Ventricular Remodeling in the Adapted Heart

描述（由申请人提供）：心肌梗死（MI）后，存在进行性心肌重构，其特征为左心室（LV）扩张、收缩功能障碍、心肌细胞肥大和基质蛋白形成增加。该项目将通过研究转录和蛋白水平的心脏保护参数，研究缺氧预处理（HP）介导的心肌重构在梗死心脏中的分子机制。我们在大鼠心肌梗死模型中建立了最佳缺氧预处理刺激（10%O2/90%NS），以启动毛细血管/小动脉形成，增加心肌血流量和心室功能。为了确定这种HP在增强心室重构信号转导过程中的作用，我们将检查(i) VEGF及其酪氨酸激酶受体VEGFR1 （Flt-1）和VEGFR2 （FIk-1）的表达，蛋白激酶C， MAP激酶- aim的表达和活性；（ii） iNOS/eNOS的表达和活性将与NFkB对这些因子的转录调节一起确定。Stat1-Aim 2 （iii）内皮细胞（EC）存活程度、坏死/凋亡程度、抗凋亡蛋白Bcl-2、survivin表达、pi -3-激酶活性、AKT/BAD磷酸化程度-Aim 3。我们的大鼠心肌梗死模型在LAD闭塞前经HP处理，在数月时间内研究心肌重构的分子机制具有显著优势。通过使用Flk1、iNOS-/-和eNOS-/-敲除小鼠，将建立FIk-1、iNOS和eNOS在心肌血管生成/重构中VEGF介导的信号传导中的必要作用。BrdU掺入法研究内皮细胞增殖，双抗体染色研究心肌细胞和内皮细胞凋亡，分别用抗大鼠CD31和抗平滑肌肌动蛋白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）介导的心肌调控HP诱导的心肌重构大鼠心肌模型。该结果将为心脏应激或冠心病患者的心脏保护提供新的治疗策略所需的新信息。

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.