需要线粒体内钙的变化。这一发现表明，目前心肌能量调节的模型之一是线粒体基质钙。我们目前正在扩大这种方法，以监测再灌注条件下心脏中活性氧的产生。4)为了扩大我们对线粒体代谢调节的分析，我们扩大了我们的研究范围，研究了线粒体的祖先——简单的细菌。我们最近完成了对被认为最接近线粒体起源的分离细菌的初步研究，反硝化副球菌（PD）。这些研究的目标是揭示这种细菌的急性能量转换调节，然后在哺乳动物线粒体中寻找类似的机制。随着人们对微生物组的兴趣日益浓厚，这些研究也应该为尚未广泛研究的细菌能量代谢的急性调节提供新的见解。在此期间，我们证明了先前描述的哺乳动物心脏的代谢稳态，即ATP和线粒体质子动机在负荷增加时的恒定自由能，存在于PD中。即PD能使其代谢率提高8倍以上，保持质子动力不变，增加ATP含量。目前，我们正在专门寻找底物氧化的酶途径，这是由细菌的工作调节的。希望这个简单的系统将为哺乳动物线粒体氧化磷酸化调控的分子机制提供深入的见解。研究能量代谢控制等生理过程的另一种经典方法是观察极端情况。在哺乳动物中，鼩鼱的静息代谢率最高，静息心率接近1000次/分钟。我们正在研究鼩鼱，希望了解这种动物是如何适应这种极端的代谢率的，这将有助于我们了解人类的状况。在西方健康科学大学的尼萨尔·达马尼博士的帮助下，我们成功地建立了世界上两个隐球菌鼩群落中的一个。我们已经完成了这种动物的基因组，其器官系统的蛋白质组学筛选以及心脏，肝脏，肾脏和骨骼肌的线粒体/组织结构的3D FIBSEM检查。这些研究揭示了在有氧骨骼肌中观察到的最广泛的线粒体网系统，而心脏结构似乎相对保守，甚至与大型动物相比。最显著的影响是在肝脏中，那里的线粒体含量基本上与心脏相当，这表明这种小动物肝脏的主要代谢能力在大型物种中不存在。这导致了一个关于哺乳动物组织中线粒体分布的新假设。在线粒体和能量利用系统可以共存的地方，如肝脏和肾脏基底外膜的内质网，线粒体被划分以满足局部需要。在像横纹肌这样需要高度分散能量的组织中，线粒体网是向几乎整个细胞输送能量所必需的。这一假说可以解释不同组织中线粒体形态的差异。5)与美国国立卫生研究院的众多项目以及与西门子医疗系统的合作，我们开发了一种低场0.55T MRI系统，用于监测人类的生理和结构功能。除了低成本，良好的图像质量和对比度以及能够在没有相关射频加热的情况下使用介入设备外，我们还利用该磁场来评估气态氧作为MRI造影剂，允许评估肺通气，向心脏输送氧气以及与血流测量相结合时心脏的潜在相对耗氧量。

The purpose of these studies is to establish a better understanding of the energy metabolism of biological tissues using modern system biology approaches. Towards this goal, the laboratory concentrates on the use of screening approaches in proteomics, metabolomics, protein structure, post-translational modifications, minimally invasive metabolic imaging information and optical spectroscopy. One of the major hypothesizes in this program is that the activity of the multi-protein Complexes that perform Oxidative Phosphorylation are coordinated in some fashion to balance the rate of ATP production with utilization in the cell. This results in the observed metabolic homeostasis where the potential energy for doing work is maintained near constant in the cell even during major alterations in workload. The following major findings were made over the last year: 1)We have expanded our transmission optical spectroscopy investigation of the functioning of mitochondria in the intact beating heart to focus on ischemic reperfusion in the mouse heart model working with the MRC group at Cambridge University. We have also validated the use of the chromophores of Complex III of oxidative phosphorylation to provide a non-invasive determination of mitochondrial membrane potential in the beating heart. Using this approach we have established the dynamics of the mitochondrial membrane potential and overall redox poise of the mitochondria during ischemia, contraction and reperfusion under a variety of conditions. Initial findings suggest a rapid recovery of the mitochondria membrane potential at reperfusion that could contribute to both reactive oxygen species generation as well as the accumulation of Ca in the mitochondria matrix. Both of these events could contribute to mitochondria damage during the reperfusion phase and methods to differentiate these complications are being evaluated (see Below). We are continuing to develop a new model to perform these optical studies on the heart, in vivo. 2) We have completed a full 3D high resolution electron microscopy screen on hearts exposed to different degrees of ischemia before the reperfusion event. These studies demonstrate highly heterogenous morphological responses while a degradation of the mitochondria reticulum, previously described by our lab as critical in energy distribution in the heart. These data may suggest that the degradation of the mitochondria reticulum may be one of the early damage events in reperfusion injury. 3) Working with Dr. Murphy, we have developed a system using RHOD2 as a fluorescent indicator of mitochondrial free calcium in the intact perfused heart that permits the monitoring of the fluorescence from RHOD2 while tracking the primary and secondary filtering of light by the heart wall. This is done by alternating a white light transmission study with a 532nm laser fluorescence study in the same heart over time. This permits the correction of primary and secondary optical filtering of the fluorescent data that has been a major problem in intact tissue studies in the past. Using this approach we have made the surprising observation that mitochondrial calcium does not increase when one of the putative major calcium transports, MCU, is knocked out but the metabolic and functional response to hormonal stimulation is unchanged. This result demonstrates that changes in intramitochondrial calcium are not required for modest increases in workload in the heart. This finding suggests that one of the current models of cardiac energy regulation by mitochondria matrix calcium. We are currently expanding this approach to monitor reactive oxygen species generation in the heart under reperfusion conditions. 