The mitochondrial permeability transition and heart failure

描述(申请人提供)：线粒体在能量产生和细胞死亡途径中都是核心的，然而关于线粒体结构-功能关系的各种问题仍然知之甚少。线粒体外膜(MOM)不仅仅是将胞浆与内膜隔开的被动屏障。它是整合代谢和细胞信号事件的场所，并提供了一种划分离散代谢状态的方法。申请人的实验室一直在使用分子遗传学方法研究MOM通透性的功能，以了解被称为电压依赖阴离子通道(VDAC)的高度丰富的通道蛋白小家族的功能，以及它们如何通过参与细胞凋亡而整合到细胞的生物能量学和细胞生存中。与这一提议相关的有两个方面：VDAC2直接与多结构域促凋亡蛋白BAK相互作用，大量证据表明VDACs是线粒体通透性转换孔(MPTP)的组成部分，是一种由钙超载和其他刺激激活的不完全特征的大电导通道。我们假设VDAC2和MPTP相互作用，并在维持心功能和细胞死亡中发挥重要作用。我们有初步的证据支持这一概念，并有兴趣通过检测VDAC2在以心脏为模型器官的突变小鼠中功能丧失和过度表达的体内后果来追求这一概念。我们的具体目标是使用基因敲除和转基因小鼠来确定VDAC2丢失和过度表达的后果，使用缺血-再灌注(I/R)损伤模型，因为众所周知，I/R涉及内源性和外源性凋亡途径。我们将通过培育VDAC2突变体到BAK或Bax缺陷小鼠、Bcl-2过表达小鼠和缺乏通透性转换孔的其他成分，特别是亲环素D的小鼠，从遗传学上测试这在多大程度上是由促和抗凋亡蛋白以及MPTP介导的。由于线粒体功能障碍是心力衰竭的一个特征，这些研究将描绘MOM通道蛋白在此疾病过程中的机制作用。心力衰竭的特征是由多种应激源引起的进行性细胞死亡。导致细胞死亡的机制正在被阐明，线粒体在这一过程中处于中心地位。这一应用旨在提供关于线粒体外膜(MOM)通透性在细胞的生物能量学和细胞通过其在细胞凋亡中的作用而存活的功能的体内数据。通透性由电压依赖性阴离子通道(VDAC)这一通道蛋白家族决定。我们先前已经证明，VDAC2的异构体直接与多域的促凋亡蛋白BAK相互作用，从而抑制其促凋亡功能。本申请提供的最新数据表明，VDAC2的表达与BAK和Bax的表达之间存在相互作用的关系。VDAC2的缺失导致BAK从MOM重新分布到内质网。此外，有大量证据表明，VDAC是线粒体通透性转换孔(MPTP)的组成部分，MPTP是一种由钙超载和其他刺激激活的不完全特征的大电导通道。由于已知MPTP的另一个重要成分亲环素D(CypD)的缺失可以在缺血/再灌流模式中保护细胞免于死亡，因此了解VDAC在心脏代谢和细胞死亡方面的功能是很重要的。因此，我们有兴趣通过以心脏为模型器官在突变小鼠中检测VDAC2功能丧失和过度表达的体内后果来追求这一点。我们的目的是使用我们培育的基因敲除和转基因小鼠来确定VDAC2基因缺失和过度表达的后果，并使用缺血再灌注(I/R)损伤模型。我们将通过将VDAC2突变体培育到BAK或Bax缺陷小鼠、Bcl2过表达小鼠和缺乏通透性转换孔其他成分的小鼠，特别是亲环素D的小鼠，从基因上测试这是由促和抗凋亡蛋白以及MPTP介导的程度。我们将从生物能量学和关于自发和诱导的凋亡易感性方面对这些突变菌株进行表征。在这两年的申请的第一年，我们将完成申请中描述的各种突变株的产生，并进行自发细胞凋亡的初步表征。在第二年，我们将通过极谱检测线粒体呼吸和呼吸链酶功能来完成生物能量学研究，并量化对缺血/再灌注的反应。预计这些目标的完成将确定心力衰竭治疗干预的目标。

DESCRIPTION (provided by applicant): Mitochondria are central in both energy production and cellular death pathways, yet a variety of questions concerning structure-function relationships of mitochondria remain poorly understood. The mitochondrial outer membrane (MOM) is more than a passive barrier separating the cytosol from the inner membrane. It is the site for integrating metabolic and cell signaling events, and provides a means for compartmentalizing discrete metabolic states. The applicant's lab has been studying the function of MOM permeability using molecular genetic approaches in order to understand the functions of a small family of highly abundant channel proteins termed Voltage-dependent Anion Channels (VDACs) and how they integrate into both the bioenergetics of the cell and cell survival through their participation in apoptosis. Two aspects are relevant to this proposal: VDAC2 directly interacts with the multi-domain pro-apoptotic protein BAK and considerable evidence suggests that VDACs are a component of the mitochondrial permeability transition pore (MPTP), an incompletely characterized large conductance channel activated by calcium overload and other stimuli. We hypothesize that VDAC2 and the MPTP interact and play an important role in maintenance of cardiac function and cell death. We have preliminary evidence for this concept and are interested in pursuing this by examining the in vivo consequences of loss of function and over-expression of VDAC2 in mutant mice using the heart as a model organ. Our specific aims are to use knockout and transgenic mice to determine the consequences of loss and over-expression of VDAC2 using an ischemia-reperfusion (I/R) injury model, since it is known that I/R involves both intrinsic