Role of mitochondria in cardiac protection

描述(申请人提供)：尽管许多研究确定了线粒体通透性转换(MPT)在缺血性损伤中的作用，线粒体通透性转换(MPT)是一种跨越基质到胞浆的非选择性孔的打开，导致线粒体功能障碍和细胞死亡，但调节MPT的拟议多蛋白复合体(即MPT孔)的调节机制仍不明确。电压依赖性阴离子通道(VDAC)和腺核苷酸转位酶(ANT)这两种蛋白质被认为是MPT孔的核心成孔单位，因为它们能够独立地形成非选择性通道，以响应与缺血/再灌注损伤相关的压力。本应用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。 以前的研究已经将VDAC和ANT定位于线粒体膜外膜和内膜之间的接触部位。针对VDAC和ANT的药物干预显示了细胞和细胞器环境中MPT的有效调节。尽管如此，不同的VDAC和ANT亚型的亚细胞器分布和同型特异性功能(S)--对于理解MPT的调节机制至关重要--几乎还没有被探索，特别是在心脏研究中。我们最近的初步发现表明，靶向抑制VDAC足以保护心脏免受缺血损伤，而蚂蚁诱导的孔洞形成可阻断药物和遗传心脏保护方案的保护作用。此外，我们的初步数据首次证明了VDAC(VDAC1-3)和ANT(ANT-2)的多种异构体在成年小鼠心脏中表达，通过免疫印迹(使用我们自最初提交以来开发的抗体)和LC/MS/MS检测到。这些发现非常令人兴奋，因为它们消除了人们普遍认为心肌VDAC和ANT执行的所有活动都可归因于VDAC1和ANT1的观点。 针对审查小组的批评意见，本A1申请已完全修改。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关键问题，这些问题对于MPT在心肌缺血损伤中的作用至关重要。我们的中心假设是，VDAC和ANT的异构体在MPT现象中以不同的方式参与，部分原因是它们在细胞器下的定位、成孔特性以及与调控蛋白(如PKC(、Bcl2和Bax))的相互作用。我们将定义不同亚细胞器中不同亚型的VDAC和ANT在正常心脏和心脏保护过程中的分布，并将严格阐明这些分子在重组脂质体形式下的成孔特性，以响应缺血/再灌注损伤中存在的已知损伤因素(包括ROS和钙离子)。我们将使用共聚焦显微镜和电子显微镜研究MPT孔亚组蛋白的表达、定位和关键成分之间的相互作用。最后，我们将利用质谱学描述MPT孔成分的翻译后修饰，并询问这些修饰在调节MPT中的作用。这些研究将为我们了解线粒体在心肌缺血损伤中的功能提供新的机制信息。

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.