INTRACELLULAR ASSEMBLY AND TARGETING OF SIGNALING MOLECULES IN HEART FAILURE

在心力衰竭中，细胞内和细胞间心肌信号通路的分子调控经常受到严重干扰。心肌细胞中的许多信号蛋白，包括G蛋白、G蛋白偶联受体、钙调节蛋白和一氧化氮合酶，都定位于肌膜小窝。心肌细胞小窝是肌膜高度特化的内陷，形成组织和调节肌节钙转运的T管系统。肌细胞小窝含有小窝蛋白-3，这是一种跨膜蛋白，为许多信号蛋白的定位提供了支架。在SCOR的最初资助期间，我们发现内皮型一氧化氮合酶(ENOS)在心肌细胞中表达，其活性受其与小窝蛋白-3相互作用的调节。在这些研究中要检验的中心假设是，一氧化氮合酶和其他关键的针对小凹的信号蛋白在心力衰竭中受到异常调节。在目标1中，我们将确定正常和衰竭心脏的心肌细胞小窝的组成和调节，并在体内和体外研究受体激活后存在于心肌细胞小窝中的信号蛋白。我们将使用激光共聚焦显微镜对小窝靶向信号蛋白进行细胞成像，并使用新开发的荧光染料二氨基荧光素确定心肌细胞中NO合成的细胞内位置。内皮型一氧化氮合酶和诱导型一氧化氮合酶都将在此背景下进行研究，分析诱导型一氧化氮合酶的定位可能为了解一氧化氮在全身脓毒症心肌抑制中的作用提供新的信息。这些对小窝成分的细胞成像研究将在其他SCOR研究人员正在研究的小鼠心力衰竭模型中进行分析。在目标2中，我们将进行荧光共振能量转移实验，以探索心肌细胞来源的NO、小窝蛋白和T管中钙结合调节蛋白之间的相互作用。在目标3中，我们将探索小窝蛋白-3在调控心肌中NO依赖的信号通路中的作用；这些研究可能对与肌营养不良综合征相关的心肌病的病理生理学有更深入的了解。在目标4中，我们将通过程序性电刺激和药物注射，以及eNOS/空小鼠的动态脑电监测，包括心率变异性分析，来表征eNOS/空小鼠的电生理表型。由于我们已经证明eNOS在体外对心肌细胞搏动频率的自主神经控制具有重要的调节作用，目标4的体内研究可能为心力衰竭导致心源性猝死的分子机制提供新的见解。

In heart failure, the molecular regulation of intracellular and intercellular myocardial signaling pathways is often profoundly perturbed. Many signaling proteins in cardiac myocytes, including G proteins, G protein- coupled receptors, calcium-regulatory proteins, and nitric oxide synthase- are localized in sarcolemmal caveolae. Cardiac myocyte caveolae represent highly specialized invaginations of the sarcolemma, and form the T-tubular system that organizes and regulates sarcomere calcium delivery. Myocyte caveolae contain the protein caveolin-3, a transmembrane protein that serves a scaffold for the localization of many signaling proteins. During the initial funding period of this SCOR, we discovered that the endothelial isoform of nitric oxide synthase (eNOS) is expressed in cardiac myocytes and that its activity is regulated by its interactions with caveolin-3. The central hypothesis to be tested in these studies is that nitric oxide synthase and other key caveolae-targeted signaling proteins are aberrantly regulated in heart failure. In Aim 1, we will determine the composition the composition and regulation of cardiac myocyte caveolae in normal and failing hearts, and characterize the signaling proteins present in cardiac myocyte caveolae following receptor activation in vivo and in vitro. We will perform cellular imaging of caveolae-targeted signaling proteins using confocal laser microscopy, and identify the intracellular sites of NO synthesis in cardiac myocytes using the newly developed fluorescent dye, diaminofluorescein. Both eNOS and iNOS will be studied in this context, analysis of iNOS localization may provide new information on the role of NO in myocardial depression in systemic sepsis. These cellular imaging studies of caveolae constituents will be analyzed in the murine heart failure models being studied by other SCOR investigators. In Aim 2, we will conduct fluorescence resonance energy transfer experiments to explore interactions between cardiac myocyte-derived NO, caveolin and Ca++-binding regulatory proteins in T-tubules. In Aim 3, we will explore the role of caveolin-3 in regulation of NO-dependent signaling pathways in the myocardium; these studies may yield insights into the pathophysiology of cardiomyopathies associated with muscular dystrophy syndromes. In Aim 4, we will characterize the electrophysiological phenotype of eNOS/null mice using programmed electrical stimulation and drug infusions, as well as ambulatory EGG monitoring in eNOS/null mice, including heart rate variability analysis. Since we have shown that eNOS importantly modulates the autonomic control of myocyte beating rate in vitro, the in vivo studies in Aim 4 may provide new insights into the molecular mechanisms of sudden cardiac death in heart failure.