Ventricular Remodeling in the Adapted Heart

描述（由申请人提供）：心肌梗死（MI）后，存在进行性心肌重塑，其特征为左心室（LV）扩张、收缩功能障碍、肌细胞肥大和基质蛋白形成增加。 本研究拟从转录和蛋白质水平研究缺氧预适应（hypoxic preconditioning，HP）介导的心肌重构的分子机制。 本研究建立了大鼠心肌梗死模型（10%O_2/90%NS）的最佳低氧预适应刺激，以促进心肌毛细血管/小动脉的形成，增加心肌血流量和心室功能。 为了确定这种HP在增强心室重塑的信号转导过程中的作用，我们将检测（i）VEGF及其酪氨酸激酶受体VEGFR 1（Flt-1）和VEGFR 2（FIk-1）的表达，蛋白激酶C、MAP激酶-Aim I的表达和活性（ii）iNOS/eNOS的表达和活性将沿着NF κ B对这些因子的转录调节，Stat 1-目标2（iii）内皮细胞（EC）存活的程度和坏死/凋亡的程度、抗凋亡蛋白Bcl-2、存活素表达、PI-3-激酶活性和AKT/BAD磷酸化的程度-目标3。 我们的大鼠MI模型在LAD阻断前进行HP，对于研究心肌重塑的分子机制具有显著的优势。 将通过使用Flk-1、iNOS-/-和eNOS-/-敲除小鼠来确定Flk-1、iNOS和eNOS在心肌血管生成/重塑中VEGF介导的信号传导中的强制性作用。 BrdU掺入法检测内皮细胞增殖，双抗体染色法检测心肌细胞和内皮细胞凋亡，抗大鼠CD 31和抗平滑肌肌动蛋白分别标记内皮细胞和平滑肌细胞测定毛细血管和小动脉密度。 本研究的结果将确定蛋白激酶C、MAP激酶、eNOS/iNOS/NO是否参与VEGF和/或受体（FIk-1/Fit-1）介导的HP诱导的大鼠MI模型心肌重构的调节。 研究结果将为保护心脏应激或冠心病患者心脏的新治疗策略提供新的信息。

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.