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传导途径的分子调节常常受到严重干扰。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，都位于肌膜小窝中。心肌细胞小窝代表肌膜的高度特化内陷，并形成组织和调节肌节钙递送的T-管系统。肌细胞小窝含有蛋白质小窝蛋白-3，一种跨膜蛋白，其为许多信号蛋白的定位提供支架。在SCOR的最初资助期间，我们发现一氧化氮合酶（eNOS）的内皮亚型在心肌细胞中表达，其活性受其与小窝蛋白-3的相互作用的调节。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小窝靶向信号蛋白在心力衰竭中受到异常调节。目的1：研究正常和衰竭心脏中心肌细胞小窝的组成、组成和调控，并在体内和体外研究受体激活后心肌细胞小窝中存在的信号蛋白。我们将使用共聚焦激光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diaminofluorescein识别心肌细胞中NO合成的细胞内位点。eNOS和iNOS的研究将在此背景下，iNOS的定位分析可能会提供新的信息，在全身性脓毒症心肌抑制的NO的作用。这些小窝成分的细胞成像研究将在其他SCOR研究者正在研究的小鼠心力衰竭模型中进行分析。目的2：通过荧光共振能量转移实验，探讨心肌细胞源性NO、小窝蛋白和T小管Ca++结合调节蛋白之间的相互作用。在目标3中，我们将探讨小窝蛋白-3在心肌NO依赖性信号通路的调节中的作用;这些研究可能会深入了解肌营养不良综合征相关心肌病的病理生理学。在目标4中，我们将使用程序化电刺激和药物输注以及eNOS/null小鼠的动态EGG监测（包括心率变异性分析）来表征eNOS/null小鼠的电生理表型。由于我们已经证明eNOS在体外重要地调节心肌细胞搏动速率的自主控制，因此Aim 4中的体内研究可能为心力衰竭中心脏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.