Ventricular Remodeling in the Adapted Heart

描述(申请人提供)：心肌梗死(MI)后，以左心室(LV)扩张、收缩功能障碍、心肌细胞肥大和基质蛋白形成增加为特征的进行性心肌重塑。该项目将通过在转录和蛋白质水平研究心肌保护参数来研究低氧预适应(HP)介导的心肌重构的分子机制(S)。我们在大鼠心肌梗死模型(10%O2/90%NS)中建立了最佳的低氧预适应刺激，以启动心肌毛细血管/小动脉的形成，增加血流量和心功能。我们将检测(I)血管内皮生长因子及其酪氨酸激酶受体VEGFR1(Flt-1)和VEGFR2(FIK-1)的表达和活性，蛋白激酶C的表达和活性；(Ii)iNOS/eNOS的表达和活性将与NFkB、STAT1-Aim 2对这些因子的转录调控一起确定(Iii)内皮细胞(EC)存活的程度和坏死/凋亡的程度，抗凋亡蛋白Bcl2，Survivin的表达，PI-3-Kinase活性，AKT/BAD磷酸化的程度-Aim 3。本实验建立的大鼠急性心肌梗死模型，在LAD结扎前给予HP处理，对研究数月来心肌重构的分子机制具有显著的优势。通过Flk1、iNOS-/-和eNOS-/-基因敲除小鼠，可以确定Fik-1、iNOS和eNOS在血管内皮生长因子介导的心肌血管生成/重塑信号转导中所起的重要作用。用BrdU掺入法检测内皮细胞的增殖，用双抗体染色检测心肌细胞和内皮细胞的凋亡，用抗大鼠CD31和抗平滑肌肌动蛋白分别标记内皮细胞和平滑肌细胞来测定毛细血管密度和微动脉密度。本研究的结果将确定蛋白激酶C、MAP激酶、eNOS/iNOS/NO是否参与了血管内皮生长因子和/或受体(Fik-1/Fit-1)介导的心肌调节HP诱导的大鼠MI模型的重构。这一结果将为新的治疗策略提供必要的新信息，以保护心脏应激或冠心病患者的心脏。

DESCRIPTION (provided by applicant): Following myocardial infarction (MI), there is a progressive myocardial remodeling characterized by left ventricular (LV) dilation, contractile dysfunction, myocyte hypertrophy and increased matrix protein formation. The proposed project will examine the molecular mechanism(s) of hypoxic preconditioning (HP)- mediated myocardial remodeling in the infarcted heart by studying cardioprotective parameters at the transcriptional and protein level. We have established the optimal hypoxic preconditioning stimulus in rat MI model (10%O2/90%NS) to initiate capillary/arteriolar formation, increased blood flow and ventricular function in the myocardium. To establish the role of such HP in potentiating the signal transduction process for ventricular remodeling we will examine (i) the expression of VEGF and its tyrosine kinase receptors VEGFR1 (Flt-1) and VEGFR2 (FIk-1), expression and activity of protein kinase C, MAP Kinases-Aim I (ii) the expression and activity of iNOS/eNOS will be determined along with the transcriptional regulation of these factors by NFkB, Stat1-Aim 2 (iii) the extent of endothelial cell (EC) survival and the extent of necrosis/apoptosis, anti-apoptotic proteins Bcl-2, survivin expression, PI-3-Kinase activity, and the extent of AKT/BAD phosphorylation -Aim 3. Our rat MI model subjected to HP before LAD occlusion has significant advantage to study the molecular mechanism of myocardial remodeling over several months. An obligatory role of FIk-1, iNOS and eNOS in VEGF mediated signaling in myocardial angiogenesis/remodeling will be established by the use of Flk1, iNOS-/- and eNOS-/- knockout mice. The endothelial cell proliferation will be studied by BrdU incorporation assay, cardiomyocyte and endothelial cell apoptosis will be studied by double antibody staining, capillary and arteriolar density will be determined by labeling endothelial and smoothe muscle cells using anti-rat CD31 and anti-smooth muscle actin respectively. The results of this study will establish whether protein kinase-C, MAP kinases, eNOS/iNOS/NO are involved in VEGF and/or receptors (FIk-1/FIt-1) mediated myocardial regulation of HP induced remodeling in rat MI model. The results will provide new information required for new therapeutic strategies to protect the heart in patients with cardiac stress or coronary heart disease.