Mitochondrial Protection by Hexokinase-II and Akt in the Heart

描述（由申请人提供）：Akt蛋白激酶在心肌细胞中提供强大的生存信号，既可以长期通过改变基因编程，也可以更强烈地通过尚未完全阐明的机制。本研究验证了活化的Akt易位到线粒体并通过磷酸化己糖激酶- ii （HK-II）、增强HK-II与通透性过渡孔（PT-pore）开放的关联和抑制的能力提供急性心脏保护的假设。我在发表的工作中产生的数据表明，在白血病抑制因子（LIF）的急性激活下，Akt易位到线粒体，磷酸化HK-II并阻止新生大鼠心室肌细胞或成年小鼠心脏分离的线粒体中H202和Ca2+诱导的pt孔打开。Akt介导的保护反应因HK-II的分离而受损。本提案的目的1寻求在akt介导的对pt孔打开的保护中建立线粒体HK-II及其磷酸化的需求。这将使用功能的丧失和获得方法进行测试，其中线粒体HK-II被HK-II解离肽降低，并通过WT HK-II的腺病毒表达增加，WT HK-II是一种突变的HK-II，缺乏其线粒体结合基元或激酶死亡的HK-II。对HK-II磷酸化的需求将通过使用HK-II突变来阻止或模拟其假定的Akt磷酸化位点的磷酸化来进行测试。为了将这些发现扩展到成人心脏，Aim #2将使用Langendorff灌注心脏模型和体内研究线粒体Akt和HK-II的调控以及HK-II的磷酸化。在IGF-1存在或不存在的情况下，或在体内，HK-II与线粒体分离以及tat融合HK-II和突变体进入心脏对灌注心脏缺血/再灌注（I/R）损伤的影响将被测试。在Aim #3中，我将确定最近发现的Akt-磷酸酶PHLPP-1是否定位于线粒体并调节线粒体Akt活性，从而调节线粒体HK-II/ pt孔打开。这将通过敲除PHLPP（使用siRNA和敲除PHLPP的小鼠）和通过腺病毒表达增加PHLPP-1表达来检验。这些实验将为HK-II作为Akt的潜在下游靶点和PHLPP作为Akt的潜在调节因子提供证据，并将为抑制I/R诱导的线粒体介导的心肌细胞死亡提供新的干预位点。

DESCRIPTION (provided by applicant): The protein kinase Akt provides strong survival signals in cardiomyocytes, both chronically through altered gene programming, and more acutely through mechanisms that have not been fully elucidated. This proposal examines the hypothesis that activated Akt translocates to mitochondria and provides acute cardioprotection via its ability to phosphorylate hexokinase-II (HK-II) and enhance HK-II association with and inhibition of permeability transition pore (PT-pore) opening. Data I have generated in my published work show that Akt translocates to mitochondria upon acute activation by leukemia inhibitory factor (LIF), phosphorylates HK-II and prevents H202 and Ca2+induced PT-pore opening in neonatal rat ventricular myocytes or mitochondria isolated from adult mouse heart. Protective responses mediated by Akt are impaired by dissociation of HK-II. Aim #1 of this proposal seeks to establish a requirement for mitochondrial HK-II and its phosphorylation in Akt-mediated protection against PT-pore opening. This will be tested using loss and gain of function approaches in which mitochondrial HK-II is decreased by an HK-II dissociating peptide, and increased by adenoviral expression of WT HK-II, a mutated HK-II lacking its mitochondrial binding motif or kinase dead HK-II. The requirement for HK-II phosphorylation will be tested by using HK-II mutated to either prevent or mimic phosphorylation at its putative Akt phosphorylation site. To extend these findings to the adult heart, Aim #2 will examine regulation of mitochondrial Akt and HK-II and the phosphorylation of HK-II using Langendorff perfused heart model and in vivo. The effects of HK-II dissociation from mitochondria and of TAT-fusion HK-II and mutants into the heart will be tested for their effects on ischemia/reperfusion (I/R) damage in perfused heart in the presence or absence of IGF-1 or in vivo. In Aim #3, I will determine whether the recently discovered Akt-phosphatase, PHLPP-1, localizes to mitochondria and regulates mitochondrial Akt activity and thus mitochondrial HK-II/PT-pore opening. This will be examined by knockdown of PHLPP (using siRNA and PHLPP knock-out mice) and by increasing PHLPP-1 expression via adenoviral expression. These experiments will provide evidence for HK-II as a potential downstream target of Akt, and PHLPP as a potential regulator of Akt, and will suggest novel sites of intervention for inhibiting I/R induced mitochondria mediated cardiomyocyte cell death.