Project 1:Diabetes, RAGE/DIAPH1 and Myocardial Infarction

项目摘要：项目1 心血管疾病(CVD)，包括糖尿病(DM)心肌对缺血损伤的敏感性增加， 是糖尿病患者发病和死亡的主要原因。我们发现了关键角色 糖尿病和心肌组织中晚期糖基化终产物受体(RAGE-基因符号Ager) 缺血/再灌注(I/R)，由于Ager的整体缺失减轻了心肌损伤，减少了氧化应激， 与野生型(WT)产仔相比，提高了ATP的功能恢复和保存。机制，由 哪种Ager缺失在糖尿病和非糖尿病(NDM)I/R心脏中提供代谢和功能保护 成为这个项目的焦点。我们的程序发现RAGE胞质结构域与 RhoGTP酶的效应因子Diaphanous-1(DIAPH1)是RAGE配体介导的细胞迁移所必需的 和CDC42/RAC-1的激活。我们最近的研究表明，DIAPH1在心肌细胞中表达 (CMS)和I/R增加DIAPH1的表达。关键的初步数据显示梗死面积和 (A)WT小鼠移植Ager缺失骨髓来源细胞后功能恢复的改善 小鼠，(B)CM特异缺失Ager/DIAPH1的小鼠，以及(C)用条件培养液灌流的WT小鼠心脏 来自Ager零或DIAPH1零的巨噬细胞(MΦS)。细胞质雄性不育和MΦS的新发现揭示DIAPH1相互作用 线粒体GTP酶丝裂原丝裂原2(MFn2)和增强MFn2‘S肌浆网系留(SR) 到线粒体(Mito)，继而mito钙调节。重要的是，我们的数据显示，高血糖， RAGE的配体以及RAGE-DIAPH1的相互作用进一步增强了DIAPH1-Mfn2驱动的MITO-SR系链。 综上所述，这些数据使我们假设RAGE/DIAPH1介导DIAPH1-Mfn2驱动的MITO- SR相互作用、钙调节改变、细胞死亡信号与I/R心脏代谢功能障碍 通过M-ΦS的细胞内机制，并通过M-Φ-CM串扰。探索综合机制。 在没有糖尿病和存在糖尿病的情况下，使用小鼠模型对I/R引起的心脏应激进行研究。我们将聘用 新型Ager和DIAPH1牙线小鼠，RAGE-DIAPH1相互作用的小分子拮抗剂，最新进展 揭示I/R损伤机制的分子技术、蛋白质组学和磁共振波谱 在DM心脏。本项目中提出的研究将确定新的机制MΦ特定的机制，以及 MΦ-CM串扰机制通过RAGE-DIAPH1调节心脏，特别是糖尿病心脏的I/R损伤。 鉴定这些机制，并测试阻断RAGE/DIAPH1的新型小分子 相互作用，将为治疗干预措施保护糖尿病心脏免受I/R损伤铺平道路。项目1将 与项目2和3以及两个核心密切合作，以实现这些目标。

Project Summary: Project 1 Cardiovascular disease (CVD), that include increased sensitivity of diabetic (DM) myocardium to ischemic injury, represents the major cause of morbidity and mortality in patients with diabetes. We have uncovered key roles for the receptor for advanced glycation end products (RAGE -gene symbol Ager) in DM and myocardial ischemia/reperfusion (I/R), as global deletion of Ager attenuated myocardial injury, reduced oxidative stress, increased functional recovery and preservation of ATP compared to wild-type (WT) littermates. Mechanisms by which Ager deletion confers metabolic and functional protection in DM and non-DM (NDM) I/R hearts will be the focus of this project. Our program discovered that the RAGE cytoplasmic domain interacts with diaphanous-1 (DIAPH1), an effector of RhoGTPases, is essential for RAGE ligand-mediated cellular migration and activation of cdc42/rac-1. Our recent studies demonstrated that DIAPH1 is expressed in cardiomyocytes (CMs) and that I/R increases expression of Diaph1. Key preliminary data reveal reduced infarct size and improved functional recovery after I/R in (a) WT mice transplanted with bone marrow derived cells from Ager null mice,(b) mice with CM specific deletion of Ager/Diaph1, and (c) WT mice hearts perfused with conditioned media from Ager null or Diaph1 null macrophages (MΦs). Novel findings in CMs and MΦs reveal that DIAPH1 interacts with mitochondrial GTPase mitofusin2 (MFN2) and augments MFN2's tethering of sarcoplasmic reticulum (SR) to mitochondria (mito) and consequently mito calcium regulation. Importantly, our data shows that high glucose, ligands of RAGE, and RAGE-DIAPH1 interaction further augments DIAPH1-MFN2 driven mito-SR tethering. Taken together, these data led us to hypothesize that RAGE/DIAPH1 mediates DIAPH1-MFN2 driven Mito- SR interactions, altered calcium regulation, cell death signaling and metabolic dysfunction in I/R hearts by cell intrinsic mechanisms in MΦs, and via MΦ-CM cross-talk. We will probe comprehensive mechanisms in cardiac stresses evoked by I/R using murine models, both in the absence and presence of DM. We will employ novel Ager and Diaph1 floxed mice, small molecule antagonists of RAGE-DIAPH1 interaction, state-of-the-art molecular techniques, proteomics, and magnetic resonance spectroscopy to uncover mechanisms of I/R injury in DM hearts.Proposed studies in this project will identify novel mechanism MΦ specific mechanism, as well as MΦ-CM cross talk mechanisms by which RAGE-DIAPH1 modulates I/R injury in hearts, particularly in DM hearts. Identification of these mechanisms, along with testing of novel small molecules that block RAGE/DIAPH1 interaction, will pave the way for therapeutic interventions to protect DM hearts from I/R injury. Project 1 will work closely with Projects 2 and 3 and the two Cores to achieve these goals.