4) To broaden our analysis of metabolic regulation in the mitochondria we have expanded our studies to study the ancestors of mitochondria, simple bacteria. We have recently completed our initial studies on isolated bacteria believed to be closest to the mitochondrial origins, paracoccus denitrificans(PD). The goal of these studies is to unravel acute energy conversion regulation in this bacterium and then look for similar mechanisms in mammalian mitochondria. With the growing interest in the microbiome, these studies should also provide new insight into the acute regulation of bacterial energy metabolism that has not been extensively studied. We demonstrated in this period that the previously described metabolic homeostasis described in the mammalian heart, that is constant free energy available in ATP as well as the mitochondria proton motive during increases is workload, exists in PD. That is PD can increase it metabolic rate by over 8 fold and maintain constant proton motive force as well as increase ATP content. Currently we are specifically looking for the enzymatic pathways in substrate oxidation that are regulated by work in the bacterium. It is hoped that this simple system will provide insight into the molecular mechanisms involved in the regulation of oxidative phosphorylation in mammalian mitochondria. 4) Another classical approach to studying physiological process like the control of energy metabolism, is to look at extreme cases. In mammals, the shrew represents one of the highest resting metabolic rates with a resting heart rate approaching 1000 beats/min. We are studying the shrew with hopes of understanding how this animal has adapted to these extreme metabolic rates will provide insight into the human condition. We have successfully established one of the two Cryptotis parva shrew colonies in the world with the help of Dr. Nissar Darmani at Western University of Health Sciences. We have completed the genome of this animal, a proteomic screen of its organ systems as well as a 3D FIBSEM examination of the mitochondria /tissue structure in heart, liver, kidney and skeletal muscle. These studies reveal the most extensive mitochondria reticulum system yet observed in the aerobic skeletal muscle while the heart structure seems relatively conserved even in comparison to larger animals. The most dramatic effect was in the liver where the mitochondria content is essentially equivalent to the heart, suggesting a major metabolic capacity in this small animals liver not present in larger species. This has led to a new hypothesis with regard to the distribution of mitochondria in mammalian tissues. Where the mitochondria and energy utilizing systems can be co-located such as the ER in liver and basolateral membrane of the kidney, the mitochondria are compartmentalized to meet the local needs. In tissues that have highly diffuse energy needs such as striated muscle, a mitochondria reticulum is necessary to deliver energy over nearly the entire cell. This hypothesis may explain the variance in mitochondria morphology in different tissues. 5) Together with numerous programs at the NIH and collaboration with Siemens Medical Systems we have developed a low field 0.55T MRI system for monitoring physiological and structural function in humans. In addition to its low cost, good image quality and contrast as well as the ability to tolerate the use of interventional devices without associated radiofrequency heating, we have exploited this magnetic field to evaluate gaseous oxygen as a MRI contrast agent permitting the evaluation of lung ventilation, delivery of oxygen to the heart and potentially the relative oxygen consumption by the heart when combined with blood flow measures.