Role of the mitochondrial ATP-binding cassette protein-1 in cellular protection

描述（申请人提供）：缺血性心脏病（IHD）是发达国家的一种主要流行病，但导致这种疾病中心肌细胞死亡的分子机制尚未完全了解。有证据表明，线粒体在调节缺血诱导的细胞损伤中起重要作用。我们以前证明过表达的线粒体蛋白，线粒体ATP结合盒蛋白-1（mABC 1）保护氧化剂诱导的细胞死亡，而其下调导致细胞死亡在基线增加。mABC 1的主要功能尚不清楚，因此尚不清楚这种蛋白质如何对氧化应激发挥保护作用。mABC 1的酵母同系物Mdl 1 p也被证明对氧化剂诱导的细胞损伤具有保护作用，并在线粒体铁稳态中发挥作用。细胞内铁积累可以是氧化应激的来源，这被认为是Mdl 1 p保护作用的潜在机制。在这项提案中，我们将跟进我们的关键观察，即mABC 1是保护氧化损伤，并建议阐明这一过程的潜在机制。此外，我们将尝试将我们的研究扩展到完整的动物，并确定mABC 1是否也可以保护心脏免受缺血性损伤。我们的主要假设是mABC 1在维持线粒体铁稳态中起作用，并且它可以保护心脏免受缺血性损伤。为了验证这一假设，我们提出了三个相互关联的具体目标。在目的1中，我们将确定mABC 1是否在维持线粒体铁稳态和产生细胞内活性氧中发挥作用。使用各种分子生物学技术调节新生大鼠心肌细胞（NRCM）中mABC 1的水平，然后测量线粒体铁和细胞内ROS水平。在目的2中，我们将确定mABC 1的保护作用是否是通过抑制线粒体通透性转换孔（mPTP）。该通道被认为是氧化剂诱导细胞死亡的关键参与者。通过改变NRCM中mABC 1的水平，我们将研究mABC 1对细胞活力和mPTP开放的影响。在目标3中，我们将制造过表达mABC 1的转基因小鼠，随后诱导缺血/再灌注（I/R）。然后将通过各种技术检查mABC 1过表达对I/R诱导的细胞死亡的影响。这些研究有望推进我们对缺血性心脏病心肌细胞死亡分子机制的认识。此外，我们研究的成功完成将对我们理解线粒体在心血管疾病中的作用产生潜在影响，并可能为IHD的治疗开辟新途径。

DESCRIPTION (provided by applicant): Ischemic heart disease (IHD) is a major epidemic throughout the developed world, yet the molecular mechanisms that lead to myocardial cell death in this disorder are not completely understood. Evidence indicates that the mitochondria play an important role in regulating ischemic-induced cellular injury. We previously demonstrated that overexpression of a mitochondrial protein, the mitochondrial ATP-binding cassette protein-1 (mABC1) protects against oxidant induced cell death, while its downregulation causes an increase in cell death at baseline. The primary function of mABC1 is unknown, thus it is unclear how this protein exerts protection against oxidant stress. The yeast homolog of mABC1, Mdl1p, has also been shown to exert protection against oxidant-induced cellular injury and plays a role in mitochondrial iron homeostasis. Intracellular iron accumulation can be a source of oxidative stress, which is thought to be the underlying mechanism for the protective effects of Mdl1p. In this proposal, we will follow up on our key observation that mABC1 is protective against oxidant injury and propose to elucidate the underlying mechanism for this process. Furthermore, we will attempt to extend our studies to intact animals and determine whether mABC1 can also protect against ischemic injury in the heart. Our main hypothesis is that mABC1 plays a role in the maintenance of the mitochondrial iron homeostasis and that it protects against ischemic damage in the heart. In order to test this hypothesis, we propose three interrelated specific aims. In Aim 1, we will determine whether mABC1 plays a role in the maintenance of mitochondrial iron homeostasis and generation of intracellular reactive oxygen species. The levels of mABC1 will be modulated in neonatal rat cardiomyocytes (NRCM) using various molecular biology techniques, followed by measurement of the mitochondria iron and intracellular ROS levels. In Aim 2, we will determine whether the protective effects of mABC1 are through inhibition of the mitochondrial permeability transition pore (mPTP). This channel is believed to be a pivotal player in oxidant-induced cell death. By altering the levels of mABC1 in NRCM, we will study the effect of mABC1 on cell viability and mPTP opening. In Aim 3, we will make transgenic mice that overexpress mABC1, followed by induction of ischemia/reperfusion (I/R). The effects of mABC1 overexpression on l/R-induced cell death will then be examined by various techniques. These lines of investigation promise to advance our knowledge of the molecular mechanisms of myocardial cell death in ischemic heart disease. Furthermore, successful completion of our studies will have potential impact on our understanding of the role of mitochondria in cardiovascular disease, and may open new avenues for the treatment of IHD.