BCL-XL AND THE HEART; THE METABOLIC BASIS TO CARDIOPROTECTION AND DISEASE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 心脏病和随之而来的心力衰竭导致西方社会的高死亡率。需要更好地了解与这种疾病相关的调节蛋白。在心脏中，抗凋亡因子Bcl-xL在缺血-再灌注期间减弱细胞死亡。这是在单个细胞和整个器官水平上观察到的。 尽管Bcl-xl促存活的机制尚不清楚，但认为Bcl-xl抑制促凋亡因子如Bax和巴克，同时还调节线粒体内膜的ATP-ADP交换。例如，抗凋亡Bcl-2蛋白（其中Bcl-xL是成员）抑制ATP的糖酵解消耗，从而在缺血期间维持ATP库并防止代谢分解。这些蛋白在心肌细胞中的过表达还维持线粒体膜电位，并防止在缺血干预期间线粒体蛋白的释放。 先前对心脏中Bcl-xL的研究集中在线粒体转运动力学、自由基产生和细胞死亡/生长测定。目前缺乏有关Bcl-xL相关代谢变化的信息，这是出乎意料的，因为Bcl-xL直接与与氧化磷酸化调节相关的线粒体膜蛋白相互作用。此外，在心脏保护和/或缺血干预期间，关于Ca 2+稳态的信息有限。已经提出了Bcl-xL对IP 3受体和线粒体Na+-Ca 2+交换的Bcl-xL依赖性调节，并且可能有助于Bcl-xL的拟议心脏保护品质。BRC旨在表征Bcl-xl过度表达对心肌细胞中细胞能量代谢和收缩功能的影响。 将使用自参考氧微传感器评估代谢，该自参考氧微传感器将评估对照和Bcl-xL+肌细胞的氧摄取。此外，将用含有胞质和线粒体荧光素酶构建体的腺病毒共感染肌细胞，以通过荧光素酶/荧光素酶反应评估细胞内ATP浓度。将使用AM酯Ca 2+染料和共聚焦显微镜测量Ca 2+稳态。 BRC的目的是解决Bcl-2蛋白的心脏保护作用是否存在代谢基础。此外，我们的目标是评估Bcl-xL上调过程中的Ca 2+转运和Ca 2+依赖性信号转导，并解决这是否有助于观察到的心脏生物能量学和细胞存活的变化。预计BRC提供的综合技术将产生有关心脏生物能量学的宝贵信息，并确定实施心脏保护的潜在治疗靶点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Heart disease and consequent heart failure contibutes to high mortality in Western society. A greater understanding of the regulatory proteins associated with this disease is required. In heart, the anti-apoptotic factor, Bcl-xL attenuates cell death during ischemia-reperfusion. This is observed at the single cell and whole organ level. Though the pro-survival mechanism of Bcl-xl in unclear, Bcl-xl is thought to inhibit pro-apoptotic factors such as Bax and Bak while also regulating ATP-ADP exchange across the inner mitochondrial membranes. For example, anti-apoptotic Bcl-2 proteins (of which Bcl-xL is a member) inhibits the glycolytic consumption of ATP thereby maintaining the ATP pool during ischemia and preventing metabolic breakdown. Overexpression of these proteins in cardiac myocytes also maintains mitochondrial membrane potential and prevents the release of mitochondrial proteins during ischemic interventions. Prior studies on Bcl-xL in the heart have focused on mitochondrial transport kinetics, free radical production and cell death/growth assays. There is a lack of information concerning the metabolic changes associated with Bcl-xL, unexpected since Bcl-xL interacts directly with mitochondrial membrane proteins associated with the regulation of oxidative phosphorylation. In addition, there is limited information on Ca2+ homeostasis during cardioprotective and/or ischemic interventions. Bcl-xL-dependent modulation of IP3-receptors and mitochondrial Na+-Ca2+ exchange by Bcl-xL has been proposed and may contribute to the proposed cardioprotective qualities of Bcl-xL. The BRC aims to characterize the effect of Bcl-xl overepxression on both cellular energy metabolism and contractile function in cardiac myocytes. Metabolism will be assessed using self-referencing oxygen microsensors that will assess the oxygen uptake of control and Bcl-xL+ myocytes. In addition, myocytes will be co-infected with adenovirus containing constructs for cytosolic and mitochondrial luciferase in order to assess intracellular ATP concentration via the luciferin/luciferase reaction. Ca2+ homeostasis will be measured using AM ester Ca2+ dyes and confocal microscopy. It is the aim of the BRC to address whether there is a metabolic basis to the cardioprotective action of Bcl-2 proteins. In addition, it is our objective to assess Ca2+ transport and Ca2+-dependent signal transduction during Bcl-xL upregulation and to address whether this contributes to the observed changes in cardiac bioenergetics and cell survival. It is anticipated that integrated technologies available at the BRC will generate invaluable information regarding cardiac bioenergetics and identify potential therapeutic targets for the implementation of cardioprotection.