Mitochondrial Protection by Hexokinase-II and Akt in the Heart

己糖激酶 II 和 Akt 对心脏的线粒体保护

• 批准号: 8269124
• 负责人: Shigeki Miyamoto
• 金额: $ 38.24万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8269124
• 关键词: 1-Phosphatidylinositol 3-Kinase; Acute; Address; Adult; Agonist; Anterior Descending Coronary Artery; Apoptosis; Binding; Biological Preservation; Blood flow; Cardiac; Cardiac Myocytes; Cell Death; Cell membrane; Cessation of life; Data; Development; Dissociation; G-Protein-Coupled Receptors; Gene Delivery; Genes; Glycolysis; Glycoproteins; Health; Heart; Heart Diseases; Heart failure; Hexokinase 2; Hour; Hydrogen Peroxide; Infarction; Inner mitochondrial membrane; Insulin-Like Growth Factor I; Intervention; Ischemia; Knockout Mice; Left; Measures; Mediating; Mediator of activation protein; Mitochondria; Modeling; Modification; Molecular; Mus; Muscle Cells; Mutate; Necrosis; Neonatal; Oxidative Stress; Pathway interactions; Peptides; Permeability; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Play; Post-Transcriptional Regulation; Protein Kinase; Publishing; Rattus; Receptor Protein-Tyrosine Kinases; Recruitment Activity; Regulation; Reperfusion Injury; Reperfusion Therapy; Reporting; Research; Role; Signal Transduction; Signaling Molecule; Site; Small Interfering RNA; Stress; Testing; Time; Transfection; Ventricular; Work; cancer cell; cell type; cellular targeting; gain of function; in vivo; in vivo Model; intervention effect; leukemia inhibitory factor; mitochondrial dysfunction; mitochondrial permeability transition pore; mutant; novel; prevent; pro-apoptotic protein; programs; protective effect; research study; response

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. PUBLIC HEALTH RELEVANCE: Cardiomyocyte death plays a crucial role in heart disease and opening of the permeability transition pore (PT-pore) in mitochondria is a major executor of cell death. The protein kinase Akt provides strong survival signals in cardiomyocytes and the mechanisms by which Akt prevents cell death have not been fully elucidated. This proposal examines the hypotheses that activated Akt translocates to mitochondria and provides acute cardioprotection via its ability to phosphorylate hexokinase-II (HK-II) and that this protective signaling is regulated by a Akt phosphatase at mitochondria.

描述(申请人提供)：蛋白激酶Akt在心肌细胞中提供强大的生存信号，这既是通过改变的基因编程实现的，也是通过尚未完全阐明的机制实现的。这一建议验证了激活的Akt移位到线粒体并通过其磷酸化己糖激酶II(HK-II)并增强HK-II与通透性转换孔(PT孔)开放的联系和抑制而提供急性心脏保护的假说。我在已发表的工作中产生的数据显示，在白血病抑制因子(LIF)急性激活时，Akt移位到线粒体，磷酸化HK-II，并阻止H202和钙离子诱导的新生大鼠心肌细胞或成年小鼠心脏线粒体PT孔的开放。Akt介导的保护性反应因HK-II的解离而受损。本提案的目的1旨在确定在Akt介导的抗PT孔开放的保护中对线粒体HK-II及其磷酸化的要求。这将通过功能丧失和功能获得的方法进行测试，在这种方法中，线粒体HK-II被HK-II解离肽减少，并通过腺病毒表达WT HK-II来增加，WT HK-II是一种突变的HK-II，缺乏其线粒体结合基序或激酶Dead HK-II。对HK-II磷酸化的需求将通过使用突变的HK-II来测试，以防止或模拟其假定的Akt磷酸化位点的磷酸化。为了将这些发现推广到成人心脏，Aim#2将使用朗恩多夫灌流心脏模型和活体检测线粒体Akt和HK-II的调节以及HK-II的磷酸化。在有或无IGF-1存在或不存在的情况下，我们将测试HK-II从线粒体解离和TAT融合HK-II和突变体进入心脏的效果，以观察它们对缺血/再灌注(I/R)损伤的影响。在目标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的活性，从而调节线粒体HK-II/PT-孔的打开。这将通过敲除PHLPP(使用siRNA和PHLPP基因敲除小鼠)和通过腺病毒表达增加PHLPP-1的表达来检验。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，并将为抑制线粒体诱导的心肌细胞死亡提供新的干预位点。公共卫生意义：心肌细胞死亡在心脏病中起着至关重要的作用，线粒体通透性转换孔(PT孔)的打开是细胞死亡的主要执行者。蛋白激酶Akt在心肌细胞中提供强大的生存信号，Akt阻止细胞死亡的机制尚未完全阐明。这项建议考察了激活Akt转位到线粒体并通过其磷酸化己糖激酶-II(HK-II)提供急性心脏保护的假设，以及这种保护信号受线粒体Akt磷酸酶调控。