and extrinic apoptotic pathways. We will genetically test to what degree this is mediated by pro- and anti-apoptotic proteins and the MPTP by breeding the VDAC2 mutants to BAK or BAX deficient mice, Bcl-2 over-expressing mice and mice lacking other components of the permeability transition pore, specifically cyclophilin D. Since mitochondrial dysfunction is a feature of heart failure, these studies will delineate the mechanistic role of MOM channel proteins in this disease process. Heart failure is characterized by progressive cell death arising from multiple stressors. The mechanisms leading to cell death are being elucidated and mitochondria are central in this process. This application is intended to provide in vivo data on the function of mitochondrial outer membrane (MOM) permeability with regard to both the bioenergetics of the cell and cell survival through its role in apoptosis. Permeability is conferred by a family of channel proteins; the Voltage-dependent Anion Channels (VDACs). We have previously demonstrated that the isoform termed VDAC2 directly interacts with the multi-domain pro-apoptotic protein BAK to suppress its pro-apoptotic function. More recent data provided in this application demonstrates that there is a reciprocal relationship between VDAC2 expression and expression of BAK and BAX. Loss of VDAC2 leads to a re-distribution of BAK from the MOM to the endoplasmic reticulum. In addition, there is considerable evidence to suggest that VDACs are a component of the mitochondrial permeability transition pore (MPTP), an incompletely characterized large conductance channel activated by calcium overload and other stimuli. Since it is known that loss of another purported component of the MPTP; cyclophilin D (CypD), confers protection from cell death in an ischemia/reperfusion paradigm, it is important to understand what functions VDACs have in regard to cardiac metabolism and cell death. Hence, we are interested in pursuing this by examining the in vivo consequences of loss of function and over-expression of VDAC2 in mutant mice using the heart as a model organ. Our aim is to use knockout and transgenic mice we have generated to determine the consequences of loss and over-expression of VDAC2 using an ischemia-reperfusion (I/R) injury model. We will genetically test to what degree this is mediated by pro- and anti-apoptotic proteins and the MPTP by breeding the VDAC2 mutants to BAK or BAX deficient mice, Bcl-2 over-expressing mice and mice lacking other components of the permeability transition pore, specifically cyclophilin D. We will characterize these mutant strains both bioenergetically and with regard to susceptibility to apoptosis, both spontaneous and induced. In the first year of this two year application we will complete the generation of the various mutant strains described in the application and carry out the intial characterization of spontaneous apoptosis. In the second year we will complete the bioenergetic studies by examining mitochondrial respiration by polarography and respiratory chain enzymatic function, and quantify the responses to ischemia/reperfusion. It is expected that completion of these goals will identify targets for therapeutic interventions in heart failure.