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，但在这种疾病中导致心肌细胞死亡的分子机制尚不完全清楚。有证据表明，线粒体在调节缺血诱导的细胞损伤中起着重要作用。我们以前证明了线粒体蛋白-线粒体ATP结合盒蛋白-1(MABC1)的过表达可以保护细胞免受氧化剂诱导的细胞死亡，而它的下调则会导致细胞死亡的增加。MABC1的主要功能尚不清楚，因此还不清楚该蛋白是如何对氧化应激起保护作用的。酵母mABC1的同源物Mdl1p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。细胞内铁的积累可能是氧化应激的一个来源，这被认为是Mdl1p保护作用的潜在机制。在这项提案中，我们将继续我们的关键观察结果，即mABC1对氧化损伤具有保护作用，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mABC1是否也可以保护心脏免受缺血损伤。我们的主要假设是，mABC1在维持线粒体铁稳态方面发挥作用，并保护心脏免受缺血性损伤。为了检验这一假设，我们提出了三个相互关联的具体目标。在目标1中，我们将确定mABC1是否在维持线粒体铁稳态和产生细胞内活性氧方面发挥作用。在新生大鼠心肌细胞(NRCM)中，mABC1的水平将通过各种分子生物学技术进行调节，随后将测量线粒体铁和细胞内ROS水平。在目标2中，我们将确定mABC1的保护作用是否通过抑制线粒体通透性转换孔(MPTP)来实现。这一通道被认为在氧化剂诱导的细胞死亡中起着关键作用。通过改变NRCM中mABC1的水平，我们将研究mABC1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mABC1的转基因小鼠，然后诱导缺血/再灌注(I/R)。MABC1超表达在L/R诱导的细胞死亡中的作用将通过各种技术来检测。这些研究有望促进我们对缺血性心脏病中心肌细胞死亡的分子机制的了解。此外，成功完成我们的研究将对我们理解线粒体在心血管疾病中的作用产生潜在的影响，并可能为IHD的治疗开辟新的途径。

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.