Role of mitochondria in cardiac protection

描述（由申请人提供）：尽管许多研究确定了线粒体通透性转换（MPT）-一种跨越基质至细胞溶质的非选择性孔的打开，导致线粒体功能障碍和细胞死亡-在缺血性损伤中的作用，但控制调节MPT（即MPT孔）的拟议多蛋白复合物的调节的离散机制仍然定义不清。两种蛋白质，电压依赖性阴离子通道（VDAC）和腺嘌呤核苷酸移位酶（ANT）已被牵连作为核心孔形成单位的MPT孔凭借其独立的能力，形成非选择性通道，以响应与缺血/再灌注损伤相关的应力。本申请的重点是确定这些分子在保护心肌缺血损伤中的基本作用。 先前的研究已经将VDAC和ANT定位于线粒体外膜和内膜之间的接触位点。靶向VDAC和ANT的药理学干预显示出在细胞和细胞器环境中对MPT的有效调节。尽管如此，亚细胞器的分布和异构体特异性功能的不同VDAC和ANT异构体的关键了解MPT的调节机制，几乎没有探索，特别是在心脏的研究。我们最近的初步研究结果表明，有针对性地抑制VDAC足以保护心脏免受缺血性损伤，ANT诱导的孔形成阻断了药理学和遗传性心脏保护方案的保护作用。此外，我们的初步数据首次证明，VDAC（VDAC 1 -3）和ANT（ANT 1 -2）的多种亚型在成年小鼠心脏中表达，这两种免疫印迹法都检测到了（使用自最初提交以来我们开发的抗体）和LC/MS/MS。这些发现非常令人兴奋，因为它们消除了广泛持有的信念，即由心肌VDAC和ANT执行的所有动作都可以归因于VDAC 1和ANT 1。 此A1应用程序已完全修改，以回应审查小组的批评。在我们的新数据以及该领域的其他发展的刺激下，修订后的提案解决了一些对MPT在心肌缺血性损伤中的作用至关重要的关键问题。我们的中心假设是VDAC和ANT的同种型以不同的方式参与MPT现象，部分原因是它们的差异亚细胞器定位、孔形成特性以及与调节蛋白（例如，PKC（Bcl-2和Bax）。我们将定义VDAC和ANT的不同亚型在正常心脏和心脏保护过程中的亚细胞器分布，并将严格阐明这些分子在重构脂质体形式中响应缺血/再灌注损伤期间存在的已知有害因素（包括ROS和Ca 2+）的成孔特性。我们将使用共聚焦和电子显微镜检查MPT孔亚蛋白质组的关键组分之间的表达，定位和相互作用。最后，我们将使用质谱描绘MPT孔组分的翻译后修饰，并询问这些修饰对调节MPT的作用。这些研究将提供新的和机制的信息，以帮助我们了解心肌缺血损伤中的线粒体功能。

DESCRIPTION (provided by applicant): Despite many investigations establishing the role of mitochondrial permeability transition (MPT)-an opening of non-selective pores spanning the matrix to the cytosol that leads to mitochondrial dysfunction and cell death-in ischemic injury, the discrete mechanisms governing modulation of a proposed multiprotein complex that regulates MPT (i.e. the MPT pore), remain poorly defined. Two proteins, the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT) have been implicated as the core pore-forming units of the MPT pore by virtue of their independent abilities to form non-selective channels in response to stresses associated with ischemia/reperfusion injury. The focus of this application is to define the fundamental roles of these molecules in protection against myocardial ischemic insult. Previous studies have mapped both VDAC and ANT localization to contact sites between outer and inner mitochondrial membranes. Pharmacological interventions targeting VDAC and ANT show effective modulation of MPT in cell and organelle settings. Despite this, the sub-organelle distribution and isoform-specific function(s) of distinct VDAC and ANT isoforms-critical to understand the regulatory mechanisms of the MPT-have been virtually unexplored, especially in cardiac research. Our recent preliminary findings suggest that targeted inhibition of VDAC is sufficient to protect the heart against ischemic insult and that induction of pore-formation by ANT blocks the protective effects of pharmacologic and genetic cardioprotective regiments. Furthermore, our preliminary data demonstrate for the first time that multiple isoforms of VDAC (VDAC1-3) and ANT (ANT1-2) are expressed in the adult mouse heart as detected by both immunoblotting (using antibodies we developed since the original submission) and LC/MS/MS. These findings are very exciting, as they dispel the widely-held belief that all actions performed by myocardial VDAC and ANT can be attributed to VDAC1 and ANT1. This A1 application has been completely revised in response to critiques by the review panel. Stimulated by our new data as well as other developments in the field, the revised proposal addresses a number of key questions that are critical to the role of MPT in myocardial ischemic injury. Our central hypothesis is that isoforms of VDAC and ANT participate in distinct manners in the phenomenon of MPT, in part owing to their differential suborganellar localization, pore-forming properties, and interactions with regulatory proteins (e.g., PKC(, Bcl-2, and Bax). We will define the sub-organelle distribution of distinct isoforms of VDAC and ANT in the normal heart and during cardioprotection and will rigorously elucidate the pore-forming properties of these molecules in the reconstituted liposome format in response to known injurious factors present during ischemia/reperfusion injury (including ROS and Ca2+). We will examine the expression, localization and interactions among critical components of the MPT pore subproteome using confocal and electron microscopy. Lastly, we will delineate post-translational modification of MPT pore components using mass spectrometry and interrogate the role of these modifications to regulate MPT. These studies will provide novel and mechanistic information to aid our understanding of mitochondrial function in myocardial ischemic injury.