S1P, Rho and Akt signaling in cardiomyocyte survival

G蛋白偶联受体的一个亚类是细胞增殖、基因表达和细胞凋亡的高效介导剂。 和细胞骨架重排。这些受体的特征是它们强烈耦合的能力， 通过G12/13蛋白，激活低分子量蛋白RhoA。这些的配体 受体包括溶血磷脂鞘氨醇-1-磷酸（S1 P）和溶血磷脂酸，以及 如凝血酶、血栓素A-2和各种趋化因子。一个统一的概念是，这些GPCR激动剂是 炎症介质，由响应于应激或损伤而激活的细胞形成或释放。 这些配体对细胞的作用可以是保护性的，也可以是有害的，这取决于所涉及的细胞类型 以及刺激的急性与慢性性质。我们假设这些GPCR的急性效应， 通过RhoA的激活和Akt的伴随激活， 心肌细胞目标#1中提出的研究将检查S1 P在缺血再灌注中的保护作用 在离体灌注心脏和心肌细胞中。保护作用将归因于激活 使用S1 P受体敲除（KO）小鼠的特异性S1 P受体亚型。RhoA，Akt， eNOS和NF κ B作为下游保护介质将在心肌细胞中从S1 P受体 和KO小鼠，并通过使用这些信号通路的抑制剂的研究。目标#2通过以下方式定义机制： RhoA介导心脏保护作用并影响细胞存活。拟议的研究将确定是否 心肌细胞中RhoA激活或过表达诱导NF κ B及其随后的变化。 下游早期基因靶标包括IAP、TNF α和IL-6家族细胞因子。转基因小鼠 可诱导的心脏RhoA表达将用于将RhoA活性的变化与心脏保护直接关联 或细胞凋亡并探索其潜在机制。目标3中提出的研究检验了这一假设 Akt通过影响线粒体功能而引起急性心脏保护。激动剂和 研究了激活Akt以诱导心脏保护的干预措施，重点是Akt对心肌细胞的作用。 线粒体渗透性转换（PT）孔和Akt直接与细胞膜组分相互作用的能力。 PT孔。使用荧光指示剂和基于FRET的Akt报告基因的生物化学和单细胞研究 被提议。Akt与己糖激酶II（HK-II）相互作用并磷酸化调节PT的可能性 特别检查孔活性。了解S1 P通过RhoA激活的生存途径 Akt可以提供一种阻止缺血性损伤引起的心肌细胞死亡的手段， 随后引发炎症和重塑。早期干预专门针对这些 因此，心肌细胞保护途径可用于预防心力衰竭的发展。

A subset of G-protein coupled receptors are highly effective mediators of cell proliferation, gene expression and cytoskeletal rearrangement. What characterizes these receptors is their ability to strongly couple, through G 12/13 proteins, to activation of the low molecular weight protein RhoA. The ligands for these receptors include the lysophospholipids sphingosine-1-phosphate (S1P) and lysophosphatidic acid, as well as thrombin, thromboxane A-2 and various chemokines. A unifying concept is that these GPCR agonists are inflammatory mediators, formed or released from cells that are activated in response to stress or injury. Effects of these ligands on cells can be either protective or deleterious depending on the cell type involved and the acute vs chronic nature of the stimulation. We hypothesize that the acute effects of these GPCRs, through activation of RhoA and concomitant activation of Akt, subserve a protective function in cardiomocytes. Studies proposed in Aim #1 will examine protective effects of S1P in ischemia reperfusion in vivo, in isolated perfused hearts and in cardiomyocytes. Protective effects will be attributed to activation of specific S1P receptor subtypes using S1P receptor knockout (KO)mice. The involvement of RhoA,Akt, eNOS and NFKB as downstream protective mediators will be assessed in cardiomyocytes from S1P receptor and KO mice, and by studies using inhibitors of these signaling pathways. Aim #2 defines mechanisms by which RhoA mediates cardioprotection and affects cell survival. Proposed studies will determine whether RhoA activation or overexpression in cardiomyocytes induces NFKB and consequent changes in its downstream early gene targets including lAPs, TNFa and 1L-6 family cytokines. Transgenic mice with inducible cardiac RhoA expression will be used to directly relate changes in RhoA activity to cardioprotection or apopotosis and explore the underlying mechanisms. Studies proposed in Aim #3 examine the hypothesis that Akt causes acute cardioprotection via effects on mitochondrial function. The ability of agonists and interventions that activate Akt to induce cardioprotection is investigated, focusing on Akt effects on the mitochondrial permeability transition (PT)pore and the ability of Akt to interact directly with components of the PT pore. Biochemical and single cell studies using fluorescent indicators and FRET based Akt reporters are proposed. The possibility that Akt interacts with and phosphorylates hexokinase II (HK-II) to regulate PT pore activity is specifically examined. Understanding the survival pathways actived by S1P through RhoA and Akt could provide a means of arresting myocyte death resulting from ischemic injury, and the subsequent initiation of inflammation and remodeling. Early intervention specifically targeted at these cardiomyocyte protective pathways could therefore be useful in preventing development of heart failure.